WO2018161921A1 - 一个表观遗传操作植物表型可塑性性状的方法 - Google Patents

一个表观遗传操作植物表型可塑性性状的方法 Download PDF

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WO2018161921A1
WO2018161921A1 PCT/CN2018/078295 CN2018078295W WO2018161921A1 WO 2018161921 A1 WO2018161921 A1 WO 2018161921A1 CN 2018078295 W CN2018078295 W CN 2018078295W WO 2018161921 A1 WO2018161921 A1 WO 2018161921A1
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seq
gene
nucleotide sequence
medium
aaice1
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French (fr)
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强胜
解洪杰
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南京农业大学
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Priority to CN201880016443.6A priority Critical patent/CN110637087B/zh
Priority to EP18764709.4A priority patent/EP3594349A4/en
Publication of WO2018161921A1 publication Critical patent/WO2018161921A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • COR cold-regulated gene
  • ICE1 is in a passivated state at room temperature due to phosphorylation, dephosphorylation is activated under low temperature conditions, and specifically binds to the MYC site on the promoter of CBF, thereby inducing expression of CBF.
  • the method comprises: quantitatively replacing the methylated cytosine site of the target gene by degenerate codon, quantitatively controlling the number of sites in which the gene can be remethylated in the organism, and quantitatively regulating the methylation of the target gene.
  • Degree and gene expression level the purpose of quantitative regulation of plant plasticity traits.
  • the plasticity property is preferably cold resistance.
  • the nucleotide sequence of the banana MabICE1 gene is SEQ ID NO: 12
  • the nucleotide sequence of the cocoa TcICE1 gene is SEQ ID NO: 13
  • the nucleotide sequence of the ⁇ CtICE1 gene is SEQ ID NO: 14
  • the nucleotide sequence of the grape VvICE1 gene is SEQ ID NO: 15
  • the nucleotide sequence of the apple MdICE1 gene is SEQ ID NO: 16
  • the nucleotide sequence of the mustard CbICE1 gene is SEQ ID NO: 17
  • the nucleotide sequence of the eggplant ScICE1 gene is SEQ ID NO: 18, tomato SlICE1
  • the nucleotide sequence of the gene is SEQ ID NO: 19
  • the nucleotide sequence of the peach PpICE1 gene is SEQ ID NO: 20
  • the nucleotide sequence of the maize ZmICE1 gene is SEQ ID NO: 21
  • the AaICE1 (DC) 1 gene SEQ ID NO: 3 was obtained after partial replacement, and the AaICE1 (DC) 2 gene SEQ ID NO: 4 was partially replaced.
  • the target gene was ligated into pBI121 by restriction endonuclease BsaI, SmaI cleavage vector pBI121 and AaICE1, AaICE1 (DC)1, AaICE1 (DC)2 gene, and the Kan resistance gene in pBI121 was replaced with Bar gene as resistance screening.
  • the banana embryogenic suspension cell line (ECS) was used as the transforming receptor, and the banana ECS was subcultured for 10 days. After centrifugation to remove the supernatant, 40 ml of the AaICE1 gene with different methylation degree was added to each cell volume of ECS.
  • the medium is divided into two periods of medium.
  • the first stage medium is PEC (1/2DKW+KNO 3 0.3g/L+AA 1000X stock solution lml/L). +Glucose 20g/L+Sucrose 10g/L+1% agar powder).
  • the culture condition is light/darkness of 16/8h per day, and the medium is changed every 30 days until After growing buds with 1-2 true leaves, transfer to the second stage of RD medium (1/2DKW+KN0 3 0.3g/L+Glucose l0g/L+Sucrose 5g/L+ different concentrations of IBA 1.0mg/ L+ different concentrations of IAA 1.0mg/L, the culture conditions were consistent with the first stage, and the medium was transferred every 30 days until the seedlings were formed.
  • AAICE1, AaICE1(DC)1, AaICE1(DC)2 gene were obtained in this experiment. Plants 12, 10 and 9 strains were screened by cold-resistant washing, and the cold tolerance of the target trait plants was increased by 1.5 °C, 3.9 °C and 2.2 °C, respectively.
  • Example 6 Obtaining AaICE1 Gene Rubber with Different Degree of Methylation of Eupatorium adenophorum
  • the callus was cultured on the callus induction medium of Ding, and cultured at 25 ° C for 7 days.
  • the growth-recovered callus was transferred to a callus induction medium supplemented with 10 mg/L silver nitrate, 5 mg/L bialaphos and 50 mg/L teicin to induce somatic embryogenesis.
  • the proliferating somatic embryos were transferred to a plant regeneration medium supplemented with 5 mg/L of bialaphos to induce plant regeneration.
  • 10, 6, and 9 rubber-resistant plants transgenic with AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes were obtained, respectively.
  • the cold resistance of the target trait rubber plants was increased by 1.6 ° C, 2.9 ° C and 2.1 ° C compared with the wild type rubber trees.
  • the epicotyl of the aseptic lemon seedlings of 30d seedlings was taken as explants, and the epicotyls were cross-cut into about 1cm and inoculated in Agrobacterium liquid for 15min, with slight oscillation during the period.
  • the filter paper was blotted dry on the hypocotyl, and placed horizontally in an additional 100 uM acetosyringone BR medium (MT + 1.0 mg / L BAP + 0.1 mg / L NAA + 3% Sucrose) co-culture in the dark at 27 ° C 3d.
  • the co-cultured epicotyls were washed once with sterile water for 4 times and then washed once with 400 mg/L of cephalosporin.
  • the plants were transplanted into a beaker containing a mixture of sterilized humus and vermiculite, covered with a gas permeable membrane, and the gas permeable membrane was gradually removed, and then transplanted into a pot containing nutrient soil.
  • 8, 8, and 6 lemon-resistant plants transgenic with AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes were obtained, respectively.
  • the cold resistance of the target trait lemon plants was increased by 1.6 ° C, 3.2 ° C and 2.3 ° C, respectively, compared with the wild type lemon.
  • MT co-culture medium (MT + 40g / L sucrose + 8g / L agar 10 20mg / L acetosyringone), co-cultured in the dark at 21 ° C for 3 days, the explants were rinsed 3 times with sterile water, filter paper suction After drying, the cells were transferred to MT minimal medium (MT + 40 g / L sucrose + 8 g / L agar + 400 mg / L cephalosporin) containing 400 mg / L cephalosporin, and hairy roots were induced in the dark at 26 ° C.
  • MT minimal medium MT + 40 g / L sucrose + 8 g / L agar + 400 mg / L cephalosporin
  • the citrus ripe fruit was broken, the seeds were taken out, and the pectin on the surface of the seed was removed by soaking for 1 min with 1 mol/L NaOH, and then rinsed with tap water; soaked in 70% alcohol for 2-3 min on a clean bench, soaked with 2% sodium hypochlorite Sterilize for 20 min, shake several times in the middle, discard the sodium hypochlorite solution, wash it with sterile water for 3 times for 5 min each time; peel the above-mentioned sterilized seeds on the ultra-clean workbench and remove the inner and outer seed coats, and place them in the MT culture.
  • the cells were incubated at 26 ° C for 20-30 days in a base tube.
  • the stainless steel long handle spoon was used to scrape the cultured Agrobacterium, and inoculated in 40 ml MT suspension medium (MT + malt extract 0.5 g / L + glutamine 1.5 g / L, pH 5.7), shake culture at 28 ° C, 180 rpm 2h.
  • the above cultured Agrobacterium liquid was diluted with an MT suspension medium to an OD600 of 0.8 for use. Place the appropriate amount of explants into the above Agrobacterium liquid (containing 100umol/L AS), infuse for 20min (slightly shake several times in the middle), pour out the bacterial solution, and put the explant material out.
  • the bacteria filter paper On the bacteria filter paper, blot the bacteria solution attached to the above, and transfer the material to the co-culture medium (MT+100uM AS+0.4mg/L 2,4-D, PH 5.7).
  • the medium is covered with a layer of filter paper. After dark culture at 22 ° C for 3 days, it was transferred to a screening medium for culture. After 4 months of screening, the regenerated adventitious buds (about 1 cm long) were cut down for greenhouse grafting.
  • Genetically transformed experiments obtained 10, 13, and 9 citrus-resistant plants transgenic with AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes, respectively. After low temperature screening, the transgenic plants with the desired traits were obtained. Compared with wild-type citrus, the cold tolerance of the trait traits increased by 1.8 °C, 3.7 °C and 2.3 °C, respectively.
  • Example 10 Obtaining different degrees of methylation of E. adenophora AaICE1 gene sugarcane
  • the field material is used as the explant, and the healthy sugarcane plant is selected and grown, and the tail part is taken.
  • the outer old leaf is peeled off layer by layer, and the young leaf tissue about 10 cm away from the top growth point is taken as the explant, 70% first.
  • MS+2,4-D lmg/L+sucrose 30g/L, pH5.8 solid medium (M1) was used as the embryogenic callus induction medium, and dark culture was carried out at 25 °C. A single colony was picked from the Agrobacterium-preserved plate and inoculated into a liquid medium of 5 ml of YEP (containing 5 mg/L of bialaphos, Rif 25 ug/m1), and cultured at 28 ° C, shaking at 200 rpm to a logarithmic growth phase (about 24 h).
  • YEP containing 5 mg/L of bialaphos, Rif 25 ug/m1
  • the culture was expanded to a 40 ml 1 YEP medium containing the same antibiotic in a 150 ml cup, cultured until the OD600 was about 0.6, transferred to a centrifuge tube, centrifuged at 5000 ° C for 5 min at 4 ° C, the supernatant was discarded, and the residue was blotted.
  • the bacterial Vir gene expression is induced as a bacterial stock solution for infecting the transformation material. Dark cultures were vigorously grown on M1 medium, and the homogenous tissues were transferred to fresh M1 medium for 4 days, and then used for transformation experiments. During the transformation, the callus was first placed on a sterile filter paper and dried on a clean bench for 1-2 hours until the surface of the tissue block was dried. With a slight contraction, it can be used for bacterial infections.
  • Example 11 Obtaining AaICE1 Gene Papaya with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to papaya genetic transformation, and introduced into papaya by Agrobacterium infection.
  • the frozen strain was smeared on a plate supplemented with 5 mg/L of bialaphos and 100 mg/L streptomycin, and then the colony was picked up to 30 ml LB liquid medium at 28 ° C, shaken at 225 rpm for 20 h, and centrifuged at 5000 rpm for 10 min to collect the cells.
  • the cells were resuspended in 30 ml of modified MS liquid medium supplemented with 100 umol/L eugenol, and the shaking was continued for 3 h, and then the broth was diluted with a modified MS liquid medium to a suitable OD600 concentration (0.6) for use.
  • the papaya young embryos pre-cultured for one month were immersed in the bacterial liquid for 20 min, and shaken several times. Then, the callus was blotted dry with a sterile filter paper, and dried in a clean bench to be inoculated. The medium was co-cultured at 25 ° C for 3 days. The callus and young embryos co-cultured for 3 days were washed 6 times with sterile water, the surface-attached cells were removed as much as possible, and excess water was blotted and inoculated with 5 mg/L bialaphos and 300 mg/L carbenicillin.
  • the selective medium was subcultured every 20 days to remove browning dead explants, and the concentration of carbenicillin was gradually reduced, and dark culture was carried out at 25 ° C to induce somatic embryogenesis.
  • 19, 23, and 21 papaya resistant plants transgenic with AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes were obtained, respectively.
  • the transgenic plants with the desired traits were obtained after low temperature screening.
  • the cold tolerance of the papaya plants with the target traits increased by 1.9 °C, 3.8 °C and 2.6 °C, respectively, compared with the wild type papaya.
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed into sputum by Agrobacterium infection.
  • Agrobacterium tumefaciens was smeared 2 days before infection, and cultured at 28 ° C for 48 h. The Agrobacterium was used for transformation in the logarithmic growth phase. The Agrobacterium tumefaciens colonies were scraped and suspended in a liquid medium, and shaken vigorously for 1 min, and then allowed to stand for 1-2 h to allow the Agrobacterium to form a suspension. Adjust the concentration of Agrobacterium, make the OD600 value of the bacterial liquid 1.0, add the EC of the pre-culture for 10-15d, let stand for 30min after shaking slightly, put it on the sterile filter paper, blot the water attached to the callus, and then transfer it. Solid medium was co-cultured.
  • the callus was picked in a triangular flask and rinsed 3 times with sterile water. The last time was washed with chloramphenicol sterile water (300 mg/L, the same below) and allowed to stand for 1 h. The Agrobacterium adhering to the callus is spread into the water, then washed once with chloramphenicol in sterile water, placed on a sterile filter paper, and the water attached to the callus is blotted and transferred to the screening medium.
  • chloramphenicol sterile water 300 mg/L, the same below
  • the expression vectors of different degrees of methylation AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to lychee genetic transformation, and introduced into lychee by Agrobacterium infection.
  • Lychee is a "yuan red” variety. Agrobacterium was stored on solid LB medium and subcultured every 1 to 2 months. Before the transformation, the single colony was placed in liquid LB medium supplemented with the corresponding antibiotic, and cultured at 28 ° C until the logarithmic growth phase, the bacterial solution was centrifuged at 4000 rpm for 10 min, resuspended in liquid callus growth medium and diluted 10 times for use. The embryogenic callus subcultured for 15 days was dried on sterile filter paper for 1 h, then placed in the prepared bacterial solution, and allowed to stand for 10 min. The sterile filter paper was used to absorb the bacterial liquid on the surface of the callus and then transferred to co-culture.
  • the medium (MS minimal medium supplemented with 2,4-D 2 mg/L, STS 5 mg/L, sucrose 50 g/L, agar 7 g/L, pH 5.8) was co-cultured for 2 days. After co-cultivation, the callus was washed 3 times with sterile water, and the liquid Zl medium was resuspended, placed on a sterile filter paper, and transferred to a Z1 medium supplemented with a certain concentration of antibiotics and antibiotics for selective culture. After 7 weeks, Resistant clones were transferred to the same selection medium for further screening.
  • MS basic medium supplemented with KT 1 mg/L, NAA 0.1 mg/L, glutamine 500 mg/L, sucrose 80 g/L agar 15 g/L, G418 100 mg/ L, 5mg/L bialaphos, Carb300mg/L, pH6.2
  • somatic embryo differentiation medium MS basic medium supplemented with KT 1 mg/L, NAA 0.1 mg/L, glutamine 500 mg/L, sucrose 80 g/L agar 15 g/L, G418 100 mg/ L, 5mg/L bialaphos, Carb300mg/L, pH6.2
  • somatic embryo differentiation medium embryoid body in mature medium
  • transfer to germination medium MS basic medium plus KT1mg/L, GA5mg/L, coconut milk 50m
  • Example 14 Obtainment of AeICE1 gene longan with different methylation degree of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to longan genetic transformation, and introduced into the longan by Agrobacterium infection.
  • the normal subcultured longan EC was inoculated on the subculture medium LMs4 for 10-15 days, and the light yellow fine granule longan EC was selected as the transgenic receptor material.
  • Agrobacterium tumefaciens was smeared 1 day before infection, and cultured at 28 °C for 24 h. The Agrobacterium was used for transformation in the logarithmic growth phase. The Agrobacterium tumefaciens colonies were scraped and suspended in a liquid medium, and shaken vigorously for 1 min, and then allowed to stand for 1 h to allow the Agrobacterium to form an aquic solution.
  • Adjust the concentration of Agrobacterium make the OD600 value of the bacterial liquid between 1-1.5, add the longan EC pre-cultured for 10-15d, let it stand for 30min after shaking slightly, and blot the water attached to the surface of the callus on the sterile pool paper. Transfer to solid medium for co-cultivation. After co-cultivation for 5 days, when the Agrobacterium grows to the plaque under the visible callus but does not grow over the callus, the callus is picked in a sterile flask and rinsed 5 times with sterile water.
  • Example 15 Obtaining AaICE1 Gene Mango with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to mango genetic transformation, and introduced into mangoes by Agrobacterium infection.
  • a single Agrobacterium colony was picked from the YEP plate with a sterile toothpick, inoculated in a 25 mg/L rifampicin YEP liquid medium, placed on a 28 ° C constant temperature shaker, shaken at 220 rpm for 24 h, and then added to the bacterial solution.
  • the new resistant YEP medium the conditions were cultured to logarithmic growth phase. The cells were precipitated by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the MS liquid medium was added and suspended for use. Take the 3-5 cm long young fruit on the mango tree, first wipe the surface of the young fruit with a cotton ball soaked in alcohol to remove surface bacteria.
  • Example 16 Obtaining AaICE1 Gene Potato with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed into potatoes and introduced into potatoes by Agrobacterium infection.
  • the detoxified test tube seedlings subcultured for 30 days were cut into stem segments containing no axillary bud length of about 0.5 cm, and precultured for 2 days.
  • the stem segments were inoculated with the spare bacterial solution for 10 min, co-cultured for 2 days, and then inoculated into callus induction medium (MS + 0.1 mg / L 2, 4-D + 1 mg / L BA + 5 mg / L bialaphos + 400 mg) /L cefotaxime sodium), after 10-20 days of culture, select uncontaminated callus and transfer to bud induction medium (MS+1mg/L ZT+1mg/L GAS+5mg/L bialaphos +400mg/ L cefotaxime sodium).
  • the obtained regenerated plants were inoculated into rooting medium for rooting culture.
  • Genetically transformed experiments obtained 15, 18, and 19 potato-resistant plants transgenic with AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes, respectively.
  • After low temperature screening transgenic plants with the desired traits were obtained.
  • the cold resistance of the target traits of potato plants increased by 1.5 °C, 3.4 °C and 2.7 °C, respectively.
  • Example 17 Obtaining AaICE1 Gene Pepper with Different Methylation Degree of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to genetic transformation of pepper and carried out by Agrobacterium infection.
  • a single Agrobacterium colony was picked from the YEP plate with a sterile toothpick, and inoculated in a liquid medium supplemented with 5 mg/L bialaphos and 25 mg/L rifampicin YEP, placed on a 28 ° C constant temperature shaker, shake culture at 220 rpm. After 24 h, the bacterial solution was added to the new resistant YEP medium and cultured to the logarithmic growth phase under the same conditions. The cells were precipitated by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the MS liquid medium was added and suspended for use.
  • the 12-day-old pepper was used as a transforming receptor for the sterile hypocotyl explants in the PD pre-culture medium (MB5+3mg/L 6-BA+1mg/L IAA+4mg/L AgN03+30g/L sucrose+ Pre-culture for 2 days on 5g/L agar), the explants were placed in liquid MS, and then transferred to the prepared Agrobacterium liquid, and immersed for 5-14 min. After the explants were drained on the sterilized filter paper, they were cultured in dark on PD medium, and 10 explants were placed in each dish, with the front side facing up.
  • the PD pre-culture medium M5+3mg/L 6-BA+1mg/L IAA+4mg/L AgN03+30g/L sucrose+ Pre-culture for 2 days on 5g/L agar
  • the explants were cultured on PD for 1-4 days at 25 ° C in the dark, then transferred to PD (containing 500 mg / L carbenicillin) selection medium for selective culture for 1-4 d, and finally transferred to PD medium ( Selective culture was carried out on a medium containing 5 mg/L of bialaphos and 500 mg/L of carbenicillin.
  • explants that differentiated adventitious buds were placed in SE medium (MB5+3mg/L 6-BA+1mg/L IAA+2mg/L GA3+4mg/L) AgN03 + 30g / L sucrose + 5g / L agar + 5mg / L bialaphos + 500mg / L Carb) on the bud elongation.
  • SE medium M5+3mg/L 6-BA+1mg/L IAA+2mg/L GA3+4mg/L
  • AgN03 30g / L sucrose + 5g / L agar + 5mg / L bialaphos + 500mg / L Carb
  • the adventitious buds grow to 2-3 cm, the elongated shoots are cut and transferred to SR (1/2MS + 0.2 mg / L IAA + 0.1 mg / L NAA + 5 mg / L bialaphos + 200 mg / L Cef) rooting.
  • Example 18 Obtaining AaICE1 Gene Sorghum with Different Methylation Degrees of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed by sorghum infection by Agrobacterium infection.
  • the appropriate amount of the preservation solution was inoculated into 20 ml of YEP night body medium, and cultured at 28 ° C and 200 rpm for 24 to 30 hours.
  • the appropriate amount of the first activated bacterial solution was inoculated into 40 ml of YEP liquid medium, and cultured at 28 ° C and 200 rpm to an OD600 value of 0.6.
  • the cells were collected by centrifugation at 6000 rpm for 10 min. 500 ul of acetosyringone and 10 ul of sillwet L-77 were added to each 50 ml of sterile water.
  • the cells obtained by centrifugation were suspended in this solution to have an OD600 value of about 0.6.
  • the sorghum seeds were immersed in 70% alcohol for 1 min, washed once with sterile water, transferred to 0.1% liters of mercury, shaken on a shaker at 150 rpm, and sterilized for 10 minutes. Finally rinse with sterile water more than 6 times. Place the sterilized seeds in a 100m1 conical flask. The amount of seeds per bottle is 100-200. Add appropriate amount of sterile water to shake at 150rpm on the shaker. After 24 hours, the seeds will germinate and grow. A bud of about 1 cm. At this time, the conical flask containing the seed was taken out and subjected to the scoring treatment.
  • the infested transformed seeds were placed in a petri dish coated with filter paper, placed in a 25 ° C culture chamber, and cultured in the dark for 48-96 hours.
  • Ticarcillin (160 mg/L) and cefotaxime sodium (150 mg/L) were added to the sterile water, and the seeds were placed in a 100 mI Erlenmeyer flask and washed more than 4 times for at least 4 minutes each time.
  • the cells were placed in a petri dish and cultured in a 25 ° C culture chamber (14 h light/10 h dark).
  • the seeds are transferred to the Hoagland medium for liquid culture, and the seedlings are cultured to the 2-4 leaf stage.
  • the seedlings are transplanted into paper cups and placed in a greenhouse for routine growth management.
  • 15, 13, 17 sorghum-resistant plants transgenic with AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes were obtained, respectively.
  • transgenic plants with the desired traits were obtained.
  • the traits of sorghum plants increased by 1.8 °C, 3.9 °C and 3.0 °C, respectively.
  • Example 19 Obtaining different degrees of methylation of E. adenophora AaICE1 gene grape
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to genetic transformation of grapes, and introduced into grapes by Agrobacterium infection.
  • the inferred grape PEM was subcultured onto fresh X6 medium and pre-incubated for one week in a dark environment at 26 ° C for transgenic manipulation.
  • 200 ⁇ l of the bacterial solution was inoculated into 20 ml of liquid LB medium (containing 5 mg/L of bialaphos).
  • 20 ⁇ l of the bacterial solution was inoculated into 20 ml of liquid LB medium (containing 5 mg/L of bialaphos), and subjected to secondary activation culture for 20 hours under the same conditions.
  • the grape PEM was inoculated into a triangular flask containing Agrobacterium liquid for 20 min, and the surface bacterial liquid was blotted with a sterile filter paper, which was subcultured into a two-layer sterile filter paper and added with about 5-6 m of X6 liquid culture.
  • Base (with 100 ⁇ M AS) in a sterile Petri dish. After co-cultivation for 48 h in dark conditions at 26 ° C, the cells were detoxified with cephalosporin and carbenicillin at a concentration of 200 mg/L for 20 min, then rinsed with sterile water for 3 times, and the grape PEM was inoculated to MS + 5 mg/L.
  • Example 20 Obtaining AaICE1 Gene Apple with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to apple genetic transformation using an ultrasonic transformation method.
  • the apple tube seedling leaves were cut into pieces of about 4 mm 2 and placed in a container containing 20 ml of ultrasonic buffer.
  • the composition of the ultrasonic buffer was: 13.7 mmo1/LNaCI, 2.7 mmo1/LKCI, 10 mmol/L Na 2 HPO 4 , 2 mmo1/LKH. 2 POP 4 , 5% DMSO, pH 7.4. Each milliliter of buffer contained 5 ug of plasmid DNA. Ultrasonic treatment was carried out for 20 min at a sound intensity of 0.5-1.0 W/cm 2 .
  • the sonicated leaves were transferred to MS regeneration medium containing 4 mg/L 6-BA+0.2 mg/L NAA for 1 week, and then transferred to a 5 mg/L bialaphos regeneration medium for 2 weeks of dark culture. Screening was performed once every 2 weeks and transferred to MS subculture medium of 0.5 mg/L 6-BA + 0.1 mg/L NAA after 2 months for a total of 3 months. Culture conditions: 16 hours of continuous light per day, light intensity of 40umol m -2 s -1 constant temperature 25 ° C subculture. Once every 4 weeks. 12, 13, and 10 AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 gene resistant plants were obtained from 300 explants. After low temperature screening, transgenic plants with the desired traits were obtained, and their cold tolerance was increased by 1.8 °C, 4.1 °C and 2.5 °C, respectively, compared with wild type plants.
  • Example 21 Obtaining of AaICE1 Gene Mustard with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of different degrees of methylation AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to islet genetic transformation, and introduced into mustard by Agrobacterium infection.
  • the solution was resuspended in 20 ml of pre-cooled MS liquid medium and used.
  • the pre-cultured hypocotyls were inoculated with the resuspended bacterial solution for 10 min, dried on filter paper, and transferred to differentiation medium for 2 days.
  • Transfer the hypocotyls after co-culture to the screening medium MS+2mg/L 6-BA+0.2mg/L NAA+5mg/L bialaphos+400mg/L Cb+3% sucrose+0.6% agar
  • the resistance screening was performed, and the screening medium was changed every 10 days.
  • Rooting culture is carried out. After the roots of the tissue culture seedlings are strong, the residual medium on the roots is washed, transplanted into the nutrient soil, and placed in an artificial climate chamber for cultivation. In the genetic transformation experiments, 18, 15, and 21 resistant plants transgenic to AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 genes were obtained, respectively. After low temperature screening, transgenic plants with the desired traits were obtained, and their cold tolerance was increased by 1.9 °C, 3.4 °C and 2.4 °C, respectively, compared with wild type plants.
  • Example 22 Obtaining AaICE1 Gene Eggplant with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to genetic transformation of eggplant, and introduced into eggplant by Agrobacterium infection.
  • Eggplant seeds are soaked in 75% alcohol for 30s, rinsed once with sterile water, then soaked in 10% NaC1O solution for 20min, rinsed 3 times with sterile water, inoculated onto 1/2MS medium, germinated at 26 ° C, light / The sterile seedlings were cultured as dark materials (16h/8h).
  • An overexpression vector constructed from Agrobacterium tumefaciens strain EHA105, plasmid vector pBI121, and ScICE1 gene.
  • the sterile seedling leaves were selected, the leaf tips were removed, and the small pieces of uniform size of 0.5 mm 2 were cut and inoculated into the pre-culture medium MS+NAA 0.2 mg/L+ZT 1.2 mg/L, and the Agrobacterium invaded after 2 days of pre-culture. Dyeing and transformation, taking Agrobacterium liquid with an OD value of 0.5, the infestation time was 10 min.
  • the infected explants were inoculated into co-culture medium (1/2MS+NAA0.2mg/L+ZT1.2mg/L+200umo1/L acetosyringone), and after dark culture for 2d, 150mg/L was used.
  • Exemite was washed with sterile water, and the surface water was absorbed by sterile filter paper.
  • the cells were inoculated into MS+NAA 0.2mg/L+ZT 1.2mg/L+150mg/L Temetin medium.
  • the induced callus medium (0.1 mg/L NAA, 3.0 mg/L ZT). It was placed at 16 h during the day, 8 h at night, and cultured under 1601 x light conditions. The explants grow for about two weeks, and grow young, lumpy, hard green callus, which is inoculated into the budding medium (adding 0.1 mg/L NAA, 4.0 mg/L ZT, 1.5 mg/L).
  • Seedlings formed by bud elongation and differentiation produced 2-3 true leaves, which were cut from the base and inoculated into 1/2MS rooting medium to induce rooting.
  • the illumination time is 16h/d
  • the temperature is 24-27°C
  • the light intensity is 1600 1x.
  • the regenerated seedlings grown in the medium have poor adaptability to the environment.
  • the triangular flask should be opened for small mouth ventilation, and the ventilation should be gradually increased according to the growth of the seedlings to improve the adaptability of the seedlings to the environment. Two weeks later, the seedlings have 4-5 true leaves, three main roots, and several lateral roots, and the bottle mouth is opened.
  • Example 23 Obtaining of different methylation degree of E. adenophora AaICE1 gene kiwifruit
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed into kiwifruit and introduced into kiwifruit by Agrobacterium LBA4404.
  • the preserved Agrobacterium strain LBA4404 was taken, and the single colony of Agrobacterium was picked and streaked into 20 ml of liquid YEP medium, and shake cultured at 200 rpm and 28 ° C for 16-24 hours. Pipette 0.5 ml of the bacterial solution into 50 ml of YEP medium containing the same antibiotic, and expand the culture in the same manner as above. When the broth was cultured until the OD600 was 0.5, the pellet was centrifuged at 4000 rpm, 4 ° C for 10 min, and suspended in an equal volume with a liquid MS medium supplemented with 100 uM of acetosyringone.
  • the kiwifruit sterile seedlings were cut into 0.5 cm square pieces under sterile conditions and attached to medium supplemented with MS as the basic medium of 3.0 mg/L 6-BA and 1.0 mg/L NAA.
  • MS as the basic medium of 3.0 mg/L 6-BA and 1.0 mg/L NAA.
  • Pre-culture for 3d After 3 days, the pre-cultured leaves were placed in the resuspended bacterial liquid, during which slight agitation occurred, so that the Agrobacterium and the leaves were fully contacted. After 10 minutes of infiltration, the excess bacterial liquid was blotted with sterile filter paper, and the back of the leaves was placed down.
  • the culture flask When the rooting seedlings grow to 5cm, the culture flask is incubated for 3-5 days under natural conditions, then gradually open the cap, loosen the lid for the first two days, open the cap half for the third and fourth days, and take the fifth and sixth days. Lower the lid, cover with half the size of the cardboard cover, remove the cardboard for the seventh and eighth days, expose the tissue culture seedlings to the natural state, refine the seedlings for 10 days, carefully remove the new seedlings with tweezers, rinse the roots with tap water. The attached agar was transplanted to the greenhouse soil for cultivation. AiICE1, AaICE1(DC)1, AaICE1(DC)2 gene kiwifruit plants 8, 11 and 12 were obtained by genetic transformation. After low temperature screening, the cold tolerance of the target plants was increased by 2.0 °C, 3.6 °C and 2.7 °C, respectively.
  • Example 24 Obtaining AaICE1 Gene Peach with Different Degree of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to peach genetic transformation, and introduced into peaches by Agrobacterium GV3101 infection.
  • a single colony of Agrobacterium tumefaciens GV3101 containing this plasmid was picked and inoculated into a 4 ml liquid medium containing 5 mg/L of bialaphos, Rif 20 mg/L, Gem 40 mg/L in YEP, and cultured at 180 rpm at 28 ° C for 36 h.
  • +NAA 1.0 mg/L + 2% cane toad 7.5 g/L agar, pH 5.8), co-cultivation in dark at 26 °C.
  • the induced callus was introduced into a differentiation medium (MS+NAA 0.05 mg/L+BA 1.0 mg/L), the light intensity was 2000-3000 lx, and the light-dark cycle was cultured at 16 h/8 h, and the culture temperature was the same as above. After that, the adventitious buds were transferred to rooting medium (1/2MS+IBA 1.0 mg/L) for rooting culture, and rooting began after 5 days.
  • Example 25 Obtaining different degrees of methylation of E. adenophora AaICE1 gene maize
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed into maize, and introduced into maize by Agrobacterium GV3101 infection.
  • the recipient materials were sown in stages on a 7d cycle.
  • the corn samples were controlled for pollination according to the test requirements. After 10-13 days of pollination, the maize immature embryos were used as transformation receptors. After disinfecting the corn cob with sodium hypochlorite, the young embryos were peeled off, and the young embryos were washed 4 times with the infecting solution (plus AS), then a certain concentration of Agrobacterium liquid was added, placed for 15 min, taken out, blotted dry with sterile filter paper, and placed.
  • Co-cultivation medium was co-cultured at 25 ° C (dark) for 2-5 days, then the young embryos were transferred to resting medium and cultured at 28 ° C for 7 days in the dark.
  • the young embryos were transferred from a resting medium to a resistance screening medium containing a marker gene, and cultured in dark. Each successive generation of attention was taken to eliminate the brown and water-stained callus, and the normally growing callus was crushed with tweezers and cultured separately.
  • tissue block is inflated, the large tissue is often peeled off, and after successive subculture screening for 3 times, the selected resistant callus is transferred to the regeneration medium, and the culture is resumed for 15 days or longer (dark culture).
  • the resistant callus was transferred to a regeneration medium for germination, and the culture condition was 28 ° C, and the light was irradiated for 12 hours at a daily light of 3000 lx.
  • the seedlings When the corn plants to be regenerated grow to three leaves, the seedlings can be transplanted into a bottle containing rooting medium and cultured indoors. When the seedlings grow thicker roots, remove the seedlings from the cans, rinse the medium with water, and transplant them in small pots mixed with nutrient soil and vermiculite (1:3), when the corn grows again. When 2-3 new leaves are used, they can be transplanted into large fields or large flower pots. After growing the three or four leaves, the leaf DNA is extracted for PCR detection. The ZmICE1 gene containing the transfer was determined. In this experiment, 10, 13, and 12 strains of AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 gene resistant plants were obtained, respectively. After low temperature screening, transgenic plants with the desired traits were obtained. Under low temperature treatment at 6 °C, the cold tolerance of the plants was increased by 13 °C, 3.0 °C and 1.7 °C, respectively.
  • Example 26 Obtaining AaICE1 Gene Cotton with Different Degree of Methylation of Eupatorium adenophorum
  • the first and second fruit nodes of each fruit branch are typically selected for transgenic manipulation.
  • the cotton bolls on these fruit nodes generally have a higher bell rate, which is beneficial to harvest more seeds; a 50 ⁇ l micro-injector is used as a tool for microinjection.
  • it should be washed with a light detergent and then rinsed with distilled water; when injecting, remove or peel off the petals and smooth the style.
  • a plant that grows normally is considered to be a plant that acquires a resistance gene.
  • 15 13 and 17 strains of AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 gene resistant plants were obtained.
  • transgenic plants with the desired traits were obtained.
  • the cold tolerance was increased by 1.7 °C, 4.2 °C and 2.9 °C, respectively.
  • Example 27 Obtaining AaICE1 Gene Peanut with Different Degree of Methylation of Eupatorium adenophorum
  • the transgenic operation is carried out.
  • the flower buds are removed every day at about 5 pm, so as to prevent the progeny from pollinating the flowers without transgenic operation, and the next season to detect the transgenic plants in the offspring cause interference.
  • the glass needles drawn by the micropipette and the dropper were respectively introduced into the plasmid DNA containing the target gene by the method of column head application and flower Cao tube injection, and the two DNA concentrations of 50 ug/ml and 100 ug/ml were used to treat the oyster sauce 523. "and "Oyster 21" two varieties. After entering the flowering period, start operation at about 6:30 every morning.
  • the smear operation is to apply 5 ul of DNA solution to the flower stigma with a micropipette.
  • the injection method uses a glass needle to inject about 5 ul into the ovary in the lower part of the calyx tube. DNA solution.
  • Each variety is processed more than 500.
  • the peanut seeds obtained by genetic transformation were sprayed on the seedlings in the greenhouse, and the peanut leaves were sprayed at 5 mg/L of bialaphos in the 2-3 leaf stage. After one week, the growth of the plants was observed, and the plants with green spots on the surface of the leaves were removed. Plants that are normally grown are considered to be resistant genes, and resistant plants are verified by PCR.
  • Example 28 Obtainment of AeICE1 Gene Mustard in Different Degrees of Methylation of Eupatorium adenophorum
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to genetic transformation of mustard, and introduced into mustard by Agrobacterium tumefaciens GV3101 infection.
  • the mustard seeds were sterilized (75% alcohol and 0.1% liters of mercury) and sown in MS medium. After 7 days, hypocotyls (0.5-0.8 cm) were cut and placed in mustard differentiation medium (MS+2 mg/L 6-BA). +0.2 mg/L NAA + 3% sucrose + 0.6% agar) was pre-incubated for 2 days.
  • Agrobacterium transformed into the plasmid containing BjICE1 gene was activated, 500 ul of activated bacterial solution was inoculated, inoculated into 50 ml of antibiotic-free YEB liquid medium, and shaken at 225 rpm on a shaking bar at 28 ° C until the active growth period of Agrobacterium (OD600 value).
  • the pre-cultured hypocotyls were inoculated with the resuspended bacterial solution for 10 min, dried on filter paper, and transferred to differentiation medium for 2 days.
  • the co-cultured hypocotyls were transferred to the mustard screening medium (MS+2 mg/L 6-BA+0.2 mg/L NAA+5 mg/L bialaphos+400 mg/L Cb+3% sucrose+0.6% agar) The resistance screening was performed, and the screening medium was changed every 10 days.
  • Example 29 Obtaining different degrees of methylation of E. adenophora AaICE1 gene in Brassica napus L.
  • the expression vectors of different degrees of methylation AaICE1, AaICE1(DC)1 and AaICE1(DC)2 genes constructed in Example 1 were transformed into Brassica napus L., and pollen-mediated transgenic experiments were carried out by pollen-mediated method.
  • the flower buds to be opened in 10-15 1-2 days on the main stem or on one branch are selected to remove the male buds, and the other buds on the selected stems or branches are removed, and then the bag is bagged.
  • the open inflorescence was bagged on the second day for the second day.
  • the pollen of the bagged plants opened on the next morning was about 0.4g, suspended in 25ml of 7.5% sucrose solution, subjected to the first sonication, and then 20 ⁇ g of plasmid DNA containing different cold tolerance populations of BrICE1 gene was added to the solution. Sub-ultrasonic treatment.
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were subjected to chrysanthemum genetic transformation, and introduced into chrysanthemum by Agrobacterium infection.
  • the preserved Agrobacterium carrying the pCAMBIA1301 plasmid of the gene of interest was inoculated in a YEB liquid medium containing 50 mg/L of rifampicin overnight. Then, they were streaked on YEB plate medium containing 5 mg/L of bialaphos and 50 mg/L of rifampicin, placed in a 28 ° C incubator, and cultured until a single colony grows. Store in a refrigerator at 4 °C for later use.
  • the explants were basically in the developmental stage of PLB, and the explants were transferred to contain 5 mg/L of bialaphos.
  • the screening medium was used for preliminary screening of anti-positive transgenic plants. By this method, 11, 10, 10 strains of AaICE1, AaICE1 (DC) 1, and AaICE1 (DC) 2 gene resistant plants were obtained, respectively. After low temperature screening, the cold tolerance of the wild type Cymbidium hybridum was increased by 2.2 ° C, 3.9 ° C and 2.8 ° C, respectively.
  • Example 39 Obtaining different degrees of methylation of E. adenophora AaICE1 gene Phalaenopsis
  • the expression vectors of the different methylation degrees AaICE1, AaICE1(DC)1, and AaICE1(DC)2 genes constructed in Example 1 were genetically transformed into Lamei, and introduced into the plum by Agrobacterium infection.
  • the cells were cultured in a constant temperature culture chamber at a temperature of 25 ⁇ 2 ° C, artificial light for 13 h / d, and the light intensity was 1500-2000 lx. After 25 days of culture, the green callus callus was selected as an explant.
  • the preserved Agrobacterium tumefaciens EHA105 was lined on the prepared YEB solid medium plate, and darkly cultured at 28 ° C for 1-2 d. Then, a single colony was picked up with a loop to inoculate 5 ml of YEB liquid medium.
  • the wild-type and in situ-substituted Arabidopsis plants were cultured normally in the light culture chamber to 3 weeks after seedling age, cold-adapted for 3 days at 4 °C, placed at 0 °C for 1 hour, and placed at -4 °C for 4 hours, then dropped every hour. 2 ° C, until -8 ° C, after 24 h of treatment, cultured at 23 ° C for 5 d, observed the wild-type and in situ replacement of Arabidopsis plants, the low temperature injury, each treatment was repeated 3 times, statistical low temperature injury index after 5 days of culture .
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of MeICE1(DC)1 and MeICE1(DC)2 genes;
  • the SacII and AsiSI restriction sites were added at both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI.
  • the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector, and the cassava was introduced by Agrobacterium-mediated method. in.
  • the Agrobacterium GV3101 expression vector stored in a 500 ul ultra-low temperature refrigerator was added to 50 ml of YEB (5 mg/L bialaphos) liquid medium, and cultured at 28 ° C, 240 rpm for 18-24 hours. Centrifuge at 4 ° C, 4000 rpm for 10 min, dilute with liquid MS to an OD value of 1.0, stand for use. After the mature 10-15d cassava somatic embryo green cotyledons were chopped, pieces of about 500 cotyledon embryos were placed in 30 ml of Agrobacterium suspension culture medium with shaking several times.
  • the callus was transferred to a stem organogenesis medium containing 5 mg/L of bialaphos and 500 mg/L of carboxybenzylomycin, and cultured at 26 ° C for 1 week.
  • the callus was transferred to a stem organogenesis medium containing 5 mg/L of bialaphos and 500 mg/L of carboxybenzylomycin, and cultured at 26 ° C for 16 weeks in the light for 2 weeks.
  • the resistant adventitious buds were picked and placed on the stem elongation medium. Incubate at 26 ° C, 16 h light. After 2-3 weeks, the stem segments which were cut out were transferred to MS medium for culture, and when the plants grew, they were cut and subcultured.
  • a stem section of about 1 cm from the lateral buds was excised and transferred to a hormone-free medium containing 5 mg/L of bialaphos for rooting experiments to eliminate false-positive plants, and the stem segments of the wild-shaped plants were used as controls. After 1-2 weeks, transgenic positive plants can root normally, while false positives and controls cannot grow roots. In situ replacement of cassava MeICE1 (DC)1, MeICE1(DC)2 gene plants 15 and 13 strains was obtained, and the cold tolerance was increased by 4.0 °C and 3.0 °C, respectively, compared with wild type cassava.
  • Example 45 CRISPR/Cas9 technology in situ replacement of different methylation levels of AcICE1 gene kiwifruit
  • the kiwifruit AcICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 10. According to the degeneracy of the codon, the theoretically methylated site of the AcICE1 gene was replaced, and the AcICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the AcICE1 gene sequence was obtained as SEQ ID NO: 51 and designated as AcICE1 (DC) 1 after complete replacement; the partial replacement AcICE1 gene sequence was SEQ ID NO: 92 and designated AcICE1 (DC) 2.
  • AcICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and kiwifruit in situ replacement. Each segment was about 500bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GTGGTAGGAGGGCTCTTCCATGG-3. ',5'-GTGGCTGCAGAGAAAATCCAAGG-3' and 5'-GATATCCACCATGTTACCGGTGG-3'; the AcICE1(DC)2 gene sgRNA sequence is 5'-GATGGTTCAAATCTTGATGGTGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the kiwifruit sterile seedlings were cut into 0.5 cm square pieces under sterile conditions and attached to medium supplemented with MS as the basic medium of 3.0 mg/L 6-BA and 1.0 mg/L NAA.
  • MS as the basic medium of 3.0 mg/L 6-BA and 1.0 mg/L NAA.
  • Pre-culture for 3d After 3 days, the pre-cultured leaves were placed in the resuspended bacterial liquid, during which slight agitation occurred, so that the Agrobacterium and the leaves were fully contacted. After 10 minutes of infiltration, the excess bacterial liquid was blotted with sterile filter paper, and the back of the leaves was placed down.
  • Example 46 CRISPR/Cas9 technology in situ replacement of different methylation degree MaICE1 gene bananas
  • the maize MaICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 11. According to the degeneracy of the codon, the theoretically methylatable site of the MaICE1 gene was replaced, and the MaICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the MAICE1 gene sequence was obtained as SEQ ID NO: 52 and designated as MaICE1 (DC) 1 after complete replacement; the MaICE1 gene sequence after partial replacement was SEQ ID NO: 93 and designated MaICE1 (DC) 2.
  • MaICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and banana in situ replacement, each segment was about 400bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GGCCTCGAAGGGGTGATGGG NGG- 3', 5'-GCAGAAGGCGGTGCCAGAGC NGG-3' and 5'-GAGGTCGACGAGTTGGACGA NGG-3'; the MaICE1(DC)2 gene sgRNA sequence is 5'-GGCCTCGAAGGGGTGATGGGCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of MaICE1 (DC)1 and MaICE1(DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI.
  • the Bar gene was ligated into pHEE401E as a resistance screening gene, and the CRISPR/Cas9 expression vector was constructed and introduced into banana by Agrobacterium GV3101.
  • the mature resistant somatic embryos were transferred to the somatic embryo germination medium, and cultured under light/dark (12h/12h) conditions until the embryos were germinated to obtain seedlings, and then the seedlings were transferred to the rooting medium, light/dark (12h).
  • the culture was carried out under conditions of /12h) to obtain a complete transformed plant.
  • bananas were replaced with MaICE1(DC)1 and MaICE1(DC)2 gene plants 21 and 15 in situ, and the cold tolerance was increased by 3.9 °C and 2.9 °C, respectively.
  • the cDNA of MbICE1 gene was cloned and the sequence was SEQ ID NO: 12. According to the degeneracy of the codon, the theoretically methylatable site of the MbICE1 gene was replaced, and the MbICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the MbICE1 gene sequence obtained after total replacement was SEQ ID NO: 53, designated MbICE1 (DC) 1; the partially substituted MbICE1 gene sequence was SEQ ID NO: 94 and designated MbICE1 (DC) 2.
  • MabICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement of banana, each segment was about 500bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GGCATTGAAGACAGACGAAACGG-3. ',5'-GCAGAAAAAGTCGGCATCTTGGG-3' and 5'-GTAGGCAGTGAAGAACTTGAAGG-3'; the MbICE1(DC)2sgRNA sequence is 5'-GCATGGACTTAAAGCCGGAG AGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the MabICE1 (DC)1 and MabICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI.
  • the Bar gene was ligated into pHEE401E as a resistance screening gene, and the CRISPR/Cas9 expression vector was constructed and introduced into the plantain by Agrobacterium GV3101.
  • Example 48 CRISPR/Cas9 technology in situ replacement of cocoa with different degrees of methylation TcICE1 gene
  • the cDNA of the cocoa TcICE1 gene was cloned and the sequence was SEQ ID NO: 13. According to the degeneracy of the codon, the theoretically methylatable site of the TcICE1 gene was replaced, and the TcICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the TcICE1 gene sequence was obtained as SEQ ID NO: 54 and designated as TcICE1 (DC) 1 after complete replacement; the TcICE1 gene sequence after partial replacement was SEQ ID NO: 95 and designated TcICE1 (DC) 2.
  • the TcICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the cocoa mature fruit was collected from the field, peeled off with 75% alcohol for 2 seconds, and then immersed in 5% NaC1O solution for 30 min. After peeling off the seed coat, it was connected to the germination medium. After 10 days, the cotyledons were taken as explants. body.
  • the cells were cultured to Agrobacterium LBA4404/PBI121 with an OD 600 of 1.37 or so, and an equal volume of PCG liquid medium (DKW+2,4-D3.0 mg/L+KT 1.0 mg/L+TDZ 0.01 mg/L+) was collected.
  • Glucose 20g/L+glutamine 250mg/L was resuspended, and the cocoa cotyledon material cultured for 10 days in the inoculation germination medium was co-cultured at 28 °C for 3 days. After co-culture, transfer to PCG solid medium (1% agar powder) containing 500mg/L Carb and 5mg/L bialaphos for bacteriostatic and resistant screening culture, and subculture on the same medium for 10 days.
  • PCG solid medium 1% agar powder
  • the medium is divided into two periods of medium.
  • the first stage medium is PEC (1/2DKW+KNO 3 0.3g/L+AA 1000X stock solution lml/L). +Glucose 20g/L+Sucrose 10g/L+1% agar powder).
  • the culture condition is light/darkness of 16/8h per day, and the medium is changed every 30 days until After growing buds with 1-2 true leaves, transfer to the second stage of RD medium (1/2DKW+KN0 3 0.3g/L+Glucose l0g/L+Sucrose 5g/L+ different concentrations of IBA 1.0mg/ L+ different concentrations of IAA 1.0mg/L, the culture conditions were consistent with the first stage, and the medium was transferred every 30 days until the seedlings were formed.
  • the TcICE1(DC)1 and TcICE1(DC)2 genes were replaced in situ. Plants 10 and 9 strains increased cold tolerance by 3.9 ° C and 2.2 ° C, respectively.
  • Example 49 CRISPR/Cas9 technology in situ replacement of different degrees of methylation of the CtICE1 gene
  • the cDNA of the CtICE1 gene was cloned and the sequence was SEQ ID NO: 14. According to the degeneracy of the codon, the theoretically methylated site of the CtICE1 gene was replaced, and the CtICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the CtICE1 gene sequence was obtained as SEQ ID NO: 55 and designated as CtICE1 (DC) 1 after partial replacement; the partial replacement CtICE1 gene sequence was SEQ ID NO: 96 and designated CtICE1 (DC) 2.
  • CtICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20 nt, and the sequence was 5'-GCTTTGATTTGGGTGAAATGGGG-3'. 5'-GCAATTAGCACTATCACTGGTGG-3' and 5'-GCTGTCATCAGTTGTTTCAATGG-3'; the CtICE1(DC)2sgRNA sequence is 5'-TGCTTGAAGTGGAAGATGAC TGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the TcICE1(DC)1, TcICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI.
  • the Bar gene was ligated into pHEE401E as a resistance screening gene, and the CRISPR/Cas9 expression vector was constructed and introduced into the sputum by Agrobacterium LBA4404.
  • Example 50 CRISPR/Cas9 technology in situ replacement of VvICE1 gene grapes with different degrees of methylation
  • VvICE1 gene cDNA was cloned and the sequence was SEQ ID NO: 15. According to the degeneracy of the codon, the theoretically methylatable site of the VvICE1 gene was replaced, and the VvICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the VvICE1 gene sequence was obtained as SEQ ID NO: 56 and designated as VvICE1 (DC) 1 after partial replacement; the VvICE1 gene sequence after partial replacement was SEQ ID NO: 97 and designated VvICE1 (DC) 2.
  • VvICE1(DC)1 was divided into 2 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • sgRNA sequence was 5'-GAGGAGGAGTGGTTGGTGAGCGG-3'. And 5'-GTGGAGGAGACTGCAAATGATGG-3'; VvICE1 (DC) 2sgRNA sequence is 5'-GTGAGCGGTGTCGAAATGAA AGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the TcICE1(DC)1, TcICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI.
  • the Bar gene was ligated into pHEE401E as a resistance screening gene, and the CRISPR/Cas9 expression vector was constructed and introduced into the grape by Agrobacterium.
  • the inferred grape PEM was subcultured onto fresh X6 medium and pre-incubated for one week in a dark environment at 26 ° C for transgenic manipulation.
  • 200 ⁇ l of the bacterial solution was inoculated into 20 ml of liquid LB medium (containing 5 mg/L of bialaphos).
  • 20 ⁇ l of the bacterial solution was inoculated into 20 ml of liquid LB medium (containing 5 mg/L of bialaphos), and subjected to secondary activation culture for 20 hours under the same conditions.
  • Example 51 Acquisition of apples with different methylation levels of MdICE1 gene by CRISPR/Cas9 technology in situ
  • the cDNA of apple MdICE1 gene was cloned and the sequence was SEQ ID NO: 16. According to the degeneracy of the codon, the theoretical methylation site of MdICE1 gene was replaced, and the MdICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the MdICE1 gene sequence was obtained as SEQ ID NO: 57 and designated as MdICE1 (DC) 1 after partial replacement; the MdICE1 gene sequence after partial replacement was SEQ ID NO: 98 and designated MdICE1 (DC) 2.
  • MdICE1 (DC) 1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500 bp, and the sgRNA with a guiding effect was designed by hand.
  • the length of sgRNA was 20 nt, and the sequence was 5'-GAAGACAAGTTGGGATTTTGAGG-3'. 5'-GCACGGTTACCGGTGGAGGAGGG-3' and 5'-GCAGCTTGGCCATTTGGGCTAGG-3'; the MdICE1 (DC) 2sgRNA sequence is 5'-GGTTGAGCAGCAAGCTTTCT GGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and MdICE1(DC)1 and MdICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Apples were genetically transformed using ultrasonic transformation.
  • the apple tube seedling leaves were cut into pieces of about 4 mm 2 and placed in a container containing 20 ml of ultrasonic buffer.
  • the composition of the ultrasonic buffer was: 13.7 mmo1/LNaCI, 2.7 mmo1/LKCI, 10 mmol/L Na 2 HPO 4 , 2 mmo1/LKH. 2 POP 4 , 5% DMSO, pH 7.4.
  • Each milliliter of buffer contained 5 ug of plasmid DNA.
  • Ultrasonic treatment was carried out for 20 min at a sound intensity of 0.5-1.0 W/cm 2 .
  • the sonicated leaves were transferred to MS regeneration medium containing 4 mg/L 6-BA+0.2 mg/L NAA for 1 week, and then transferred to a 5 mg/L bialaphos regeneration medium for 2 weeks of dark culture. Screening was performed once every 2 weeks and transferred to MS subculture medium of 0.5 mg/L 6-BA + 0.1 mg/L NAA after 2 months for a total of 3 months. Culture conditions: 16 hours of continuous light per day, light intensity of 40umol m -2 s -1 constant temperature 25 ° C subculture. Once every 4 weeks. Sixteen and 17 in situ replacement MdICE1 (DC)1 and MdICE1 (DC)2 gene resistant plants were obtained from 400 explants. Compared with wild-type plants, their cold tolerance increased by 4.1 ° C and 2.5 ° C, respectively.
  • Example 52 CRISPR/Cas9 technology in situ replacement of different methylation levels of CbICE1 gene mustard
  • the cDNA of the CbICE1 gene was cloned, and the sequence was SEQ ID NO: 17. According to the degeneracy of the codon, the theoretically methylated site of the CbICE1 gene was replaced, and the CbICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete replacement CbICE1 gene sequence is SEQ ID NO: 58, designated CbICE1 (DC) 1; the partially substituted CbICE1 gene sequence is SEQ ID NO: 99, designated CbICE1 (DC) 2.
  • CbICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GAAGATCTTGTGGATGTGGTGGG-3'. 5'-GTTCAGATTGGAGGAGGGAAGGG-3' and 5'-GTGAACATTCATATGTTCTGTGG-3'; the CbICE1(DC)2sgRNA sequence is 5'-GAAGATCTTGTGGATGTGGTGGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and CbICE1(DC)1 and CbICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Rooting culture is carried out. After the roots of the tissue culture seedlings are strong, the residual medium on the roots is washed, transplanted into the nutrient soil, and placed in an artificial climate chamber for cultivation. In the genetic transformation experiments, 22 and 25 in situ replacement MdICE1(DC)1 and CbICE1(DC)2 gene resistant plants were obtained. Compared with wild type plants, their cold tolerance was increased by 3.4 ° C and 2.4 ° C, respectively.
  • Example 53 CRISPR/Cas9 technology in situ replacement of eggplant with different degree of methylation ScICE1 gene
  • the cDNA of the eggplant ScICE1 gene was cloned and the sequence was SEQ ID NO: 18. According to the degeneracy of the codon, the theoretically methylated site of the ScICE1 gene was replaced, and the ScICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites. After the complete replacement, the ScICE1 gene sequence was SEQ ID NO: 59 and designated as ScICE1 (DC) 1; after partial replacement, the ScICE1 gene sequence was SEQ ID NO: 100 and designated as ScICE1 (DC) 2.
  • the ScICE1(DC)1 was divided into two segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with a guiding effect was designed by hand.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GTACAGTTCGATGGGATTCGGGG-3'. And 5'-GGTTGTCCCAAAGATAACCAAGG-3'; the ScICE1(DC)2sgRNA sequence is 5'-GTAATCTGACTGATAGAAAA AGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of ScICE1(DC)1, ScICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the explants were washed with sterile water of Temetin, and the surface water was absorbed by sterile filter paper.
  • the cells were inoculated into MS+NAA 0.2 mg/L+ZT1.2 mg/L+150 mg/L Temetin medium, and the culture was restored.
  • the induced callus medium (0.1 mg/L NAA, 3.0 mg/L ZT). It was placed at 16 h during the day, 8 h at night, and cultured under 1601 x light conditions.
  • the explants grow for about two weeks, and grow young, lumpy, hard green callus, which is inoculated into the budding medium (adding 0.1 mg/L NAA, 4.0 mg/L ZT, 1.5 mg/L).
  • Seedlings formed by bud elongation and differentiation produced 2-3 true leaves, which were cut from the base and inoculated into 1/2MS rooting medium to induce rooting.
  • the illumination time is 16h/d
  • the temperature is 24-27°C
  • the light intensity is 1600 1x.
  • the regenerated seedlings grown in the medium have poor adaptability to the environment.
  • the triangular flask should be opened for small mouth ventilation, and the ventilation should be gradually increased according to the growth of the seedlings to improve the adaptability of the seedlings to the environment. Two weeks later, the seedlings have 4-5 true leaves, three main roots, and several lateral roots, and the bottle mouth is opened.
  • Example 54 CRISPR/Cas9 technology in situ replacement of tomato with different methylation degree SlICE1 gene
  • the tomato SlICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 19. According to the degeneracy of the codon, the theoretically methylatable site of the SlICE1 gene was replaced, and the SlICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the sequence of the SlICE1 gene after obtaining all substitutions is SEQ ID NO: 60, designated as SlICE1 (DC) 1; after partial replacement, the sequence of the SlICE1 gene is SEQ ID NO: 101, designated as SlICE1 (DC) 2.
  • SlICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GGTTGTTGTTAATAGCCTTTTGG-3'. 5'-GTGGATGATACTGTTAAGAA TGG-3' and 5'-GCTGTTATCAGCTGCTTCAATGG-3';
  • the SlICE1 (DC) 2sgRNA sequence is 5'-GTTGTTGTTAATAGCCTTTTGGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of SlICE1(DC)1, SlICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Tomato varieties with higher germination rate on 1/2MS minimal medium were selected as materials for this experiment.
  • the seedlings were grown on 1/2 MS minimal medium and grew to a height of about 5-6 cm (about 18 days), the cotyledons of the sterile seedlings were cut for use as a recipient material for transformation.
  • a small amount of Agrobacterium tumefaciens pBLGC containing the expression vector of interest was taken from the glycerol tube, and cultured on a YEP solid medium (5 mg/L bialaphos, 20 mg/L Rif), and cultured at 28 ° C in the dark.
  • a single colony (about 36-48 h) having a diameter of about 1 mm was grown.
  • Single colonies were transferred to the same solid medium plate and cultured until the vigorous growth period (about 36 h).
  • Single colonies of Agrobacterium were picked from YEP plates, inoculated into YEP liquid medium containing 5 mg/L bialaphos, 20 mg/L Rif, mixed, and cultured at 28 ° C, 180-200 rpm overnight (about 16- 18h).
  • the next day the inoculum was inoculated in 20 ml of YEP liquid medium, shaken at 28 ° C, shaking at 180 rpm for 3-4 hours, and the Agrobacterium was cultured to log phase (OD600 was about 0.6); then, the cells were collected by centrifugation at 4000 rpm for 15 min at 4 ° C.
  • the daughter leaves were transferred to screening medium (Ms+6-BA 1 mg/L+IAA 0.2 mg/L+5 mg/L bialaphos +500 mg/L carbenicillin); every three days Change the medium once until no Agrobacterium appears. After 14 days, some callus or cluster regeneration buds were observed in the vicinity of the incision. At this time, the regenerated shoots were divided into individual plants as much as possible, and screening was continued under the pressure of 5 mg/L of bialaphos. After about 4 weeks, the cells were transferred to a differentiation medium containing 5 mg/L of bialaphos and 500 mg/L of carbenicillin for about 1 month; the obtained resistant buds were further screened, and the carbenicillin was selected during the screening.
  • screening medium Ms+6-BA 1 mg/L+IAA 0.2 mg/L+5 mg/L bialaphos +500 mg/L carbenicillin
  • Example 55 Acquisition of in situ replacement of PpICE1 gene peaches with different degrees of methylation by CRISPR/Cas9 technology
  • the PpICE1 gene cDNA was cloned and the sequence was SEQ ID NO: 20. According to the degeneracy of the codon, the theoretically methylated site of the PpICE1 gene was replaced, and the PpICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the PpICE1 gene sequence was obtained as SEQ ID NO: 61 and designated as PpICE1 (DC) 1 after partial replacement; the PpICE1 gene sequence after partial replacement was SEQ ID NO: 102 and designated PpICE1 (DC) 2.
  • PpICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GTCAAGATTGTTGAAAACCGAGG-3'. 5'-GGCAAAGTTCTTCCTTGATACGG-3' and 5'-GTACTGCTTTTATCTGATCCGGG-3'; the PpICE1(DC)2sgRNA sequence is 5'-GTCAAGATTGTTGAAAACCGAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of SlICE1(DC)1, SlICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • a single colony of Agrobacterium tumefaciens GV3101 containing this plasmid was picked and inoculated into a 4 ml liquid medium containing 5 mg/L of bialaphos, Rif 20 mg/L, Gem 40 mg/L in YEP, and cultured at 180 rpm at 28 ° C for 36 h.
  • +NAA 1.0 mg/L + 2% cane toad 7.5 g/L agar, pH 5.8), co-cultivation in dark at 26 °C.
  • the induced callus was introduced into a differentiation medium (MS+NAA 0.05 mg/L+BA 1.0 mg/L), the light intensity was 2000-3000 lx, and the light-dark cycle was cultured at 16 h/8 h, and the culture temperature was the same as above. After that, the adventitious buds were transferred to rooting medium (1/2MS+IBA 1.0 mg/L) for rooting culture, and rooting began after 5 days.
  • 11 and 9 strains were substituted for PpICE1(DC)1 and PpICE1(DC)2 gene-resistant plants. Compared with wild peach plants, their cold tolerance increased by 4.0 ° C and 2.1 ° C, respectively.
  • Example 56 Acquisition of in situ replacement of different methylation degree ZmICE1 gene maize by CRISPR/Cas9 technology
  • the cDNA of maize ZmICE1 gene was cloned and the sequence was SEQ ID NO: 21. According to the degeneracy of the codon, the theoretically methylated site of ZmICE1 gene was replaced, and the ZmICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the ZmICE1 gene sequence was obtained as SEQ ID NO: 62 and designated as ZmICE1 (DC) 1 after partial replacement; the ZmICE1 gene sequence after partial replacement was SEQ ID NO: 103 and designated ZmICE1 (DC) 2. .
  • the ZmICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 400bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GGGGCTACCTGGGCTCCGACGGG-3'. , 5'-GAAGATCAGCAAGATGGATAGGG-3' and 5'-GCTGAGTGCGCGGATGGTCCTGG-3';
  • ZmICE1 (DC) 2sgRNA sequence is 5'-GTGGCCGCGGCCGCGGAGGAGGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and ZmICE1(DC)1 and ZmICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the recipient materials were sown in stages on a 7d cycle.
  • the corn samples were controlled for pollination according to the test requirements. After 10-13 days of pollination, the maize immature embryos were used as transformation receptors. After disinfecting the corn cob with sodium hypochlorite, peel off the immature embryo (be careful when peeling the young embryo, do not insult the embryo), wash the young embryo 4 times with the infecting solution (plus AS), then add a certain concentration of Agrobacterium liquid, place 10 After -30 min, remove it, blot it with sterile filter paper, put it on the co-culture medium, co-culture for 2-5 days at 25 ° C (dark), then transfer the immature embryos to resting medium, and darkly culture at 28 °C.
  • the young embryos were transferred from a resting medium to a resistance screening medium containing a marker gene, and cultured in dark. Each successive generation of attention was taken to eliminate the brown and water-stained callus, and the normally growing callus was crushed with tweezers and cultured separately. During the sub-screening process, it is often observed that the contamination or the return of Agrobacterium is immediately removed, and the uncontaminated material continues to be cultured. In addition, unorganized tissue pieces that have been browned contaminated are often removed or transferred to new, identical media.
  • the tissue block After the tissue block is inflated, the large tissue is often peeled off, and after successive subculture screening for 3 times, the selected resistant callus is transferred to the regeneration medium, and the culture is resumed for 15 days or longer (dark culture).
  • the resistant callus was transferred to a regeneration medium for germination, and the culture condition was 28 ° C, and the light was irradiated for 12 hours at a daily light of 3000 lx.
  • the seedlings can be transplanted into a bottle containing rooting medium and cultured indoors.
  • the seedlings grow thicker roots, remove the seedlings from the cans, rinse the medium with water, and transplant them in small pots mixed with nutrient soil and vermiculite (1:3), when the corn grows again. When 2-3 new leaves are used, they can be transplanted into large fields or large flower pots. After growing the three or four leaves, the leaf DNA is extracted for PCR detection. The ZmICE1 gene containing the transfer was determined. In this experiment, 14 and 12 strains were used to replace ZmICE1 (DC)1 and ZmICE1 (DC)2 gene resistant plants in situ. Under the condition of low temperature treatment at 6 °C, the cold tolerance was increased by 3.3 °C and 1.8 °C, respectively, compared with wild plants.
  • Example 57 Acquisition of cotton with different degrees of methylation GhICE1 gene by CRISPR/Cas9 technology in situ
  • the cDNA of the cotton GhICE1 gene was cloned and the sequence was SEQ ID NO: 22. According to the degeneracy of the codon, the theoretically methylated site of the GhICE1 gene was replaced, and the GhICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete replacement GhICE1 gene sequence was SEQ ID NO: 63 and designated GhICE1 (DC) 1; the partially replaced GhICE1 gene sequence was SEQ ID NO: 104 and designated GhICE1 (DC) 2.
  • GhICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500 bp, and the sgRNA with a guiding effect was designed by hand.
  • the length of sgRNA was 20 nt, and the sequence was 5'-GTCAAGATGGTTGAAAACGGAGG-3'. , 5'-GCCCTTTCCTTTTACCCTTTTGG-3' and 5'-GTAAACATCCACATGTTCTGTGG-3';
  • GhICE1 (DC) 2sgRNA sequence is 5'-GTCAAGATGGTTGAAAACGGAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and GhICE1(DC)1 and GhICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the first and second fruit nodes of each fruit branch are typically selected for transgenic manipulation.
  • the cotton bolls on these fruit nodes generally have a higher bell rate, which is beneficial to harvest more seeds; a 50 ⁇ l micro-injector is used as a tool for microinjection.
  • it should be washed with a light detergent and then rinsed with distilled water; when injecting, remove or peel off the petals and smooth the style.
  • a plant that grows normally is considered to be a plant that acquires a resistance gene.
  • 24 and 25 in situ replacement GhICE1 (DC)1 and GhICE1 (DC)2 gene resistant plants were obtained. Under the condition of low temperature treatment at 4 °C, the cold tolerance was increased by 4.6 °C and 2.8 °C, respectively, compared with the wild type cotton plants.
  • Example 58 Acquisition of peanuts with different degrees of methylation degree AhICE1 gene by CRISPR/Cas9 technology in situ
  • the cDNA of peanut AhICE1 gene was cloned and the sequence was SEQ ID NO: 23. According to the degeneracy of the codon, the theoretically methylated site of the AhICE1 gene was replaced, and the AhICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete AhICE1 gene sequence was obtained as SEQ ID NO: 64 and designated as AhICE1 (DC) 1; the partially substituted AhICE1 gene sequence was SEQ ID NO: 105 and designated AhICE1 (DC) 2.
  • the length of the sgRNA is 20 nt
  • the sgRNA sequences of the AhICE1 (DC) 1 and AhICE1 (DC) 2 genes are both 5'-GCCGTCCAAAAATCTTATGGCGG-3', which guides the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and GhICE1(DC)1 and GhICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the transgenic operation is carried out.
  • the flower buds are removed every day at about 5 pm, so as to prevent the progeny from pollinating the flowers without transgenic operation, and the next season to detect the transgenic plants in the offspring cause interference.
  • the glass needles drawn by the micropipette and the dropper were respectively introduced into the plasmid DNA containing the target gene by the method of column head application and flower Cao tube injection, and the two DNA concentrations of 50 ug/ml and 100 ug/ml were used to treat the oyster sauce 523. "and "Oyster 21" two varieties. After entering the flowering period, start operation at about 6:30 every morning.
  • the smear operation is to apply 5 ul of DNA solution to the flower stigma with a micropipette.
  • the injection method uses a glass needle to inject about 5 ul into the ovary in the lower part of the calyx tube. DNA solution.
  • Each variety is processed more than 500.
  • the peanut seeds obtained by genetic transformation were sprayed on the seedlings in the greenhouse, and the peanut leaves were sprayed at 5 mg/L of bialaphos in the 2-3 leaf stage. After one week, the growth of the plants was observed, and the plants with green spots on the surface of the leaves were removed. Plants that are normally grown are considered to be resistant genes, and resistant plants are verified by PCR. Genetic transformation showed 12 and 15 in situ replacement of AhICE1 (DC)1 and AhICE1 (DC)2 gene resistant plants. Compared with wild-type peanut plants, their cold tolerance was increased by 2.0 ° C and 1.3 ° C, respectively.
  • Example 59 CRISPR/Cas9 technology in situ replacement of BjICE1 gene mustard with different degrees of methylation
  • the cDNA of Brassica juncea BjICE1 gene was cloned, and the sequence was SEQ ID NO: 24. According to the degeneracy of the codon, the theoretically methylated site of the BjICE1 gene was replaced, and the BjICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites. After the complete replacement, the BjICE1 gene sequence was SEQ ID NO: 65 and designated as BjICE1 (DC) 1; after partial replacement, the BjICE1 gene sequence was SEQ ID NO: 106 and designated BjICE1 (DC) 2.
  • the BjICE1(DC)1 was divided into two segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GTTCGGTTCGTTGACGCAGCTGG-3'. And 5'-GTTCAGAACGGAGGAGGTAAAGG-3'; BjICE1 (DC) 2sgRNA sequence is 5'-GCTCCGGTTTCGATGGGGTTCGG-3', which guides the cleavage of DNA by Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and BjICE1(DC)1 and BjICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the mustard seeds were sterilized (75% alcohol and 0.1% liters of mercury) and sown in MS medium. After 7 days, hypocotyls (0.5-0.8 cm) were cut and placed in mustard differentiation medium (MS+2 mg/L 6-BA). +0.2 mg/L NAA + 3% sucrose + 0.6% agar) was pre-incubated for 2 days.
  • Agrobacterium transformed into the plasmid containing BjICE1 gene was activated, 500 ul of activated bacterial solution was inoculated, inoculated into 50 ml of antibiotic-free YEB liquid medium, and shaken at 225 rpm on a shaking bar at 28 ° C until the active growth period of Agrobacterium (OD600 value).
  • the pre-cultured hypocotyls were inoculated with the resuspended bacterial solution for 10 min, dried on filter paper, and transferred to differentiation medium for 2 days.
  • the co-cultured hypocotyls were transferred to the mustard screening medium (MS+2 mg/L 6-BA+0.2 mg/L NAA+5 mg/L bialaphos+400 mg/L Cb+3% sucrose+0.6% agar) The resistance screening was performed, and the screening medium was changed every 10 days.
  • Example 60 CRISPR/Cas9 technology in situ replacement of different methylation degree BrICE1 gene rapeseed
  • the cloned Bracine 1 gene cDNA was sequenced as SEQ ID NO: 25. According to the degeneracy of the codon, the theoretically methylatable site of the BrICE1 gene was replaced, and the BrICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the BRICE1 gene sequence was obtained as SEQ ID NO: 66 and designated as BrICE1 (DC) 1 after complete replacement; the partial replacement BRICE1 gene sequence was SEQ ID NO: 107 and designated BrICE1 (DC) 2.
  • BrICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp. The sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20 nt, and the sequence was 5'-GTTCGGTTCGTTGACGCAGCTGG-3'. 5'-GACTCTTTCTTGCCGTGTCAAGG-3' and 5'-GCCGTCATCAGCTGTTTCAACGG-3'; the BrICE1(DC)2sgRNA sequence is 5'-GTCGAGGCTAAAAGCCTGAGAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the BrICE1 (DC)1 and BrICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the rapeseed transgenic experiment was carried out by pollen-mediated method. After entering the flowering period of rapeseed, select the flower buds to be opened in 10-15 1-2d on the main stem or on one branch, and remove the other flower buds on the selected stem or branch, and then bag. At the same time, in the same variety, the open inflorescence was bagged on the second day for the second day. The pollen of the bagged plants opened on the next morning was about 0.4g, suspended in 25ml of 7.5% sucrose solution, subjected to the first sonication, and then 20 ⁇ g of plasmid DNA containing different cold tolerance populations of BrICE1 gene was added to the solution. Sub-ultrasonic treatment.
  • Example 61 CRISPR/Cas9 technology in situ replacement of different degrees of methylation CdICE1 gene chrysanthemum
  • the cDNA of the chrysanthemum CdICE1 gene was cloned, and the sequence was SEQ ID NO:26. According to the degeneracy of the codon, the theoretically methylatable site of the CdICE1 gene was replaced, and the CdICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the CdICE1 gene sequence was obtained as SEQ ID NO: 67 and designated as CdICE1 (DC) 1 after partial replacement; the CdICE1 gene sequence after partial replacement was SEQ ID NO: 108 and designated CdICE1 (DC) 2.
  • CdICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GGGTTGTTGGAAATGAAGCTAGG-3'. 5'-GTTGGTGGTGTAGGCGTCAATGG-3' and 5'-GCAGTGCTTCTAGAGACTGCAGG-3'; the CdICE1 (DC) 2sgRNA sequence is 5'-GTAACTAGTCTCCATATACTCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and CdICE1(DC)1 and CdICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the preserved Agrobacterium carrying the pCAMBIA1301 plasmid of the gene of interest was inoculated in a YEB liquid medium containing 50 mg/L of rifampicin overnight. Then, they were streaked on YEB plate medium containing 5 mg/L of bialaphos and 50 mg/L of rifampicin, and placed in a constant temperature incubator at 28 ° C. After incubation to a single colony, the plate was placed on 4 °C refrigerator is reserved for use.
  • Single Agrobacterium colonies were picked from the above YEB plate medium, inoculated into 20 ml of YEB liquid medium supplemented with 5 mg/L of bialaphos and 50 mg/L of rifampicin, and cultured at 28 ° C with shaking at 180 rpm. To logarithmic growth period. After two activations, the bacterial solution was inoculated to a YEB liquid medium containing only 100 pmol/L of RS at a ratio of 1:100. The culture was shaken for 4-6 h, and when the OD600 value reached about 0.4-0.6, it was used for genetic transformation.
  • the leaves at the upper end of the cultured sterile chrysanthemum plants were taken as explants, and cut into 8 mm*8 mm pieces with sterile scissors, and placed in embryogenic callus-inducing medium (Ms+0.5 mg/ Pre-culture was carried out in L 6-BA + 1.5 mg / L NAA + sucrose 30 g / L + agar powder 5 g / L). After pre-incubation for 3 days, pour the bacterial solution for dip dye into a sterile flask, rinse the pre-cultured leaves with sterile water 1-2 times, blot dry with sterile filter paper, and then directly infiltrate into a triangular flask. 25min.
  • the impregnated leaves were inoculated on the co-culture medium and co-cultured for 2 days at 25 ⁇ 2 °C. The colonies grow around the leaves, the leaves are removed, rinsed with sterile water for 2-3 times, and the filter paper is blotted dry.
  • the culture medium was transferred to an embryogenic callus-inducing selection medium (addition of 300 mg/L Cef) until the resistant embryos were completely differentiated. After screening for 15-20 days in the differentiation culture, a small part of the chrysanthemum leaf incision began to differentiate.
  • the regenerated plants in the medium grow to about 2 cm, they are transferred from the mother chrysanthemum with sterile scissors to the MS minimal medium without any plant growth hormone for rooting. At 25 ° C, the light intensity is 1200- The culture was carried out under a photoperiod of 2400 lx at 16 h/8 h. On the sixth day, the base of the root began to germinate. Genetic transformation showed that 8 and 10 strains of CdICE1(DC)1 and CdICE1(DC)2 resistant plants were replaced in situ. Compared with wild-type chrysanthemum plants, their cold tolerance was increased by 5.0 ° C and 2.6 ° C, respectively.
  • Example 62 CRISPR/Cas9 technology in situ replacement of different methylation degree ThICE1 gene small salt mustard
  • ThICE1 gene of the small salt mustard was cloned and the sequence was SEQ ID NO: 27. According to the degeneracy of the codon, the theoretically methylated site of the ThICE1 gene was replaced, and the ThICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites in the medium.
  • the complete replacement of the ThICE1 gene sequence was SEQ ID NO: 68, designated as ThICE1 (DC) 1; and after partial replacement, the ThICE1 gene sequence was SEQ ID NO: 109 and designated ThICE1 (DC) 2.
  • the TICE1(DC)1 was divided into two segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GCTAGAAACGAAGGCTGAGA AGG-3. ' and 5'-GGAGGAAGAAGCTTAATGAT AGG-3';
  • the ThICE1 (DC) 2sgRNA sequence is 5'-GGGTTTGGGAGTCCTGCAAATGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of ThICE1 (DC)1 and ThICE1(DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the small salt mustard seeds were placed in a test tube, soaked in sterile water, and placed in a refrigerator at 4 ° C for one month.
  • 70% of the culture chamber is placed for one and a half months.
  • the petiole was cut, washed with water, disinfected with 70% ethanol, disinfected with 2% (V/V) sodium hypochlorite for 10 min, and then washed with sterile water for 4 times before use as an explant.
  • the explants were incubated on pre-culture medium MS + 2.5 mg / L 6-BA + 0.1 mg / L NAA (pH 5.8), temperature 22 ° C, relative humidity 70%, dark culture for 3d.
  • Single colonies were picked from the plates, inoculated into 5 ml of LB liquid medium supplemented with 5 mg/L of bialaphos, and shaken at 28 ° C, 180 rpm until the OD600 was 0.6.
  • the explants were immersed in the bacterial solution for 5 min, and then placed on a sterile filter paper to absorb the attached bacterial liquid.
  • the explants were incubated with co-cultivation medium MS + 2.5 mg / L 6-BA + 0.1 mg / L NAA (pH 5.8), and cultured at 25 ° C in the dark.
  • Co-cultured explants were transferred to MS + 2.5 mg/L 6-BA + 0.1 mg/L NAA + 5 mg/L bialaphos 300 mg/L Cef (pH 5.8) selection medium for 2 weeks. After 4 days of culture, the explants were rinsed once every 3 days with sterile water and replaced with fresh selection medium.
  • the resistant callus produced by the explant-transformed cells was transferred to a selection medium and subcultured.
  • the adventitious bud grows to 1 cm, it is cut and connected to the rooting medium 1/2 MS + 5 mg / L bialaphos 300 mg / LCef (pH 5.8), and adventitious roots grow after 15 days.
  • 13 and 12 strains of in situ replacement of ThICE1 (DC) 1 and ThICE1 (DC) 2 gene resistant plants were obtained. Compared with wild-type small salt mustard plants, their cold tolerance increased by 3.2 ° C and 1.7 ° C, respectively.
  • Example 63 Acquisition of in situ replacement of different methylation degree DcICE1 gene wild carrot by CRISPR/Cas9 technique
  • the cDNA of the wild carrot DcICE1 gene was cloned, and the sequence was SEQ ID NO: 28. According to the degeneracy of the codon, the theoretically methylatable site of the DcICE1 gene was replaced, and the DcICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete DcICE1 gene sequence was obtained as SEQ ID NO: 69 and designated as DcICE1 (DC) 1; after partial replacement, the DcICE1 gene sequence was SEQ ID NO: 110 and designated as DcICE1 (DC) 2.
  • the DcICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20 nt, and the sequence was 5'-GGTTCTCTAATAATTATCTTGGG-3'. , 5'-GATCGGAAGGGAAAGAAGAAGGG-3' and 5'-GTCTCTTACTTTCCACCATGAGG-3'; the DcICE1 (DC) 2sgRNA sequence is 5'-GGTTGGTGTCGGGTTTAACCGGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the DcICE1 (DC)1 and DcICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • a single colony of Agrobacterium tumefaciens engineered bacteria was inoculated into 50 ml of LB liquid medium containing 5 mg/L of bialaphos, and cultured at 28 ° C under shaking at 200 rpm in the dark to an OD600 of about 0.6.
  • Sterile seedlings were placed on filter paper and the hypocotyls were cut into 0.5 cm long sections using a sterile, sharp scalpel for genetic transformation. Place the hypocotyl of the processed wild carrot sterile seedlings into the prepared dip solution on the ultra-clean workbench for 15-20min, gently oscillate in the middle to ensure that all hypocotyl segments can be fully infiltrated. The explants were removed and the excess bacterial solution was aspirated on sterile filter paper. Place on the co-cultivation medium and incubate for 2-3 days at 26 ° C in the dark.
  • the explants were taken out, placed in a sterile empty flask, rinsed 2-3 times with sterile water, rinsed 1-2 times with sterile water containing 500 mg/L Carb, and blotted with sterile filter paper.
  • Moisture transferred to B5 antibacterial medium supplemented with a certain concentration of antibiotics, callus culture at 26 ° C, light 16h / d conditions, subculture 2-3 times; to grow callus and then into differentiation culture
  • On the base embryoid bodies and buds are induced. After the induced buds grow for about 40 days, the shoots are cut to 2-3 cm and transferred to the rooting medium to induce rooting; when the normal roots grow and 3-4 true leaves are grown, the resistant shoots are removed.
  • the root medium was washed with tap water, planted in a nutrient bowl containing vermiculite and charcoal (2:1), covered with a plastic film, and given a condition of 26 ° C, light 16 h / d; 7 d open the ventilation of the plastic sheet
  • the mouth is properly smelted, and after the resistant seedlings are gradually adapted to the external growth environment conditions, they can be routinely managed under the greenhouse.
  • 7 and 10 strains of DcICE1 (DC)1 and DcICE1 (DC)2 gene-resistant plants were replaced in situ. Compared with wild-type wild carrot plants, their cold tolerance increased by 3.1 ° C and 2.1 ° C, respectively.
  • Example 64 Acquisition of in situ replacement of GmICE1 gene soybeans with different degrees of methylation by CRISPR/Cas9 technology
  • the soybean GmICE1 gene cDNA was cloned and the sequence was SEQ ID NO:29. According to the degeneracy of the codon, the theoretically methylatable site of the GmICE1 gene was replaced, and the GmICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete replacement GmICE1 gene sequence was SEQ ID NO: 70 and designated GmICE1 (DC) 1; after partial replacement, the GmICE1 gene sequence was SEQ ID NO: 111 and designated GmICE1 (DC) 2.
  • GmICE1(DC)1 was divided into 2 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GAAAAGGGTGGGTTGGGCCCCGG-3'. And 5'-GTCTGTCATTGAGCTTCTTCCGG-3'; GmICE1(DC) 2 sgRNA sequence is 5'-GAGGTGAGAGGGGGAGCAGGAGG-3', which guides the site-specific cleavage of DNA by Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and GmICE1(DC)1 and GmICE1(DC)2 genes of different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • liquid germination medium (sucrose 20g / L, pH 5.8), the volume of the medium is about twice the volume of the seed, in order to completely cover the seeds and Provide plenty of moisture.
  • LGM liquid germination medium
  • the lid was closed, it was placed in an incubator for 16 hours, and the culture conditions were 23 ° C throughout the day, and it was treated in the dark.
  • a three-knife stroke was made parallel to the central axis at the position of the embryo tip, and immersed in a petri dish containing the Agrobacterium suspension for 30 min. After the infestation time, the explants were transferred to a tin box with sterile filter paper using sterilized forceps, and the explants were placed horizontally and the incisions were upward.
  • Lightly add LCCM Bs salt powder 0.321g/L, sucrose 30g/L, MES3.9g/L, pH 5.4, BAP 1.67mg/L, GA3 0.25mg/L, Cys 400mg/L, DTT 154.2) Mg/L, As 39.24 mg/L).
  • the iron box After closing the iron box, it was placed in a smart incubator for 3 days, and the culture condition was 23 ° C throughout the day, and it was treated in the dark. After 3 days of co-cultivation, the explants were removed with sterilized forceps on a clean bench and the cotyledons were removed with a scalpel, but the hypocotyls were retained. After excision, the explants were placed in a bud induction medium (SIM) (BS salt powder 3.21 g/L, sucrose 30 g/L, MES 0.59 g/L, Noble Agar 8 g/L, BAP 1.67 mg/L, Tic 250 mg).
  • SIM bud induction medium
  • RM rooting medium
  • MS Salt powder 4.43g / L, sucrose 20g / L, MES 0.59g / L, Phytagel 3g / L, pH 5.6, Asp 50mg / L, L-Glu 50mg / L, Tic 125mg / L, Cef 50mg / L, IBA 1mg Continue to train in /L). After 1-2 weeks, when the bottom of the seedling grows a certain number of the most roots, it is taken out from the RM medium.
  • Example 65 Acquisition of in situ replacement of different methylation degree RsICE1 gene radish by CRISPR/Cas9 technology
  • the radish RsICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 30. According to the degeneracy of the codon, the theoretically methylatable site of the RsICE1 gene was replaced, and the RsICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the sequence of the RsICE1 gene after obtaining all substitutions was SEQ ID NO: 71, designated RsICE1 (DC) 1; after partial replacement, the sequence of the RsICE1 gene was SEQ ID NO: 112, designated RsICE1 (DC) 2.
  • RsICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 400bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GATTTCAGATTCCTCGGTGGTGG-3'. 5'-GGAGGAAGAAGCTTAATGATAGG-3' and 5'-GCTGGCTACCATGAAAGCTTTGG-3'; the RsICE1(DC)2sgRNA sequence is 5'-GATTTCAGATTCCTCGGTGGAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the RsICE1(DC)1, RsICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the seedlings of the sterile seedlings with the stalks of the stalks were pre-cultured for 2 days, inoculated with EHA105 Agrobacterium bacterium with an OD600 of 0.3-0.6 for 5-7 min, and then co-cultured for 5 days, then The explants were transferred to MS+6-BA 6mg ⁇ L -1 +NAA 0.05mg ⁇ L -1 +Cef 500mg ⁇ L -1 for 7 days delay screening, then the explants were transferred to MS + 6-BA 6mg ⁇ L -1 + NAA 0.05mg ⁇ L -1 + 5mg ⁇ L -1 bialaphos Cef 500mg ⁇ L for 10 days on a medium resistance screening -1, for every 10 days Subculture is transferred once.
  • Agrobacterium-mediated genetic transformation was used to obtain 6 or 5 strains of RsICE1(DC)1 and RsICE1(DC)2 resistant plants in situ. Compared with wild-type radish plants, their cold tolerance was increased by 4.5 ° C and 2.4 ° C, respectively.
  • Example 66 CRISPR/Cas9 technology in situ replacement of different degrees of methylation TaICE1 gene wheat
  • the cDNA of TaICE1 gene was cloned and the sequence was SEQ ID NO: 31. According to the degeneracy of the codon, the theoretically methylated site of TaICE1 gene was replaced, and the TaICE1 gene was quantitatively replaced according to the target trait. Different degrees of methylation sites. After the complete replacement, the TaICE1 gene sequence was SEQ ID NO: 72 and designated TaICE1 (DC) 1; after partial replacement, the TaICE1 gene sequence was SEQ ID NO: 113 and designated TaICE1 (DC) 2.
  • the TaICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 400bp. The sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GCGTGGACGCCTCCTCCTTGGGG-3'. 5'-GCTGCAGGGTGGGCGTCGACGGG-3' and 5'-GCCGTCATCAGCTGCTTCGATGG-3'; the TaICE1(DC)2sgRNA sequence is 5'-GTAGGCCGGGGGCGCGGCGGCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and TaICE1(DC)1 and TaICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the wheat ears 12-14 days after the flowering were collected, and the immature seeds with the same size in the middle of the wheat ears were taken.
  • the young embryos were inoculated on the surface of the callus induction medium, and cultured at 25 ° C in the dark. After 20 days, they were transferred to the same medium. Generation culture.
  • the immature embryos were inoculated on MC medium and cultured for 15-20 days. After the callus formed by the immature embryos was completely developed, it was placed in a refrigerator at 4 ° C, cold treated for about 2 months, and then subcultured at room temperature. In the middle of August, the callus was transferred to MC+acetosyringone (AS) 0.1mmol/L medium, and the transformation was started after 5-7d.
  • AS acetosyringone
  • the cells were transferred to regeneration medium MD+IAA 1 mg/L+ZT 1 mg/L, and then seedlings were formed.
  • Agrobacterium-mediated genetic transformation was used to obtain 23 and 25 in situ replacements of TaICE1(DC)1 and TaICE1(DC)2 resistant plants. Compared with wild-type wheat plants, the cold tolerance was increased by 4.8 ° C and 3.3 ° C under -10 ° C treatment conditions, respectively.
  • Example 67 Acquisition of in situ replacement of different methylation degree HvICE1 gene barley by CRISPR/Cas9 technology
  • HvICE1 The cDNA of HmICE1 gene was cloned and the sequence was SEQ ID NO: 32. According to the degeneracy of the codon, the theoretically methylated site of HvICE1 gene was replaced, and the HvICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the HvICE1 gene sequence was obtained as SEQ ID NO: 73 and designated as HvICE1 (DC) 1 after complete replacement; the HvICE1 gene sequence after partial replacement was SEQ ID NO: 114 and designated HvICE1 (DC) 2.
  • the length of the sgRNA is 20 nt
  • the sgRNA sequences of the HvICE1 (DC) 1 and HvICE1 (DC) 2 genes are both 5'-GATGCCGGCCAAGAACCTGATGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and HvICE1(DC)1 and HvICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the size of the immature embryos of barley ears is guaranteed to be between 1.5mm and 2mm in diameter, usually about 14 days after pollination.
  • the immature seeds and wheat ears are completely separated, the seeds are placed in an Erlenmeyer flask, and placed in a clean bench. The following steps are performed in a sterile clean bench. 70% alcohol is simply rinsed for 30s, washed three times with sterile water, then treated with 50% sodium hypochlorite for 4 minutes, and washed with sterile water for 3-4 times to minimize the residue of sodium hypochlorite. Hold the upper end of the seed about 1/3 with the camera, and make the immature embryos face up.
  • the immature embryos were transferred to the BCS callus screening medium (containing 5 mg/L bialaphos and 160 mg/L Timentin) with the scutellum facing down, and cultured in the dark for 6-8 weeks at 24 °C. Replace the fresh medium every 2 weeks.
  • BCT transition medium containing 5 mg/L bialaphos and 160 mg/L Timentin.
  • the callus After two weeks of transitional culture, the callus will show signs of regeneration, and if not, replace the fresh medium every two weeks. After significant signs of regeneration on the callus, transfer to BCR callus regeneration medium (containing 5 mg/L bialaphos and 160 mg/L Timentin). At 24 ° C, the whole light was cultured and the fresh medium was changed every two weeks. Choose a plant culture bottle instead of a glass culture dish to increase the space for plant regeneration. After the callus grows to a certain size on the selection medium, it is transferred to B13M culture, and cultured under low light at 24 °C. The medium was changed every two weeks, and when the regenerated stem buds grew, they were transferred to a test tube for cultivation.
  • BCR callus regeneration medium containing 5 mg/L bialaphos and 160 mg/L Timentin
  • the seedlings were carefully transferred to a glass cultured at a length of 15 cm, and the permeable plastic membrane was sealed, and the medium in the test tube was a rooting medium.
  • 9 and 7 strains were used to replace HvICE1 (DC)1 and HvICE1 (DC)2 gene-resistant plants in situ. Compared with wild-type barley plants, the cold resistance was increased by 2.4 ° C and 1.7 ° C under -10 ° C treatment conditions, respectively.
  • Example 68 Acquisition of rubber tree with different methylation degree HbICE1 gene in situ by CRISPR/Cas9 technology
  • the HbICE1 gene cDNA was cloned and the sequence was SEQ ID NO: 33. According to the degeneracy of the codon, the theoretically methylated site of the HbICE1 gene was replaced, and the HbICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the HbICE1 gene sequence was obtained as SEQ ID NO: 74 and designated as HbICE1 (DC) 1 after partial replacement; the HbICE1 gene sequence after partial replacement was SEQ ID NO: 115 and designated HbICE1 (DC) 2.
  • HbICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GAATCAGGCTTCAAATTTGATGG-3'. 5'-GGATGAATACTTGTAGATTGAGG-3' and 5'-GTTTATTGCTCTCCACTATGAGG-3'; the HbICE1(DC)2sgRNA sequence is 5'-GAATCAGGCTTCAAATTTGATGG-3', which directs the Cas9 protein to cut-point DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and HbICE1(DC)1 and HbICE1(DC)2 genes of different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the rubber tree mononuclear edged anthers were inoculated on the embryogenic callus induction medium and cultured for 30 days for Agrobacterium infection.
  • the callus was inoculated in the EHA105 suspension for 5 min, then transferred to a medium supplemented with 5 uM acetosyringone and 10 mg/L silver nitrate, and cultured at 22 ° C for 2 d. Each process is set to 3 repetitions. After co-cultivation, the callus was washed with sterile water and then transferred to 50 mg/L tequila for 1 min. After being dried by sterile filter paper, it was transferred to 10 mg/L silver nitrate and 500 mg/L.
  • the callus was cultured on the callus induction medium of Ding, and cultured at 25 ° C for 7 days.
  • the growth-recovered callus was transferred to callus induction medium supplemented with 10 mg/L silver nitrate, 5 mg/L bialaphos and 50 mg/L teicin to induce somatic embryogenesis.
  • the proliferating somatic embryos were transferred to a plant regeneration medium supplemented with 5 mg/L of bialaphos to induce plant regeneration.
  • Sixty-nine strains of HbICE1 (DC)1 and HbICE1(DC)2 gene-resistant plants were replaced by genetic transformation experiments. Compared with the wild type rubber tree, its cold resistance was increased by 2.9 ° C and 1.8 ° C, respectively.
  • Example 69 CRISPR/Cas9 technology in situ replacement of different methylation degree JrICE1 gene walnuts
  • the cDNA of the JrICE1 gene of the walnut was cloned and the sequence was SEQ ID NO: 34. According to the degeneracy of the codon, the theoretically methylated site of the JrICE1 gene was replaced, and the JrICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete replacement JrICE1 gene sequence is SEQ ID NO: 75, designated JrICE1 (DC) 1; after partial replacement, the JrICE1 gene sequence is SEQ ID NO: 116, designated JrICE1 (DC) 2.
  • the JrICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 400bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GCTAACACCAGCAACAGTTGCGG-3'. , 5'-GGAATGCCCTGTTTATGAACAGG-3' and 5'-GTTCATGGAGAACGATAAGGCGG-3'; JrICE1 (DC) 2sgRNA sequence is 5'-GAGGACAAAGTGGGCTTGGGAGG-3', which guides the cleavage of DNA by Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and JrICE1(DC)1 and JrICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Agrobacterium carrying the transformation plasmid frozen at -80 °C was streaked on solid YEB plate medium containing 5 mg/L of bialaphos and cultured overnight at 28 °C. Single colonies on the plates were picked, inoculated into liquid YEB medium containing 5 mg/L of bialaphos, and shaken (200 rpm) overnight at 28 ° C under a constant temperature shaker.
  • the pre-cultured walnut stem segments were mixed with the Agrobacterium liquid solution obtained above for 30 min, and the infected stem segments were blotted with sterile filter paper and inoculated into a co-cultivation medium. It is DKW+6-BA0.5mg/L+IBA0.1mg/L+AS (acetosyringone) 200 ⁇ mol/L+sucrose 30g/L+ agar 6.0mg/L, the pH is adjusted to 5.8 before autoclaving, and the co-culture time is set to 3. Days, darkness.
  • the culture conditions were a culture temperature of 25 ⁇ 1 °C.
  • the screening medium combination is DKW + 6-BA 0.5 mg / L + IBA 0.1 mg / L + 5 mg / L bialaphos + Cef 500 mol / L + sucrose 30 g / L + Agar 6.0 mg / L, 5 weeks after the adventitious bud germination.
  • the culture conditions were a culture temperature of 25 ⁇ 1 ° C, an illumination time of 16 h/d, and an illumination intensity of 2000-2500 lx.
  • 12 and 10 strains were substituted for JrICE1 (DC)1 and JrICE1 (DC)2 gene-resistant plants. Compared with wild walnut plants, their cold tolerance increased by 3.4 ° C and 2.1 ° C, respectively.
  • Example 70 CRISPR/Cas9 technology in situ replacement of LsICE1 gene lettuce with different degrees of methylation
  • the cDNA of LsICE1 gene was cloned and the sequence was SEQ ID NO: 35. According to the degeneracy of the codon, the theoretically methylated site of the LsICE1 gene was replaced, and the LsICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the LsICE1 gene sequence was obtained as SEQ ID NO: 76 and designated as LsICE1 (DC) 1 after partial replacement; the partial replacement LsICE1 gene sequence was SEQ ID NO: 117 and designated LsICE1 (DC) 2.
  • the LsICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GGGAATTGGGAGGTACAAATTGG-3'. , 5'-GTTGGGCTAGGGATCGCGGTTGG-3' and 5'-GCCTCCTACTGCAAAACATATGG-3';
  • LsICE1 (DC) 2sgRNA sequence is 5'-GGAGAAAGAAGCTAAACGACCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the LsICE1 (DC)1 and LsICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the lettuce cotyledon explants infected with Agrobacterium were co-cultured with Agrobacterium in differentiation medium (MS+6-BA 0.5 mg/L+NAA 0.20 mg/L) and transferred to screening medium (Sm8). Resistance screening was performed on +5 mg/L bialaphos Cef 200 mg/L, subcultured once a week. After one week of culture, the base of the cotyledon can gradually grow a small amount of dense callus and slowly turn green. After about one week, small buds can be formed, and the small buds gradually differentiate into young leaves to form small buds. A plurality of small shoots are generally formed on one callus.
  • Example 71 Acquisition of rice with different degrees of methylation OsICE1 gene by CRISPR/Cas9 technology in situ
  • the rice OsICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 36. According to the degeneracy of the codon, the theoretically methylated site of the OsICE1 gene was replaced, and the OsICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete OsICE1 gene sequence was obtained as SEQ ID NO: 77, designated as OsICE1 (DC) 1; and after partial replacement, the OsICE1 gene sequence was SEQ ID NO: 118 and designated OsICE1 (DC) 2.
  • OsICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GTGGTGGTGGTGGCGCGCATGGG-3'. , 5'-GGCGGTCGTTGAGCTTCTTCCGG-3' and 5'-GCCCCACTGGACAACAGCCAAGG-3';
  • OsICE1 (DC) 2sgRNA sequence is 5'-GCGTCCCAAATGCCGGAGTTCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and OsICE1(DC)1 and OsICE1(DC)2 genes of different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the mature seeds of Japanese clear rice were selected and disinfected by mercury-elevation method (soaked in 0.1% mercuric chloride for 15 min, washed with sterilized water), placed on a clean bench to dry, and then placed in callus induction medium ( 4.4g/L MS+2.5mg/L 2,4-D+600mg/L casein+30g/L sucrose+5g/L plant gel, KOH adjusted to pH5.8, autoclaved) for callus culture Incubate at 28 ° C for 2 weeks.
  • callus induction medium 4.4g/L MS+2.5mg/L 2,4-D+600mg/L casein+30g/L sucrose+5g/L plant gel, KOH adjusted to pH5.8, autoclaved
  • Agrobacterium GV3101 carrying the plasmid of the target gene was inoculated into 5 ml of YEB liquid medium containing 5 mg/L of bialaphos, cultured at 28 °C to the late phase of logarithmic growth phase, and then expanded in a volume of 1:100, at OD600.
  • Agrobacterium cells were harvested at 0.10 and resuspended in the infestation medium.
  • the callus of rice was inoculated according to the conventional method. After co-culture, it was placed on a subculture medium containing 5 mg/L of bialaphos. After dark culture at 12 °C for 12-16 days, it was differentiated on the re-differentiation medium. A positive seedling resistant to bialaphos.
  • Example 72 CRISPR/Cas9 technology in situ replacement of different methylation levels of PaICE1 gene Phalaenopsis
  • the cDNA of Paline1 gene of Phalaenopsis was cloned and the sequence was SEQ ID NO: 37. According to the degeneracy of the codon, the theoretically methylated site of PaICE1 gene was replaced, and the PaICE1 gene was quantitatively replaced according to the target trait requirement. Different degrees of methylation sites.
  • the PaICE1 gene sequence obtained after total replacement was SEQ ID NO: 78, designated PaICE1 (DC) 1; the partially substituted PaICE1 gene sequence was SEQ ID NO: 119, designated PaICE1 (DC) 2.
  • the PaICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 400bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20nt, and the sequence was 5'-GTTTGTGGGGGAAGAAGGACTGG-3'. 5'-GGGATGGTGAGTTTGAGAAGCGG-3' and 5'-GAATAGTAGTCTTACCGGAGGGG-3'; the PaICE1(DC)2sgRNA sequence is 5'-GCCGGGAGCTCCTTAAACCAGGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and PaICE1(DC)1 and PaICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Phalaenopsis protocorm was pre-cultured for 3 days in pre-culture liquid medium (6-BA 1.0 mg/L+NAA 5.0 mg/L+2,4-D 2.0 mg/L+AS 100 ⁇ mol/L).
  • Agrobacterium tumefaciens EHA105 for transformation was cultured in LB (p H7.0) medium containing 5 mg/L bialaphos and acetosyringone 100 ⁇ mol/L at 28 ° C, 150 r/min for 24 h, in the same LB medium.
  • the protocorm was then washed 3 times with liquid medium containing 5.0 mg/L meropenem, respectively, on a solid medium (with KT 3.0 mg/L) supplemented with meropenem 5.0 mg/L and myxomycin 100.0 mg/L. Alternate culture, transfer once every 3d to completely remove Agrobacterium.
  • the detoxified protocorm was transferred to a solid medium containing 5 mg/L of bialaphos and KT 3.0 mg/L, and cultured every 2 weeks, and then transferred to the same medium for subculture.
  • the protocorm was cultured on a protocorm-like (PLBs) induction medium containing 5 mg/L of bialaphos (addition of TDZ and 2,4-D each 1.0 mg/L), and a new one was developed on the cut surface.
  • PLBs are differentiated into seedlings.
  • the plants were transferred to a proliferation medium (6-BA 5.0 mg/L) for cultivation.
  • the incision is moved to the rooting medium (Hyponex No.1 30.0g/L+NAA0.3mg/L), and when each plant grows 4-5 new roots, the length is 2-4cm.
  • the bottle is planted.
  • Example 73 CRISPR/Cas9 technology in situ replacement of different methylation levels of PmICE1 gene
  • the cloned PmICE1 gene cDNA has the sequence of SEQ ID NO: 38. According to the degeneracy of the codon, the theoretically methylated site of the PmICE1 gene is replaced, and the PmICE1 gene is quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the PmICE1 gene sequence obtained after all substitutions was SEQ ID NO: 79, designated PmICE1 (DC) 1; the partial replacement PmICE1 gene sequence was SEQ ID NO: 120, designated PmICE1 (DC) 2.
  • the length of the sgRNA is 20 nt
  • the sgRNA sequences of the PmICE1 (DC) 1 and PmICE1 (DC) 2 genes are both 5'-GCGTCGAGTTCCCAACGACTCGG-3', which guides the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different methylation degree PmICE1 (DC)1, PmICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Agrobacterium EHA105 stored in a -80 ° C refrigerator was inoculated onto YEB plate medium containing 5 mg/L of bialaphos and secreted for 2-3 days. After the colony grows, a single colony is picked and inoculated in 30 ml of YEB liquid medium containing 5 mg/L of bialaphos for the first activation, and shaken at 28 ° C for 1-2 d on a shaker until the OD600 value is about Take off when it is 0.8-1.0.
  • the first activated 50 u1 broth was inoculated in 50 ml of YEB liquid medium containing no bialaphos for the second activation, and cultured at 28 ° C under a common shaker to an OD600 value of 0.4.
  • the cotyledons of the semi-expanded lobes were used as explants, and the cotyledons were cut into cubes of 1 cm*1 cm size for pre-culture for 3 days.
  • the activated Agrobacterium tumefaciens EHA105 was centrifuged at 4 ° C, 6000 rpm, and the cells were collected.
  • the cells were resuspended in liquid MS+AS 100 umol/L until the OD600 value was 0.4, and the callus was infested for 10 min. After co-cultivation for 2 d, the liquid was used.
  • MS+Cef 1000umol/L degerming after washing 5 times in sterile water, placed in the screening medium Ms+BA1.0mg/L+NAA0.5mg/L+KT0.5mg/L+IBA2.0mg/L+2 Selective culture was carried out in 4-D 0.2 mg/L + 5 mg/L bialaphos + Cef 1000 umol/L. The regenerated plants were transferred to MS+6-BA1.0 mg/L+NAA 0.1 mg/L.
  • Example 74 CRISPR/Cas9 technology in situ replacement of different degrees of methylation CsICE1 gene tea
  • the cDNA of the CsICE1 gene was cloned and the sequence was SEQ ID NO: 39. According to the degeneracy of the codon, the theoretically methylated site of the CsICE1 gene was replaced, and the CsICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the CsICE1 gene sequence was obtained as SEQ ID NO: 80 and designated as CsICE1 (DC) 1 after partial replacement; the partial replacement CsICE1 gene sequence was SEQ ID NO: 121 and designated CsICE1 (DC) 2.
  • CsICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt and the sequence was 5'-GCAATGTACAAGGGTATAGTGGG-3'. 5'-GTTTATGGAGAACAATAAGGTGG-3' and 5'-GCGGTACTCTTGGATTCAGCTGG-3'; the CsICE1(DC)2sgRNA sequence is 5'-GGGATTGAGGACGGAAGAAATGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and CsICE1(DC)1 and CsICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the cells were cultured in a constant temperature culture chamber at a temperature of 25 ⁇ 2 ° C, artificial light for 13 h / d, and the light intensity was 1500-2000 lx. After 25 days of culture, the green callus callus was selected as an explant.
  • the preserved Agrobacterium tumefaciens EHA105 was lined on the prepared YEB solid medium plate, and darkly cultured at 28 ° C for 1-2 d. Then, a single colony was picked up with a loop to inoculate 5 ml of YEB liquid medium.
  • the pre-cultured cotyledon callus was placed in the spare Agrobacterium tumefaciens solution, and the bacterial liquid was immersed in the experimental material. After infestation for 15 minutes, the excess bacterial liquid was absorbed by a sterile filter paper and inoculated to add 150 umol/L.
  • the YEB co-cultivation medium of AS was cultured in the dark at 25 ° C for 3 days. After co-cultivation, rinse the material 3-4 times with sterile water, dry the filter paper to absorb the surface moisture of the material, transfer the callus to the pre-culture medium, delay the selection for culture for 3 days, and then transfer to the additional 100umol/ LCef and 100umol/LSpe in sterile medium were cultured in the dark.
  • the explants were transferred to a screening medium supplemented with 100 umol/LSpe for resistance bud screening after 15 days. After 20 days of screening culture, viable resistant shoots were transferred to a new additional 100 umol/LSpe differentiation medium for culture. After cultured for 30 days in the differentiation medium, the callus of the cotyledon differentiated into seedlings, and the whole resistant plants were obtained after being transferred to the rooting medium for 2 weeks. Genetic transformation showed that 6 and 7 strains were replaced with CsICE1(DC)1 and CsICE1(DC)2 gene-resistant plants in situ. Compared with wild-type plants, their cold tolerance increased by 3.3 ° C and 2.0 ° C, respectively.
  • Example 75 CRISPR/Cas9 technology in situ replacement of different methylation levels of AtICE1 gene Arabidopsis thaliana
  • the Arabidopsis thaliana AtICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 40. According to the degeneracy of the codon, the theoretically methylated site of the AtICE1 gene was replaced, and the AtICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites in the medium.
  • the full-replacement AtICE1 gene sequence was SEQ ID NO: 81 and designated as AtICE1 (DC) 1; the partially replaced AtICE1 gene sequence was SEQ ID NO: 122 and designated AtICE1 (DC) 2.
  • AtICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20 nt, and the sequence was 5'-GCTCCTATTTCGATGGGGTTTGG-3'. , 5'-GATAAATGAGAGCGGTAAAGCGG-3' and 5'-GCAGTGCTTTTCGATACAGCAGG-3';
  • the AtICE1 (DC) 2sgRNA sequence is 5'-GAAGATCTTGTGGATGTGGTTGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of AtICE1 (DC)1 and AtICE1(DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the preserved Agrobacterium tumefaciens GV3101 was lined on the prepared YEB solid medium plate and darkly cultured at 28 ° C for 1-2 d. Then, a single colony was picked up with a loop to inoculate 5 ml of YEB liquid medium. After shaking at 28 ° C, 220 rpm for 24 h, 1 ml of the bacterial solution was aspirated into the YEB liquid medium under aseptic conditions, and cultured at 28 ° C, 220 rpm for 4-6 h, and the OD value of the bacterial liquid was measured at 600 nm. About -0.8 can be used for Arabidopsis infection.
  • Non-transgenic seeds can not grow normally, only 2 cotyledons can grow, and the growth of roots is also severely inhibited, generally dying 10 days later.
  • the screened plants were transplanted to vermiculite: peat soil: perlite was 9:3:1 in the matrix, covering the film for 2-3 days.
  • Genetic transformation showed 26 and 27 in situ replacement of AtICE1 (DC)1 and AtICE1 (DC)2 gene resistant plants. Compared with the wild-type Arabidopsis plant Col-0, the cold tolerance was increased by 2.4 ° C and 1.5 ° C, respectively.
  • Example 76 Acquisition of in vitro replacement of different methylation degree BrcICE1 gene phthalocyanine by CRISPR/Cas9 technology
  • the cDNA of the BrincICE gene of phthalocyanine was cloned, and the sequence was SEQ ID NO: 41. According to the degeneracy of the codon, the theoretical methylation site of the BrrICE1 gene was replaced, and the BrrICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the BracICE1 gene sequence after obtaining all substitutions was SEQ ID NO: 82 and designated as BrrICE1 (DC) 1; after partial replacement, the BrrICE1 gene sequence was SEQ ID NO: 123 and designated as BrrICE1 (DC) 2.
  • the BRRICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20 nt and the sequence was 5'-GTCGAGGCTAAAAGCCTGAGAGG-3'. 5'-GTTCAGAACGGAGGAGGTAAAGG-3' and 5'-GCCAGAGGGAGAGATCCATTTGG-3'; the BrrICE1 (DC) 2sgRNA sequence is 5'-GCAACACCCTTATGCGGAGGTGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the BrrICE1 (DC)1 and BrrICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the phthalocyanine seeds were sterilized with 1% sodium hypochlorite for 20 min and then washed 4-5 times with sterile water. The seeds were then placed on budding medium (M S medium containing 20 g/L sucrose, pH 5.8), incubated at 24 ° C, 16 h / 8 h light, and light intensity 60-80 uEm -2 s -1 for 5 days.
  • budding medium M S medium containing 20 g/L sucrose, pH 5.8
  • the preserved Agrobacterium tumefaciens EHA101 was lined on the prepared YEB solid medium plate, and darkly cultured at 28 ° C for 1-2 d, then single colonies were picked up in a 5 ml YEB liquid medium with an inoculating loop.
  • Agrobacterium inoculation was performed by injecting a bacterial suspension (0.5-1.0 ul) into the root of the cotyledon petiole and cocultivating for 3 days on the M S medium.
  • callus induction medium Ms + 20uM 6-BA + 3% sucrose + 500mg / L carboxybenzylomycin + 5mg / L bialaphos, PH 5.8
  • the culture conditions are the same as above .
  • the cut surface of the petiole of the phthalocyanine grows from the exposed vascular bundle tissue to form the callus in a short period of time.
  • the callus meristem is formed from the callus and transferred to the stem elongation medium.
  • Example 77 CRISPR/Cas9 technology in situ replacement of different methylation levels of the ItICE1 gene indigo
  • the indigo ItICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 42. According to the degeneracy of the codon, the theoretically methylated site of the ItICE1 gene was replaced, and the ItICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete ItICE1 gene sequence was obtained as SEQ ID NO: 83 and designated as ItICE1 (DC) 1 after partial replacement; the partial replacement ItICE1 gene sequence was SEQ ID NO: 124 and designated as ItICE1 (DC) 2.
  • ItICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 500bp, and the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GAGCCGAACCCCATGGAGATCGG-3'. 5'-GCTGAGCGATGATGGAGAGATGG-3' and 5'-GCTGGCTACCATGAAAGCTTTGG-3';
  • the ItICE1 (DC) 2sgRNA sequence is 5'-GATGGACGAGACGGGGATTGAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of ItICE1(DC)1, ItICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • Indigo seeds were immersed in 70% alcohol for 45s-lmin, then immersed in 0.1% HgCI 2 for 15min, washed with sterile water for 3-5 times, inoculated on MS basic medium, and cultured at 25 °C for 12-15d.
  • the light intensity is 2000 lx and the photoperiod is 16 h/d.
  • the cells were collected by centrifugation at 4000 r/min for 10 min.
  • the cotyledon stalk was immersed in the Agrobacterium-infected bacterial liquid prepared above, and shaken at 100 rpm for 10 min, the bacterial liquid was discarded, and the cotyledon petiole was removed and dried with a sterile filter paper, and the co-culture medium (MS basic medium + 3 mg/L 6) was inserted.
  • MS basic medium + 3 mg/L 6 the co-culture medium
  • the cotyledon petioles are removed for sterilization, washed first with sterile water, then immersed in sterile water containing Cef 500 mg/L for 20 min, and then washed with sterile water. After 5 passes, the residual water was thoroughly blotted on a sterile filter paper.
  • the seedlings obtained by the expansion were rooted for 25 days, and when the seedlings grew an appropriate amount of roots, the lid of the sterile plant culture flask was opened and placed indoors for 2 days.
  • the nutrient soil, sand and field soil were arranged in a ratio of 1:1:1 as the culture medium for transforming the seedlings.
  • the tissue culture seedlings were carefully taken out, the medium adhered to the roots was washed with water, and the plants were transplanted in a seeding pot and placed in a greenhouse for cultivation.
  • Genetic transformation showed 12 and 10 genetically transformed ItICE1 (DC)1 and ItICE1(DC)2 gene resistant plants, respectively. Compared with wild-type indigo plants, their cold tolerance increased by 3.3 ° C and 1.9 ° C, respectively.
  • Example 78 CRISPR/Cas9 technology in situ replacement of different degrees of methylation EcICE1 gene red peony
  • the erythrocyte EcICE1 gene cDNA was cloned with the sequence of SEQ ID NO: 43. According to the degeneracy of the codon, the theoretically methylatable site of the EcICE1 gene was replaced, and the EcICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the complete EcICE1 gene sequence was obtained as SEQ ID NO: 84 and designated as EcICE1 (DC) 1; the partial replacement EcICE1 gene sequence was SEQ ID NO: 125 and designated EcICE1 (DC) 2.
  • the EcICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp. The sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20 nt, and the sequence was 5'-GGCGGTTCCGAGCTGGGCTCCGG-3'. 5'-GGATGAAAATTAGTCGAAGGGGG-3' and 5'-GTCTGCGTGCACAAAACATGCGG-3'; the EcICE1(DC)2sgRNA sequence is 5'-GGGCTTTTTGGCTCGGTGCAGGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of EcICE1(DC)1, EcICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the preserved GV3101 strains were taken out from the low-temperature refrigerator, and 5-6 lines were gently drawn on the surface of the prepared medium by the scribing method in the ultra-clean workbench.
  • the culture dish was placed at 25-27 ° C for dark culture for 24-36 h. Then, a single colony on the surface of the culture medium in the culture dish was picked up by the inoculation needle and placed in a liquid medium of 15 ml centrifuge tube, and suspension culture was carried out for 12-18 h at 27 ° C and a 180 rpm constant temperature shaker.
  • the bacteria-removing solution was placed in a 100m1 triangular flask at a ratio of 1/40, and the culture was continued for about 6 hours.
  • the OD600 value is 0.5, it is used as an infection treatment.
  • the cultivar DH201-2 was used as the material, and the upper and middle stem segments of the plants cultured on the rooting medium for 30 days were cultured on the preculture medium for 6 days.
  • the red carp explants were immersed in the flask for 30 minutes, then the plant material was taken out and inoculated on the plant co-cultivation medium (100 uM acetosyringone, pH 5.8) for 3 days.
  • the infected stem segments were then transferred to an induction regeneration medium (Ms + 0.05 mg/L TDZ + 0.5 mg/L NAA + 5 mg/L bialaphos + 300 mg/L cephalosporin, pH 5.8).
  • Example 79 CRISPR/Cas9 technology in situ replacement of JcICE1 gene jatropha with different degrees of methylation
  • the JcICE1 gene cDNA of jatropha was cloned and the sequence was SEQ ID NO: 44. According to the degeneracy of the codon, the theoretically methylated site of the JcICE1 gene was replaced, and the JcICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites. After the complete replacement, the JcICE1 gene sequence was SEQ ID NO: 85 and designated JcICE1 (DC) 1; after partial replacement, the JcICE1 gene sequence was SEQ ID NO: 126 and designated JcICE1 (DC) 2.
  • the JcICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of the sgRNA was 20 nt, and the sequence was 5'-GGTTCGATTTGGGTGACATTGGG-3'. , 5'-GGCAAAGCTTATCCTTGATACGG-3' and 5'-GTCTCCTGCTCTCCACTATGAGG-3'; JcICE1 (DC) 2sgRNA sequence is 5'-GAAACGTGTCCTGGGTTTGCAGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and JcICE1(DC)1, JcICE1(DC)2 genes with different degrees of methylation;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the transformed shoots of Jatropha curcas leaves should be carried out in the dark, and transferred to light culture after about 2 weeks.
  • the direct induction medium of adventitious buds is SIO-I: MS + TDZ 1.0 mg / L + 5 mg / L of dialanine Phosphorus + GA3 0.5 mg / I, Cb 500 mg / L was added to the induction medium. After more buds were formed, they were transferred to adventitious bud proliferation medium MS+BA 0.3mg/L+IBA 0.01mg/L in light culture, and 5mg/L bialaphos was added to the medium, Cb 500mg/ L.
  • Rooting plants can be obtained in about 30 days. Then take the seedlings with better rooting, use the principle of gradual progress to refine the seedlings, and gradually open the bottle cap. After completely opening the cap for 2-3 days, the rooting shoots were taken out, the medium was carefully washed away, the old leaves and dead leaves were removed, and they were planted in a sterilized frog stone matrix. At the beginning, you can cover the ground part with a film to prevent excessive transpiration, avoid direct sunlight, and increase the survival rate.
  • Example 80 CRISPR/Cas9 technology in situ replacement of different degrees of methylation PiaICE1 gene European spruce
  • the cDNA of the European spruce PiaICE1 gene was cloned, and the sequence was SEQ ID NO: 45. According to the degeneracy of the codon, the theoretically methylated site of the PiaICE1 gene was replaced, and the PiaICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites in the medium.
  • the PiaICE1 gene sequence after obtaining all substitutions was SEQ ID NO: 86, designated PiaICE1 (DC) 1; after partial replacement, the PiaICE1 gene sequence was SEQ ID NO: 127, designated PiaICE1 (DC) 2.
  • PiaICE1(DC)1 was divided into 2 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by artificially.
  • the length of sgRNA was 20 nt and the sequence was 5'-GGAGTTGGCCTGCGAGGGGTTGG-3'. And 5'-GGACTCGAACCTGCGAAGCTGGG-3'; PiaICE1 (DC) 2sgRNA sequence is 5'-GTTCAAGGCAATGCTGGACGCGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of PiaICE1 (DC)1 and PiaICE1(DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • 0.5m1 bacterial solution was transferred to 25m1 YEB+5mg/L bialaphos in liquid medium for 6h.
  • the cells were collected by centrifugation at 5000rpm for 10min, and the same amount of liquid induction medium was taken. Suspended cells.
  • the pre-cultured stem segments were immersed in the prepared leachable bacterial solution, shaken gently, soaked for 15 min, and the stem segments were removed, and the excess bacterial liquid was aspirated on the sterile filter paper, and then returned to the induction medium for co-cultivation (addition of 80 uM) AS).
  • the stem segments were washed 3 times with liquid induction medium to remove excess Agrobacterium. And transferred to the primary screening medium (DCR + 6-BA 1.0 mg / L + TDZ 0.001 mg / L + NAA 0.1 mg / L + Cef 200 mg / L + bialaphos 5 mg / L, pH 5.8) on the culture.
  • the washed stem segments were transferred to the primary screening medium for about 20 days, and transferred to the secondary screening medium (DCR+Cef 200 mg/L + bialaphos 5 mg/L, pH 5.8) when a few stem segments began to grow. . It was subcultured once on the secondary screening medium for 25 days.
  • a total of 186 Km resistant buds were obtained on the primary screening medium.
  • the first obtained Km resistant buds were subcultured twice on the secondary screening medium. During the subculture process, a large number of buds were whitened, and the buds remained green. Resistant to buds for Km.
  • a total of 39 Km resistant shoots were obtained in this study.
  • Adventitious buds with a height of about 1-5 cm were transferred to rooting medium (1/4 MS basic medium supplemented with IBA 0.2 mg/L, NAA 0.1 mg/L, sucrose 296, agar powder 0.6%, pH 5.8), after inoculation for 7 days, The base of the adventitious bud begins to grow root primordium, and after 25 days the root primordium can be elongated to 1-2 cm.
  • Genetic transformation showed 10 and 13 in situ replacement of PiaICE1 (DC)1 and PiaICE1 (DC)2 gene resistant plants. Compared with wild-type European spruce plants, their cold tolerance increased by 3.9 ° C and
  • Example 81 Acquisition of in vitro replacement of different methylation degree PsICE1 gene sweet poplar by CRISPR/Cas9 technology
  • the cDNA of the sweet poplar PsICE1 gene was cloned, and the sequence was SEQ ID NO: 46. According to the degeneracy of the codon, the theoretically methylated site of the PsICE1 gene was replaced, and the PsICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the PsICE1 gene sequence after obtaining all substitutions was SEQ ID NO: 87 and designated as PsICE1 (DC) 1; the partial replacement PsICE1 gene sequence was SEQ ID NO: 128 and designated PsICE1 (DC) 2.
  • the PsICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement. Each segment was about 500 bp.
  • the sgRNA with a guiding effect was designed by hand.
  • the length of the sgRNA was 20 nt, and the sequence was 5'-GGTTTCTTCCAACCCTTCAAAGG-3'. 5'-GGTCGTGGTGAGGATCAAAAGGG-3' and 5'-GCAGTCCTACTGGATTCAGCTGG-3'; the PsICE1(DC)2sgRNA sequence is 5'-GCAGCTAGAGGAAAGAACTTGGG-3', which directs the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage vector pHEE401E and different degrees of methylation of PsICE1(DC)1, PsICE1(DC)2 gene;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the Agrobacterium GV3101 strain stored at -70 ° C was thawed on ice, and an appropriate amount of the bacterial solution was applied to the LB medium plate containing the corresponding antibiotic, and cultured at 28 ° C until a single colony was produced, about 2 d.
  • Single colonies were picked from the plates, inoculated into 5 ml LB liquid medium supplemented with spc 100 mg/L, GM 50 mg/L, cultured at 28 ° C, shaking at 220 rpm for 24 h, and then 0.4 ml of the bacterial solution was removed from the solution to 20 ml containing the corresponding antibiotics.
  • the culture was carried out for about 6 hours until the OD600 value was 0.5.
  • the bacterial cells were collected at 5000 rpm for 10 min to collect the cells, and then the cells were resuspended in an equal volume of 1/2 MS liquid medium, and the broth was diluted to an OD600 value of 0.5. Used for infestation. Add 20 uM acetosyringone, gently shake at 25 °C (90 rpm) for 30 min; select the sweet potato tissue culture seedlings with the same growth potential (4-6 weeks), take 2, 3 stem segments, trim off the leaves and axillary buds, quickly with a blade Cut it into 1-1.5m stem segments, transfer to the prepared dip solution, gently shake at 25 ° C (250ml flask, 90rpm) for 30min, remove the stem segment, use filter paper to dry the residual bacterial solution, and transfer to the free
  • the antibiotic culture medium is dark cultured; the co-culture medium is a differentiation medium, and the co-culture is a dark culture method.
  • the stem segments were placed on a screening medium (callus induction medium + cef 200 mg/L + 5 mg/L bialaphos, pH 5.6) for screening culture, and at both ends of the stem segment after 15-20 days. A green resistant callus will grow. After the stem segments of the cultured cells are completely grown with small shoots, they are transferred to the shoot elongation medium (MS+6-BA 0.2 mg/L+TDZ 0.01 mg/L+cef 200 mg/L+5 mg/L dialanine). Phosphorus, pH 5.6), bud elongation medium contains a lower concentration of hormones, which is conducive to the rapid growth of resistant buds.
  • a screening medium callus induction medium + cef 200 mg/L + 5 mg/L bialaphos, pH 5.6
  • Example 82 CRISPR/Cas9 technology in situ replacement of different degrees of methylation of PtICE1 gene
  • the cDNA of the PtICE1 gene was cloned and the sequence was SEQ ID NO: 47. According to the degeneracy of the codon, the theoretically methylated site of the PtICE1 gene was replaced, and the PtICE1 gene was quantitatively replaced according to the desired trait. Different degrees of methylation sites.
  • the PtICE1 gene sequence obtained after total replacement was SEQ ID NO: 88, designated PtICE1 (DC) 1; the partial replacement PtICE1 gene sequence was SEQ ID NO: 129, designated PtICE1 (DC) 2.
  • PtICE1(DC)1 was divided into 3 segments for CRISPR/Cas9 vector construction and in situ replacement, each segment was about 400bp, and the sgRNA with guiding effect was designed by artificially.
  • the length of sgRNA was 20nt, and the sequence was 5'-GTTTCATCTCTGTTAAACAGGGG-3'. , 5'-GATGTTAGCTTTGATGGGTCGGG-3' and 5'-GCTTCCAGCTAAGAATTTGATGG-3'; PtICE1 (DC) 2sgRNA sequence is 5'-GTTTCATCTCTGTTAAACAGGGG-3', which directs Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the PtICE1 (DC)1 and PtICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the Yang genetic transformation experiment method was the same as the sweet potato genetic transformation experiment (Example 39).
  • 18 and 22 strains of in situ replacement of PtICE1(DC)1 and PtICE1(DC)2 genes were obtained.
  • their cold tolerance increased by 3.7 ° C and 2.2 ° C, respectively.
  • Example 83 Acquisition of PopICE1 Gene Arabidopsis thaliana with different degrees of methylation in vitro by CRISPR/Cas9 technology
  • the PopICE1 gene cDNA was cloned and the sequence was SEQ ID NO: 48. According to the degeneracy of the codon, the theoretically methylatable site of the PopICE1 gene was replaced, and the quantitative replacement was performed according to the target trait requirement. Different degrees of methylation sites in the PopICE1 gene.
  • the entire PostICE1 gene sequence was obtained as SEQ ID NO: 89 and designated as PopICE1 (DC) 1; the partial replacement PopICE1 gene sequence was SEQ ID NO: 130 and designated PopICE1 (DC) 2.
  • the length of the sgRNA is 20 nt
  • the sgRNA sequences of the PopICE1 (DC) 1 and PopICE1 (DC) 2 genes are both 5'-GCTCCTATTTCGATGGGGTT NGG-3', which guides the cleavage of DNA by the Cas9 protein.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the PtICE1 (DC)1 and PtICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.
  • the preserved Agrobacterium tumefaciens GV3101 was lined on the prepared YEB solid medium plate and darkly cultured at 28 ° C for 1-2 d. Then, a single colony was picked up with a loop to inoculate 5 ml of YEB liquid medium. After shaking at 28 ° C, 220 rpm for 24 h, 1 ml of the bacterial solution was aspirated into the YEB liquid medium under aseptic conditions, and cultured at 28 ° C, 220 rpm for 4-6 h, and the OD value of the bacterial liquid was measured at 600 nm. About -0.8 can be used for Arabidopsis infection.
  • Non-transgenic seeds can not grow normally, only two cotyledons can grow, and root growth is also severely inhibited.
  • the screened plants were transplanted to vermiculite: peat soil: perlite was 9:3:1 in the matrix, covering the film for 2-3 days.
  • Genetic transformation showed that 14, 19, 17 in situ replacement of PopICE1, PopICE1 (DC) 1, and PopICE1 (DC) 2 gene resistant plants were obtained.
  • the cold tolerance was increased by 0.8 ° C, 2.0 ° C and 1.5 ° C, respectively.
  • Example 84 CRISPR/Cas9 technology in situ extraction of PwICE1 gene Arabidopsis thaliana with different degrees of methylation
  • the PwICE1 gene cDNA of the Phyllostachys pubescens L. was cloned, and the sequence was SEQ ID NO: 49. According to the degeneracy of the codon, the theoretically methylatable site of the PwICE1 gene was replaced, and the quantitative replacement was performed according to the target trait requirement. Different degrees of methylation sites in the PwICE1 gene.
  • the PwICE1 gene sequence obtained after total replacement was SEQ ID NO: 90 and designated PwICE1 (DC) 1; the partial replacement PwICE1 gene sequence was SEQ ID NO: 131 and designated PwICE1 (DC) 2.
  • the length of the sgRNA is 20 nt
  • the PwICE1 (DC) 1 and PwICE1 (DC) 2 gene sequences are both 5'-GCTCCTATTTCGATGGGGTT NGG-3', which guides the Cas9 protein to site-directed cleavage of DNA.
  • the promoter selects the YAO promoter, and the target gene is ligated into the zCas9 sequence by restriction endonuclease BsaI, KpnI cleavage of the vector pHEE401E and different degrees of methylation of the PtICE1 (DC)1 and PtICE1 (DC)2 genes;
  • the SacII and AsiSI restriction sites were added to both ends of the gene, and the pHEE401E and Bar genes were digested by SacII and AsiSI, and the Bar gene was ligated into pHEE401E as a resistance screening gene to construct a CRISPR/Cas9 expression vector.

Abstract

提供一种表观遗传操作植物表型可塑性性状的方法，通过简并密码子定量替换目的基因的可甲基化位点，通过设计合成基因，进行转基因操作；或利用CRISPR/Cas9技术构建植物表达载体进行原位在体基因简并密码子定量替换可甲基化位点，定量调控外源或原位在体目的基因甲基化程度和基因表达水平，达到定量调控目标植物的可塑性性状。通过定量调节ICE1基因的甲基化程度培育不同抗寒能力植物，为通过表观遗传技术培育抗寒作物提供了新的选择，为降低低温伤害对作物产量和品质等的影响提供了技术方法。

Description

一个表观遗传操作植物表型可塑性性状的方法 技术领域
本发明属于分子生物学与植物遗传工程领域，涉及一个表观遗传操作植物表型可塑性性状的方法。
背景技术
DNA甲基化是基因组DNA的一种重要表观遗传形式，是指在DNA甲基转移酶(DNA methyltransferase，DNMT)的催化下，以S-腺苷甲硫氨酸(S-adenosylmethionine，SAM)为甲基供体，将甲基转移到特定的碱基上的过程。DNA甲基化现象广泛存在于细菌、植物和动物中，参与生物体的多种生物学过程。DNA甲基化在植物中具有物种、组织、器官、年龄特异性，参与遗传功能的调控，包括转录、复制、DNA修复、转基因和细胞分化，在植物生长发育及进化过程中起着重要的调节作用。Jullien等人(2012)在对拟南芥(Arabidopsis thaliana)有性生殖过程中的DNA甲基化动态变化进行研究时发现，在雌配子发生过程中，维持甲基化方式几乎不发生，而受精后在胚胎中从头甲基化与维持甲基化明显发生，并且维持一定甲基化水平直到成株，这种DNA甲基化动态变化周期对于拟南芥的繁殖具有一定意义。高盐处理可以导致水稻基因组DNA甲基化状态的改变(韩雅楠等，2010)。Tan等(2010)研究表明，盐胁迫可诱导玉米ABA负调控因子ZmPP2C内含子区甲基化水平提高，致使其表达量显著下调；而诱导参与活性氧代谢的ZmGST去甲基化，使得其表达水平增加。这些研究表明，逆境胁迫通过改变植物DNA表观遗传修饰状态，调控基因表达和植物生长发育，从而应对逆境胁迫。
作为表观遗传修饰主要方式之一，DNA甲基化已成为研究热点。虽然人们对DNA甲基化的研究开展了大量的工作，但主要集中在甲基化发生机制、甲基化生物学功能及甲基化检测方法等方面，但如何通过甲基化的改变活化基因表达的研究尚处于起步阶段，研究还不够深入。上述研究多在体内环境中进行，体外环境下甲基化是否可以被调控仍未得到验证，而转基因植物中发生的甲基化沉默多与DNA甲基化相关，上述因素导致了DNA甲基化在转基因作物培育中的应用。
低温是经常发生且危害严重的逆境因素之一，许多植物在受到非冷害低温处理后，其抗冷性将会增强，这就是所谓的冷驯化现象(Thomashow，1999)。但是，热带和亚热带起源的植物对温度条件要求比较高，无法适应骤冷温度(0-15℃)。低温伤害严重影响香蕉(Xu et al., 2002)、棉花(Zhang&Zhang，2006)、玉米(Ma et al.,2006)和水稻(Quan et al.,2006)等的产量和品质。在1991-1992、1992-1993年以及最近的1999-2000年等几个严寒冬春，华南地区都因特强寒潮的入侵而导致了大面积的蕉园受到毁灭性的破坏，使广大蕉农蒙受极大的损失(Xu et al.,2002)。棉花整个生长发育过程中，最适宜的温度为20～30℃，当日平均温度低于15℃而造成对棉花生长发育的影响时，称为冷害(Yin,2002)。2001年7月31日-8月3日连续低温、降水，最低温连续11-13℃，导致昌吉、石河子、奎屯26.7×104hm 2棉花减产40％-50％，纤维品质下降1-2级，经济损失20×10 8元(Zhang&Zhang，2006)。由于东北地区大部气温较低，不少年份玉米生长季积温不足(≥10℃)，玉米生长发育较慢，成熟期推迟，导致玉米在秋霜前不能正常成熟，从而发生低温冷害，严重冷害年份(如1957、1969、1972和1976年等)玉米减产15％以上(Ma et al.,2006)。我国大部分稻区都受到低温冷害的威胁，冷害发生频繁。吉林省延边地区水稻冷害发生年有1954、1956、1957、1969、1971、1972、1974、1976、1979、1980、1986、1988、1993、1995、1998、2002、2003和2006年(Quan et al.,2006)。
驯化过程中新产生的蛋白即为低温诱导蛋白，因低温驯化而表达的基因就称为低温诱导基因(cold-regulated gene，COR)。植物在低温胁迫下COR基因的转录水平会提高，而这些基因的表达又能被ABA所诱导，因此曾经有专家预测是低温信号引起植物体内ABA的大量合成，后者再诱导下游的其它基因表达，该通路称为ABA依赖信号途径(Skriver et al.,1990)。之后的研究发现COR的表达能够在ABA合成基因发生突变的植株内表达，从而证实了独立于ABA途径通路的存在，称ABA非依赖信号途径。1994年Yamaguchi和Skinozaki首次在拟南芥rd29A基因的启动子区域中发现了一个9bp(TACCGACAT)的DRE元件(dehydration responseve element)，同年又从COR15a基因的启动子区域鉴定出另一5bp(CCGAC)的DNA调控元件CRT(C-repeat)。这两个元件均含有CCGAC核心序列，即LTRE(low-temperature responsive element)元件。CRT/DRE或其核心序列CCGAC普遍存在于低温诱导基因的启动子中，可促使低温、干旱及高盐等条件下基因的表达。第一个CRT/DRE结合因子为CBF1转录因子，后来又利用探针分离了CBF2和CBF3。除了模式植物拟南芥之外，人们也从别的物种克隆了许多类CBF基因。由于CBF的启动区不含CRT/DRE序列和CCGAC基序，故该基因家族不存在自我调节现象，其上游必然有诱导其表达的物质。根据CBF的表达特性及其功能预测，Gilmour等(1998)提出了ICE-CBF通路模型。ICE1(inducer of CBF expression 1)是唯一已经鉴定直接作用于CBF启动子的转录因子，它是控制CBF基因的一个主开关。DNA结合实验表明它能与CBF基因启动 子的MYC识别序列CANNTG(即Gilmour预测的ICE盒)结合(Chinnusamy et al.,2003)，而ICE盒正是激活CBF基因转录的重要顺式作用元件(Zarka et al.,2003)。ICE1在常温下由于磷酸化而处于钝化状态，在低温条件下发生脱磷酸化被活化，并特异性地结合到CBF的启动子上的MYC位点，从而诱导CBF的表达。
Jaglo Ohoson(1998)发现，CBF1在转基因拟南芥中组成型超表达后，可使含有CRT/DRE调控元件的COR基因在未经低温诱导的条件下表达，说明CBF1是拟南芥低温驯化反应的重要调控因子，它通过调控COR基因的表达水平，从而提高拟南芥的抗冻性。CBFs基因强表达后，抗冻性得到一定提高，但使转基因拟南芥植株生长缓慢，花期延迟(Gilmour et al.,2004)。Fernando等(2003)研究表明，拟南芥cbf2突变体在冷胁迫抗性，CBF1和CBF3的表达增强，耐旱、耐盐能力提高，表明CBF2是CBF1和CBF3表达的一个负调节子。ICE1的转基因拟南芥植株，与未转基因拟南芥植株相比，转基因拟南芥植株的耐寒能力有明显提高(Chinnusamy et al.,2003；Chinnusamy et al.,2006)。郑银英等(2009)也将克隆到的拟南芥ICE1基因转入烟草，结果表明瞬时低温冻害下，转基因烟草存活率明显高于对照烟草植株，说明ICE1基因可以提高低温敏感植物的抗寒性。突变体的转录组学研究表明，ICE1表达缺陷造成50％的低温诱导基因不能被低温激活。但前人关于转ICE1基因提高受体植株耐冷能力的研究，并不能控制提高受体植株耐冷能力的程度。
发明内容
本发明提供了一种基因甲基化定量调控的表观遗传操作技术，通过简并密码子定量替换可甲基化位点后设计合成基因，或利用CRISPR/Cas9技术进行原位基因简并密码子定量替换遗传操作，在转基因受体或目标植物在体定量调控目的基因的甲基化程度，实现基因转录和表达水平的调控，达到表观遗传操作植物表型可塑性性状的效果。
一个表观遗传操作植物表型可塑性性状的方法,通过简并密码子定量替换目的基因的可甲基化位点后设计合成基因，进行转基因操作；或利用CRISPR/Cas9技术构建植物表达载体进行原位在体基因简并密码子定量替换可甲基化位点后，定量调控原位在体目的基因的甲基化程度，实现基因表达水平调控，达到定量调控植物的可塑性性状。
所述的方法优选：通过简并密码子定量替换目的基因的可甲基化胞嘧啶位点，定量控制基因在生物体中可被重新甲基化的位点数，达到定量调控目的基因甲基化程度和基因表达水平，实现定量调控植物的可塑性性状的目的。
所述的方法还优选：设计合成密码子定量替换的目的基因，并进行转基因遗传操作，定量调控外源目的基因在受体中的甲基化程度和基因表达水平，达到定量调控植物的可塑性性状。
所述的方法还优选：利用CRISPR/Cas9编辑技术构建植物表达载体进行原位在体基因简并密码子定量替换目的基因可甲基化胞嘧啶位点，定量调控原位在体目的基因的甲基化程度和基因表达水平，达到定量调控改造植物的可塑性性状。
本发明所述的方法中，所述的可塑性性状优选耐寒性。
本发明所述的方法中，所述的目的基因优选ICE1基因。
所述的ICE1基因选自紫茎泽兰AaICE1基因，木薯MeICE1基因，猕猴桃AcICE1基因，野生蕉MaICE1基因，芭蕉MabICE1基因，可可TcICE1基因，枳CtICE1基因，葡萄VvICE1基因，苹果MdICE1基因，芥CbICE1基因，茄子ScICE1基因，番茄SlICE1基因，桃PpICE1基因，玉米ZmICE1基因，棉花GhICE1基因，花生AhICE1基因，芥菜BjICE1基因，油菜BrICE1基因，菊花CdICE1基因，小盐芥ThICE1基因，野胡萝卜DcICE1基因，大豆GmICE1基因，萝卜RsICE1基因，小麦TaICE1基因，大麦HvICE1基因，橡胶树HbICE1基因，胡桃JrICE1基因，莴苣LsICE1基因，水稻OsICE1基因，蝴蝶兰PaICE1基因，梅花PmICE1基因，茶CsICE1基因，拟南芥AtICE1基因，芜菁BrrICE1基因，菘蓝ItICE1基因，赤桉EcICE1基因，麻风树JcICE1基因，欧洲云杉PiaICE1基因，甜杨PsICE1基因，杨PtICE1基因，穿叶眼子菜PopICE1基因，竹叶眼子菜PwICE1基因；其中，紫茎泽兰AaICE1基因核苷酸序列为SEQ ID NO：1，木薯MeICE1基因核苷酸序列为SEQ ID NO：9，猕猴桃AcICE1基因核苷酸序列为SEQ ID NO：10，野生蕉MaICE1基因核苷酸序列为SEQ ID NO：11，芭蕉MabICE1基因核苷酸序列为SEQ ID NO：12，可可TcICE1基因核苷酸序列为SEQ ID NO：13，枳CtICE1基因核苷酸序列为SEQ ID NO：14，葡萄VvICE1基因核苷酸序列为SEQ ID NO：15，苹果MdICE1基因核苷酸序列为SEQ ID NO：16，芥CbICE1基因核苷酸序列为SEQ ID NO：17，茄子ScICE1基因核苷酸序列为SEQ ID NO：18，番茄SlICE1基因核苷酸序列为SEQ ID NO：19，桃PpICE1基因核苷酸序列为SEQ ID NO：20，玉米ZmICE1基因核苷酸序列为SEQ ID NO：21，棉花GhICE1基因核苷酸序列为SEQ ID NO：22，花生AhICE1基因核苷酸序列为SEQ ID NO：23，芥菜BjICE1基因核苷酸序列为SEQ ID NO：24，油菜BrICE1基因核苷酸序列为SEQ ID NO：25，菊花CdICE1基因核苷酸序列为SEQ ID NO：26，小盐芥ThICE1基因核苷酸序列为SEQ ID NO：27，野胡萝卜DcICE1基因核苷酸序列为SEQ ID NO：28，大豆GmICE1基因核苷酸序列为SEQ ID NO：29，萝卜RsICE1基因核苷酸序列为SEQ ID NO：30，小麦TaICE1基因核苷酸序列为SEQ ID NO：31，大麦HvICE1基因核苷酸序 列为SEQ ID NO：32，橡胶树HbICE1基因核苷酸序列为SEQ ID NO：33，胡桃JrICE1基因核苷酸序列为SEQ ID NO：34，莴苣LsICE1基因核苷酸序列为SEQ ID NO：35，水稻OsICE1基因核苷酸序列为SEQ ID NO：36，蝴蝶兰PaICE1基因核苷酸序列为SEQ ID NO：37，梅花PmICE1基因核苷酸序列为SEQ ID NO：38，茶CsICE1基因核苷酸序列为SEQ ID NO：39，拟南芥AtICE1基因核苷酸序列为SEQ ID NO：40，芜菁BrrICE1基因核苷酸序列为SEQ ID NO：41，菘蓝ItICE1基因核苷酸序列为SEQ ID NO：42，赤桉EcICE1基因核苷酸序列为SEQ ID NO：43，麻风树JcICE1基因核苷酸序列为SEQ ID NO：44，欧洲云杉PiaICE1基因核苷酸序列为SEQ ID NO：45，甜杨PsICE1基因核苷酸序列为SEQ ID NO：46，杨PtICE1基因核苷酸序列为SEQ ID NO：47，穿叶眼子菜PopICE1基因核苷酸序列为SEQ ID NO：48，竹叶眼子菜PwICE1基因核苷酸序列为SEQ ID NO：49。
所述的通过简并密码子全部替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：3，木薯：SEQ ID NO：50，猕猴桃：SEQ ID NO：51，野生蕉：SEQ ID NO：52，芭蕉：SEQ ID NO：53，可可：SEQ ID NO：54，枳：SEQ ID NO：55，葡萄：SEQ ID NO：56，苹果：SEQ ID NO：57，芥：SEQ ID NO：58，茄子：SEQ ID NO：59，番茄：SEQ ID NO：60，桃：SEQ ID NO：61，玉米：SEQ ID NO：62，棉花：SEQ ID NO：63，花生：SEQ ID NO：64，芥菜：SEQ ID NO：65，油菜：SEQ ID NO：66，菊花：SEQ ID NO：67，小盐芥：SEQ ID NO：68，野胡萝卜：SEQ ID NO：69，大豆：SEQ ID NO：70，萝卜：SEQ ID NO：71，小麦：SEQ ID NO：72，大麦：SEQ ID NO：73，橡胶树：SEQ ID NO：74，胡桃：SEQ ID NO：75，莴苣：SEQ ID NO：76，水稻：SEQ ID NO：77，蝴蝶兰：SEQ ID NO：78，梅花：SEQ ID NO：79，茶：SEQ ID NO：80，拟南芥：SEQ ID NO：81，芜菁：SEQ ID NO：82，菘蓝：SEQ ID NO：83，赤桉：SEQ ID NO：84，麻风树：SEQ ID NO：85，欧洲云杉：SEQ ID NO：86，甜杨：SEQ ID NO：87，杨：SEQ ID NO：88，穿叶眼子菜：SEQ ID NO：89，竹叶眼子菜：SEQ ID NO：90；通过简并密码子部分替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰 ：SEQ ID NO：4，木薯：SEQ ID NO：91，猕猴桃：SEQ ID NO：92，野生蕉：SEQ ID NO：93，芭蕉：SEQ ID NO：94，可可：SEQ ID NO：95，枳：SEQ ID NO：96，葡萄：SEQ ID NO：97，苹果：SEQ ID NO：98，芥：SEQ ID NO：99，茄子：SEQ ID NO：100，番茄：SEQ ID NO：101，桃：SEQ ID NO：102，玉米：SEQ ID NO：103，棉花：SEQ ID NO：104，花生：SEQ ID NO：105，芥菜：SEQ ID NO：106，油菜：SEQ ID NO：107，菊花：SEQ ID NO：108，小盐芥：SEQ ID NO：109，野胡萝卜：SEQ ID NO：110，大豆：SEQ ID NO：111，萝卜：SEQ ID NO：112，小麦：SEQ ID NO：113，大麦：SEQ ID NO：114， 橡胶树：SEQ ID NO：115，胡桃：SEQ ID NO：116，莴苣：SEQ ID NO：117，水稻：SEQ ID NO：118，蝴蝶兰：SEQ ID NO：119，梅花：SEQ ID NO：120，茶：SEQ ID NO：121，拟南芥：SEQ ID NO：122，芜菁：SEQ ID NO：123，菘蓝：SEQ ID NO：124，赤桉：SEQ ID NO：125，麻风树：SEQ ID NO：126，欧洲云杉：SEQ ID NO：127，甜杨：SEQ ID NO：128，杨：SEQ ID NO：129，穿叶眼子菜：SEQ ID NO：130，竹叶眼子菜：SEQ ID NO：131。
作为本发明方法的优选，合成或CRISPR/Cas9原位进行简并密码子全部或部分替换可甲基化的胞嘧啶后的AaICE1基因，将其转化到水稻、木薯、香蕉、番茄、可可、橡胶、柠檬、甜橙、柑橘、甘蔗、番木瓜、枇杷、荔枝、龙眼、芒果、马铃薯、辣椒、高粱、葡萄、苹果、芥、茄子、猕猴桃、桃、玉米、棉花、花生、芥菜、油菜、菊花、胡萝卜、大豆、萝卜、小麦、大麦、胡桃、莴苣、大花蕙兰、蝴蝶兰、腊梅、茶、赤桉植株中，获得抗寒性增强的转基因植株；所述的全部替换可甲基化的胞嘧啶后的AaICE1基因序列为SEQ ID NO：3，部分替换可甲基化的胞嘧啶后的AaICE1基因序列为SEQ ID NO：4。
一种耐寒基因，选自全部或部分替换可甲基化的胞嘧啶后的ICE1基因。
其中，野生型ICE1基因选自紫茎泽兰AaICE1基因，木薯MeICE1基因，猕猴桃AcICE1基因，野生蕉MaICE1基因，芭蕉MabICE1基因，可可TcICE1基因，枳CtICE1基因，葡萄VvICE1基因，苹果MdICE1基因，芥CbICE1基因，茄子ScICE1基因，番茄SlICE1基因，桃PpICE1基因，玉米ZmICE1基因，棉花GhICE1基因，花生AhICE1基因，芥菜BjICE1基因，油菜BrICE1基因，菊花CdICE1基因，小盐芥ThICE1基因，野胡萝卜DcICE1基因，大豆GmICE1基因，萝卜RsICE1基因，小麦TaICE1基因，大麦HvICE1基因，橡胶树HbICE1基因，胡桃JrICE1基因，莴苣LsICE1基因，水稻OsICE1基因，蝴蝶兰PaICE1基因，梅花PmICE1基因，茶CsICE1基因，拟南芥AtICE1基因，芜菁BrrICE1基因，菘蓝ItICE1基因，赤桉EcICE1基因，麻风树JcICE1基因，欧洲云杉PiaICE1基因，甜杨PsICE1基因，杨PtICE1基因，穿叶眼子菜PopICE1基因，竹叶眼子菜PwICE1基因；其中，紫茎泽兰AaICE1基因核苷酸序列为SEQ ID NO：1，木薯MeICE1基因核苷酸序列为SEQ ID NO：9，猕猴桃AcICE1基因核苷酸序列为SEQ ID NO：10，野生蕉MaICE1基因核苷酸序列为SEQ ID NO：11，芭蕉MabICE1基因核苷酸序列为SEQ ID NO：12，可可TcICE1基因核苷酸序列为SEQ ID NO：13，枳CtICE1基因核苷酸序列为SEQ ID NO：14，葡萄VvICE1基因核苷酸序列为SEQ ID NO：15，苹果MdICE1基因核苷酸序列为SEQ ID NO：16，芥CbICE1基因核苷酸序列为SEQ ID NO：17，茄子ScICE1基因核苷酸序列为SEQ ID NO： 18，番茄SlICE1基因核苷酸序列为SEQ ID NO：19，桃PpICE1基因核苷酸序列为SEQ ID NO：20，玉米ZmICE1基因核苷酸序列为SEQ ID NO：21，棉花GhICE1基因核苷酸序列为SEQ ID NO：22，花生AhICE1基因核苷酸序列为SEQ ID NO：23，芥菜BjICE1基因核苷酸序列为SEQ ID NO：24，油菜BrICE1基因核苷酸序列为SEQ ID NO：25，菊花CdICE1基因核苷酸序列为SEQ ID NO：26，小盐芥ThICE1基因核苷酸序列为SEQ ID NO：27，野胡萝卜DcICE1基因核苷酸序列为SEQ ID NO：28，大豆GmICE1基因核苷酸序列为SEQ ID NO：29，萝卜RsICE1基因核苷酸序列为SEQ ID NO：30，小麦TaICE1基因核苷酸序列为SEQ ID NO：31，大麦HvICE1基因核苷酸序列为SEQ ID NO：32，橡胶树HbICE1基因核苷酸序列为SEQ ID NO：33，胡桃JrICE1基因核苷酸序列为SEQ ID NO：34，莴苣LsICE1基因核苷酸序列为SEQ ID NO：35，水稻OsICE1基因核苷酸序列为SEQ ID NO：36，蝴蝶兰PaICE1基因核苷酸序列为SEQ ID NO：37，梅花PmICE1基因核苷酸序列为SEQ ID NO：38，茶CsICE1基因核苷酸序列为SEQ ID NO：39，拟南芥AtICE1基因核苷酸序列为SEQ ID NO：40，芜菁BrrICE1基因核苷酸序列为SEQ ID NO：41，菘蓝ItICE1基因核苷酸序列为SEQ ID NO：42，赤桉EcICE1基因核苷酸序列为SEQ ID NO：43，麻风树JcICE1基因核苷酸序列为SEQ ID NO：44，欧洲云杉PiaICE1基因核苷酸序列为SEQ ID NO：45，甜杨PsICE1基因核苷酸序列为SEQ ID NO：46，杨PtICE1基因核苷酸序列为SEQ ID NO：47，穿叶眼子菜PopICE1基因核苷酸序列为SEQ ID NO：48，竹叶眼子菜PwICE1基因核苷酸序列为SEQ ID NO：49；
所述的通过简并密码子全部替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：3，木薯：SEQ ID NO：50，猕猴桃：SEQ ID NO：51，野生蕉：SEQ ID NO：52，芭蕉：SEQ ID NO：53，可可：SEQ ID NO：54，枳：SEQ ID NO：55，葡萄：SEQ ID NO：56，苹果：SEQ ID NO：57，芥：SEQ ID NO：58，茄子：SEQ ID NO：59，番茄：SEQ ID NO：60，桃：SEQ ID NO：61，玉米：SEQ ID NO：62，棉花：SEQ ID NO：63，花生：SEQ ID NO：64，芥菜：SEQ ID NO：65，油菜：SEQ ID NO：66，菊花：SEQ ID NO：67，小盐芥：SEQ ID NO：68，野胡萝卜：SEQ ID NO：69，大豆：SEQ ID NO：70，萝卜：SEQ ID NO：71，小麦：SEQ ID NO：72，大麦：SEQ ID NO：73，橡胶树：SEQ ID NO：74，胡桃：SEQ ID NO：75，莴苣：SEQ ID NO：76，水稻：SEQ ID NO：77，蝴蝶兰：SEQ ID NO：78，梅花：SEQ ID NO：79，茶：SEQ ID NO：80，拟南芥：SEQ ID NO：81，芜菁：SEQ ID NO：82，菘蓝：SEQ ID NO：83，赤桉：SEQ ID NO：84，麻风树：SEQ ID NO：85，欧洲云杉：SEQ ID NO：86，甜杨：SEQ ID NO：87，杨：SEQ ID NO：88，穿叶眼子菜：SEQ ID NO：89，竹叶眼子菜：SEQ ID NO：90；
通过简并密码子部分替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰 ：SEQ ID NO：4，木薯：SEQ ID NO：91，猕猴桃：SEQ ID NO：92，野生蕉：SEQ ID NO：93，芭蕉：SEQ ID NO：94，可可：SEQ ID NO：95，枳：SEQ ID NO：96，葡萄：SEQ ID NO：97，苹果：SEQ ID NO：98，芥：SEQ ID NO：99，茄子：SEQ ID NO：100，番茄：SEQ ID NO：101，桃：SEQ ID NO：102，玉米：SEQ ID NO：103，棉花：SEQ ID NO：104，花生：SEQ ID NO：105，芥菜：SEQ ID NO：106，油菜：SEQ ID NO：107，菊花：SEQ ID NO：108，小盐芥：SEQ ID NO：109，野胡萝卜：SEQ ID NO：110，大豆：SEQ ID NO：111，萝卜：SEQ ID NO：112，小麦：SEQ ID NO：113，大麦：SEQ ID NO：114，橡胶树：SEQ ID NO：115，胡桃：SEQ ID NO：116，莴苣：SEQ ID NO：117，水稻：SEQ ID NO：118，蝴蝶兰：SEQ ID NO：119，梅花：SEQ ID NO：120，茶：SEQ ID NO：121，拟南芥：SEQ ID NO：122，芜菁：SEQ ID NO：123，菘蓝：SEQ ID NO：124，赤桉：SEQ ID NO：125，麻风树：SEQ ID NO：126，欧洲云杉：SEQ ID NO：127，甜杨：SEQ ID NO：128，杨：SEQ ID NO：129，穿叶眼子菜：SEQ ID NO：130，竹叶眼子菜：SEQ ID NO：131。
本发明还提供了一种包含ICE1基因的CRISPR/Cas9表达载体，所述表达载体为植物转化质粒。
利用ICE1基因进一步构建包含表达构件的CRISPR/Cas9转化质粒时，Cas9的启动子可为CaMV 35S、U6、T7及YAO启动子等。表达构件可以通过农杆菌(如Agrobacterium菌株GV3101)转入整合到植物的基因组中，稳定遗传和表达。
利用本发明提供的基因甲基化定量调控的遗传操作技术，基因甲基化位点可在转基因或基因编辑材料间进行遗传，从而达到对植物性状控制的目的。
利用不同甲基化程度ICE1基因制备不同耐冷性植株，可通过含有ICE1表达构件的植物转化质粒获得。各种植物的转化方法和步骤有所不同，同种植物不同品种也可能存在差异。但是植物转化的技术和方法是已知和成熟的。通常通过农杆菌导入植物的未成熟胚、成熟胚、未分化的愈伤组织或原生质体，然后根据标记基因进行筛选，获得转化植株，经过生根培养基培养就可以获得可以种植的种苗。也可以通过花粉介导法，通过农杆菌导入植物的花粉从而获得转基因种子。具体制备步骤如下：
(1)利用DNA重组技术，构建含有所述ICE1基因的CRISPR/Cas9转化质粒；
(2)将步骤(1)中构建的植物转化质粒通过农杆菌浸染方法或花粉介导法导入植物组织，利用标记基因进行筛选含有ICE1基因的转基因植株；
本发明适用于所有植物，包括双子叶和单子叶植物。植物可选取香蕉、枇杷、木薯、木瓜、 龙眼、水稻、玉米、棉花、小麦、高粱、大豆、油菜、马铃薯、甘薯、大麦、甘蔗、烟草、桃、梨、橡胶树、芜菁、菘蓝、蝴蝶兰、茶、梅花、赤桉、蔬菜(青菜、白菜、黄瓜、芹菜、辣椒、茄子)等。
本发明基于以下发明点提出：本发明研究表明，转不同耐冷性种群具有不同甲基化水平的AaICE1基因对拟南芥耐冷能力有不同程度的提高，而种群耐冷性与AaICE1基因的甲基化程度显著负相关，说明可以通过控制基因的甲基化程度来控制转基因后受体植株的耐冷能力。本发明中，我们发现ICE的表达受其基因序列中胞嘧啶甲基化水平的调节，甲基化程度越高其表达量越低，反之，则越高。
本发明相比现有技术具有以下优点：第一，基因在遗传操作过程中，会在受体生物中发生甲基化，有时会导致遗传操作的目标基因沉默，难以实现其通过甲基化调节基因表达的效应。本发明通过针对不同甲基化水平基因的调节基因表达的功能进行简并密码子定量替换胞嘧啶碱基的设计合成，或利用CRISPR/Cas9基因编辑技术进行原位基因简并密码子定量替换遗传操作，其大部分甲基化位点可以在受体植物中恢复甲基化，从而可以保证来源目的基因的甲基化位点和水平，通过筛选可以获得与供体基因相应的甲基化水平效应，实现表观遗传学的体外遗传操作。第二，本发明基于自然界的生物基因的天然甲基化并利用其表观遗传多样性的甲基化水平和位点，进行克隆并基于简并密码子定量替换胞嘧啶碱基的人工模拟合成，再进行遗传操作，培养转基因植物。可以保证转基因植物的目的基因重新甲基化的定量化，使遗传改良植物具有更强的适应能力。第三，通过CRISPR/Cas9基因编辑技术进行简并密码子定量替换，定量调控目标植物目的基因的甲基化位点和基因表达，定量原位在体调控改造目标植物可塑性性状。这为通过基因编辑在体原位表观遗传操作改良生物可塑性性状提供了技术途径。由于基因是本种来源，在培育更优良的作物后的生态环境和食品安全风险要低于不同生物类别的异源基因。第四，本发明利用植物天然耐寒性种质资源为基础，虽然ICE1基因是通过CBF基因发挥作用，转CBF基因后的植株耐寒能力有一定提高，但植株生长受抑制，实际应用价值较低；而ICE1基因作为植物组成型表达的基因，超表达后可明显提高植株耐寒能力，且对植株表型没有影响。不过，在不了解ICE1基因受甲基化程度控制基因表达量之前，培育转ICE1基因植物的耐冷程度是盲目的也是有限的，而本发明可以利用不同甲基化水平的ICE1基因定量实现耐冷能力的定量化提高，特别是可以将CBF冷响应转录通路调控植物耐冷效应最大化，培育更高耐冷能力的植物。还可以使得转基因植物在耐冷能力方面保持多样性。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
实施例1.转紫茎泽兰不同甲基化程度AaICE1基因水稻的获得
克隆紫茎泽兰AaICE1基因cDNA序列，序列为SEQ ID NO：1，根据密码子的简并性特征，将AaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换AaICE1基因中的不同程度的甲基化位点。获得全部替换后AaICE1基因序列为SEQ ID NO：3，命名为AaICE1(DC)1；部分替换后AaICE1基因序列为SEQ ID NO：4，命名为AaICE1(DC)2。通过限制性内切酶BsaI、SmaI切割载体pBI121和AaICE1、AaICE1(DC)1、AaICE1(DC)2基因，将目的基因连入pBI121，用Bar基因替换pBI121中Kan抗性基因，作为抗性筛选基因，构建不同甲基化程度的pBI121-AaICE1表达载体。通过农杆菌介导法导入水稻中。
选取日本晴水稻成熟种子(去壳)用升汞法(0.1％升汞浸泡15min，灭菌水清洗干净)消毒后，置于超净台上晾干，然后将胚放置于愈伤诱导培养基(4.4g/L MS+2.5mg/L 2,4-D+600mg/L干酪素+30g/L蔗糖+5g/L植物凝胶，KOH调至pH5.8，高压灭菌)上进行愈伤培养，28℃培养2周。将携带质粒pBI121-AaICE1的农杆菌GV3101接种到5ml含5mg/L双丙氨磷(Bialaphos)的YEB液体培养基中，28℃摇菌培养至对数生长期晚期，再以1:100体积扩大培养，在OD600为0.10时收集农杆菌菌体并重悬于侵染培养基中。按常规方法侵染水稻的愈伤组织，经共培养侵染后，置于含500mg/L Carb和5mg/L双丙氨磷的传代培养基上，28℃暗培养12-16d后，转移至5mg/L双丙氨磷的继代培养基上继续筛选培养，在再分化培养基上分化得到双丙氨磷抗性的阳性苗。转基因实验分别获得了11、15、13株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。5℃冷处理条件下，转基因后代植株耐冷能力有所分化，筛选后可获得目的性状，与野生型日本晴植株相比，其耐冷性分别提高了1.4℃、3.0℃和2.0℃。
实施例2.转紫茎泽兰不同甲基化程度AaICE1基因木薯的获得
根据密码子的简并性特征，将紫茎泽兰AaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换AaICE1基因中的不同程度的甲基化位点。获得全部替换后AaICE1(DC)1基因SEQ ID NO：3，部分替换AaICE1(DC)2基因SEQ ID NO：4。通过限制性内切酶BsaI、SmaI切割载体pBI121和AaICE1、AaICE1(DC)1、AaICE1(DC)2基因，将目的基因连入pBI121，用Bar基因替换pBI121中Kan抗性基因，作为抗性筛选基因，构建不同甲基化程度的pBI121-AaICE1表达载体。通过农杆菌GV3101导入木薯中。
取500ul超低温冰箱保存的农杆菌GV3101表达载体，加入到50ml YEB(添加5mg/L双丙氨磷)液体培养基中，28℃，240rpm，培养18-24h。4℃，4000rpm离心10min，用液体MS稀释到OD值1.0，待用。将成熟10-15d的木薯体胚绿色子叶切碎后，放大约500个子叶胚的碎块在30ml的农杆菌悬浮培养液中，期间振荡几次。45min后，吸去菌液，将子叶块在超净工作台中放置20min或用灭过菌的滤纸吸掉多于的液体，转移到茎器官发生培养基上，于26℃，16h光照共培养3d。分别在转化材料共培养了3d后，将愈伤取出，用无菌水冲洗3次，然后用添加500mg/L Carb和MS(pH5.3)培养基再冲洗2次，在超净工作台中放置20min或用无菌滤纸把残留的液体吸干。转移愈伤到含5mg/L双丙氨磷的茎器官发生培养基上，26℃暗培养3周。挑取抗性不定芽，放在茎伸长培养基上。26℃，16h光照培养。2-3周后，切下生长出的茎段转移到MS培养基上进行培养，当植株长大时，将其切开继代。切取从侧芽中长出的大约1cm的茎段，转移到包含5mg/L双丙氨磷的无激素培养基上进行生根实验，以消除假阳性植株，用野生形植株的茎段做对照。1-2周后，转基因阳性植株可以正常生根，而假阳性和对照则死亡。共获得转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因植株18、15和13株，转基因后代经低温筛选后可获得目的性状植株，耐寒性较野生型木薯分别提高了1.8℃、3.7℃和2.5℃。
实施例3.转紫茎泽兰不同甲基化程度AaICE1基因香蕉的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行香蕉遗传转化，通过农杆菌GV3101导入香蕉中。
以香蕉胚性悬浮细胞系(ECS)为转化受体，取在继代培养10d的香蕉ECS，离心去上清后，每1mL细胞密实体积ECS加入40ml含有不同甲基化程度AaICE1基因pBI121-AaICE1质粒的农杆菌GV3101，27℃，黑暗静置1-2h，然后将其进行共培养。共培养条件为：黑暗条件下，27℃，50rpm转速下振荡培养24h，然后将培养物静置去上清，再加入40ml的液体培养基，将转速提高至110rpm，连续共培养7d。将共培养完成后的培养物静置去上清，加入香蕉ECS液体筛选培养基40ml，27℃，转速100rpm振荡培养，每10-14d继代一次，连续继代3代以上。继代的方法是：取前一代0.1-0.5ml细胞密实体积的ECS加入到新鲜液体筛选培养基中继续振荡培养。同时，将每代继代剩余的ESC以及原共培养完成后的ESC用半固体筛选培养基进行筛选作为对照。采用半固体筛选时，先用含5mg/L双丙氨磷的M2培养基洗涤2-3次，无菌纸吸干多余培养基，转接到半固体筛选培养基上进行胚诱导，每个月更换一次体胚诱导培养基直到胚成熟。取液体筛选培养3代以后的ECS静置去上清，然后将其均匀铺于体胚诱 导培养基M3上，黑暗培养2-3个月，每个月更换一次体胚诱导培养基直到胚成熟，将成熟的抗性体胚转移至体胚萌发培养基上，光/暗(12h/12h)条件下培养至体胚萌发得到小苗，然后将小苗转移至生根培养基上，光/暗(12h/12h)条件下进行培养，即获得完整的转化植株。本实验共获得转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因植株22、21和15株，耐寒性筛选后获得相应的转基因植株，耐寒性分别提高了1.7℃、3.9℃和2.9℃。
实施例4.转紫茎泽兰不同甲基化程度AaICE1基因番茄的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行番茄遗传转化，通过农杆菌侵染对番茄进行遗传转化。
番茄实生苗在1/2MS基本培养基上长到约5-6cm高时，切取其无菌苗的子叶用作转化的受体材料。从甘油管中取少量含有目的表达载体的根癌农杆菌pBLGC菌液，于YEP固体培养基(添加5mg/L双丙氨磷，20mg/L Rif)上划线培养，28℃避光培养至长出直径约1mm大小的单菌落(约36-48h)。取单菌落转接于同样的固体培养基平板，培养至生长旺盛期(约36h)。从YEP平板上挑取农杆菌单菌落，接种到含5mg/L双丙氨磷，20mg/L Rif的YEP液体培养基中，混均，28℃，180-200rpm摇菌培养过夜(约16-18h)。次日以1％接种量接于20m1YEP液体培养基，28℃，180rpm振荡培养3-4小时，使农杆菌培养至对数期(OD600约为0.6)；然后4℃，4000rpm离心15min收集菌体，弃上清，用YEP液体培养基稀释原菌液的3-5倍，并加入200um乙酰丁香酮，28℃，180-200rpm摇菌1-2h。取完全展开的绿色子叶片，用无菌手术刀在子叶上划些伤口，在备好的农杆菌悬浮液中浸染10-20min左右，取出叶片，用无菌滤纸吸干，转入到固体分化培养基(Ms+6-BA 2mg/L+IAA 0.2mg/L)中，于黑暗、23-25℃条件下共培养2-3天(视菌体生长情况而定)。共培养三天后，将子叶片转入筛选培养基(Ms+6-BA 1mg/L+IAA0.2mg/L+5mg/L双丙氨磷+500mg/L羧苄青霉素)上；每隔三天换一次培养基，直至没有农杆菌出现。14天后可观察到有的切口附近长出愈伤或丛生再生芽，这时将再生芽尽可能分成单株，继续在5mg/L双丙氨磷压力下进行筛选。大约4周后，转入含5mg/L双丙氨磷和500mg/L羧苄青霉素的分化培养基中筛选约1个月；对获得的抗性芽(丛)进一步筛选，筛选期间羧苄青霉素一直保持500mg/L。两个月左右，再生芽可长到2-3cm，这时可将存活的幼苗转入生根培养基(1/2MS，IBA lmg/L，蔗糖30g/L)分化培养1-2周。生根时则去除羧苄青霉素。最后将根系发育良好的植株移栽入盆土中。转基因实验分别获得了13、18、11株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的抗性植株。低温筛选后获得目的性状转基因植株，与野生型植株相比，其耐寒性分别提高了1.9℃、4.1℃和2.4℃。
实施例5.转紫茎泽兰不同甲基化程度AaICE1基因可可的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行可可遗传转化，通过农杆菌LBA4404导入可可植株中。
从田间采集可可成熟果实，剥去果皮75％酒精侵泡数秒后，再用体积百分数为5％NaC1O溶液浸泡30min，剥去种皮后接入萌发培养基中，10天后取其子叶作为外植体。将培养至OD 600＝1.37左右的农杆菌LBA4404/PBI121收集菌体，用等体积PCG液体培养基(DKW+2,4-D3.0mg/L+KT 1.0mg/L+TDZ 0.01mg/L+Glucose 20g/L+glutamine 250mg/L)重悬，侵染萌发培养基中培养10d的可可子叶材料，28℃暗共培养3天。共培养后转接到含500mg/L Carb和5mg/L双丙氨磷的PCG固体培养基(1％琼脂粉)上进行抑菌及抗性筛选培养，10天于相同培养基上继代一次，在PCG培养基中培养20天后转入含Carb 500mg/L和5mg/L双丙氨磷的SCG培养基(KW+2,4-D 3mg/L+KT lmg/L+Glucose 20g/L+10％CW+1％琼脂粉)中培养20天，再转入含Car 500mg/L和5mg/L双丙氨磷的ED培养基(DKW+Sucrose 30g/L+Glucose1g/L+1％琼脂粉)中培养直到长出胚状体。同时经常观察污染或农杆菌复出的立即移出未污染材料继续培养。将成熟胚转移到胚发育的培养基中，该培养基分为二个时期的培养基，第一阶段培养基为PEC(1/2DKW+KNO 3 0.3g/L+AA 1000X stock solution lml/L+Glucose 20g/L+Sucrose 10g/L+1％琼脂粉)，在第一阶段的培养中，培养条件为每天的光照时期是光/暗为16/8h，每30天换一次培养基，直到长出带1-2片真叶的芽后，转入第二阶段的RD培养基(1/2DKW+KN0 3 0.3g/L+Glucose l0g/L+Sucrose 5g/L+不同浓度的IBA 1.0mg/L+不同浓度的IAA 1.0mg/L中，培养条件和第一阶段一致，每30天转一次培养基，直到成苗。本实验共获得转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因植株12、10和9株，经耐寒洗筛选后，目的性状植株耐寒性分别提高了1.5℃、3.9℃和2.2℃。
实施例6.转紫茎泽兰不同甲基化程度AaICE1基因橡胶的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行橡胶遗传转化，通过农杆菌EHA105导入橡胶植株中。
将橡胶树单核靠边期花药接种于胚性愈伤组织诱导培养基上，培养30d后用于农杆菌侵染。将愈伤组织在EHA105悬浮液中侵染5min后，转入添加5uM乙酰丁香酮和10m g/L硝酸银共培养基上培养，22℃暗培养2d。每个处理设置3次重复。共培养后的愈伤组织视其长菌情况用无菌水洗涤后转入50mg/L特泯丁浸泡lmin，置无菌滤纸吸干后转移到添加10mg/L硝 酸银和500mg/L特泯丁的愈伤诱导培养基上进行恢复培养，25℃暗培养7d。恢复生长的愈伤组织转入添加10mg/L硝酸银、5mg/L双丙氨磷和50mg/L特泯丁的愈伤诱导培养基诱导体细胞胚胎发生。最后，将增殖后体细胞胚转入添加5mg/L双丙氨磷的植株再生培养基诱导植株再生。遗传转化实验分别获得了10、6、9株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的橡胶抗性植株。低温筛选后，目的性状橡胶植株与野生型橡胶树相比，其耐寒性分别提高了1.6℃、2.9℃和2.1℃。
实施例7.转紫茎泽兰不同甲基化程度AaICE1基因柠檬的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行柠檬遗传转化，通过农杆菌EHA105导入柠檬中。
超低温保存的工程菌液接种在加有5mg/L双丙氨磷和50mg/L利福平的LB平板上划线培养，28℃活化36h左右，挑单菌落于LB液体培养基中150rpm摇床过夜培养。取菌液分装于已灭菌的离心管中，4000rpm离心10min，去上清液，用MT液体培养基悬浮培养2h后稀释至OD600约0.5左右进行侵染。取30d苗龄无菌柠檬实生苗的上胚轴为外植体，上胚轴横切成1cm左右，置于农杆菌菌液中侵染15min，期间轻微振荡。滤纸吸干上胚轴上的菌液，水平放置于附加100uM的乙酰丁香酮BR培养基(MT+1.0mg/L BAP+0.1mg/L NAA+3％Sucrose)中27℃黑暗条件下共培养3d。共培养后的上胚轴用无菌水洗4次后，用400mg/L的头孢霉素洗一次。无菌滤纸吸干水分后转入附加5mg/L双丙氨磷和400mg/L的头孢霉素的BR培养基中，光下培养。当获得的抗性芽伸长到1cm左右时，自基部切下，转移到附加5mg/L双丙氨磷的培养基中继续生长并诱导生根。根发育充分后，开瓶炼苗1-2d，植株从培养基中取出，自来水洗净根上的琼脂。移栽到装有灭菌腐质土和蛭石混合物的烧杯中，其上用透气膜覆盖，逐步去掉透气膜后，移栽到装有营养土的花盆中。遗传转化实验分别获得了8、8、6株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的柠檬抗性植株。低温筛选后，目的性状柠檬植株与野生型柠檬相比，其耐寒性分别提高了1.6℃、3.2℃和2.3℃。
实施例8.转紫茎泽兰不同甲基化程度AaICE1基因甜橙的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行甜橙遗传转化，通过发根农杆菌MSU440导入甜橙中。
转化受体材料主要为“早金”甜橙。成熟甜橙果实中取出种子，自来水冲洗干净后于1M NaOH溶液中浸泡10min去除果胶，自来水漂洗3-5次；超净台上3％的次氯酸钠溶液浸泡消毒15-20min，灭菌蒸馏水漂洗3次，每次5min；消毒后的种子在超净工作台上剥去内外种皮 后播种于MT播种培养基(MT+25g/L蔗糖+8g/L琼脂)上，26℃暗培养4W后取出黄化苗光照至上胚轴稍转绿(约需5-7d)后切取无菌苗上胚轴茎段用于农杆菌侵染。取-80℃保存的发根农杆菌MSU440菌株在含50mg/L壮观霉素的LB固体培养基上划线，28℃暗处倒置培养36-48h获单菌落；挑取单菌落于新的添加50mg/L壮观霉索的LB固体培养基上划线，同样条件下培养36-48h，用无菌手术刀片刮取所有菌体于不加抗生素的MT液体培养基中，200rpm，28℃振荡培养1.5-2h后调节菌液OD600至0.6-0.8后备用。上胚轴切段浸没于发根农杆菌菌液中侵染20min，期问摇动数次；侵染结束后在灭菌滤纸上吸干外植体上多余菌液，转至垫有灭菌滤纸的MT共培养培养基(MT+40g/L蔗糖+8g/L琼脂十20mg/L乙酰丁香酮)上，21℃暗处共培养3d后将外植体用无菌水漂洗3次，滤纸吸干后转至含400mg/L头孢霉素的MT基本培养基(MT+40g/L蔗糖+8g/L琼脂+400mg/L头孢霉素)上，26℃暗处培养诱导毛状根。将毛状根切成长约1.5cm根段于MT生芽培养基(MT+40g/L蔗糖+8g/L琼脂+1mg/L BA)上26℃，16h/8h光/暗条件下诱导生芽，当芽体生长至0.5cm左右时将芽体切下转到MT伸长培养基(MT+30g/L蔗糖十8g/L琼脂+0.1mg/L BA+0.1mg/L IAA+0.25mg/L GA3)上，待芽体伸长至I cm左右时转至MT生根培养基(1/2MT+25g/L蔗糖+8g/L琼脂+0.5mg/L NAA+0.1mg/L IBA+0.5g/L活性炭)上诱导生根。遗传转化实验分别获得了14、10、11株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的甜橙抗性植株。低温筛选后，目的性状甜橙植株与野生型甜橙相比，其耐寒性分别提高了2.0℃、3.8℃和2.5℃。
实施例9.转紫茎泽兰不同甲基化程度AaICE1基因柑橘的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行柑橘遗传转化，通过农杆菌EHA105导入柑橘中。
将柑橘成熟果实破碎，取出种子，用1mol/L NaOH浸泡10min去除种子表面的果胶，再用自来水漂洗干净；在超净工作台上用70％酒精浸泡2-3min，用2％的次氯酸钠浸泡灭菌20min，中间摇动数次，弃去次氯酸钠溶液，用无菌水洗涤3次，每次5min；将上述灭菌的种子在超净工作台上剥去内外种皮，置于装有MT培养基的试管中，26℃暗培养20-30d。将黄化的上胚轴或茎段在光照下(26℃，14/10h光周期)培养10-15d，然后取上胚轴或茎段用于转化实验。将保存在超低温冰箱中的农杆菌在含有双丙氨磷5mg/L和利福平40mg/L的YEB固体培养基上划线，28℃暗培养2d。挑取单克隆接种在含有40mg/L Rif和5mg/L双丙氨磷的YEB液体培养基中震荡培养24h(200rpm，28℃)，再次在YEB固体培养基上划线培养，48h后，用不锈钢长柄药匙刮取培养好的农杆菌，接种在40ml MT悬浮培养基(MT+麦芽提取物 0.5g/L+谷氨酞胺1.5g/L，PH 5.7)里，于28℃，180rpm振荡培养2h。用MT悬浮培养基将上述培养好的农杆菌菌液稀释OD600为0.8备用。将适量处理好的外植体放入上述农杆菌液中(含有100umol/L的AS)，侵染20min后(中间轻轻晃动几次)，倒出菌液，将外植体材料置于灭菌滤纸上，吸干上面附着的菌液，再将材料转移到共培养培养基上(MT+100uM AS+0.4mg/L 2,4-D，PH 5.7)，共培养基上覆盖一层滤纸，22℃暗培养3d后，转入筛选培养基上进行培养。筛选4个月后，将再生的不定芽(长1cm左右)切下来进行温室嫁接。遗传转化实验分别获得了10、13、9株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的柑橘抗性植株。低温筛选后获得目的性状转基因植株，目的性状柑橘植株与野生型柑橘相比，其耐寒性分别提高了1.8℃、3.7℃和2.3℃。
实施例10.转紫茎泽兰不同甲基化程度AaICE1基因甘蔗的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行甘蔗遗传转化，通过农杆菌侵染导入甘蔗中。
以田间材料为外植体，选生长健壮甘蔗植株，取其尾梢部分，逐层剥去外部老叶，取其距顶端生长点约l0cm左右的幼叶组织作为外植体，先用70％乙醇浸泡30s后，再用0.1％的升汞水溶液处理l0min，后用无菌水洗3-4遍后，吸干水珠，将其横切成0.2-0.5mm厚的薄片接种于固体培养基上。以MS+2,4-D lmg/L+蔗糖30g/L，pH5.8固体培养基(M1)作为胚性愈伤组织诱导培养基，25℃暗培养。从农杆菌保存平板上挑取单菌落接种到5m1YEP(含5mg/L双丙氨磷、Rif 25ug/m1)液体培养基中，于28℃，200rpm振荡培养至对数生长期(约24h)。于150m1三角瓶中扩大培养到40m1含相同抗生素的YEP培养基中，培养至OD600为0.6左右，转移到离心管中，4℃5000rpm离心5min，倒掉上清，吸干残液。用含150umol/L AS的等体积MR液体培养基(1/5MS大量元素+MS其他成分+2,4-D 1mg/L+AS 150umol/L+l0mmol/L果糖+l0mmol/L葡萄糖+30g/L蔗糖，pH5.3)重悬细菌，于28℃，2000rpm培养2h。诱导细菌Vir基因表达，作为感染转化材料的菌液原液。选取在M1培养基上暗培养生长旺盛，整齐一致的愈伪组织转移到新鲜M1培养基上培养4d，再用于转化试验。转化时，先将愈伤组织置于无菌滤纸上在超净工作台上进行吹干处理1-2h，至组织块表面干燥。稍有收缩，即可用于细菌感染。将菌液浸泡感染后10min后的材料转移到滤纸上，在超净工作台上吹晾到材料表面干爽、无水膜，并把材料组织切成0.3-0.5cm的小块，然后转到含有100umol/L AS的M1固体培养基上，23℃左右置黑暗共培养3d。共培养后先用无菌水洗涤3-4次，再用MS液体培养基洗一次，置滤纸上于超净工作台内吹晾到干爽。共培养后洗净、晾干的转化材料接种到含 500mg/L羧苄青霉素和5mg/L双丙氨磷的M2固体培养基上进行筛选培养。以后每20-30d于相同的培养基上继代一次，同时经常观察发现污染或农杆菌复出的立即移出未污染材料继续培养，另外，经常把出现褐化污染的组织块剔出或转移好的或未被污染的组织块到新的相同培养基上。组织块膨大后，经常把人块组织剥小，小芽出现后也即时将小芽转到培养基上，以保证足够的选择压力。抗性幼苗长到4-5cm高时，转含5mg/L双丙氨磷、Cef 300mg/L的M3培养基上诱导生根。遗传转化实验分别获得了15、12、17株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的甘蔗抗性植株。低温筛选后获得目的性状转基因植株，目的性状甘蔗植株与野生型柑橘相比，其耐寒性分别提高了2.4℃、4.6℃和3.3℃。
实施例11.转紫茎泽兰不同甲基化程度AaICE1基因番木瓜的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行番木瓜遗传转化，通过农杆菌侵染导入番木瓜中。
选取健康、无病毒植株上7-8成熟番木瓜果实，自来水洗净后，用70％的酒精擦洗1遍，在超净工作台上用消毒过的水果刀将果实剖开，将种子取出放在70％的酒精里浸泡50s，然后用15％(v/v)的次氯酸钠溶液消毒20min，无菌水洗4-5次，然后滤纸吸干待用。用无菌解剖刀和镊子将番木瓜种子剥开，将完整的幼胚接种到诱导培养基上(改良MS+10mg/L2,4-D+2mg/LKT+0.5mg/LBA+400mg/L谷氨酰胺+30g/L蔗糖，pH为6.2)。于25℃、黑暗条件下进行愈伤组织诱导，每4周继代1次。将成熟体胚接种到MS+0.1mg/L NAA+0.5mg/LBA+7g/L琼脂粉+30g/L蔗糖的体胚萌发培养基上，于25℃进行周期性光照培养(10h/d光照，14h/d黑暗)。将冻存菌株在添加5mg/L双丙氨磷和100mg/L链霉素的平板上划板，然后挑单菌落到30ml LB液体培养基28℃，225rpm摇20h,5000rpm 10min离心收集菌体，用30ml添加100umol/L乙酞丁香酮的改良MS液体培养基重悬菌体，继续摇3h，然后用改良MS液体培养基稀释菌液至适宜的OD600浓度(0.6)备用。将预培养一个月的番木瓜幼胚浸没在菌液中20min，期间摇动几次，然后将愈伤组织用无菌滤纸吸干表面菌液，并在超净台内适度吹干，接种于诱导培养基上，于25℃共培养3天。共培养3d的愈伤组织和幼胚用无菌水冲洗6次，尽可能去除表面附着的菌体，吸干多余水份后接种于含5mg/L双丙氨磷和300mg/L羧苄青霉素的选择性培养基上，每20天继代一次，去除褐变死亡的外植体，并逐步降低羧苄青霉素浓度，于25℃暗培养，诱导体胚发生。遗传转化实验分别获得了19、23、21株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的番木瓜抗性植株。低温筛选后获得目的性状转基因植株，目的性状番木瓜植株与野生型番木瓜相比，其耐寒性分别提高了1.9℃、3.8℃和2.6℃。
实施例12.转紫茎泽兰不同甲基化程度AaICE1基因枇杷的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行枇杷遗传转化，通过农杆菌侵染导入枇杷中。
感染前2d根癌农杆菌划平板，28℃下暗培养48h，待农杆菌处于对数生长期即可用于转化。刮取根癌农杆菌菌落悬浮于液体培养基中，剧烈震荡lmin后，静置1-2h，让农杆菌形成悬浊液。调整农杆菌浓度，使菌液OD600值为1.0，加入预培养10-15d的批把EC，略微摇动后静置30min，置于无菌滤纸上吸干愈伤组织上附着的水分后，转入固体培养基共培养。共培养l0d后，挑取愈伤组织于三角锥瓶中，用无菌水冲洗3次，最后1次用羧苄霉素无菌水(300mg/L，下同)冲洗并静置1h，让粘附于愈伤组织上的农杆菌扩散到水中，然后再用羧苄霉素无菌水冲洗1次，置于无菌滤纸上吸干愈伤组织上附着的水分后转移到筛选培养基上培养(筛选培养基为附加1 mg/L 2,4-D、0.25 mg/L KT、l0mg/L潮霉素、5 mg/L双丙氨磷和300mg/L羧苄霉素的MS培养基)。当再生植株长至2-3厘米高并具有4-7个叶片时，打开瓶盖，在自然光照条件下炼苗3-4d，轻轻取出洗净后，移至泥碳土与蛙石1:1的基质中，浇透水，盖上有缺口的烧杯，以控制水份蒸发。数日后，逐渐揭去烧杯，以免湿度过大，造成移栽苗腐烂。遗传转化实验分别获得了12、13、11株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的枇杷抗性植株。低温筛选后获得目的性状转基因植株，目的性状枇杷植株与野生型枇杷相比，其耐寒性分别提高了1.5℃、3.6℃和2.2℃。
实施例13.转紫茎泽兰不同甲基化程度AaICE1基因荔枝的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行荔枝遗传转化，通过农杆菌侵染导入荔枝中。
荔枝为“元红”品种。农杆菌于固体LB培养基上保存，每l-2个月继代一次。转化前挑单菌落于附加相应抗生素的液体LB培养基中，28℃振荡培养直到对数生长期，将菌液4000rpm离心10min，用液体愈伤组织生长培养基重悬并稀释10倍备用。将继代培养15天的胚性愈伤组织于无菌滤纸上干燥1h，然后投入准备好的菌液中，静置l0min，无菌滤纸吸干愈伤组织表面的菌液后转至共培养培养基(MS基本培养基附加2,4-D 2mg/L，STS 5mg/L，蔗糖50g/L，琼脂7g/L，pH5.8)中共培养2天。共培养后，将愈伤组织用无菌水冲洗3次，液体Zl培养基重悬，平铺于无菌滤纸上，转入附加一定浓度抗生素和抗菌素的Z1培养基进行选择培养，7周后，将抗性克隆转至相同的选择培养基中进一步筛选。纯化的抗性愈伤组织转入体胚分化培养基(MS基本培养基附加KT 1mg/L，NAA0.1mg/L，谷氨酰胺500mg/L，蔗糖 80g/L琼脂15g/L，G418 100mg/L，5mg/L双丙氨磷，Carb300mg/L，pH6.2)诱导体胚分化，胚状体在成熟培养基(MS基本培养基附加椰乳50ml/L，水解酪蛋白500mg/L，蔗糖50g/L，琼脂9g/L，pH5.8)中培养2个月后，移入萌发培养基(MS基本培养基附加KT1mg/L，GA5mg/L，椰乳50ml/L，谷氨酰胺200mg/L，蔗糖30g/L，琼脂7g/L，G418 50mg/L，5mg/L双丙氨磷，pH5.8)萌发成苗。遗传转化实验分别获得了12、13、11株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的荔枝抗性植株。低温筛选后获得目的性状转基因植株，目的性状荔枝植株与野生型荔枝相比，其耐寒性分别提高了1.7℃、3.9℃和2.7℃。
实施例14.转紫茎泽兰不同甲基化程度AaICE1基因龙眼的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行龙眼遗传转化，通过农杆菌侵染导入龙眼中。
将正常继代培养的龙眼EC接种于继代培养基LMs4上预培养10-15d，选取浅黄色细小颖粒状龙眼EC用作转基因受体材料。感染前1d根癌农杆菌划平板，28℃下暗培养24h，待农杆菌处于对数生长期即可用于转化。刮取根癌农杆菌菌落悬浮于液体培养基中，剧烈震荡1min后，静置1h，让农杆菌形成悬蚀液。调整农杆菌浓度，使菌液OD600值在1-1.5之间，加入预培养10-15d的龙眼EC，略微摇动后静置30min，于无菌池纸上吸干愈伤组织表面附着的水分后，转入固体培养基共培养。共培养5d后，待农杆菌生长至可见愈伤组织下的菌斑但未长满愈伤组织时，挑取愈伤组织于无菌三角瓶中，用无菌水冲洗5次，最后1次用羧苄霉素无菌水(300mg/L)冲洗并静置1h，让粘附于愈伤组织上的农杆菌充分扩散到水中，然后再用羧苄霉素无菌水(300mg/L冲洗一遍，置于无菌滤纸上吸干愈伤组织上附着的水分后转移到筛选培养基上培养。遗传转化实验分别获得了8、10、7株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的龙眼抗性植株。低温筛选后获得目的性状转基因植株，目的性状龙眼植株与野生型龙眼相比，其耐寒性分别提高了1.4℃、3.5℃和2.3℃。
实施例15.转紫茎泽兰不同甲基化程度AaICE1基因芒果的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行芒果遗传转化，通过农杆菌侵染导入芒果中。
用无菌牙签从YEP平板上挑取农杆菌单菌落，接种在加有25mg/L利福平YEP液体培养基中，置于28℃恒温摇床上，220rpm震荡培养24h后，再将菌液加入到新的抗性YEP培养基中，同样条件培养至对数生长期。5000rpm离心5min沉淀菌体，弃去上清液，加入MS液体培养基悬浮，待用。取芒果树上3-5cm长的幼果，首先用酒精浸泡过的棉球拭擦幼果表面， 以除去表面细菌。然后用流水冲洗几分钟，放置超净工作台上用的次氯酸钠溶液+Tween20(3滴/L)浸泡，期间不断的搅动。接着用无菌水冲洗一次，每次5min。将经过表面消毒灭菌的幼果放在无菌滤纸上，沿着幼果的纵轴方向用灭菌手术刀片将幼胚均匀切开，然后用小镊子将幼胚夹出，接种于培养基(MS+2,4-D 1mg/L+6-BA 1.5mg/L)上，并使纵切面与培养基接触。取备用菌液，将幼胚用备用菌液侵染10min，共培养2d后，接种于愈伤诱导培养基上，培养10-20d后选择未污染的愈伤组织转入芽诱导培养基上。在所有处理的愈伤组织诱导过程中，用于启动培养的培养基中附加几抗坏血酸，并在接种后每隔2d更换一次培养基，共更换3次，以降低幼胚分泌的多酚类物质对愈伤诱导的影响。将获得到的再生植株，接种于生根培养基中进行生根培养。遗传转化实验分别获得了12、11、14株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的芒果抗性植株。低温筛选后获得目的性状转基因植株，目的性状芒果植株与野生型芒果相比，其耐寒性分别提高了1.9℃、3.9℃和2.5℃。
实施例16.转紫茎泽兰不同甲基化程度AaICE1基因马铃薯的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行马铃薯遗传转化，通过农杆菌侵染导入马铃薯中。
挑取单菌落接种于含25mg/1的氯霉素和5mg/L双丙氨磷的50ml的YEB液体培养基中，28℃，140rpm震荡培养过夜，然后取5ml接种于50ml上述YEB液体培养基中，振荡培养至OD600＝0.5，备用。取一株带有4-5个茎节的马铃薯脱毒试管苗，在无菌条件下切成0.5cm左右的单节茎段，放于液体培养基(MS+3mg/L 6-BA+8％蔗糖)中。将继代培养30d的脱毒试管苗剪成不含腋芽长0.5cm左右的茎段，预培养2d。将茎段用备用菌液侵染10min，共培养2d后，接种于愈伤诱导培养基(MS+0.1mg/L 2，4-D+1mg/L BA+5mg/L双丙氨磷+400mg/L头孢噻肟钠)上，培养10-20d后选择未污染的愈伤组织转入芽诱导培养基(MS+1mg/L ZT+1mg/L GAS+5mg/L双丙氨磷+400mg/L头孢噻肟钠)上。将获得到的再生植株，接种于生根培养基中进行生根培养。遗传转化实验分别获得了15、18、19株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的马铃薯抗性植株。低温筛选后获得目的性状转基因植株，目的性状马铃薯植株与野生型植株相比，其耐寒性分别提高了1.5℃、3.4℃和2.7℃。
实施例17.转紫茎泽兰不同甲基化程度AaICE1基因辣椒的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行辣椒遗传转化，通过农杆菌侵染进行。
用无菌牙签从YEP平板上挑取农杆菌单菌落，接种在加有5mg/L双丙氨磷及25mg/L利 福平YEP液体培养基中，置于28℃恒温摇床上，220rpm震荡培养24h后，再将菌液加入到新的抗性YEP培养基中，同样条件培养至对数生长期。5000rpm离心5min沉淀菌体，弃去上清液，加入MS液体培养基悬浮，待用。选颗粒饱满的辣椒种子，用水清洗数次，70％乙醇消毒30s，无菌水冲洗1次，0.1％HgCl 2浸泡5-8min，无菌水冲洗3-4次。接种于1/2MS培养基上，于25℃、光照强度2000lx，14h/d暗的光照条件下培养出无菌苗。剪取12d龄的辣椒无菌苗下胚轴外植体为转化受体，在PD预培养基(MB5+3mg/L 6-BA+1mg/L IAA+4mg/L AgN03+30g/L蔗糖+5g/L琼脂)上预培养2d，将外植体置于液体MS中，再统一转移至己准备好的农杆菌菌液中，浸没5-14min。外植体在灭菌滤纸上沥干菌液后，置PD培养基上暗培养，每皿放10个外植体，正面朝上。将外植体在黑暗条件下25℃于PD上培养1-4d后，再转入PD(含500mg/L羧苄青霉素)选择培养基上进行选择培养1-4d，最后转移到PD培养基(含5mg/L双丙氨磷、500mg/L羧苄青霉素)上进行选择培养。5天转接一次，经15天左右的培养，将分化出不定芽的外植体接于SE培养基(MB5+3mg/L 6-BA+1mg/L IAA+2mg/L GA3+4mg/L AgN03+30g/L蔗糖+5g/L琼脂+5mg/L双丙氨磷+500mg/L Carb)上使芽伸长。待不定芽长至2-3cm时切下伸长芽移至SR(1/2MS+0.2mg/L IAA+0.1mg/L NAA+5mg/L双丙氨磷+200mg/L Cef)上生根。除共培养外，其他培养阶段均在25℃，14h/d光照，光照强度2000lx条件下。遗传转化实验分别获得了9、11、8株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的辣椒抗性植株。低温筛选后获得目的性状转基因植株，目的性状辣椒植株与野生型植株相比，其耐寒性分别提高了2.1℃、4.5℃和3.6℃。
实施例18.转紫茎泽兰不同甲基化程度AaICE1基因高粱的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体，通过农杆菌侵染对高粱进行遗传转化。
取适量保存菌液接种于20ml的YEP夜体培养基中，在28℃200rpm震荡培养24-30h。取适量第一次活化后的菌液接种于40ml的YEP液体培养基中，28℃200rpm震荡培养至OD600值为0.6。6000rpm离心10min收集菌体。每50ml无菌水加入500ul乙酰丁香酮，10ul sillwet L-77配成溶液。用此溶液悬浮离心得到的菌体，使OD600值为0.6左右备用。高粱种子于70％酒精中浸泡1min，用无菌水洗涤一次后，转移至0.1％的升汞中，使其在摇床上150rpm震荡，消毒10分钟。最后用无菌水漂洗6次以上。将灭过菌的种子放于100m1的锥形瓶中，每瓶种子量为100-200粒，加无菌水适量，使其在摇床上150rpm震荡，24小时左右后，种子将萌发，长出1cm左右的芽。此时取出装有种子的锥形瓶，进行划胚处理。对划伤 种子用备用农杆菌侵染液浸泡后，抽滤30min，有利于农杆菌侵入生长点细胞。将侵染转化过的种子摆在铺有滤纸的培养皿中，放在25℃的培养室中，暗培养48-96小时。在无菌水中添加替卡西林(160mg/L)和头孢噻肟钠(150mg/L)，将种子放于100mI的锥形瓶中，洗涤4次以上，每次至少4min。后摆于培养皿中，25℃的培养室(14h光照/10h黑暗)中培养。当种子生根后，移至Hoagland培养液中液体培养壮苗，培养至2-4叶期，将幼苗移栽至纸杯中，放于温室培养，进行常规生长管理。遗传转化实验分别获得了15、13、17株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的高粱抗性植株。低温筛选后获得目的性状转基因植株，目的性状高粱植株与野生型植株相比，其耐寒性分别提高了1.8℃、3.9℃和3.0℃。
实施例19.转紫茎泽兰不同甲基化程度AaICE1基因葡萄的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行葡萄遗传转化，通过农杆菌侵染导入葡萄中。
将诱导扩繁的葡萄PEM继代到新鲜的X6培养基上，26℃暗环境下预培养一周以供转基因操作。取200μl菌液接种于20ml液体LB培养基(含5mg/L双丙氨磷)中。28℃、180rpm培养24h后取20μl该菌液接种于20ml液体LB培养基(含5mg/L双丙氨磷)中，相同条件下进行二次活化培养20h。将菌液转移到灭菌的50ml离心管中，5000×g离心8min，去上清，再用重悬缓冲液(MS培养基+20g/L蔗糖+100μM AS)重悬离心两次。在重悬缓冲液中28℃、180rpm孵育1h，用重悬液稀释调整菌液浓度为OD 600＝0.3-0.4。将葡萄PEM放入盛有农杆菌液的三角瓶中侵染20min，用无菌滤纸吸干表面菌液，将其继代到放入两层灭菌滤纸并添加约5-6m的X6液体培养基(附加100μM AS)的无菌培养皿中。26℃黑暗条件下共培养48h后，用浓度均为200mg/L的头孢霉素、羧苄青霉素溶液中脱菌20min，再用无菌水冲洗3遍，将葡萄PEM接种于MS+5mg/L双丙氨磷+200mg/L Cef+200mg/L Carb+10mg/L Hyg+30g/L蔗糖+3g/L植物凝胶培养基上，PEM诱导出的抗性SE接种于MS+0.2mg/L KT+0.1mg/L NOA+10mg/L Hyg+30g/L蔗糖+3g/L植物凝胶。26℃黑暗条件下培养，每三周继代一次，直至抗性体细胞胚长出。葡萄遗传转化共获得17、19、14株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因植株，低温筛选后获得目的性状转基因植株，耐寒性分别提高了1.4℃、3.0℃和2.1℃。
实施例20.转紫茎泽兰不同甲基化程度AaICE1基因苹果的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行苹果遗传转化，使用超声波转化法进行。
将苹果试管苗叶片剪成4mm 2左右碎片，放入含有20ml超声缓冲液的容器中，超声缓冲液 的成分为：13.7mmo1/LNaCI，2.7mmo1/LKCI，l0mmol/LNa 2HPO 4，2mmo1/LKH 2POP 4，5％DMSO，pH 7.4。每毫升缓冲液含质粒DNA 5ug。在声强为0.5-1.0W/cm 2进行20min超声波处理。将超声处理后的叶片转入含有4mg/L6-BA+0.2mg/LNAA的MS再生培养基上恢复生长1周，然后转入含有5mg/L双丙氨磷再生培养基上进行2周暗培养筛选，每2周转移1次，2个月后转移到0.5mg/L6-BA+0.1mg/LNAA的MS继代培养基上这一过程共持续3个月。培养条件：每天连续16h光照，光照强度为40umol m -2 s -1恒温25℃继代保存。每4周继代一次。分别从300个外植体叶片上获得了12、13、10株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，与野生型植株相比，其耐寒性分别提高了1.8℃、4.1℃和2.5℃。
实施例21.转紫茎泽兰不同甲基化程度AaICE1基因芥的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行芥遗传转化，通过农杆菌侵染导入芥中。
芥种子消毒(70％的酒精和1％次氯酸钠)后播种于MS培养基，一周后切取下胚轴，置于分化培养基(MS+2mg/L 6-BA+0.2mg/L NAA+3％蔗糖+0.6％琼脂)上预培养2d。将保存的农杆菌EHA105活化，取500ul活化菌液，接种到50ml YEB液体培养基中，28℃、220rpm振荡培养，至OD600值为0.5，4℃离心(4000r/min，10min)，弃上清液，重悬于20ml预冷的MS液体培养基，备用。用重悬菌液侵染预培养后的下胚轴10min，滤纸上干燥后转移至分化培养基上，共培养2d。将共培养后的下胚轴转移至筛选培养基(MS+2mg/L 6-BA+0.2mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行抗性筛选，每l0d更换一次筛选培养基。将多次筛选获得的抗性芽转移到生根培养基(MS+2mg/L 6-BA+0.4mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行生根培养。待组培苗根系强壮后，洗净根系上残留培养基，移栽到营养土中，置于人工气候室进行培养。遗传转化实验分别获得了18、15、21株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的抗性植株。低温筛选后获得目的性状转基因植株，与野生型植株相比，其耐寒性分别提高了1.9℃、3.4℃和2.4℃。
实施例22.转紫茎泽兰不同甲基化程度AaICE1基因茄子的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行茄子遗传转化，通过农杆菌侵染导入茄子中。
茄子种子用75％酒精浸泡30s，无菌水冲洗1次，然后用10％的NaC1O溶液浸泡20min，无菌水冲洗3次后，接种到1/2MS培养基上萌发，于26℃，光照/黑暗(16h/8h)条件下培养 成无菌苗作为供试材料。由根癌农杆菌菌株EHA105、质粒载体pBI121，ScICE1基因构建成的超表达载体。选取无菌苗子叶，去除叶尖，切成0.5mm 2大小均匀的小块，接种于预培养培养基MS+NAA 0.2mg/L+ZT 1.2mg/L中，预培养2d后进行农杆菌侵染转化，取OD值为0.5的农杆菌菌液，侵染时间为10min。将侵染后的外植体接种于共培养培养基(1/2MS+NAA0.2mg/L+ZT1.2mg/L+200umo1/L乙酰丁香酮)中，暗培养2d后，用含150mg/L特美汀的无菌水清洗外植体，无菌滤纸吸干表面水分，接种于MS+NAA 0.2mg/L+ZT 1.2mg/L+150mg/L特美汀培养基中，恢复培养后置于诱导愈伤组织培养基中(添加0.lmg/L NAA，3.0mg/L ZT)。置于白天16h，夜间8h，16001x光照条件下培养。外植体生长两周左右，长出幼嫩、团状、坚硬的绿色愈伤组织，将其接种到生芽培养基(添加0.lmg/L NAA，4.0mg/L ZT，1.5mg/L 6-BA，12.0mg/L AgN0 3)中诱导生芽。芽伸长分化形成的幼苗长出2-3片真叶，将其从基部切断，接种到1/2MS生根培养基中诱导生根。光照时间16h/d，温度24-27℃，光照强度1600 1x。生长在培养基中的再生苗对环境适应能力差，应将三角瓶揭开小口通风，并根据幼苗生长情况逐步加大通风量，提高幼苗对环境适应能力。两周后，幼苗有4-5片真叶，三条主根，若干条侧根，揭开瓶口，同时注意保证幼苗水分充足，继续培养5d后即可移栽。遗传转化实验分别获得了8、10、7株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的抗性植株。低温筛选后获得目的性状转基因植株，与野生型植株相比，其耐寒性分别提高了1.5℃、3.0℃和2.2℃。
实施例23.转紫茎泽兰不同甲基化程度AaICE1基因猕猴桃的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行猕猴桃遗传转化，通过农杆菌LBA4404导入猕猴桃中。
取保存的农杆菌菌种LBA4404，划线后挑取农杆菌单菌落，入20ml液体YEP培养基中，于200rpm，28℃条件下振荡培养16-24h。吸取0.5ml菌液，入50ml含同样抗生素的YEP培养液中，条件同上扩大培养。待菌液培养至OD600为0.5时，于4000rpm，4℃离心10min沉淀，用加有100uM乙酰丁香酮的液体MS培养基等体积悬浮备用。将猕猴桃无菌苗幼嫩的叶片，在无菌条件下切成0.5cm见方的小块，接在以MS为基本培养基附加3.0mg/L 6-BA和1.0mg/L NAA的培养基上预培养3d。3d后，将预培养的叶片放入重悬好的菌液中，期间轻微震荡，使农杆菌与叶片充分接触，浸染10min后用无菌滤纸吸干多余菌液，叶片背面向下放置在含100uM AS的再生培养基上，28℃暗培养2d，注意在共培养的培养基上垫上一层无菌滤纸，防止农杆菌生长过旺。将经过共培养的叶片外植体用无菌水冲洗4-5次，用无菌滤纸吸去残留的无菌水，接种到含有500mg/L Carb和5mg/L双丙氨磷的再生培养基上，一个月继代一 次，待分化芽长至2-3cm，切下，插入生根培养基中。5-10d可见不定根长出。待生根幼苗长至5cm时，培养瓶放在自然条件下培养3-5d，然后逐渐的打开瓶盖，前两天将盖子松动，第三、四天打开瓶盖一半，第五、六天取下盖子，用瓶盖一半大小的硬纸板盖上，第七、八天取下硬纸板，使组培苗暴露在自然状态下，炼苗10d，用镊子小心的取出新生苗，自来水冲洗根上面附着的琼脂，移栽至温室土壤中培养。遗传转化共获转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因猕猴桃植株8、11和12株，低温筛选后，目的植株耐寒性分别提高了2.0℃、3.6℃和2.7℃。
实施例24.转紫茎泽兰不同甲基化程度AaICE1基因桃的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行桃遗传转化，通过农杆菌GV3101侵染导入桃中。
挑选果型正常，大小均一，无伤无虫果，先后用洗衣粉水搓洗，自来水冲洗，浸泡于0.2％升汞中10min，取出后用无菌水冲洗5次，无菌去皮，剥开果核，取出幼胚，接种在愈伤组织诱导培养基MS+2,4-D 0.5mg/L+BA 0.75mg/L+NAA1.0mg/L+2％蔗糖+7.5g/L琼脂上，pH 5.8，26℃暗中诱导愈伤组织。挑取包含此质粒的根癌农杆菌GV3101单菌落，接种到含5mg/L双丙氨磷、Rif 20mg/L、Gem 40mg/L的YEP 4ml液体培养基中，180rpm 28℃暗培养36h。取此菌液1ml加入50ml新鲜YEP液体培养基及200umol/L的乙酰丁香酮相同条件下培养12h，后取此菌液40ml于5000rpm 4℃离心10min，沉淀加入液体MS，180rpm 28℃继续振荡暗培养2h，至对数生长期，备用。预培养后的外植体直接浸入农杆菌悬浮液中浸泡，后取出用无菌滤纸吸去多余菌液，放入培养基(MS+2,4-D 0.5mg/L+BA 0.75mg/L+NAA 1.0mg/L+2％蔗搪+7.5g/L琼脂，pH5.8)，26℃暗中共培养。将诱导出的愈伤组织接入分化培养基(MS+NAA 0.05mg/L+BA 1.0mg/L)，光照强度2000-3000lx，光暗周期16h/8h下培养，培养温度同上。之后将不定芽移入生根培养基(1/2MS+IBA 1.0mg/L)上进行生根培养，5d后开始生根。桃转基因实验分别获得了7、9、9株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，与野生桃植株相比，其耐寒性分别提高了1.8℃、3.7℃和2.0℃。
实施例25.转紫茎泽兰不同甲基化程度AaICE1基因玉米的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行玉米遗传转化，通过农杆菌GV3101侵染导入玉米中。
温室条件下对受体材料以7d为周期分期播种，玉米试材按试验需要进行控制授粉，授粉 后10-13d，取玉米幼胚作为转化受体。用次氯酸钠消毒玉米棒后，剥出幼胚，用侵染液(加AS)清洗幼胚4遍，然后加入一定浓度的农杆菌菌液，放置15min，取出后用灭菌滤纸吸干，放到共培养培养基上，在25℃(黑暗)共培养2-5d，然后将幼胚转移到静息培养基，在28℃条件下暗培养7d。侵染和共培养环节菌液浓度OD500＝0.3-0.5，侵染时间10min、共培养时间3d。从静息培养基上把幼胚移入含有标记基因的抗性筛选培养基上，暗培养。每次继代注意淘汰呈褐色和水渍化的愈伤组织，并把生长正常的愈伤组织用镊子夹碎，分开选择培养。在继代筛选过程中注意经常观察发现污染或农杆菌复出的立即移出，未污染材料继续培养。另外，经常把出现褐化污染的组织块剔出或转移好的未被污染的组织块到新的相同培养基上。组织块膨大后，经常把大块组织剥小，连续继代筛选3次后，将选择到的抗性愈伤组织转到再生培养基上，恢复培养15d或更长时间(暗培养)。再将抗性愈伤组织转到再生培养基上发芽，培养条件为28℃，每日3000lx光强下，光照12h。待再生的玉米植株长到3片叶时，可将幼苗移植到含生根培养基的瓶中，在室内培养。当幼苗长出较粗的根后，将幼苗从罐头瓶中取出，用水冲净培养基，移栽于混有营养土和蛭石(1：3)的小花盆中，当玉米又长出2-3片新叶时，可将其移入大田或大花盆中，待长出三四叶后提取叶片DNA进行PCR检测。确定含有转入的ZmICE1基因。本实验分别获得了10、13、12株AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，6℃低温处理条件下，与野生植株相比其耐寒性分别提高了13℃、3.0℃和1.7℃。
实施例26.转紫茎泽兰不同甲基化程度AaICE1基因棉花的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行棉花遗传转化。
取构建好的植物表达载体农杆菌菌液，大量提取植物表达载体质粒DNA，选择次日将开放的花蕾进行自交。由于棉花是常异交植物，天然异交率在10％左右，因而外来花粉往往会造成品种间的混杂。在开花前一天，可见花冠快速伸长，黄色或乳白色的花冠呈指状突出于花蕾，次日即开放成为花朵。选择这样的花蕾，于指状花冠的前端用细线扎紧，并将细线的另一端系于铃柄，作为收获时的标记；在开花后20-24h左右即次日，选择果枝和花位较好的幼子房作为转化对象。一般选择每个果枝的第一和第二个果节位的花朵进行转基因操作。这些果节上的棉铃一般成铃率较高，有利于收获较多的种子；用50μl微量进样器作为微注射的工具。每次使用前和使用后，应以淡洗涤剂清洗，再用蒸馏水漂清；注射时，摘除或剥去花瓣，抹平花柱。在剥除花瓣时，注意不能损伤幼子房的表皮层，以免增加脱落率；用右手持微量注射器，左手 轻扶摘除花瓣后的幼子房，从抹平花柱处沿子房的纵轴方向进针至子房长度的约三分之二处，并后退至约三分之一处。花粉管通道法转化得到的棉花种子收获后于在温室盆栽幼苗，在2-3叶期，以5mg/L双丙氨磷喷洒棉花叶片。一周后观察植株生长状况，拔除叶片表面有退绿斑点的植株。生长正常的被认为是获得抗性基因的植株。本实验分别获得了15、13、17株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，4℃低温处理条件下与野生型棉花植株相比，其耐寒性分别提高了1.7℃、4.2℃和2.9℃。
实施例27.转紫茎泽兰不同甲基化程度AaICE1基因花生的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行花生遗传转化。
在花生进入盛花期时，进行转基因操作。在进入盛花期前，每天下午5点左右摘掉花蕾，以免后代有没经过转基因操作的花授粉结实，下季检测后代中的转基因植株时造成干扰。分别用微量移液器和滴管拉成的玻璃针，采用柱头涂抹和花曹管注射的方法导入含目的基因的质粒DNA，选用50ug/ml和100ug/ml两个DNA浓度处理“汕油523”和“汕油21”两个品种。进入盛花期后，每天早上6点半左右开始进行操作，涂抹操作用微量移液器吸取5ul DNA溶液涂于花朵柱头上；注射方法用玻璃针在花萼管的下部向子房方向注入约5ul的DNA溶液。每一个品种处理500朵以上。遗传转化得到的花生种子收获后于在温室盆栽幼苗，在2-3叶期，以5mg/L双丙氨磷)喷洒花生叶片。一周后观察植株生长状况，拔除叶片表面有退绿斑点的植株。生长正常的被认为是获得抗性基因的植株，抗性植株进行PCR验证。遗传转化分别获得了8、14、15株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，与野生型花生植株相比，其耐寒性分别提高了1.3℃、℃、2.8℃和1.9℃。
实施例28.转紫茎泽兰不同甲基化程度AaICE1基因芥菜的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行芥菜遗传转化，通过农杆菌GV3101侵染导入芥菜中。
将芥菜种子消毒(75％的酒精和0.1％升汞)后播种于MS培养基，7d后切取下胚轴(0.5-0.8cm)，置于芥菜分化培养基(MS+2mg/L 6-BA+0.2mg/L NAA+3％蔗糖+0.6％琼脂)上预培养2d。将已转入含BjICE1基因质粒的农杆菌活化，侵染时取500ul活化菌液，接种到50ml无抗生素的YEB液体培养基中，28℃摇床225rpm振荡培养至农杆菌生长活跃期(OD600值约为0.5-0.6)，4℃离心(4000r/min，10min)后，弃上清液，重悬于20ml预冷的MS液体培养基，备用。用重悬菌液侵染预培养后的下胚轴10min，滤纸上干燥后转移至分化培养基上， 共培养2d。将共培养后的下胚轴转移至芥菜筛选培养基(MS+2mg/L 6-BA+0.2mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行抗性筛选，每l0d更换一次筛选培养基.将多次筛选获得的抗性芽转移到芥菜生根培养基(MS+2mg/L 6-BA+0.4mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行生根培养。待组培苗根系强壮后，洗净根系上残留培养基，移栽到营养土中，置于人工气候室(温度：25士2℃，光照强度：20001x，相对湿度70％，光照/黑暗＝16/8h)进行培养。遗传转化分别获得了24、21株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状转基因植株，与野生型芥菜植株相比，其耐寒性分别提高了1.8℃、4.0℃和2.9℃。
实施例29.转紫茎泽兰不同甲基化程度AaICE1基因油菜的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行油菜遗传转化，以花粉介导法进行油菜转基因实验
油菜盛花期后，选取主茎或一次分枝上10-15个1-2d内将要开放的花蕾徒手去雄，并摘除所选茎或分枝上其他花蕾，然后套袋。同时在同一品种中选取第2天将开放的花序进行套袋，以备第2天取粉用。翌日上午当天开放的套袋植株的花粉约0.4g，悬浮在25ml 7.5％的蔗糖溶液中，进行第1次超声波处理，然后在溶液中加入20μg含不同耐冷性种群BrICE1基因的质粒DNA进行第2次超声波处理。第2次处理后，在溶液中加入10μl 1/10 000(W/V)硼酸，然后用处理过的花粉授在前一天去雄的油菜柱头上，套袋并挂牌，同时标注授粉花蕾数。授粉后5-6d去袋，使处理的授粉株充分发育。采收种子。采收的种子种植后，至两片子叶伸展开时喷施5mg/L双丙氨磷溶液，光照培养7d后，未转基因植株叶边缘变黄，而转基因植株正常生长。遗传转化分别获得了33、37株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型油菜植株相比，-10℃处理条件下其耐寒性分别提高了1.5℃、4.2℃和2.6℃。
实施例30.转紫茎泽兰不同甲基化程度AaICE1基因菊花的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行菊花遗传转化，通过农杆菌侵染导入菊花中。
取保存的带有目的基因的pCAMBIA1301质粒的农杆菌，接种于含有50mg/L利福平的YEB液体培养基中过夜培养。然后，分别划线接种于含5mg/L双丙氨磷有、50mg/L利福平的YEB平板培养基上，倒置于28℃恒温培养箱中，培养至单菌落长出后，将平板于4℃冰箱保存备用。在上述YEB平板培养基上分别挑取农杆菌单菌落，接种于添加有5mg/L双丙氨磷、50mg/L 利福平的20ml的YEB液体培养基中，在28℃下，180rpm振荡培养至对数生长期。活化两次后按1:100的比例将菌液接种于只含有100pmol/L RS的YEB液体培养基中。振荡培养4-6h，当其OD600值达到0.4-0.6左右时，用于遗传转化。取培养的无菌菊花植株上端的叶片作为外植体，用灭过菌的剪刀将其剪成8mm*8mm左右的小块，放入胚性愈伤组织诱导的培养基(Ms+0.5mg/L 6-BA+1.5mg/L NAA+蔗糖30g/L+琼脂粉5g/L)中进行预培养。预培养3d后，将浸染用的菌液倒入无菌的三角瓶中，把预培养的叶片用无菌水冲洗1-2次，用无菌滤纸吸干，然后放入三角瓶中直接浸染25min。浸染后取出，用无菌滤纸吸除过量菌液。将浸染后的叶片接种于共培养的培养基上，在25±2℃条件下共培养2天。待叶片周围长出菌落，将叶片取出，用无菌水冲洗2-3次，无歯滤纸吸干。转入胚性愈伤组织诱导的筛选培养基(300mg/L Cef)中进行培养，直到抗性体胚完全分化出来。筛选分化培养上培养15-20d后，小部分菊花叶片切口处开始分化。当培养基中的再生植株长到2cm左右时，用无菌的剪刀从菊花母体上剥离转移到不添加任何植物生长激素的MS基本培养基中进行生根，在25℃下，光照强度为1200-2400lx，16h/8h的光周期下进行培养。第6天开始根基部就开始萌发根。遗传转化分别获得了9、11、10株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状植株，与野生型菊花植株相比，其耐寒性分别提高了2.2℃、4.4℃和2.9℃。
实施例31.转紫茎泽兰不同甲基化程度AaICE1基因胡萝卜的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行胡萝卜遗传转化，通过农杆菌侵染导入胡萝卜中。
挑取根癌农杆菌工程菌单菌落接种于50ml含5mg/L双丙氨磷的LB液体培养基中，28℃，黑暗条件下200rpm振荡培养至OD600约为0.6。在超净操作台上将根癌农杆菌培养液转入无菌的50ml离心管中，4℃，8000rpm离心5min，弃上清，用等体积的无菌MS液体培养基悬浮菌体，待用。在高压灭菌过的培养皿中放2-3张无菌滤纸，加适量B5液体培养基使之湿润。取无菌苗于滤纸上，用无菌、锋利的解剖刀把下胚轴切成0.5cm长的小段，用于遗传转化。于超净工作台上将处理好的野胡萝卜无菌苗的下胚轴放入制备好的浸染液中，置15-20min，中间轻轻振荡，以保证各下胚轴小段都能够充分浸染。取出外植体，于无菌滤纸上吸去多余的菌液。摆放于共培养培养基上，26℃、黑暗下培养2-3d。共培养后，取出外植体，放入无菌的空三角瓶中，用无菌水漂洗2-3次；用含500mg/L Carb的无菌水冲洗1-2遍，无菌滤纸吸干水分，转接到附加一定浓度抗生素的B5抑菌培养基上，于26℃、光照16h/d条件下进行愈伤培养，继代2-3次；待长出愈伤组织后转入分化培养基上，诱导胚状体和芽。在 诱导出的芽生长约40d后，苗长至2-3cm时切下，转入生根培养基上诱导生根；待长出正常根、长出3-4片真叶时，将抗性苗移出，用自来水冲去根部的培养基，栽于装有蛭石和草炭(2:1)的营养钵中，用塑料薄膜覆盖，给予26℃、光照16h/d的条件；7d后打开塑料布的通风口进行适当炼苗，待抗性苗逐渐适应外部生长环境条件后，既可在温室下进行常规管理。转基因实验分别获得了9、12、10株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型野胡萝卜植株相比，其耐寒性分别提高了1.5℃、3.0℃和2.0℃。
实施例32.转紫茎泽兰不同甲基化程度AaICE1基因大豆的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行大豆遗传转化，通过农杆菌侵染导入大豆中。
将消毒后的大豆种子倒入到一个无菌塑料罐中，加入液体萌发培养基(LGM)(蔗糖20g/L，pH 5.8)，培养基体积大约是种子体积的2倍，以便完全覆盖种子及提供充足的水分。盖上盖子后在培养箱中放置16h，培养条件为全天23℃，黑暗处理。将40m1农杆菌重悬液倒入一个无菌培养皿中，检查播种大豆后的LGM、舍弃污染的。从种子胚方向，用解剖刀先斜切再沿中轴线将大豆种子剖开，去皮。在胚尖位置处平行于中轴轻划三刀，浸泡到装有重悬农杆菌液的培养皿中30min。侵染时间到后，用已灭菌的镊子将外植体转移到铺有无菌滤纸的铁盒中，外植体水平放置、切口向上。往铁盒中轻轻加入LCCM(Bs盐粉末0.321g/L，蔗糖30g/L，MES3.9g/L，pH 5.4，BAP 1.67mg/L，GA3 0.25mg/L，Cys 400mg/L，DTT 154.2mg/L，As 39.24mg/L)。封闭铁盒后于智能培养箱中放置3天，培养条件为全天23℃，黑暗处理。共培养3天后，于超净工作台上用已灭菌的镊子将外植体取出，并用手术刀将子叶切除掉，但保留下胚轴。切好后将外植体放入芽诱导培养基(SIM)(BS盐粉末3.21g/L、蔗糖30g/L、MES 0.59g/L、Noble Agar 8g/L，BAP 1.67mg/L、Tic 250mg/L、Cef 100mg/L，PH 5.7)中，每皿放置7个外植体，外植体的顶端必须高于培养基平面，下胚轴部分则必须插入培养基中。将两个一次性塑料培养皿的底部重合，使用微孔通气型医用胶带进行密封。将新的培养皿放到人工气候室中恢复培养14天，培养条件为全天25℃，16/8昼夜比。将长出幼芽的外植体转到芽伸长培养基(SEM)(MS盐粉末4.43g/L、蔗糖30g/L、MES 0.59g/L、Noble Agar 8g/L、pH 5.7、Asp 50mg/L、Glu 50mg/L、IAA 0.lmg/L、GA3 0.5mg/L、ZR lmg/L、Tic 250mg/L、)中SIM中，舍弃没有诱导出丛生芽的外植体。仍然置于人工气候室中培养，培养条件为全天25℃、16/8昼夜比。一般每2周更换一次新鲜的SEM，每次更换培养基时都要丢弃死亡的外植体。进行5次SEM后就可以结束本批次的继代培养。在幼芽的伸长过程中，将芽从外植体上切下来，然后放置到培养 皿中并用IBA(1mg/ml)浸泡其底部30s，之后将其转移到生根培养基(RM)(MS盐粉末4.43g/L、蔗糖20g/L、MES 0.59g/L、Phytagel 3g/L、pH 5.6、Asp 50mg/L、L-Glu 50mg/L，Tic 125mg/L，Cef 50mg/L，IBA 1mg/L)中继续培养。1-2周后，当苗的底部长出一定数最的根时，将其从RM培养基中取出。用自来水冲洗根部后再移植到土壤中。幼苗移栽到土壤中后需要先在人工气候室中生长一周左右以适应自然的环境，然后再转移到温室中培养。转基因实验分别获得了6、9、9株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。与野生型大豆植株相比，其耐寒性分别提高了2.3℃、4.0℃和2.8℃。
实施例33.转紫茎泽兰不同甲基化程度AaICE1基因萝卜的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行萝卜遗传转化，通过农杆菌侵染导入萝卜中。
切取萝卜材料四天苗龄无菌苗带柄子叶外植体先进行2天预培养，用OD600为0.3-0.6的EHA105农杆菌菌液侵染5-7min后，再进行5天共培养，然后将外植体转接到MS+6-BA 6mg·L -1+NAA 0.05mg·L -1+Cef 500mg·L -1的培养基上进行7天延迟筛选，再将外植体转接到MS+6-BA 6mg·L -1+NAA 0.05mg·L -1+5mg·L -1双丙氨磷Cef 500mg·L -1的培养基上进行10天的抗性筛选，每10天进行一次转接继代培养。利用农杆菌介导的遗传转化方法，分别获得了11、16、13株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型萝卜植株相比，其耐寒性分别提高了1.7℃、4.1℃和2.9℃。
实施例34.转紫茎泽兰不同甲基化程度AaICE1基因小麦的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行小麦遗传转化，通过农杆菌侵染导入小麦中。
采集扬花后12-14d的麦穗，取小麦幼穗中部大小一致的未成熟子粒，将幼胚接种在愈伤组织诱导培养基表面，25℃下暗培养，20d后转入相同培养基进行继代培养。将幼胚接种在MC培养基上，培养15-20d，待幼胚形成的愈伤组织发育完全后，放人4℃冰箱，冷处理2个月左右，然后在常温下继代培养。8月上中旬将愈伤组织转入MC+乙酰丁香酮(AS)0.1mmol/L培养基中，培养5-7d开始转化。将幼胚愈伤组织转入工程菌感染液中0.5-1.0h，然后转移到MD培养基+AS 0.1mmol/L，共培养2d，用含有0.5g/L头孢唑林钠的无菌水冲洗愈伤组织，然后用无菌卫生纸吸干多余的液体，将愈伤组织转MD+500mg/L Carb+5mg/L双丙氨磷培养基进行筛选，筛选出的抗性愈伤组织转入ME分化培养基+0.5g/L头孢唑林钠+5mg/L双丙氨磷。在5mg/L双丙氨磷下通过6-8周的选择培养后，转入再生培养基MD+IAA 1mg/L+ZT 1mg/L， 再生成苗。利用农杆菌介导的遗传转化方法，分别获得了19、12、18株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后获得目的性状植株，与野生型小麦植株相比，-10℃处理条件下，其耐寒性分别提高了2.2℃、4.2℃和3.3℃。
实施例35.转紫茎泽兰不同甲基化程度AaICE1基因大麦的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行大麦遗传转化，通过农杆菌侵染导入大麦中。
检查大麦幼穗未成熟胚的大小，幼胚大小保证为直径1.5mm-2mm之间，一般是在授粉大约14天左右。完整的将未成熟的种子和麦穗分离，将种子置于锥形瓶中，并放置于超净台内，以下步骤均在无菌超净台内进行。70％酒精简单冲洗30s，用无菌水清洗三次，再用50％次氯酸钠处理4min，无菌水冲洗3-4次，尽量减少次氯酸钠的残留。用摄子夹住种子下端约1/3处，使未成熟胚朝上。用超细尖头摄子从种子中部刺透，并掀起去除部分种皮，露出未成熟胚。用镊子尽量去除胚轴，同时保持盾片的完整性，分离未成熟胚，并置于BCI愈伤诱导培养基上，小盾片这边朝上，约25个/培养皿。用200ul的枪头吸取待接种菌液，滴在未成熟胚上，还可以用刀片或者接种环蘸取菌液，湿润胚的表面。若未成熟胚上的菌液太多，需要在培养基表面来回拖动未成熟胚，去除其表面过量的菌液，若只是湿润，直接把胚转移到新鲜的BCI培养基上，小盾片朝下切面朝上。将培养皿封口，24℃条件下，黑暗培养72h。若有农杆菌过量生长的未成熟胚，需要将其放入小烧杯，用无菌水和含有160mg/L Timentin抗生素的无菌水冲洗数次，并用滤纸将表面液体吸干。将未成熟胚转移至见BCS愈伤筛选培养基上(含有5mg/L双丙氨磷和160mg/L Timentin)盾片朝下，24℃条件下，黑暗培养6-8周。每隔2周更换新鲜培养基，转移愈伤的时候注意尽量不要将未成熟胚分化成的愈伤分开。愈伤在选择培养基上长到一定大小之后，转移到BCT过渡培养基(含有5mg/L双丙氨磷和160mg/L Timentin)。24℃条件下，低光照培养，光照条件可以在培养皿上面盖一张纸来达到。两周的过渡培养之后，愈伤会出现再生的迹象，如果没有出现，每隔两周更换新鲜培养基。愈伤上出现明显的再生迹象之后，转移至BCR愈伤再生培养基(含有5mg/L双丙氨磷和160mg/L Timentin)。24℃条件下，全光照培养，每隔两周更换新鲜培养基。选择植物培养瓶代替玻璃培养皿，增加植株再生的空间。愈伤在选择培养基上长到一定大小之后，转移到B13M培养，24℃条件下，低光照培养。每两周更换一次培养基，当再生的茎芽长大后转移至试管中培养。在茎芽发育到2-3cm，将幼苗小心地转移到长15cm的玻璃培养，透气滤菌塑料膜封口，试管中的培养基为生根培养基。遗传转化实验分别获得了14、19、15株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性 植株。低温筛选后，与野生型大麦植株相比，-10℃处理条件下其耐寒性分别提高了1.8℃、3.4℃和2.7℃。
实施例36.转紫茎泽兰不同甲基化程度AaICE1基因胡桃的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行胡桃遗传转化，通过农杆菌侵染导入胡桃中。
取-80℃冻存的携带有转化质粒的农杆菌在含有5mg/L双丙氨磷的固体YEB平板培养基上划线，28℃培养过夜。挑取平板上的单菌落，接种到含有5mg/L双丙氨磷的液体YEB培养基中，置于28℃恒温摇床振荡(200rpm)过夜。用紫外可见光分光光度计测定其在600nm波长下的吸光值，待OD600＝0.5-0.8时，倒入30ml的离心管中，常温下6000rpm离心5-6min，去上清液，加入液体DKW培养基至30ml，涡旋混合5min，用作侵染备用。取预培养良好的胡桃茎段与上述得到的农杆菌菌液混合，侵染时间为30min，将侵染后的茎段用无菌滤纸吸干，接种到共培养培养基中，共培养基组合为DKW+6-BA0.5mg/L+IBA0.1mg/L+AS(乙酰丁香酮)200μmol/L+蔗糖30g/L+琼脂6.0mg/L，高压灭菌前pH调至5.8，共培养时间设置3天，黑暗处理。培养条件为培养温度25±1℃。共培养3天后，转接到筛选培养基中，筛选培养基组合为DKW+6-BA0.5mg/L+IBA0.1mg/L+5mg/L双丙氨磷+Cef 500mol/L+蔗糖30g/L+琼脂6.0mg/L，5周后不定芽萌发。培养条件为培养温度25±1℃，光照时间16h/d，光照强度为2000-2500lx。遗传转化实验分别获得了11、12、17株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。与野生型胡桃植株相比，其耐寒性分别提高了1.7℃、3.8℃和2.7℃。
实施例37.转紫茎泽兰不同甲基化程度AaICE1基因莴苣的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行莴苣遗传转化，通过农杆菌侵染导入莴苣中。
在含有Kam和Rif的LB平板上挑取携带有不同甲基化程度LsICE1基因的农杆菌单菌落，接种于添加5mg/L双丙氨磷的LB液体培养基中，28℃，150rpm摇床过夜培养养至对数中期(OD600＝0.4-0.8)。取10ml菌液分装于已灭菌的10ml离心管中，3000rpm离心3min，去上清液，用灭菌去离子水悬浮，稀释至OD600值为0.5左右作为侵染使用。经农杆菌侵染的莴苣子叶外植体，与农杆菌一起在分化培养基(MS+6-BA 0.5mg/L+NAA 0.20mg/L)上共培养后，转入到筛选培养基(Sm8+5mg/L双丙氨磷+Cef 200mg/L)上进行抗性筛选，每周继代一次。经过一周培养后，子叶的基部能逐渐长出少量致密的愈伤组织并慢慢转为绿色，大约一周后便能形成小芽点，小芽点逐渐分化出幼叶，形成小芽。一个愈伤组织上一般能形成多个小芽。当 芽长至2-3cm时切下，插入到生根培养基(1/2Ms+NAA 0.05mg/L+Cef 200mg/L)中诱导生根，2-3周后移栽到育苗钵。通过这种方法分别获得了17、18、20株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型莴苣植株相比，其耐寒性分别提高了2.0℃、3.8℃和2.5℃。
实施例38.转紫茎泽兰不同甲基化程度AaICE1基因大花蕙兰的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行大花蕙兰遗传转化，通过农杆菌侵染导入大花蕙兰中。
从甘油冻存的工程菌株取20u1菌液稀释到200u1后涂布到含有5mg/L双丙氨磷的YEB固体培养基，20h后挑选单菌落接种于含有5mg/L双丙氨磷YEB液体培养基，28℃振荡培养16h左右，用pH值5.2的YEB液体培养基将菌液稀释到OD600值为0.4。将培养的无菌苗切除叶梢和根，保留基部约0.5cm高，并纵切为2-4块组织切块(0.3-0.5cm)，经过2d预培养后(加入浓度为100mmol/ml的AS溶液)，侵入菌液10min，取出组织切块并用无菌滤纸吸干菌液，接种到共培养基，当共培养3d后，分挑取外植体，用含有50mg/L的Cef的MS液体培养基清洗外植体，并在含有50mg/L的Cef的PLB诱导培养基继续培养，以消除农杆菌。将外植体转移到含有50mg/L的Cef的PLB诱导培养基继续培养到20-30d，外植体基本上处于PLB形成发育时期，再将外植体转移到含有5mg/L双丙氨磷的筛选培养基，进行抗阳性转基因植株的初步筛选。通过这种方法分别获得了11、10、10株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型大花蕙兰植株相比，其耐寒性分别提高了2.2℃、3.9℃和2.8℃。
实施例39.转紫茎泽兰不同甲基化程度AaICE1基因蝴蝶兰的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行蝴蝶兰遗传转化，通过农杆菌侵染导入蝴蝶兰中。
将蝴蝶兰原球茎在预培养液体培养基(添加6-BA1.0mg/L+NAA5.0mg/L+2,4-D 2.0mg/L+AS 100μmol/L)中，预培养3d。用于转化的农杆菌EHA105在含有5mg/L双丙氨磷利福平50.0mg/L、和乙酰丁香酮100μmol/L的LB(p H7.0)培养基中28℃、150r/min震荡培养24h，在同上LB培养基中加入20％(v/v)上述菌液，活化5h，测O.D600≈0.60；6000r/min离心3min，用灭菌液体培养基(加AS 100μmol/L)重悬并稀释至原体积，直接用于对原球茎的遗传转化。侵染15min，原球茎接种于固体培养基(加6-BA1.0mg/L+NAA 5.0mg/L+2,4-D2.0mg/L+AS 100μmol/L)上共培养3d。然后用含有5.0mg/L美罗培南的液体培养基冲洗原 球茎3次，分别在添加美罗培南5.0mg/L和噻孢霉素100.0mg/L的固体培养基(添加KT 3.0mg/L)上交替培养，3d转接一次，至完全去除农杆菌。脱菌的原球茎转至含有5mg/L双丙氨磷和KT 3.0mg/L的固体培养基上选择培养，每2周后转至同样培养基上继代培养。45d后将原球茎在含5mg/L双丙氨磷的类原球茎状体(PLBs)诱导培养基(添加TDZ和2,4-D各1.0mg/L)上培养，切面上长出新的PLBs并分化成苗。将植株转至增殖培养基(6-BA 5.0mg/L)中培养。丛生小苗高达到3-5cm时，切开移到生根培养基(Hyponex No.1 30.0g/L+NAA0.3mg/L)，当每株长出4-5条新根，长达2-4cm时，出瓶栽植。转基因实验分别获得了15、18、20株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后的目的性状植株与野生型蝴蝶兰植株相比，其耐寒性分别提高了2.0℃、3.6℃和2.7℃。
实施例40.转紫茎泽兰不同甲基化程度AaICE1基因腊梅的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行腊梅遗传转化，通过农杆菌侵染导入腊梅中。
将保存于-80℃冰箱的农杆菌EHA105接种到含有5mg/L双丙氨磷的YEB平板培养基上，暗培养2-3d。待菌落长出后，挑取单菌落接种于含5mg/L双丙氨磷的30ml YEB液体培养基中进行第一次活化，在28℃下于摇床上振荡培养1-2d，到OD600值约为0.8-1.0时取下备用。将第一次活化的50u1菌液接种于不含双丙氨磷的50m1 YEB液体培养基中进行第二次活化，在28℃下于普通摇床振荡培养至OD600值为0.4。本实验采用腊梅半展开的子叶为外植体，将子叶切成1cm*1cm大小的方块进行预培养3d。将活化后的农杆菌EHA105菌液在4℃，6000rpm下离心后收集菌体，加入液体MS+AS100umol/L重悬到OD600值为0.4，侵染愈伤组织10min，共培养2d后，用液体MS+Cef 1000umol/L脱菌，在无菌水冲洗5次后，放于筛选培养基Ms+BA1.0mg/L+NAA0.5mg/L+KT0.5mg/L+IBA2.0mg/L+2,4-D0.2mg/L+5mg/L双丙氨磷+Cef1000umol/L中进行选择培养。再生植株转移至MS+6-BA1.0mg/L+NAA0.1mg/L上，当苗生长到3-4cm高时即转入1/2MS+NAA0.1mg/L中进行生根培养。转基因实验分别获得了8、14、13株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型腊梅相比，其耐寒性分别提高了1.7℃、4.2℃和3.0℃。
实施例41.转紫茎泽兰不同甲基化程度AaICE1基因茶的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行茶遗传转化，通过农杆菌侵染导入茶中。
选取8-9月份未成熟茶果，经蒸馏水清洗，剥去果皮，取出尚未成熟的乳白或略带棕色的 种子，剥开外种皮，在无菌条件下，75％乙醇表面消毒30s后，用0.1％HgCl 2浸泡3min，再用无菌水漂洗6-8次。将子叶切成长度为0.5cm大小的方块，接种到固体培养基改良ER+0.1mg/L+1.0mg/L6-BA+0.1g/L氯霉素+30g/L蔗糖上。接种后在温度为25±2℃的恒温培养室内培养，人工光照13h/d，光照强度1500-2000lx。培养25d后选择转绿的子叶愈伤组织作为外植体。取保存的根癌农杆菌EHA105在已制备好的YEB固体培养基平板上画线，并在28℃条件下暗培养1-2d后，用接种环挑取单菌落接种于5ml YEB液体培养基中，于28℃、220rpm振荡培养24h后，在无菌条件下吸取1ml菌液到含200uml/L乙酰丁香酮的液体培养基中，于28℃、220rpm振荡培养4-6h后，在600nm处测得菌液OD值为0.6-0.8左右即可用于外植体浸染。在超净工作台上选择已经转绿的子叶愈伤组织作为外植体接种于预培养基改良ER+0.2mg/LIBA+30g/L蔗糖上，25℃暗条件下预培养3d。将预培养后的子叶愈伤组织放入备用的根癌农杆菌菌液中，菌液浸没实验材料，侵染15min后，用无菌滤纸吸干表面多余的菌液，接种到添加150umol/L AS的YEB共培养培养基上，25℃条件下黑暗培养3d。共培养后，用无菌水冲洗材料3-4次，无菌滤纸吸干材料表面水分，将愈伤组织转接到预培养基中，延后选择培养3d，之后转接到含附加100umol/LCef和100umol/LSpe的除菌培养基上黑暗培养。每3d转接一次，15d以后外植体转至附加100umol/LSpe的筛选培养基中进行抗性芽筛选。筛选培养20d后，将有活力的抗性芽转移至新的附加5mg/L双丙氨磷100umol/LSpe分化培养基中培养。在分化培养基中培养30d，子叶愈伤组织分化出幼苗，转入生根培养基培养2周后得到完整的抗性植株。遗传转化分别获得了12、16、17株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。低温筛选后，与野生型植株相比，其耐寒性分别提高了2.3℃、4.3℃和3.2℃。
实施例42.转紫茎泽兰不同甲基化程度AaICE1基因赤桉的获得
将实施例1中构建的不同甲基化程度AaICE1、AaICE1(DC)1、AaICE1(DC)2基因的表达载体进行赤桉遗传转化，通过农杆菌侵染导入赤桉中。
从低温冰箱中取出保存的GV3101菌种，在超净工作台中采用划线法在已配好的培养基表面轻轻连续画线5-6条。将培养皿倒置于25-27℃进行暗培养24-36h。然后用接种针挑取培养皿中培养基表面的单个菌落放于15m1离心管液体培养基上，在27℃、180rpm恒温摇床上进行悬浮培养12-18h。取菌液按1/40的比例放入100m1的三角瓶，继续培养6个小时左右。待OD600值为0.5时，用作侵染处理。以尾赤按栽培无性系DH201-2为材料，取自生根培养基上培养30天植株的上部和中部的茎段在预培养基上培养6d。于超净工作台中，将赤桉外植体放入三 角瓶中的菌液浸泡30min，然后取出植物材料，接种到植物共培养基(100uM乙酰丁香酮，PH 5.8)上培养3d。之后将浸染后的茎段转入到(Ms+0.05mg/L TDZ+0.5mg/L NAA+5mg/L双丙氨磷+300mg/L头孢霉素，PH 5.8)的诱导再生培养基上培养15天后，形成愈伤组织，除去褐变和疏松、颜色发黄的愈伤，将颜色红绿、生长良好的愈伤组织转移到同样的培养基上，转移三次以便除去农杆菌，然后转移到只有双丙氨磷的培养基上，可得到再生芽。将再生芽转移到壮苗培养基上培养20天后，再转入到生根培养基中生根培养。转基因实验分别获得了14、19、11株转AaICE1、AaICE1(DC)1、AaICE1(DC)2基因抗性植株。与野生型尾赤按植株相比，其耐寒性分别提高了3.7℃和2.3℃。
实施例43.CRISPR/Cas9技术原位替换紫茎泽兰不同甲基化程度AaICE1基因拟南芥的获得
紫茎泽兰AaICE1基因序列(SEQ ID NO：1)，根据密码子的简并性特征，将AaICE1基因中理论上可甲基化的位点进行替换后，获得全部替换后AaICE1(DC)1基因(SEQ ID NO：3)及部分替换AaICE1(DC)2基因(SEQ ID NO：4)。将AaICE1分成3段进行CRISPR/Cas9载体构建和转基因，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA序列分别为5’-GCTCCTATTTCGATGGGGTT TGG-3’，5’-GATAAATGAGAGCGGTAAAG CGG-3’和5’-GCAGTGCTTTTCGATACAGC AGG-3’，引导Cas9蛋白对DNA的定点切割，sgRNA的长度一般应为20nt。启动子可选CaMV 35S、U6、T7及YAO等，本实验选择效率较高的YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的AaICE1、AaICE1(DC)1、AaICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过花粉管介导法导入拟南芥中。
将盛花期拟南芥的花表面部分浸泡在农杆菌悬浮液(OD＝0.8)中5分钟，同时轻轻旋转。将浸染后植株套袋保持高度的湿润状态，暗室培养24小时。种子成熟，角果自然开裂后可以收种子。收集到的拟南芥种子于4℃春化2天，种子消毒后在含有5mg/L双丙氨磷的平板上进行培养。成功转入重组质粒的种子能够正常生长，非转基因种子不能正常生长，仅能长出2片子叶，根的生长也受到严重抑制而死亡。将筛选到的植株移植到蛭石：泥炭土：珍珠岩为9:3:1的基质中，覆盖薄膜2-3天。分子验证分别得到转AaICE1、AaICE1(DC)1和AaICE1(DC)2基因的拟南芥植株19、18和26棵。
将野生型和原位替换后的拟南芥植株在光照培养室内正常培养至苗龄3周后，4℃冷适应3天，0℃放置1小时，-4℃放置4小时，然后每小时下降2℃，直至-8℃，处理24h后，23 ℃培养5d，观察野生型和原位替换后的拟南芥植株的低温伤害情况，每个处理重复3次，统计培养5d后的低温伤害指数。原位替换AaICE1、AaICE1(DC)1和AaICE1(DC)2基因拟南芥低温伤害指数分别为0.59、0.14和0.36，与野生型Col-0相比，耐寒性分别提高1.8℃、3.5℃和2.4℃。
实施例44.CRISPR/Cas9技术原位替换不同甲基化程度MeICE1基因木薯的获得
克隆木薯MeICE1基因，获得基因cDNA序列为SEQ ID NO：9，根据密码子的简并性特征，将MeICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换MeICE1基因中的不同程度的甲基化位点。获得全部替换后MeICE1基因序列为SEQ ID NO：50，命名为MeICE1(DC)1；部分替换后MeICE1基因序列为SEQ ID NO：91，命名为MeICE1(DC)2。将MeICE1(DC)1分成3段进行CRISPR/Cas9载体构建和木薯原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCCTCACCTTTGCTAAATAGAGG-3’，5’-GTAAACCCTGGACCATTTTCAGG-3’和5’-GCCTATCATTGAGCTTCTTCCGG-3’；MeICE1(DC)2基因sgRNA序列为5’-GGTTTTGAGTGCATAAAGTAGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的MeICE1(DC)1、MeICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌介导法导入木薯中。
取500ul超低温冰箱保存的农杆菌GV3101表达载体，加入到50ml YEB(添加5mg/L双丙氨磷)液体培养基中，28℃，240rpm，培养18-24h。4℃，4000rpm离心10min，用液体MS稀释到OD值1.0，待用。将成熟10-15d的木薯体胚绿色子叶切碎后，放大约500个子叶胚的碎块在30ml的农杆菌悬浮培养液中，期间振荡几次。45min后，吸去菌液，将子叶块在超净工作台中放置20min或用灭过菌的滤纸吸掉多于的液体，转移到茎器官发生培养基上，于26℃，16h光照共培养3d。分别在转化材料共培养了3d后，将愈伤取出，用无菌水冲洗3次，然后用添加500mg/1羧苄霉素液体MS(pH5.3)培养基再冲洗2次，在超净工作台中放置20min或用无菌滤纸把残留的液体吸干。转移愈伤到含5mg/L双丙氨磷和500mg/L羧苄霉素的茎器官发生培养基上，26℃暗培养1周。将愈伤转移到包含5mg/L双丙氨磷和500mg/L羧苄霉素的茎器官发生培养基上，26℃，16h光照培养2周。挑取抗性不定芽，放在茎伸长培养基上。26℃，16h光照培养。2-3周后，切下生长出的茎段转移到MS培养基上进行 培养，当植株长大时，将其切开继代。切取从侧芽中长出的大约1cm的茎段，转移到包含5mg/L双丙氨磷的无激素培养基上进行生根实验，以消除假阳性植株，用野生形植株的茎段做对照。1-2周后，转基因阳性植株可以正常生根，而假阳性和对照则不能长出根。共获得原位替换木薯MeICE1(DC)1、MeICE1(DC)2基因植株15和13株，耐寒性较野生型木薯分别提高了4.0℃和3.0℃。
实施例45.CRISPR/Cas9技术原位替换不同甲基化程度AcICE1基因猕猴桃获得
克隆猕猴桃AcICE1基因cDNA，序列为SEQ ID NO：10，根据密码子的简并性特征，将AcICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换AcICE1基因中的不同程度的甲基化位点。获得全部替换后AcICE1基因序列为SEQ ID NO：51，命名为AcICE1(DC)1；部分替换后AcICE1基因序列为SEQ ID NO：92，命名为AcICE1(DC)2。将AcICE1(DC)1分成3段进行CRISPR/Cas9载体构建和猕猴桃原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTGGTAGGAGGGCTCTTCCATGG-3’，5’-GTGGCTGCAGAGAAAATCCAAGG-3’和5’-GATATCCACCATGTTACCGGTGG-3’；AcICE1(DC)2基因sgRNA序列为5’-GATGGTTCAAATCTTGATGGTGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的AcICE1(DC)1、AcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌LBA4404导入猕猴桃中。
取保存的农杆菌菌种LBA4404，划线后挑取农杆菌单菌落，入20ml含5mg/L双丙氨磷液体YEP培养基中，于200rpm，28℃条件下振荡培养16-24h。吸取0.5ml菌液，入50ml含5mg/L双丙氨磷的YEP培养液中，条件同上扩大培养。待菌液培养至OD600为0.5时，于4000rpm，4℃离心10min沉淀，用加有100uM乙酰丁香酮的液体MS培养基等体积悬浮备用。将猕猴桃无菌苗幼嫩的叶片，在无菌条件下切成0.5cm见方的小块，接在以MS为基本培养基附加3.0mg/L 6-BA和1.0mg/L NAA的培养基上预培养3d。3d后，将预培养的叶片放入重悬好的菌液中，期间轻微震荡，使农杆菌与叶片充分接触，浸染10min后用无菌滤纸吸干多余菌液，叶片背面向下放置在含100uM AS的再生培养基上，28℃暗培养2d，注意在共培养的培养基上垫上一层无菌滤纸，防止农杆菌生长过旺。将经过共培养的叶片外植体用无菌水冲洗4-5次，用无菌滤纸吸去残留的无菌水，接种到含有5mg/L双丙氨磷，400mg/L Carb的再生培养基上，一个月继代一次，待分化芽长至2-3cm，切下，插入生根培养基中。5-10 d可见不定根长出。待生根幼苗长至5cm时，培养瓶放在自然条件下培养3-5d，然后逐渐的打开瓶盖，前两天将盖子松动，第三、四天打开瓶盖一半，第五、六天取下盖子，用瓶盖一半大小的硬纸板盖上，第七、八天取下硬纸板，使组培苗暴露在自然状态下，炼苗10d，用镊子小心的取出新生苗，自来水冲洗根上面附着的琼脂，移栽至温室土壤中培养。遗传转化共获原位替换猕猴桃AcICE1(DC)1、AcICE1(DC)2转基因植株11和12株，耐寒性分别提高了3.6℃和2.4℃。
实施例46.CRISPR/Cas9技术原位替换不同甲基化程度MaICE1基因香蕉的获得
克隆香蕉MaICE1基因cDNA，序列为SEQ ID NO：11，根据密码子的简并性特征，将MaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换MaICE1基因中的不同程度的甲基化位点。获得全部替换后MaICE1基因序列为SEQ ID NO：52，命名为MaICE1(DC)1；部分替换后MaICE1基因序列为SEQ ID NO：93，命名为MaICE1(DC)2。将MaICE1(DC)1分成3段进行CRISPR/Cas9载体构建和香蕉原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGCCTCGAAGGGGTGATGGG NGG-3’，5’-GCAGAAGGCGGTGCCAGAGC NGG-3’和5’-GAGGTCGACGAGTTGGACGA NGG-3’；MaICE1(DC)2基因sgRNA序列为5’-GGCCTCGAAGGGGTGATGGGCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的MaICE1(DC)1、MaICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌GV3101导入香蕉中。
以香蕉胚性悬浮细胞系(ECS)为转化受体，取在继代培养10d的香蕉ECS，离心去上清后，每1mL细胞密实体积ECS加入40ml含有不同甲基化程度MaICE1基因的农杆菌GV3101，27℃，黑暗静置1-2h，然后将其进行共培养。共培养条件为：黑暗条件下，27℃，50rpm转速下振荡培养24h，然后将培养物静置去上清，再加入40ml的液体培养基，将转速提高至110rpm，连续共培养7d。将共培养完成后的培养物静置去上清，加入香蕉ECS液体筛选培养基40ml，27℃，转速100rpm振荡培养，每10-14d继代一次，连续继代3代以上。继代的方法是：取前一代0.1-0.5ml细胞密实体积的ECS加入到新鲜液体筛选培养基中继续振荡培养。同时，将每代继代剩余的ESC以及原共培养完成后的ESC用半固体筛选培养基进行筛选作为对照。采用半固体筛选时，先用含头孢霉素500mg/L的M2培养基洗涤2-3次，无菌纸吸干多余培养基，转接到半固体筛选培养基上进行胚诱导，每个月更换一次体胚诱导培养基直到胚成熟。 取液体筛选培养3代以后的ECS静置去上清，然后将其均匀铺于体胚诱导培养基M3上，黑暗培养2-3个月，每个月更换一次体胚诱导培养基直到胚成熟，将成熟的抗性体胚转移至体胚萌发培养基上，光/暗(12h/12h)条件下培养至体胚萌发得到小苗，然后将小苗转移至生根培养基上，光/暗(12h/12h)条件下进行培养，即获得完整的转化植株。本实验共获得香蕉原位替换MaICE1(DC)1、MaICE1(DC)2基因植株21和15株，耐寒性分别提高了3.9℃和2.9℃。
实施例47.CRISPR/Cas9技术原位替换不同甲基化程度MabICE1基因芭蕉的获得
克隆芭蕉MbICE1基因cDNA，序列为SEQ ID NO：12，根据密码子的简并性特征，将MbICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换MbICE1基因中的不同程度的甲基化位点。获得全部替换后MbICE1基因序列为SEQ ID NO：53，命名为MbICE1(DC)1；部分替换后MbICE1基因序列为SEQ ID NO：94，命名为MbICE1(DC)2。将MabICE1(DC)1分成3段进行CRISPR/Cas9载体构建和芭蕉原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGCATTGAAGACAGACGAAACGG-3’，5’-GCAGAAAAAGTCGGCATCTTGGG-3’和5’-GTAGGCAGTGAAGAACTTGAAGG-3’；MbICE1(DC)2sgRNA序列为5’-GCATGGACTTAAAGCCGGAG AGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的MabICE1(DC)1、MabICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌GV3101导入芭蕉中。芭蕉遗传转化实验操作方法同香蕉遗传转化实验(实施例11)。遗传转化实验中，共获得原位替换MabICE1(DC)1、MabICE1(DC)2基因植株13和15株。与野生型芭蕉植株相比，其耐寒性分别提高了4.2℃和2.6℃。
实施例48.CRISPR/Cas9技术原位替换不同甲基化程度TcICE1基因可可的获得
克隆可可TcICE1基因cDNA，序列为SEQ ID NO：13，根据密码子的简并性特征，将TcICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换TcICE1基因中的不同程度的甲基化位点。获得全部替换后TcICE1基因序列为SEQ ID NO：54，命名为TcICE1(DC)1；部分替换后TcICE1基因序列为SEQ ID NO：95，命名为TcICE1(DC)2。将TcICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCAGCAAAATATTATCTTGATGG-3’，5’-GGAGGGAATTTCGGGTTGTTNGG-3’和5’-GATAAAAACGAAAAGAGAAGGGG-3’；TcICE1(DC)2sgRNA 序列为5’-GGAGGGAATTTCGGGTTGTTCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的TcICE1(DC)1、TcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌LBA4404导入可可中。
从田间采集可可成熟果实，剥去果皮75％酒精侵泡数秒后，再用体积百分数为5％NaC1O溶液浸泡30min，剥去种皮后接入萌发培养基中，10天后取其子叶作为外植体。将培养至OD 600＝1.37左右的农杆菌LBA4404/PBI121收集菌体，用等体积PCG液体培养基(DKW+2,4-D3.0mg/L+KT 1.0mg/L+TDZ 0.01mg/L+Glucose 20g/L+glutamine 250mg/L)重悬，侵染萌发培养基中培养10d的可可子叶材料，28℃暗共培养3天。共培养后转接到含500mg/L Carb和5mg/L双丙氨磷的PCG固体培养基(1％琼脂粉)上进行抑菌及抗性筛选培养，10天于相同培养基上继代一次，在PCG培养基中培养20天后转入含Carb 500mg/L和5mg/L双丙氨磷的SCG培养基(KW+2,4-D 3mg/L+KT lmg/L+Glucose 20g/L+10％CW+1％琼脂粉)中培养20天，再转入含Car 500mg/L和5mg/L双丙氨磷的ED培养基(DKW+Sucrose 30g/L+Glucose1g/L+1％琼脂粉)中培养直到长出胚状体。同时经常观察污染或农杆菌复出的立即移出未污染材料继续培养。将成熟胚转移到胚发育的培养基中，该培养基分为二个时期的培养基，第一阶段培养基为PEC(1/2DKW+KNO 3 0.3g/L+AA 1000X stock solution lml/L+Glucose 20g/L+Sucrose 10g/L+1％琼脂粉)，在第一阶段的培养中，培养条件为每天的光照时期是光/暗为16/8h，每30天换一次培养基，直到长出带1-2片真叶的芽后，转入第二阶段的RD培养基(1/2DKW+KN0 3 0.3g/L+Glucose l0g/L+Sucrose 5g/L+不同浓度的IBA 1.0mg/L+不同浓度的IAA 1.0mg/L中，培养条件和第一阶段一致，每30天转一次培养基，直到成苗。本实验共获得原位替换TcICE1(DC)1、TcICE1(DC)2基因植株10和9株，耐寒性分别提高了3.9℃和2.2℃。
实施例49.CRISPR/Cas9技术原位替换不同甲基化程度CtICE1基因枳的获得
克隆枳CtICE1基因cDNA，序列为SEQ ID NO：14，根据密码子的简并性特征，将CtICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换CtICE1基因中的不同程度的甲基化位点。获得全部替换后CtICE1基因序列为SEQ ID NO：55，命名为CtICE1(DC)1；部分替换后CtICE1基因序列为SEQ ID NO：96，命名为CtICE1(DC)2。将CtICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的 sgRNA，sgRNA的长度为20nt，序列分别为5’-GCTTTGATTTGGGTGAAATGGGG-3’，5’-GCAATTAGCACTATCACTGGTGG-3’和5’-GCTGTCATCAGTTGTTTCAATGG-3’；CtICE1(DC)2sgRNA序列为5’-TGCTTGAAGTGGAAGATGAC TGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的TcICE1(DC)1、TcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌LBA4404导入枳中。
将枳果实剖开取种，自来水洗净后，再用1mol/LNaOH去果胶，冲净后在超净工作台中用70％的酒精晃洗1min左右，用2％的次氯酸钠浸泡18min灭菌，中间可晃动数次。然后倒掉次氯酸钠，用无菌水冲洗三次，每次5-6min。将灭好的种子在无菌条件下剥去种皮，置于装有固体培养基的试管中进行暗培养，培养条件为24℃。培养15-30d后，转入光照培养(24℃，14/10h光周期)，然后取上胚轴进行转化。上胚轴或茎段切割方式采用斜切方法进行，斜切成0.5-1cm左右长的茎段，尽量避开节点。将摇好的农杆菌菌液用含20mg/L AS的LM悬浮培养基稀释，将菌液的OD 600值调至0.8。再将切割好的外植体材料浸泡在已制备好的农杆菌菌液里，侵染20min，中间可摇动几次。侵染完毕，用无菌滤纸吸干外植体表面菌液，再接种到共培养培养基(MT+BA1.0mg/L+IAA0.5mg/L+AS 20mg/L)上，21-23℃暗培养3d。共培养培养基中可铺一层无菌滤纸，以降低农杆菌污染的几率。已转化的外植体共培养三天后，用400mg/L头孢霉素洗一次，无菌水冲3-5次，直至洗净表面农杆菌，无菌吸水纸吸干后转入附加5mg/L双丙氨磷和400mg/L头孢霉素的筛选培养基(MT+BA 1.0mg/L+IAA 0.5mg/L+5mg/L双丙氨磷+Cef 400mg/L)上，25℃暗培养两周后再转到光照/黑暗(16/8h)条件下培养。每20d左右继代一次，继代时除去非切口处再生芽体。当抗性芽生长至0.5cm左右时，转到伸长培养基(MT+BA 0.5m/L+AA 0.5mg/L+GA 0.5mg/L+5mg/L双丙氨磷+Cef 400mg/L)上促其伸长。当抗性芽长至1-2cm时，转移芽至生根培养基(MT+BA 0.5mg/L+IAA 0.5mg/L+活性炭0.5g/L)上诱导生根。遗传转化共获得原位替换CtICE1(DC)1、CtICE1(DC)2基因植株20和20株，耐寒性分别提高了3.8℃和1.7℃。
实施例50.CRISPR/Cas9技术原位替换不同甲基化程度VvICE1基因葡萄的获得
克隆葡萄VvICE1基因cDNA，序列为SEQ ID NO：15，根据密码子的简并性特征，将VvICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换VvICE1基因中的不同程度的甲基化位点。获得全部替换后VvICE1基因序列为SEQ ID NO：56，命名为VvICE1(DC)1； 部分替换后VvICE1基因序列为SEQ ID NO：97，命名为VvICE1(DC)2。将VvICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GAGGAGGAGTGGTTGGTGAGCGG-3’和5’-GTGGAGGAGACTGCAAATGATGG-3’；VvICE1(DC)2sgRNA序列为5’-GTGAGCGGTGTCGAAATGAA AGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的TcICE1(DC)1、TcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体，通过农杆菌导入葡萄中。
将诱导扩繁的葡萄PEM继代到新鲜的X6培养基上，26℃暗环境下预培养一周以供转基因操作。取200μl菌液接种于20ml液体LB培养基(含5mg/L双丙氨磷)中。28℃、180rpm培养24h后取20μl该菌液接种于20ml液体LB培养基(含5mg/L双丙氨磷)中，相同条件下进行二次活化培养20h。将菌液转移到灭菌的50ml离心管中，5000×g离心8min，去上清，再用重悬缓冲液(MS培养基+20g/L蔗糖+100μM AS)重悬离心两次。在重悬缓冲液中28℃、180rpm孵育1h，用重悬液稀释调整菌液浓度为OD 600＝0.3-0.4。将葡萄PEM放入盛有农杆菌液的三角瓶中侵染20min，用无菌滤纸吸干表面菌液，将其继代到放入两层灭菌滤纸并添加约5-6m的X6液体培养基(附加100μM AS)的无菌培养皿中。26℃黑暗条件下共培养48h后，用浓度均为200mg/L的头孢霉素、羧苄青霉素溶液中脱菌20min，再用无菌水冲洗3遍，将葡萄PEM接种于MS+5mg/L双丙氨磷+200mg/L Cef+200mg/L Carb+10mg/L Hyg+30g/L蔗糖+3g/L植物凝胶培养基上，PEM诱导出的抗性SE接种于MS+0.2mg/L KT+0.1mg/L NOA+10mg/L Hyg+30g/L蔗糖+3g/L植物凝胶。26℃黑暗条件下培养，每三周继代一次，直至抗性体细胞胚长出。葡萄遗传转化共获得原位替换WwICE1(DC)1、WwICE1(DC)2基因植株10和9株，耐寒性分别提高了3.3℃和1.7℃。
实施例51.CRISPR/Cas9技术原位替换不同甲基化程度MdICE1基因苹果的获得
克隆苹果MdICE1基因cDNA，序列为SEQ ID NO：16，根据密码子的简并性特征，将MdICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换MdICE1基因中的不同程度的甲基化位点。获得全部替换后MdICE1基因序列为SEQ ID NO：57，命名为MdICE1(DC)1；部分替换后MdICE1基因序列为SEQ ID NO：98，命名为MdICE1(DC)2。将MdICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的 sgRNA，sgRNA的长度为20nt，序列分别为5’-GAAGACAAGTTGGGATTTTGAGG-3’，5’-GCACGGTTACCGGTGGAGGAGGG-3’和5’-GCAGCTTGGCCATTTGGGCTAGG-3’；MdICE1(DC)2sgRNA序列为5’-GGTTGAGCAGCAAGCTTTCT GGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的MdICE1(DC)1、MdICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
使用超声波转化法对苹果进行遗传转化。将苹果试管苗叶片剪成4mm 2左右碎片，放入含有20ml超声缓冲液的容器中，超声缓冲液的成分为：13.7mmo1/LNaCI，2.7mmo1/LKCI，l0mmol/LNa 2HPO 4，2mmo1/LKH 2POP 4，5％DMSO，pH 7.4。每毫升缓冲液含质粒DNA 5ug。在声强为0.5-1.0W/cm 2进行20min超声波处理。将超声处理后的叶片转入含有4mg/L6-BA+0.2mg/LNAA的MS再生培养基上恢复生长1周，然后转入含有5mg/L双丙氨磷再生培养基上进行2周暗培养筛选，每2周转移1次，2个月后转移到0.5mg/L6-BA+0.1mg/LNAA的MS继代培养基上这一过程共持续3个月。培养条件：每天连续16h光照，光照强度为40umol m -2s -1恒温25℃继代保存。每4周继代一次。分别从400个外植体叶片上获得了16、17个原位替换MdICE1(DC)1、MdICE1(DC)2基因抗性植株。与野生型植株相比，其耐寒性分别提高了4.1℃和2.5℃。
实施例52.CRISPR/Cas9技术原位替换不同甲基化程度CbICE1基因芥的获得
克隆芥CbICE1基因cDNA，序列为SEQ ID NO：17，根据密码子的简并性特征，将CbICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换CbICE1基因中的不同程度的甲基化位点。获得全部替换后CbICE1基因序列为SEQ ID NO：58，命名为CbICE1(DC)1；部分替换后CbICE1基因序列为SEQ ID NO：99，命名为CbICE1(DC)2。将CbICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GAAGATCTTGTGGATGTGGTGGG-3’，5’-GTTCAGATTGGAGGAGGGAAGGG-3’和5’-GTGAACATTCATATGTTCTGTGG-3’；CbICE1(DC)2sgRNA序列为5’-GAAGATCTTGTGGATGTGGTGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的CbICE1(DC)1、CbICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E 作为抗性筛选基因，构建CRISPR/Cas9表达载体。
芥种子消毒(70％的酒精和1％次氯酸钠)后播种于MS培养基，一周后切取下胚轴，置于分化培养基(MS+2mg/L 6-BA+0.2mg/L NAA+3％蔗糖+0.6％琼脂)上预培养2d。将保存的农杆菌EHA105活化，取500ul活化菌液，接种到50ml YEB液体培养基中，28℃、220rpm振荡培养，至OD600值为0.5，4℃离心(4000r/min，10min)，弃上清液，重悬于20ml预冷的MS液体培养基，备用。用重悬菌液侵染预培养后的下胚轴10min，滤纸上干燥后转移至分化培养基上，共培养2d。将共培养后的下胚轴转移至筛选培养基(MS+2mg/L 6-BA+0.2mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行抗性筛选，每l0d更换一次筛选培养基。将多次筛选获得的抗性芽转移到生根培养基(MS+2mg/L 6-BA+0.4mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行生根培养。待组培苗根系强壮后，洗净根系上残留培养基，移栽到营养土中，置于人工气候室进行培养。遗传转化实验分别获得了22、25个原位替换MdICE1(DC)1、CbICE1(DC)2基因抗性植株。与野生型植株相比，其耐寒性分别提高了3.4℃和2.4℃。
实施例53.CRISPR/Cas9技术原位替换不同甲基化程度ScICE1基因茄子的获得
克隆茄子ScICE1基因cDNA，序列为SEQ ID NO：18，根据密码子的简并性特征，将ScICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换ScICE1基因中的不同程度的甲基化位点。获得全部替换后ScICE1基因序列为SEQ ID NO：59，命名为ScICE1(DC)1；部分替换后ScICE1基因序列为SEQ ID NO：100，命名为ScICE1(DC)2。将ScICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTACAGTTCGATGGGATTCGGGG-3’和5’-GGTTGTCCCAAAGATAACCAAGG-3’；ScICE1(DC)2sgRNA序列为5’-GTAATCTGACTGATAGAAAA AGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的ScICE1(DC)1、ScICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
遗传转化以白撮茄为受体，将种子用75％酒精浸泡30s，无菌水冲洗1次，然后用10％的NaC1O溶液浸泡20min，无菌水冲洗3次后，接种到1/2MS培养基上萌发，于26℃，光照/黑暗(16h/8h)条件下培养成无菌苗作为供试材料。由根癌农杆菌菌株EHA105、质粒载体pBI121， ScICE1基因构建成的超表达载体。选取无菌苗子叶，去除叶尖，切成0.5mm 2大小均匀的小块，接种于预培养培养基MS+NAA 0.2mg/L+ZT 1.2mg/L中，预培养2d后进行农杆菌侵染转化，取OD值为0.5的农杆菌菌液，侵染时间为10min。将侵染后的外植体接种于共培养培养基(1/2MS+NAA0.2mg/L+ZT 1.2mg/L+200umo1/L乙酰丁香酮)中，暗培养2d后，用含150mg/L特美汀的无菌水清洗外植体，无菌滤纸吸干表面水分，接种于MS+NAA 0.2mg/L+ZT1.2mg/L+150mg/L特美汀培养基中，恢复培养后置于诱导愈伤组织培养基中(添加0.lmg/L NAA，3.0mg/L ZT)。置于白天16h，夜间8h，16001x光照条件下培养。外植体生长两周左右，长出幼嫩、团状、坚硬的绿色愈伤组织，将其接种到生芽培养基(添加0.lmg/L NAA，4.0mg/L ZT，1.5mg/L 6-BA，12.0mg/L AgN0 3)中诱导生芽。芽伸长分化形成的幼苗长出2-3片真叶，将其从基部切断，接种到1/2MS生根培养基中诱导生根。光照时间16h/d，温度24-27℃，光照强度1600 1x。生长在培养基中的再生苗对环境适应能力差，应将三角瓶揭开小口通风，并根据幼苗生长情况逐步加大通风量，提高幼苗对环境适应能力。两周后，幼苗有4-5片真叶，三条主根，若干条侧根，揭开瓶口，同时注意保证幼苗水分充足，继续培养5d后即可移栽。遗传转化实验分别获得了13、10个原位替换ScICE1(DC)1、ScICE1(DC)2基因抗性植株。与野生型植株相比，其耐寒性分别提高了3.0℃和2.2℃。
实施例54.CRISPR/Cas9技术原位替换不同甲基化程度SlICE1基因番茄的获得
克隆番茄SlICE1基因cDNA，序列为SEQ ID NO：19，根据密码子的简并性特征，将SlICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换SlICE1基因中的不同程度的甲基化位点。获得全部替换后SlICE1基因序列为SEQ ID NO：60，命名为SlICE1(DC)1；部分替换后SlICE1基因序列为SEQ ID NO：101，命名为SlICE1(DC)2。将SlICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGTTGTTGTTAATAGCCTTTTGG-3’，5’-GTGGATGATACTGTTAAGAA TGG-3’和5’-GCTGTTATCAGCTGCTTCAATGG-3’；SlICE1(DC)2sgRNA序列为5’-GTTGTTGTTAATAGCCTTTTGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的SlICE1(DC)1、SlICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
选择在1/2MS基本培养基上、发芽率较高的番茄品种作为本实验的材料。当实生苗在1/2MS 基本培养基上，长到约5-6cm高时(约18天)，切取其无菌苗的子叶用作转化的受体材料。从甘油管中取少量含有目的表达载体的根癌农杆菌pBLGC菌液，于YEP固体培养基(添加5mg/L双丙氨磷，20mg/L Rif)上划线培养，28℃避光培养至长出直径约1mm大小的单菌落(约36-48h)。取单菌落转接于同样的固体培养基平板，培养至生长旺盛期(约36h)。从YEP平板上挑取农杆菌单菌落，接种到含5mg/L双丙氨磷，20mg/L Rif的YEP液体培养基中，混均，28℃，180-200rpm摇菌培养过夜(约16-18h)。次日以1％接种量接于20m1YEP液体培养基，28℃，180rpm振荡培养3-4小时，使农杆菌培养至对数期(OD600约为0.6)；然后4℃，4000rpm离心15min收集菌体，弃上清，用YEP液体培养基稀释原菌液的3-5倍，并加入200um乙酰丁香酮，28℃，180-200rpm摇菌1-2h。取完全展开的绿色子叶片，用无菌手术刀在子叶上划些伤口，在备好的农杆菌悬浮液中浸染10-20min左右，取出叶片，用无菌滤纸吸干，转入到固体分化培养基(Ms+6-BA 2mg/L+IAA 0.2mg/L)中，于黑暗、23-25℃条件下共培养2-3天(视菌体生长情况而定)。共培养三天后，将子叶片转入筛选培养基(Ms+6-BA 1mg/L+IAA 0.2mg/L+5mg/L双丙氨磷+500mg/L羧苄青霉素)上；每隔三天换一次培养基，直至没有农杆菌出现。14天后可观察到有的切口附近长出愈伤或丛生再生芽，这时将再生芽尽可能分成单株，继续在5mg/L双丙氨磷压力下进行筛选。大约4周后，转入含5mg/L双丙氨磷和500mg/L羧苄青霉素的分化培养基中筛选约1个月；对获得的抗性芽(丛)进一步筛选，筛选期间羧苄青霉素一直保持500mg/L。两个月左右，再生芽可长到2-3cm，这时可将存活的幼苗转入生根培养基(1/2MS，IBA lmg/L，蔗糖30g/L)分化培养1-2周。生根时则去除羧苄青霉素。最后将根系发育良好的植株移栽入盆土中。转基因实验分别获得了22、21株原位替换SlICE1(DC)1、SlICE1(DC)2基因抗性植株。与野生型植株相比，其耐寒性分别提高了4.1℃和2.4℃。
实施例55.CRISPR/Cas9技术原位替换不同甲基化程度PpICE1基因桃的获得
克隆桃PpICE1基因cDNA，序列为SEQ ID NO：20，根据密码子的简并性特征，将PpICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PpICE1基因中的不同程度的甲基化位点。获得全部替换后PpICE1基因序列为SEQ ID NO：61，命名为PpICE1(DC)1；部分替换后PpICE1基因序列为SEQ ID NO：102，命名为PpICE1(DC)2。将PpICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTCAAGATTGTTGAAAACCGAGG-3’，5’-GGCAAAGTTCTTCCTTGATACGG-3’和5’-GTACTGCTTTTATCTGATCCGGG-3’；PpICE1(DC)2sgRNA序列为5’-GTCAAGATTGTTGAAAACCGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子 选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的SlICE1(DC)1、SlICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
挑选果型正常，大小均一，无伤无虫果，先后用洗衣粉水搓洗，自来水冲洗，浸泡于0.2％升汞中10min，取出后用无菌水冲洗5次，无菌去皮，剥开果核，取出幼胚，接种在愈伤组织诱导培养基MS+2,4-D 0.5mg/L+BA 0.75mg/L+NAA1.0mg/L+2％蔗糖+7.5g/L琼脂上，pH 5.8，26℃暗中诱导愈伤组织。挑取包含此质粒的根癌农杆菌GV3101单菌落，接种到含5mg/L双丙氨磷、Rif 20mg/L、Gem 40mg/L的YEP 4ml液体培养基中，180rpm 28℃暗培养36h。取此菌液1ml加入50ml新鲜YEP液体培养基及200umol/L的乙酰丁香酮相同条件下培养12h，后取此菌液40ml于5000rpm 4℃离心10min，沉淀加入液体MS，180rpm 28℃继续振荡暗培养2h，至对数生长期，备用。预培养后的外植体直接浸入农杆菌悬浮液中浸泡，后取出用无菌滤纸吸去多余菌液，放入培养基(MS+2,4-D 0.5mg/L+BA 0.75mg/L+NAA 1.0mg/L+2％蔗搪+7.5g/L琼脂，pH5.8)，26℃暗中共培养。将诱导出的愈伤组织接入分化培养基(MS+NAA 0.05mg/L+BA 1.0mg/L)，光照强度2000-3000lx，光暗周期16h/8h下培养，培养温度同上。之后将不定芽移入生根培养基(1/2MS+IBA 1.0mg/L)上进行生根培养，5d后开始生根。桃转基因实验分别获得了11、9株原位替换PpICE1(DC)1、PpICE1(DC)2基因抗性植株。与野生桃植株相比，其耐寒性分别提高了4.0℃和2.1℃。
实施例56.CRISPR/Cas9技术原位替换不同甲基化程度ZmICE1基因玉米的获得
克隆玉米ZmICE1基因cDNA，序列为SEQ ID NO：21，根据密码子的简并性特征，将ZmICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换ZmICE1基因中的不同程度的甲基化位点。获得全部替换后ZmICE1基因序列为SEQ ID NO：62，命名为ZmICE1(DC)1；部分替换后ZmICE1基因序列为SEQ ID NO：103，命名为ZmICE1(DC)2。。将ZmICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGGGCTACCTGGGCTCCGACGGG-3’，5’-GAAGATCAGCAAGATGGATAGGG-3’和5’-GCTGAGTGCGCGGATGGTCCTGG-3’；ZmICE1(DC)2sgRNA序列为5’-GTGGCCGCGGCCGCGGAGGAGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的ZmICE1(DC)1、ZmICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、 AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
温室条件下对受体材料以7d为周期分期播种，玉米试材按试验需要进行控制授粉，授粉后10-13d，取玉米幼胚作为转化受体。用次氯酸钠消毒玉米棒后，剥出幼胚(剥幼胚时要谨慎，不要创伤胚)，用侵染液(加AS)清洗幼胚4遍，然后加入一定浓度的农杆菌菌液，放置10-30min，取出后用灭菌滤纸吸干，放到共培养培养基上，在25℃(黑暗)共培养2-5d，然后将幼胚转移到静息培养基，在28℃条件下暗培养7d。侵染和共培养环节菌液浓度OD500＝0.3-0.5，侵染时间10min、共培养时间3d。从静息培养基上把幼胚移入含有标记基因的抗性筛选培养基上，暗培养。每次继代注意淘汰呈褐色和水渍化的愈伤组织，并把生长正常的愈伤组织用镊子夹碎，分开选择培养。在继代筛选过程中注意经常观察发现污染或农杆菌复出的立即移出，未污染材料继续培养。另外，经常把出现褐化污染的组织块剔出或转移好的未被污染的组织块到新的相同培养基上。组织块膨大后，经常把大块组织剥小，连续继代筛选3次后，将选择到的抗性愈伤组织转到再生培养基上，恢复培养15d或更长时间(暗培养)。再将抗性愈伤组织转到再生培养基上发芽，培养条件为28℃，每日3000lx光强下，光照12h。待再生的玉米植株长到3片叶时，可将幼苗移植到含生根培养基的瓶中，在室内培养。当幼苗长出较粗的根后，将幼苗从罐头瓶中取出，用水冲净培养基，移栽于混有营养土和蛭石(1：3)的小花盆中，当玉米又长出2-3片新叶时，可将其移入大田或大花盆中，待长出三四叶后提取叶片DNA进行PCR检测。确定含有转入的ZmICE1基因。本实验分别获得了14、12株原位替换ZmICE1(DC)1、ZmICE1(DC)2基因抗性植株。6℃低温处理条件下，与野生植株相比，其耐寒性分别提高了3.3℃和1.8℃。
实施例57.CRISPR/Cas9技术原位替换不同甲基化程度GhICE1基因棉花的获得
克隆棉花GhICE1基因cDNA，序列为SEQ ID NO：22，根据密码子的简并性特征，将GhICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换GhICE1基因中的不同程度的甲基化位点。获得全部替换后GhICE1基因序列为SEQ ID NO：63，命名为GhICE1(DC)1；部分替换后GhICE1基因序列为SEQ ID NO：104，命名为GhICE1(DC)2。将GhICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTCAAGATGGTTGAAAACGGAGG-3’，5’-GCCCTTTCCTTTTACCCTTTTGG-3’和5’-GTAAACATCCACATGTTCTGTGG-3’；GhICE1(DC)2sgRNA序列为5’-GTCAAGATGGTTGAAAACGGAGG-3’，引导Cas9蛋白对DNA的定点切割。启 动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的GhICE1(DC)1、GhICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取构建好的植物表达载体农杆菌菌液，大量提取植物表达载体质粒DNA，选择次日将开放的花蕾进行自交。由于棉花是常异交植物，天然异交率在10％左右，因而外来花粉往往会造成品种间的混杂。在开花前一天，可见花冠快速伸长，黄色或乳白色的花冠呈指状突出于花蕾，次日即开放成为花朵。选择这样的花蕾，于指状花冠的前端用细线扎紧，并将细线的另一端系于铃柄，作为收获时的标记；在开花后20-24h左右即次日，选择果枝和花位较好的幼子房作为转化对象。一般选择每个果枝的第一和第二个果节位的花朵进行转基因操作。这些果节上的棉铃一般成铃率较高，有利于收获较多的种子；用50μl微量进样器作为微注射的工具。每次使用前和使用后，应以淡洗涤剂清洗，再用蒸馏水漂清；注射时，摘除或剥去花瓣，抹平花柱。在剥除花瓣时，注意不能损伤幼子房的表皮层，以免增加脱落率；用右手持微量注射器，左手轻扶摘除花瓣后的幼子房，从抹平花柱处沿子房的纵轴方向进针至子房长度的约三分之二处，并后退至约三分之一处。花粉管通道法转化得到的棉花种子收获后于在温室盆栽幼苗，在2-3叶期，以5mg/L双丙氨磷喷洒棉花叶片。一周后观察植株生长状况，拔除叶片表面有退绿斑点的植株。生长正常的被认为是获得抗性基因的植株。本实验分别获得了24、25株原位替换GhICE1(DC)1、GhICE1(DC)2基因抗性植株。4℃低温处理条件下，与野生型棉花植株相比，其耐寒性分别提高了4.6℃和2.8℃。
实施例58.CRISPR/Cas9技术原位替换不同甲基化程度AhICE1基因花生的获得
克隆花生AhICE1基因cDNA，序列为SEQ ID NO：23，根据密码子的简并性特征，将AhICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换AhICE1基因中的不同程度的甲基化位点。获得全部替换后AhICE1基因序列为SEQ ID NO：64，命名为AhICE1(DC)1；部分替换后AhICE1基因序列为SEQ ID NO：105，命名为AhICE1(DC)2。通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，AhICE1(DC)1和AhICE1(DC)2基因sgRNA序列均为5’-GCCGTCCAAAAATCTTATGGCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的GhICE1(DC)1、GhICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性 筛选基因，构建CRISPR/Cas9表达载体。
在花生进入盛花期时，进行转基因操作。在进入盛花期前，每天下午5点左右摘掉花蕾，以免后代有没经过转基因操作的花授粉结实，下季检测后代中的转基因植株时造成干扰。分别用微量移液器和滴管拉成的玻璃针，采用柱头涂抹和花曹管注射的方法导入含目的基因的质粒DNA，选用50ug/ml和100ug/ml两个DNA浓度处理“汕油523”和“汕油21”两个品种。进入盛花期后，每天早上6点半左右开始进行操作，涂抹操作用微量移液器吸取5ul DNA溶液涂于花朵柱头上；注射方法用玻璃针在花萼管的下部向子房方向注入约5ul的DNA溶液。每一个品种处理500朵以上。遗传转化得到的花生种子收获后于在温室盆栽幼苗，在2-3叶期，以5mg/L双丙氨磷)喷洒花生叶片。一周后观察植株生长状况，拔除叶片表面有退绿斑点的植株。生长正常的被认为是获得抗性基因的植株，抗性植株进行PCR验证。遗传转化分别获得了12、15株原位替换AhICE1(DC)1、AhICE1(DC)2基因抗性植株。与野生型花生植株相比，其耐寒性分别提高了2.0℃和1.3℃。
实施例59.CRISPR/Cas9技术原位替换不同甲基化程度BjICE1基因芥菜的获得
克隆芥菜BjICE1基因cDNA，序列为SEQ ID NO：24，根据密码子的简并性特征，将BjICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换BjICE1基因中的不同程度的甲基化位点。获得全部替换后BjICE1基因序列为SEQ ID NO：65，命名为BjICE1(DC)1；部分替换后BjICE1基因序列为SEQ ID NO：106，命名为BjICE1(DC)2。将BjICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTTCGGTTCGTTGACGCAGCTGG-3’和5’-GTTCAGAACGGAGGAGGTAAAGG-3’；BjICE1(DC)2sgRNA序列为5’-GCTCCGGTTTCGATGGGGTTCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的BjICE1(DC)1、BjICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将芥菜种子消毒(75％的酒精和0.1％升汞)后播种于MS培养基，7d后切取下胚轴(0.5-0.8cm)，置于芥菜分化培养基(MS+2mg/L 6-BA+0.2mg/L NAA+3％蔗糖+0.6％琼脂)上预培养2d。将已转入含BjICE1基因质粒的农杆菌活化，侵染时取500ul活化菌液，接种到50ml无抗生素的YEB液体培养基中，28℃摇床225rpm振荡培养至农杆菌生长活跃期(OD600值约为 0.5-0.6)，4℃离心(4000r/min，10min)后，弃上清液，重悬于20ml预冷的MS液体培养基，备用。用重悬菌液侵染预培养后的下胚轴10min，滤纸上干燥后转移至分化培养基上，共培养2d。将共培养后的下胚轴转移至芥菜筛选培养基(MS+2mg/L 6-BA+0.2mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行抗性筛选，每l0d更换一次筛选培养基.将多次筛选获得的抗性芽转移到芥菜生根培养基(MS+2mg/L 6-BA+0.4mg/L NAA+5mg/L双丙氨磷+400mg/L Cb+3％蔗糖+0.6％琼脂)上进行生根培养。待组培苗根系强壮后，洗净根系上残留培养基，移栽到营养土中，置于人工气候室(温度：25士2℃，光照强度：20001x，相对湿度70％，光照/黑暗＝16/8h)进行培养。遗传转化分别获得了24、21株原位替换BjICE1(DC)1、BjICE1(DC)2基因抗性植株。与野生型芥菜植株相比，其耐寒性分别提高了4.6℃和2.7℃。
实施例60.CRISPR/Cas9技术原位替换不同甲基化程度BrICE1基因油菜的获得
克隆油菜BrICE1基因cDNA，序列为SEQ ID NO：25，根据密码子的简并性特征，将BrICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换BrICE1基因中的不同程度的甲基化位点。获得全部替换后BrICE1基因序列为SEQ ID NO：66，命名为BrICE1(DC)1；部分替换后BrICE1基因序列为SEQ ID NO：107，命名为BrICE1(DC)2。将BrICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTTCGGTTCGTTGACGCAGCTGG-3’，5’-GACTCTTTCTTGCCGTGTCAAGG-3’和5’-GCCGTCATCAGCTGTTTCAACGG-3’；BrICE1(DC)2sgRNA序列为5’-GTCGAGGCTAAAAGCCTGAGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的BrICE1(DC)1、BrICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
以花粉介导法进行油菜转基因实验。进入油菜盛花期后，选取主茎或一次分枝上10-15个1-2d内将要开放的花蕾徒手去雄，并摘除所选茎或分枝上其他花蕾，然后套袋。同时在同一品种中选取第2天将开放的花序进行套袋，以备第2天取粉用。翌日上午当天开放的套袋植株的花粉约0.4g，悬浮在25ml 7.5％的蔗糖溶液中，进行第1次超声波处理，然后在溶液中加入20μg含不同耐冷性种群BrICE1基因的质粒DNA进行第2次超声波处理。第2次处理后，在溶液中加入10μl 1/10 000(W/V)硼酸，然后用处理过的花粉授在前一天去雄的油菜柱头上，套袋并挂牌，同时标注授粉花蕾数。授粉后5-6d去袋，使处理的授粉株充分发育。采收 种子。采收的种子种植后，至两片子叶伸展开时喷施5mg/L双丙氨磷溶液，光照培养7d后，未转基因植株叶边缘变黄，而转基因植株正常生长。遗传转化分别获得了33、37株原位替换BrICE1(DC)1、BrICE1(DC)2基因抗性植株。与野生型油菜植株相比，-10℃处理条件下，其耐寒性分别提高了4.4℃和2.7℃。
实施例61.CRISPR/Cas9技术原位替换不同甲基化程度CdICE1基因菊花的获得
克隆菊花CdICE1基因cDNA，序列为SEQ ID NO：26，根据密码子的简并性特征，将CdICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换CdICE1基因中的不同程度的甲基化位点。获得全部替换后CdICE1基因序列为SEQ ID NO：67，命名为CdICE1(DC)1；部分替换后CdICE1基因序列为SEQ ID NO：108，命名为CdICE1(DC)2。将CdICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGGTTGTTGGAAATGAAGCTAGG-3’，5’-GTTGGTGGTGTAGGCGTCAATGG-3’和5’-GCAGTGCTTCTAGAGACTGCAGG-3’；CdICE1(DC)2sgRNA序列为5’-GTAACTAGTCTCCGTATACTCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的CdICE1(DC)1、CdICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取保存的带有目的基因的pCAMBIA1301质粒的农杆菌，接种于含有50mg/L利福平的YEB液体培养基中过夜培养。然后，分别划线接种于含5mg/L双丙氨磷、50mg/L利福平的YEB平板培养基上，倒置于28℃恒温培养箱中，培养至单菌落长出后，将平板于4℃冰箱保存备用。在上述YEB平板培养基上分别挑取农杆菌单菌落，接种于添加有5mg/L双丙氨磷、50mg/L利福平的20ml的YEB液体培养基中，在28℃下，180rpm振荡培养至对数生长期。活化两次后按1:100的比例将菌液接种于只含有100pmol/L RS的YEB液体培养基中。振荡培养4-6h，当其OD600值达到0.4-0.6左右时，用于遗传转化。取培养的无菌菊花植株上端的叶片作为外植体，用灭过菌的剪刀将其剪成8mm*8mm左右的小块，放入胚性愈伤组织诱导的培养基(Ms+0.5mg/L 6-BA+1.5mg/L NAA+蔗糖30g/L+琼脂粉5g/L)中进行预培养。预培养3d后，将浸染用的菌液倒入无菌的三角瓶中，把预培养的叶片用无菌水冲洗1-2次，用无菌滤纸吸干，然后放入三角瓶中直接浸染25min。浸染后取出，用无菌滤纸吸除过量菌液。将浸染后的叶片接种于共培养的培养基上，在25±2℃条件下共培养2天。待叶片周围长出菌落，将叶片取出， 用无菌水冲洗2-3次，无歯滤纸吸干。转入胚性愈伤组织诱导的筛选培养基(添加300mg/L Cef)中进行培养，直到抗性体胚完全分化出来。筛选分化培养上培养15-20d后，小部分菊花叶片切口处开始分化。当培养基中的再生植株长到2cm左右时，用无菌的剪刀从菊花母体上剥离转移到不添加任何植物生长激素的MS基本培养基中进行生根，在25℃下，光照强度为1200-2400lx，16h/8h的光周期下进行培养。第6天开始根基部就开始萌发根。遗传转化分别获得了8、10株原位替换CdICE1(DC)1、CdICE1(DC)2基因抗性植株。与野生型菊花植株相比，其耐寒性分别提高了5.0℃和2.6℃。
实施例62.CRISPR/Cas9技术原位替换不同甲基化程度ThICE1基因小盐芥的获得
克隆小盐芥ThICE1基因cDNA，序列为SEQ ID NO：27，根据密码子的简并性特征，将ThICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换ThICE1基因中的不同程度的甲基化位点。获得全部替换后ThICE1基因序列为SEQ ID NO：68，命名为ThICE1(DC)1；部分替换后ThICE1基因序列为SEQ ID NO：109，命名为ThICE1(DC)2。将ThICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCTAGAAACGAAGGCTGAGA AGG-3’和5’-GGAGGAAGAAGCTTAATGAT AGG-3’；ThICE1(DC)2sgRNA序列为5’-GGGTTTGGGAGTCCTGCAAATGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的ThICE1(DC)1、ThICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将小盐芥种子置于试管中，用无菌水浸泡，放置4℃冰箱一个月。将小盐芥种子均匀点播在培养基质中(营养土：蛭石：珍珠岩＝4:3:1)。然后在光照16h/d，光照强度2000lx，温度25℃，相对湿，70％的培养室中放置一个半月。剪下叶柄，清水洗净后用70％乙醇消毒，2％(V/V)的次氯酸钠消毒10min，再用无菌水冲洗4次后作为外植体。将外植体接至预培养培养基MS+2.5mg/L 6-BA+0.1mg/L NAA(PH5.8)上，温度22℃，相对湿度70％，暗培养3d。从平板上挑取单菌落，接种到5ml附加5mg/L双丙氨磷的LB液体培养基中，28℃，180rpm，摇培至OD600为0.6。取OD600为0.6菌液，按1％比例转入液体培养基中，28℃，180rpm，OD600＝0.3即可用于转化。外植体浸入菌液中5min后置于无菌滤纸上吸去附着的菌液。将外植体接至共培养培养基MS+2.5mg/L 6-BA+0.1mg/L NAA(PH5.8)上，25℃暗培养。将共培养外植体转至MS+2.5 mg/L 6-BA+0.1mg/L NAA+5mg/L双丙氨磷300mg/L Cef(PH5.8)选择培养基上2周。选择培养4d后用无菌水每隔3d冲洗一次外植体，并换用新鲜选择培养基。选择培养2周后，将外植体转化细胞产生的抗性愈伤组织转至选择培养基上继代扩繁培养。待不定芽长到1cm时，切下，接至生根培养基1/2MS+5mg/L双丙氨磷300mg/LCef(PH5.8)上，15d后长出不定根。转基因实验分别获得了13、12株原位替换ThICE1(DC)1、ThICE1(DC)2基因抗性植株。与野生型小盐芥植株相比，其耐寒性分别提高了3.2℃和1.7℃。
实施例63.CRISPR/Cas9技术原位替换不同甲基化程度DcICE1基因野胡萝卜的获得
克隆野胡萝卜DcICE1基因cDNA，序列为SEQ ID NO：28，根据密码子的简并性特征，将DcICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换DcICE1基因中的不同程度的甲基化位点。获得全部替换后DcICE1基因序列为SEQ ID NO：69，命名为DcICE1(DC)1；部分替换后DcICE1基因序列为SEQ ID NO：110，命名为DcICE1(DC)2。将DcICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGTTCTCTAATAATTATCTTGGG-3’，5’-GATCGGAAGGGAAAGAAGAAGGG-3’和5’-GTCTCTTACTTTCCACCATGAGG-3’；DcICE1(DC)2sgRNA序列为5’-GGTTGGTGTCGGGTTTAACCGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的DcICE1(DC)1、DcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
挑取根癌农杆菌工程菌单菌落接种于50ml含5mg/L双丙氨磷的LB液体培养基中，28℃，黑暗条件下200rpm振荡培养至OD600约为0.6。在超净操作台上将根癌农杆菌培养液转入无菌的50ml离心管中，4℃，8000rpm离心5min，弃上清，用等体积的无菌MS液体培养基悬浮菌体，待用。在高压灭菌过的培养皿中放2-3张无菌滤纸，加适量B5液体培养基使之湿润。取无菌苗于滤纸上，用无菌、锋利的解剖刀把下胚轴切成0.5cm长的小段，用于遗传转化。于超净工作台上将处理好的野胡萝卜无菌苗的下胚轴放入制备好的浸染液中，置15-20min，中间轻轻振荡，以保证各下胚轴小段都能够充分浸染。取出外植体，于无菌滤纸上吸去多余的菌液。摆放于共培养培养基上，26℃、黑暗下培养2-3d。共培养后，取出外植体，放入无菌的空三角瓶中，用无菌水漂洗2-3次；用含500mg/L Carb的无菌水冲洗1-2遍，无菌滤纸吸干水分，转接到附加一定浓度抗生素的B5抑菌培养基上，于26℃、光照16h/d条 件下进行愈伤培养，继代2-3次；待长出愈伤组织后转入分化培养基上，诱导胚状体和芽。在诱导出的芽生长约40d后，苗长至2-3cm时切下，转入生根培养基上诱导生根；待长出正常根、长出3-4片真叶时，将抗性苗移出，用自来水冲去根部的培养基，栽于装有蛭石和草炭(2:1)的营养钵中，用塑料薄膜覆盖，给予26℃、光照16h/d的条件；7d后打开塑料布的通风口进行适当炼苗，待抗性苗逐渐适应外部生长环境条件后，既可在温室下进行常规管理。转基因实验分别获得了7、10株原位替换DcICE1(DC)1、DcICE1(DC)2基因抗性植株。与野生型野胡萝卜植株相比，其耐寒性分别提高了3.1℃和2.1℃。
实施例64.CRISPR/Cas9技术原位替换不同甲基化程度GmICE1基因大豆的获得
克隆大豆GmICE1基因cDNA，序列为SEQ ID NO：29，根据密码子的简并性特征，将GmICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换GmICE1基因中的不同程度的甲基化位点。获得全部替换后GmICE1基因序列为SEQ ID NO：70，命名为GmICE1(DC)1；部分替换后GmICE1基因序列为SEQ ID NO：111，命名为GmICE1(DC)2。将GmICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GAAAAGGGTGGGTTGGGCCCCGG-3’和5’-GTCTGTCATTGAGCTTCTTCCGG-3’；GmICE1(DC) 2sgRNA序列为5’-GAGGTGAGAGGGGGAGCAGGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的GmICE1(DC)1、GmICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将消毒后的大豆种子倒入到一个无菌塑料罐中，加入液体萌发培养基(LGM)(蔗糖20g/L，pH 5.8)，培养基体积大约是种子体积的2倍，以便完全覆盖种子及提供充足的水分。盖上盖子后在培养箱中放置16h，培养条件为全天23℃，黑暗处理。将40m1农杆菌重悬液倒入一个无菌培养皿中，检查播种大豆后的LGM、舍弃污染的。从种子胚方向，用解剖刀先斜切再沿中轴线将大豆种子剖开，去皮。在胚尖位置处平行于中轴轻划三刀，浸泡到装有重悬农杆菌液的培养皿中30min。侵染时间到后，用已灭菌的镊子将外植体转移到铺有无菌滤纸的铁盒中，外植体水平放置、切口向上。往铁盒中轻轻加入LCCM(Bs盐粉末0.321g/L，蔗糖30g/L，MES3.9g/L，pH 5.4，BAP 1.67mg/L，GA3 0.25mg/L，Cys 400mg/L，DTT 154.2mg/L，As 39.24mg/L)。封闭铁盒后于智能培养箱中放置3天，培养条件为全天23℃，黑暗处理。共培养3天后，于 超净工作台上用已灭菌的镊子将外植体取出，并用手术刀将子叶切除掉，但保留下胚轴。切好后将外植体放入芽诱导培养基(SIM)(BS盐粉末3.21g/L、蔗糖30g/L、MES 0.59g/L、Noble Agar 8g/L，BAP 1.67mg/L、Tic 250mg/L、Cef 100mg/L，PH 5.7)中，每皿放置7个外植体，外植体的顶端必须高于培养基平面，下胚轴部分则必须插入培养基中。将两个一次性塑料培养皿的底部重合，使用微孔通气型医用胶带进行密封。将新的培养皿放到人工气候室中恢复培养14天，培养条件为全天25℃，16/8昼夜比。将长出幼芽的外植体转到芽伸长培养基(SEM)(MS盐粉末4.43g/L、蔗糖30g/L、MES 0.59g/L、Noble Agar 8g/L、pH 5.7、Asp 50mg/L、Glu 50mg/L、IAA 0.lmg/L、GA3 0.5mg/L、ZR lmg/L、Tic 250mg/L、)中SIM中，舍弃没有诱导出丛生芽的外植体。仍然置于人工气候室中培养，培养条件为全天25℃、16/8昼夜比。一般每2周更换一次新鲜的SEM，每次更换培养基时都要丢弃死亡的外植体。进行5次SEM后就可以结束本批次的继代培养。在幼芽的伸长过程中，将芽从外植体上切下来，然后放置到培养皿中并用IBA(1mg/ml)浸泡其底部30s，之后将其转移到生根培养基(RM)(MS盐粉末4.43g/L、蔗糖20g/L、MES 0.59g/L、Phytagel 3g/L、pH 5.6、Asp 50mg/L、L-Glu 50mg/L，Tic 125mg/L，Cef 50mg/L，IBA 1mg/L)中继续培养。1-2周后，当苗的底部长出一定数最的根时，将其从RM培养基中取出。用自来水冲洗根部后再移植到土壤中。幼苗移栽到土壤中后需要先在人工气候室中生长一周左右以适应自然的环境，然后再转移到温室中培养。转基因实验分别获得了11、9株原位替换GmICE1(DC)1、GmICE1(DC)2基因抗性植株。与野生型大豆植株相比，其耐寒性分别提高了4.2℃和3.0℃。
实施例65.CRISPR/Cas9技术原位替换不同甲基化程度RsICE1基因萝卜的获得
克隆萝卜RsICE1基因cDNA，序列为SEQ ID NO：30，根据密码子的简并性特征，将RsICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换RsICE1基因中的不同程度的甲基化位点。获得全部替换后RsICE1基因序列为SEQ ID NO：71，命名为RsICE1(DC)1；部分替换后RsICE1基因序列为SEQ ID NO：112，命名为RsICE1(DC)2。将RsICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GATTTCAGATTCCTCGGTGGTGG-3’，5’-GGAGGAAGAAGCTTAATGATAGG-3’和5’-GCTGGCTACCATGAAAGCTTTGG-3’；RsICE1(DC)2sgRNA序列为5’-GATTTCAGATTCCTCGGTGGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的RsICE1(DC)1、RsICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、 AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
切取萝卜材料四天苗龄无菌苗带柄子叶外植体先进行2天预培养，用OD600为0.3-0.6的EHA105农杆菌菌液侵染5-7min后，再进行5天共培养，然后将外植体转接到MS+6-BA 6mg·L -1+NAA 0.05mg·L -1+Cef 500mg·L -1的培养基上进行7天延迟筛选，再将外植体转接到MS+6-BA 6mg·L -1+NAA 0.05mg·L -1+5mg·L -1双丙氨磷Cef 500mg·L -1的培养基上进行10天的抗性筛选，每10天进行一次转接继代培养。利用农杆菌介导的遗传转化方法，分别获得了6、5株原位替换RsICE1(DC)1、RsICE1(DC)2基因抗性植株。与野生型萝卜植株相比，其耐寒性分别提高了4.5℃和2.4℃。
实施例66.CRISPR/Cas9技术原位替换不同甲基化程度TaICE1基因小麦的获得
克隆小麦TaICE1基因cDNA，序列为SEQ ID NO：31，根据密码子的简并性特征，将TaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换TaICE1基因中的不同程度的甲基化位点。获得全部替换后TaICE1基因序列为SEQ ID NO：72，命名为TaICE1(DC)1；部分替换后TaICE1基因序列为SEQ ID NO：113，命名为TaICE1(DC)2。将TaICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCGTGGACGCCTCCTCCTTGGGG-3’，5’-GCTGCAGGGTGGGCGTCGACGGG-3’和5’-GCCGTCATCAGCTGCTTCGATGG-3’；TaICE1(DC)2sgRNA序列为5’-GTAGGCCGGGGGCGCGGCGGCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的TaICE1(DC)1、TaICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
采集扬花后12-14d的麦穗，取小麦幼穗中部大小一致的未成熟子粒，将幼胚接种在愈伤组织诱导培养基表面，25℃下暗培养，20d后转入相同培养基进行继代培养。将幼胚接种在MC培养基上，培养15-20d，待幼胚形成的愈伤组织发育完全后，放人4℃冰箱，冷处理2个月左右，然后在常温下继代培养。8月上中旬将愈伤组织转入MC+乙酰丁香酮(AS)0.1mmol/L培养基中，培养5-7d开始转化。将幼胚愈伤组织转入工程菌感染液中0.5-1.0h，然后转移到MD培养基+AS 0.1mmol/L，共培养2d，用含有0.5g/L头孢唑林钠的无菌水冲洗愈伤组织，然后用无菌卫生纸吸干多余的液体，将愈伤组织转MD+500mg/L Carb+5mg/L双丙氨磷培养 基进行筛选，筛选出的抗性愈伤组织转入ME分化培养基+0.5g/L头孢唑林钠+5mg/L双丙氨磷。在5mg/L双丙氨磷下通过6-8周的选择培养后，转入再生培养基MD+IAA 1mg/L+ZT 1mg/L，再生成苗。利用农杆菌介导的遗传转化方法，分别获得了23、25株原位替换TaICE1(DC)1、TaICE1(DC)2基因抗性植株。与野生型小麦植株相比，-10℃处理条件下，其耐寒性分别提高了4.8℃和3.3℃。
实施例67.CRISPR/Cas9技术原位替换不同甲基化程度HvICE1基因大麦的获得
克隆大麦HvICE1基因cDNA，序列为SEQ ID NO：32，根据密码子的简并性特征，将HvICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换HvICE1基因中的不同程度的甲基化位点。获得全部替换后HvICE1基因序列为SEQ ID NO：73，命名为HvICE1(DC)1；部分替换后HvICE1基因序列为SEQ ID NO：114，命名为HvICE1(DC)2。通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，HvICE1(DC)1和HvICE1(DC)2基因sgRNA序列均为5’-GATGCCGGCCAAGAACCTGATGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的HvICE1(DC)1、HvICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
检查大麦幼穗未成熟胚的大小，幼胚大小保证为直径1.5mm-2mm之间，一般是在授粉大约14天左右。完整的将未成熟的种子和麦穗分离，将种子置于锥形瓶中，并放置于超净台内，以下步骤均在无菌超净台内进行。70％酒精简单冲洗30s，用无菌水清洗三次，再用50％次氯酸钠处理4min，无菌水冲洗3-4次，尽量减少次氯酸钠的残留。用摄子夹住种子下端约1/3处，使未成熟胚朝上。用超细尖头摄子从种子中部刺透，并掀起去除部分种皮，露出未成熟胚。用镊子尽量去除胚轴，同时保持盾片的完整性，分离未成熟胚，并置于BCI愈伤诱导培养基上，小盾片这边朝上，约25个/培养皿。用200ul的枪头吸取待接种菌液，滴在未成熟胚上，还可以用刀片或者接种环蘸取菌液，湿润胚的表面。若未成熟胚上的菌液太多，需要在培养基表面来回拖动未成熟胚，去除其表面过量的菌液，若只是湿润，直接把胚转移到新鲜的BCI培养基上，小盾片朝下切面朝上。将培养皿封口，24℃条件下，黑暗培养72h。若有农杆菌过量生长的未成熟胚，需要将其放入小烧杯，用无菌水和含有160mg/L Timentin抗生素的无菌水冲洗数次，并用滤纸将表面液体吸干。将未成熟胚转移至见BCS愈伤筛选培养基上(含有5mg/L双丙氨磷和160mg/L Timentin)盾片朝下，24℃条件下，黑暗培养6-8周。每隔2周更换新 鲜培养基，转移愈伤的时候注意尽量不要将未成熟胚分化成的愈伤分开。愈伤在选择培养基上长到一定大小之后，转移到BCT过渡培养基(含有5mg/L双丙氨磷和160mg/L Timentin)。24℃条件下，低光照培养，光照条件可以在培养皿上面盖一张纸来达到。两周的过渡培养之后，愈伤会出现再生的迹象，如果没有出现，每隔两周更换新鲜培养基。愈伤上出现明显的再生迹象之后，转移至BCR愈伤再生培养基(含有5mg/L双丙氨磷和160mg/L Timentin)。24℃条件下，全光照培养，每隔两周更换新鲜培养基。选择植物培养瓶代替玻璃培养皿，增加植株再生的空间。愈伤在选择培养基上长到一定大小之后，转移到B13M培养，24℃条件下，低光照培养。每两周更换一次培养基，当再生的茎芽长大后转移至试管中培养。在茎芽发育到2-3cm，将幼苗小心地转移到长15cm的玻璃培养，透气滤菌塑料膜封口，试管中的培养基为生根培养基。遗传转化实验分别获得了9、7株原位替换HvICE1(DC)1、HvICE1(DC)2基因抗性植株。与野生型大麦植株相比，-10℃处理条件下，其耐寒性分别提高了2.4℃和1.7℃。
实施例68.CRISPR/Cas9技术原位替换不同甲基化程度HbICE1基因橡胶树的获得
克隆橡胶树HbICE1基因cDNA，序列为SEQ ID NO：33，根据密码子的简并性特征，将HbICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换HbICE1基因中的不同程度的甲基化位点。获得全部替换后HbICE1基因序列为SEQ ID NO：74，命名为HbICE1(DC)1；部分替换后HbICE1基因序列为SEQ ID NO：115，命名为HbICE1(DC)2。将HbICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GAATCAGGCTTCAAATTTGATGG-3’，5’-GGATGAATACTTGTAGATTGAGG-3’和5’-GTTTATTGCTCTCCACTATGAGG-3’；HbICE1(DC)2sgRNA序列为5’-GAATCAGGCTTCAAATTTGATGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的HbICE1(DC)1、HbICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将橡胶树单核靠边期花药接种于胚性愈伤组织诱导培养基上，培养30d后用于农杆菌侵染。将愈伤组织在EHA105悬浮液中侵染5min后，转入添加5uM乙酰丁香酮和10m g/L硝酸银共培养基上培养，22℃暗培养2d。每个处理设置3次重复。共培养后的愈伤组织视其长菌情况用无菌水洗涤后转入50mg/L特泯丁浸泡lmin，置无菌滤纸吸干后转移到添加10mg/L硝酸银和500mg/L特泯丁的愈伤诱导培养基上进行恢复培养，25℃暗培养7d。恢复生长的愈伤 组织转入添加10mg/L硝酸银、5mg/L双丙氨磷和50mg/L特泯丁的愈伤诱导培养基诱导体细胞胚胎发生。最后，将增殖后体细胞胚转入添加5mg/L双丙氨磷的植株再生培养基诱导植株再生。遗传转化实验分别获得了6、9株原位替换HbICE1(DC)1、HbICE1(DC)2基因抗性植株。与野生型橡胶树相比，其耐寒性分别提高了2.9℃和1.8℃。
实施例69.CRISPR/Cas9技术原位替换不同甲基化程度JrICE1基因胡桃的获得
克隆胡桃JrICE1基因cDNA，序列为SEQ ID NO：34，根据密码子的简并性特征，将JrICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换JrICE1基因中的不同程度的甲基化位点。获得全部替换后JrICE1基因序列为SEQ ID NO：75，命名为JrICE1(DC)1；部分替换后JrICE1基因序列为SEQ ID NO：116，命名为JrICE1(DC)2。将JrICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCTAACACCAGCAACAGTTGCGG-3’，5’-GGAATGCCCTGTTTATGAACAGG-3’和5’-GTTCATGGAGAACGATAAGGCGG-3’；JrICE1(DC)2sgRNA序列为5’-GAGGACAAAGTGGGCTTGGGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的JrICE1(DC)1、JrICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取-80℃冻存的携带有转化质粒的农杆菌在含有5mg/L双丙氨磷的固体YEB平板培养基上划线，28℃培养过夜。挑取平板上的单菌落，接种到含有5mg/L双丙氨磷的液体YEB培养基中，置于28℃恒温摇床振荡(200rpm)过夜。用紫外可见光分光光度计测定其在600nm波长下的吸光值，待OD600＝0.5-0.8时，倒入30ml的离心管中，常温下6000rpm离心5-6min，去上清液，加入液体DKW培养基至30ml，涡旋混合5min，用作侵染备用。取预培养良好的胡桃茎段与上述得到的农杆菌菌液混合，侵染时间为30min，将侵染后的茎段用无菌滤纸吸干，接种到共培养培养基中，共培养基组合为DKW+6-BA0.5mg/L+IBA0.1mg/L+AS(乙酰丁香酮)200μmol/L+蔗糖30g/L+琼脂6.0mg/L，高压灭菌前pH调至5.8，共培养时间设置3天，黑暗处理。培养条件为培养温度25±1℃。共培养3天后，转接到筛选培养基中，筛选培养基组合为DKW+6-BA0.5mg/L+IBA0.1mg/L+5mg/L双丙氨磷+Cef 500mol/L+蔗糖30g/L+琼脂6.0mg/L，5周后不定芽萌发。培养条件为培养温度25±1℃，光照时间16h/d，光照强度为2000-2500lx。遗传转化实验分别获得了12、10株原位替换JrICE1(DC)1、JrICE1(DC)2基因抗性植株。与野 生型胡桃植株相比，其耐寒性分别提高了3.4℃和2.1℃。
实施例70.CRISPR/Cas9技术原位替换不同甲基化程度LsICE1基因莴苣的获得
克隆莴苣LsICE1基因cDNA，序列为SEQ ID NO：35，根据密码子的简并性特征，将LsICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换LsICE1基因中的不同程度的甲基化位点。获得全部替换后LsICE1基因序列为SEQ ID NO：76，命名为LsICE1(DC)1；部分替换后LsICE1基因序列为SEQ ID NO：117，命名为LsICE1(DC)2。将LsICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGGAATTGGGAGGTACAAATTGG-3’，5’-GTTGGGCTAGGGATCGCGGTTGG-3’和5’-GCCTCCTACTGCAAAACATATGG-3’；LsICE1(DC)2sgRNA序列为5’-GGAGAAAGAAGCTAAACGACCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的LsICE1(DC)1、LsICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
在含有Kam和Rif的LB平板上挑取携带有不同甲基化程度LsICE1基因的农杆菌单菌落，接种于添加5mg/L双丙氨磷的LB液体培养基中，28℃，150rpm摇床过夜培养养至对数中期(OD600＝0.4-0.8)。取10ml菌液分装于已灭菌的10ml离心管中，3000rpm离心3min，去上清液，用灭菌去离子水悬浮，稀释至OD600值为0.5左右作为侵染使用。经农杆菌侵染的莴苣子叶外植体，与农杆菌一起在分化培养基(MS+6-BA 0.5mg/L+NAA 0.20mg/L)上共培养后，转入到筛选培养基(Sm8+5mg/L双丙氨磷Cef 200mg/L)上进行抗性筛选，每周继代一次。经过一周培养后，子叶的基部能逐渐长出少量致密的愈伤组织并慢慢转为绿色，大约一周后便能形成小芽点，小芽点逐渐分化出幼叶，形成小芽。一个愈伤组织上一般能形成多个小芽。当芽长至2-3cm时切下，插入到生根培养基(1/2Ms+NAA 0.05mg/L+Cef 200mg/L)中诱导生根，2-3周后移栽到育苗钵。通过这种方法分别获得了28、24株原位替换LsICE1(DC)1、LsICE1(DC)2基因抗性植株。与野生型莴苣植株相比，其耐寒性分别提高了3.2℃和1.5℃。
实施例71.CRISPR/Cas9技术原位替换不同甲基化程度OsICE1基因水稻的获得
克隆水稻OsICE1基因cDNA，序列为SEQ ID NO：36，根据密码子的简并性特征，将OsICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换OsICE1基因中的不同程度的甲基化位点。获得全部替换后OsICE1基因序列为SEQ ID NO：77，命名为OsICE1(DC)1； 部分替换后OsICE1基因序列为SEQ ID NO：118，命名为OsICE1(DC)2。将OsICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTGGTGGTGGTGGCGCGCATGGG-3’，5’-GGCGGTCGTTGAGCTTCTTCCGG-3’和5’-GCCCCACTGGACAACAGCCAAGG-3’；OsICE1(DC)2sgRNA序列为5’-GCGTCCCAAATGCCGGAGTTCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的OsICE1(DC)1、OsICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
选取日本晴水稻成熟种子(去壳)用升汞法(0.1％升汞浸泡15min，灭菌水清洗干净)消毒后，置于超净台上晾干，然后将胚放置于愈伤诱导培养基(4.4g/L MS+2.5mg/L 2,4-D+600mg/L干酪素+30g/L蔗糖+5g/L植物凝胶，KOH调至pH5.8，高压灭菌)上进行愈伤培养，28℃培养2周。将携带目的基因质粒的农杆菌GV3101接种到5ml含5mg/L双丙氨磷的YEB液体培养基中，28℃摇菌培养至对数生长期晚期，再以1:100体积扩大培养，在OD600为0.10时收集农杆菌菌体并重悬于侵染培养基中。按常规方法侵染水稻的愈伤组织，经共培养侵染后，置于含5mg/L双丙氨磷的传代培养基上，28℃暗培养12-16d后在再分化培养基上分化得到双丙氨磷抗性的阳性苗。转基因实验分别获得了17、14株原位替换OsICE1(DC)1、OsICE1(DC)2基因抗性植株。5℃冷处理条件下，与野生型日本晴植株相比，其耐寒性分别提高了3.1℃和2.0℃。
实施例72.CRISPR/Cas9技术原位替换不同甲基化程度PaICE1基因蝴蝶兰的获得
克隆蝴蝶兰PaICE1基因cDNA，序列为SEQ ID NO：37，根据密码子的简并性特征，将PaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PaICE1基因中的不同程度的甲基化位点。获得全部替换后PaICE1基因序列为SEQ ID NO：78，命名为PaICE1(DC)1；部分替换后PaICE1基因序列为SEQ ID NO：119，命名为PaICE1(DC)2。将PaICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTTTGTGGGGGAAGAAGGACTGG-3’，5’-GGGATGGTGAGTTTGAGAAGCGG-3’和5’-GAATAGTAGTCTTACCGGAGGGG-3’；PaICE1(DC)2sgRNA序列为5’-GCCGGGAGCTCCTTAAACCAGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的 PaICE1(DC)1、PaICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将蝴蝶兰原球茎在预培养液体培养基(添加6-BA1.0mg/L+NAA5.0mg/L+2,4-D 2.0mg/L+AS 100μmol/L)中，预培养3d。用于转化的农杆菌EHA105在含有5mg/L双丙氨磷和乙酰丁香酮100μmol/L的LB(p H7.0)培养基中28℃、150r/min震荡培养24h，在同上LB培养基中加入20％(v/v)上述菌液，活化5h，测O.D600≈0.60；6000r/min离心3min，用灭菌液体培养基(加AS 100μmol/L)重悬并稀释至原体积，直接用于对原球茎的遗传转化。侵染15min，原球茎接种于固体培养基(加6-BA1.0mg/L+NAA 5.0mg/L+2,4-D 2.0mg/L+AS100μmol/L)上共培养3d。然后用含有5.0mg/L美罗培南的液体培养基冲洗原球茎3次，分别在添加美罗培南5.0mg/L和噻孢霉素100.0mg/L的固体培养基(添加KT 3.0mg/L)上交替培养，3d转接一次，至完全去除农杆菌。脱菌的原球茎转至含有5mg/L双丙氨磷和KT 3.0mg/L的固体培养基上选择培养，每2周后转至同样培养基上继代培养。45d后将原球茎在含5mg/L双丙氨磷的类原球茎状体(PLBs)诱导培养基(添加TDZ和2,4-D各1.0mg/L)上培养，切面上长出新的PLBs并分化成苗。将植株转至增殖培养基(6-BA 5.0mg/L)中培养。丛生小苗高达到3-5cm时，切开移到生根培养基(Hyponex No.1 30.0g/L+NAA0.3mg/L)，当每株长出4-5条新根，长达2-4cm时，出瓶栽植。转基因实验分别获得了19、20株原位替换PaICE1(DC)1、PaICE1(DC)2基因抗性植株。与野生型蝴蝶兰植株相比，其耐寒性分别提高了3.6℃和2.1℃。
实施例73.CRISPR/Cas9技术原位替换不同甲基化程度PmICE1基因腊梅的获得
克隆腊梅PmICE1基因cDNA，序列为SEQ ID NO：38，根据密码子的简并性特征，将PmICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PmICE1基因中的不同程度的甲基化位点。获得全部替换后PmICE1基因序列为SEQ ID NO：79，命名为PmICE1(DC)1；部分替换后PmICE1基因序列为SEQ ID NO：120，命名为PmICE1(DC)2。通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，PmICE1(DC)1和PmICE1(DC)2基因sgRNA序列均为5’-GCGTCGAGTTCCCAACGACTCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PmICE1(DC)1、PmICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性 筛选基因，构建CRISPR/Cas9表达载体。
将保存于-80℃冰箱的农杆菌EHA105接种到含有5mg/L双丙氨磷的YEB平板培养基上，暗培养2-3d。待菌落长出后，挑取单菌落接种于含5mg/L双丙氨磷的30ml YEB液体培养基中进行第一次活化，在28℃下于摇床上振荡培养1-2d，到OD600值约为0.8-1.0时取下备用。将第一次活化的50u1菌液接种于不含双丙氨磷的50m1 YEB液体培养基中进行第二次活化，在28℃下于普通摇床振荡培养至OD600值为0.4。本实验采用腊梅半展开的子叶为外植体，将子叶切成1cm*1cm大小的方块进行预培养3d。将活化后的农杆菌EHA105菌液在4℃，6000rpm下离心后收集菌体，加入液体MS+AS100umol/L重悬到OD600值为0.4，侵染愈伤组织10min，共培养2d后，用液体MS+Cef 1000umol/L脱菌，在无菌水冲洗5次后，放于筛选培养基Ms+BA1.0mg/L+NAA0.5mg/L+KT0.5mg/L+IBA2.0mg/L+2,4-D0.2mg/L+5mg/L双丙氨磷+Cef1000umol/L中进行选择培养。再生植株转移至MS+6-BA1.0mg/L+NAA0.1mg/L上，当苗生长到3-4cm高时即转入1/2MS+NAA0.1mg/L中进行生根培养。转基因实验分别获得了11、13株原位替换PmICE1(DC)1、PmICE1(DC)2基因抗性植株。与野生型腊梅相比，其耐寒性分别提高了2.2℃和1.5℃。
实施例74.CRISPR/Cas9技术原位替换不同甲基化程度CsICE1基因茶的获得
克隆茶CsICE1基因cDNA，序列为SEQ ID NO：39，根据密码子的简并性特征，将CsICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换CsICE1基因中的不同程度的甲基化位点。获得全部替换后CsICE1基因序列为SEQ ID NO：80，命名为CsICE1(DC)1；部分替换后CsICE1基因序列为SEQ ID NO：121，命名为CsICE1(DC)2。将CsICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCAATGTACAAGGGTATAGTGGG-3’，5’-GTTTATGGAGAACAATAAGGTGG-3’和5’-GCGGTACTCTTGGATTCAGCTGG-3’；CsICE1(DC)2sgRNA序列为5’-GGGATTGAGGACGGAAGAAATGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的CsICE1(DC)1、CsICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
选取8-9月份未成熟茶果，经蒸馏水清洗，剥去果皮，取出尚未成熟的乳白或略带棕色的种子，剥开外种皮，在无菌条件下，75％乙醇表面消毒30s后，用0.1％HgCl 2浸泡3min，再用 无菌水漂洗6-8次。将子叶切成长度为0.5cm大小的方块，接种到固体培养基改良ER+0.1mg/L+1.0mg/L6-BA+0.1g/L氯霉素+30g/L蔗糖上。接种后在温度为25±2℃的恒温培养室内培养，人工光照13h/d，光照强度1500-2000lx。培养25d后选择转绿的子叶愈伤组织作为外植体。取保存的根癌农杆菌EHA105在已制备好的YEB固体培养基平板上画线，并在28℃条件下暗培养1-2d后，用接种环挑取单菌落接种于5ml YEB液体培养基中，于28℃、220rpm振荡培养24h后，在无菌条件下吸取1ml菌液到含200uml/L乙酰丁香酮的液体培养基中，于28℃、220rpm振荡培养4-6h后，在600nm处测得菌液OD值为0.6-0.8左右即可用于外植体浸染。在超净工作台上选择已经转绿的子叶愈伤组织作为外植体接种于预培养基改良ER+0.2mg/LIBA+30g/L蔗糖上，25℃暗条件下预培养3d。将预培养后的子叶愈伤组织放入备用的根癌农杆菌菌液中，菌液浸没实验材料，侵染15min后，用无菌滤纸吸干表面多余的菌液，接种到添加150umol/L AS的YEB共培养培养基上，25℃条件下黑暗培养3d。共培养后，用无菌水冲洗材料3-4次，无菌滤纸吸干材料表面水分，将愈伤组织转接到预培养基中，延后选择培养3d，之后转接到含附加100umol/LCef和100umol/LSpe的除菌培养基上黑暗培养。每3d转接一次，15d以后外植体转至附加100umol/LSpe的筛选培养基中进行抗性芽筛选。筛选培养20d后，将有活力的抗性芽转移至新的附加100umol/LSpe分化培养基中培养。在分化培养基中培养30d，子叶愈伤组织分化出幼苗，转入生根培养基培养2周后得到完整的抗性植株。遗传转化分别获得了6、7株原位替换CsICE1(DC)1、CsICE1(DC)2基因抗性植株。与野生型植株相比，其耐寒性分别提高了3.3℃和2.0℃。
实施例75.CRISPR/Cas9技术原位替换不同甲基化程度AtICE1基因拟南芥的获得
克隆拟南芥AtICE1基因cDNA，序列为SEQ ID NO：40，根据密码子的简并性特征，将AtICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换AtICE1基因中的不同程度的甲基化位点。获得全部替换后AtICE1基因序列为SEQ ID NO：81，命名为AtICE1(DC)1；部分替换后AtICE1基因序列为SEQ ID NO：122，命名为AtICE1(DC)2。将AtICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GCTCCTATTTCGATGGGGTTTGG-3’，5’-GATAAATGAGAGCGGTAAAGCGG-3’和5’-GCAGTGCTTTTCGATACAGCAGG-3’；AtICE1(DC)2sgRNA序列为5’-GAAGATCTTGTGGATGTGGTTGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的AtICE1(DC)1、AtICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、 AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取保存的根癌农杆菌GV3101在已制备好的YEB固体培养基平板上画线，并在28℃条件下暗培养1-2d后，用接种环挑取单菌落接种于5ml YEB液体培养基中，于28℃、220rpm振荡培养24h后，在无菌条件下吸取1ml菌液到YEB液体培养基中，于28℃、220rpm振荡培养4-6h后，在600nm处测得菌液OD值为0.6-0.8左右即可用于拟南芥侵染。将盛花期拟南芥的花表面部分浸泡在农杆菌悬浮液(OD＝0.8)中5分钟，同时轻轻旋转。将浸染后植株套袋保持高度的湿润状态，暗室培养24小时。种子成熟，角果自然开裂后可以收种子。收集到的拟南芥种子于4℃春化2天，种子消毒后在含有5mg/L双丙氨磷的平板上进行培养。成功转入重组质粒的种子能够在抗性培养及上正常生长出4片以上真叶。非转基因种子不能正常生长，仅能长出2片子叶，根的生长也受到严重抑制，一般萌发10天以后死亡。将筛选到的植株移植到蛭石：泥炭土：珍珠岩为9:3:1的基质中，覆盖薄膜2-3天。遗传转化分别获得了26、27株原位替换AtICE1(DC)1、AtICE1(DC)2基因抗性植株。与野生型拟南芥植株Col-0相比，其耐寒性分别提高了2.4℃和1.5℃。
实施例76.CRISPR/Cas9技术原位替换不同甲基化程度BrrICE1基因芜菁的获得
克隆芜菁BrrICE1基因cDNA，序列为SEQ ID NO：41，根据密码子的简并性特征，将BrrICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换BrrICE1基因中的不同程度的甲基化位点。获得全部替换后BrrICE1基因序列为SEQ ID NO：82，命名为BrrICE1(DC)1；部分替换后BrrICE1基因序列为SEQ ID NO：123，命名为BrrICE1(DC)2。将BrrICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTCGAGGCTAAAAGCCTGAGAGG-3’，5’-GTTCAGAACGGAGGAGGTAAAGG-3’和5’-GCCAGAGGGAGAGATCCATTTGG-3’；BrrICE1(DC)2sgRNA序列为5’-GCAACACCCTTATGCGGAGGTGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的BrrICE1(DC)1、BrrICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
芜菁种子用1％次氯酸钠消毒20min，然后用无菌水洗涤4-5次。然后将种子放在出芽培养基(含20g/L蔗糖的M S培养基，PH 5.8)上，24℃，16h/8h光照，光照强度60-80uEm -2s -1培 养5天。取保存的根癌农杆菌EHA101在已制备好的YEB固体培养基平板上画线，并在28℃条件下暗培养1-2d后，用接种环挑取单菌落接种于5ml YEB液体培养基中，于28℃、220rpm振荡培养24h后，在无菌条件下吸取1ml菌液到YEB液体培养基中，于28℃、220rpm振荡培养4-6h后，在600nm处测得菌液OD值为2.0左右即可用于外植体浸染。农杆菌接种用注射器将细菌悬浮液(0.5-1.0ul)注射到子叶柄根部，在M S培养基上共培养3d。然后转移至愈伤组织诱导培养基(Ms+20uM 6-BA+3％蔗糖+500mg/L羧苄霉素+5mg/L双丙氨磷，PH 5.8)上培养2-3周，培养条件同上。以这种方式培养，芜菁子叶柄的切面在一段短时间内由曝露的维管束组织发育形成愈伤组织，4周后由此愈伤组织形成茎分生组织，转移至茎伸长培养基上(Ms+3％蔗糖+500mg/L羧苄霉素+5mg/L双丙氨磷，PH 5.8)，1-2周后转移至生根培养基上(Ms+3％蔗糖+2mg/L IAA+500mg/L羧苄霉素，PH 5.8)培养。2-3周后转移至温室培养。遗传转化分别获得了14、17株原位替换BrrICE1(DC)1、BrrICE1(DC)2基因抗性植株。与野生型芜菁植株相比，其耐寒性分别提高了3.8℃和2.1℃。
实施例77.CRISPR/Cas9技术原位替换不同甲基化程度ItICE1基因菘蓝的获得
克隆菘蓝ItICE1基因cDNA，序列为SEQ ID NO：42，根据密码子的简并性特征，将ItICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换ItICE1基因中的不同程度的甲基化位点。获得全部替换后ItICE1基因序列为SEQ ID NO：83，命名为ItICE1(DC)1；部分替换后ItICE1基因序列为SEQ ID NO：124，命名为ItICE1(DC)2。将ItICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GAGCCGAACCCCATGGAGATCGG-3’，5’-GCTGAGCGATGATGGAGAGATGG-3’和5’-GCTGGCTACCATGAAAGCTTTGG-3’；ItICE1(DC)2sgRNA序列为5’-GATGGACGAGACGGGGATTGAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的ItICE1(DC)1、ItICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
将菘蓝菜种子用70％酒精浸泡消毒45s-lmin后，用0.1％HgCI 2浸泡消毒15min，用无菌水清洗3-5次，接种于MS基本培养基上，25℃培养12-15d，光照强度为2000lx，光周期为16h/d。从活化后保存的含有目的表达载体的农杆菌平板上挑取单菌落接种于25m1 LB液体培养基中(含有5mg/L双丙氨磷)，28℃避光振荡培养至OD 600＝0.1-0.3，4000r/min离心l0min 收集菌体，去上清，将菌体重悬于相同体积的MS液体培养基中，并加入100umol/L乙酰丁香酮，用于外植体的侵染。将无菌种子萌发长至4-5cm的幼苗取出，切取菘蓝子叶柄(去掉生长点)为外植体，子叶叶柄朝下插入预培养基(MS基本培养基+3rng/L 6-BA+0.2mg/L NAA+2mg/L ZT，PH 5.8)中，25士1℃光照下预培养ld。将子叶柄浸入上述制备的农杆菌感染菌液中，100rpm摇床振荡l 0min后，弃去菌液取出子叶柄用无菌滤纸吸干，插入共培养基(MS基本培养基+3mg/L 6-BA+0.2mg/L NAA+2mg/L ZT+100umol/L乙酰丁香酮，PH 5.8)中，28℃暗培养2-3d。至共培养基中长出可见农杆菌菌落时，取出子叶柄进行除菌，先用无菌水清洗一遍，然后用含Cef 500mg/L的无菌水振荡浸泡20min，再用无菌水清洗3-5遍后，置于灭菌的滤纸上充分吸干残留水份。将子叶柄转到分化培养基(MS基本培养基+3mg/L 6-BA+0.2mg/L NAA+2mg/L ZT+0.5g/L AgN0 3+5mg/L双丙氨磷+200mg/L Cef+300mg/L Carb，PH 5.8)上进行分化筛选培养(25℃，光照16h/d)，25天后开始有芽的分化。将分化培养中分化出的菘蓝绿芽切下，置于扩繁培养基(MS基本培养基+3mg/L 6-BA+0.2mg/L NAA+6mg/L 500mg/L Cef，PH 5.8)中进行继代培养3-4次(每两周继代一次)。将扩繁得到的幼苗进行生根培养25d，待幼苗长出适量根，打开无菌植株培养瓶的盖子，室内放置2天。由营养土、沙土及田间泥土按1:1:1的比例配置作为转化幼苗的培养基质。小心取出组培苗，用水洗清根上附着的培养基，将植株移栽于播种盆中，置于温室培养。遗传转化分别获得了12、10株遗传转化ItICE1(DC)1、ItICE1(DC)2基因抗性植株。与野生型菘蓝植株相比，其耐寒性分别提高了3.3℃和1.9℃。
实施例78.CRISPR/Cas9技术原位替换不同甲基化程度EcICE1基因赤桉的获得
克隆赤桉EcICE1基因cDNA，序列为SEQ ID NO：43，根据密码子的简并性特征，将EcICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换EcICE1基因中的不同程度的甲基化位点。获得全部替换后EcICE1基因序列为SEQ ID NO：84，命名为EcICE1(DC)1；部分替换后EcICE1基因序列为SEQ ID NO：125，命名为EcICE1(DC)2。将EcICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGCGGTTCCGAGCTGGGCTCCGG-3’，5’-GGATGAAAATTAGTCGAAGGGGG-3’和5’-GTCTGCGTGCACAAAACATGCGG-3’；EcICE1(DC)2sgRNA序列为5’-GGGCTTTTTGGCTCGGTGCAGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的EcICE1(DC)1、EcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E 作为抗性筛选基因，构建CRISPR/Cas9表达载体。
从低温冰箱中取出保存的GV3101菌种，在超净工作台中采用划线法在已配好的培养基表面轻轻连续画线5-6条。将培养皿倒置于25-27℃进行暗培养24-36h。然后用接种针挑取培养皿中培养基表面的单个菌落放于15m1离心管液体培养基上，在27℃、180rpm恒温摇床上进行悬浮培养12-18h。取菌液按1/40的比例放入100m1的三角瓶，继续培养6个小时左右。待OD600值为0.5时，用作侵染处理。以尾赤按栽培无性系DH201-2为材料，取自生根培养基上培养30天植株的上部和中部的茎段在预培养基上培养6d。于超净工作台中，将赤桉外植体放入三角瓶中的菌液浸泡30min，然后取出植物材料，接种到植物共培养基(100uM乙酰丁香酮，PH 5.8)上培养3d。之后将浸染后的茎段转入到(Ms+0.05mg/L TDZ+0.5mg/L NAA+5mg/L双丙氨磷+300mg/L头孢霉素，PH 5.8)的诱导再生培养基上培养15天后，形成愈伤组织，除去褐变和疏松、颜色发黄的愈伤，将颜色红绿、生长良好的愈伤组织转移到同样的培养基上，转移三次以便除去农杆菌，然后转移到只有双丙氨磷的培养基上，可得到再生芽。将再生芽转移到壮苗培养基上培养20天后，再转入到生根培养基中生根培养。转基因实验分别获得了9、11株原位替换EcICE1(DC)1、EcICE1(DC)2基因抗性植株。与野生型尾赤按植株相比，其耐寒性分别提高了3.7℃和2.3℃。
实施例79CRISPR/Cas9技术原位替换不同甲基化程度JcICE1基因麻风树的获得
克隆麻风树JcICE1基因cDNA，序列为SEQ ID NO：44，根据密码子的简并性特征，将JcICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换JcICE1基因中的不同程度的甲基化位点。获得全部替换后JcICE1基因序列为SEQ ID NO：85，命名为JcICE1(DC)1；部分替换后JcICE1基因序列为SEQ ID NO：126，命名为JcICE1(DC)2。将JcICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGTTCGATTTGGGTGACATTGGG-3’，5’-GGCAAAGCTTATCCTTGATACGG-3’和5’-GTCTCCTGCTCTCCACTATGAGG-3’；JcICE1(DC)2sgRNA序列为5’-GAAACGTGTCCTGGGTTTGCAGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的JcICE1(DC)1、JcICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取培养室内盆栽的两年生麻风树实生苗的幼嫩叶片(离生长点第3-5片叶片)，消毒后接 种于愈伤诱导或直接不定芽诱导培养基上预培养3天，切成3mm*3mm大小方块，而后使用浓度为OD600＝1.0的菌液(含100uM AS)侵染10min，之后共培养3天(共培养培养基中添加100uM AS)pH 5.3。共培养3天后，用添加有500mg/L Cb的液体MS培养基洗菌3-5遍后。转化后的麻疯树叶片直接出芽阶段需要在黑暗下进行，约2周后转入光照培养，不定芽直接诱导培养基为SIO-I：MS+TDZ 1.0mg/L+5mg/L双丙氨磷+GA3 0.5mg/I，诱导培养基中添加Cb 500mg/L。待有较多芽点形成后，转入不定芽增殖伸长培养基MS+BA 0.3mg/L+IBA 0.01mg/L中光照培养，培养基中添加5mg/L双丙氨磷，Cb 500mg/L。20-30天后，将伸长较好的不定芽转入生根培养基1/2MS+IBA 2.0mg/L中光照下生根培养，培养基中不添加任何抗生素。约30天后即可得到生根植株。然后取生根较好的小苗，采用循序渐进的原则进行炼苗，逐步打开瓶盖。完全打开瓶盖2-3天后，取出生根苗，小心地清洗掉培养基，去除老叶、死叶，种植于己灭菌的蛙石基质中。开始的时候可以用薄膜罩住地上部分，防止蒸腾作用过强，避免阳光直射，提高成活率。同时，也要注意薄膜内的湿度，防止湿度过大，幼苗发生腐烂、死亡。遗传转化分别获得了7、8株原位替换JcICE1(DC)1、JcICE1(DC)2基因抗性植株。与野生型麻风树植株相比，其耐寒性分别提高了3.2℃和1.8℃。
实施例80.CRISPR/Cas9技术原位替换不同甲基化程度PiaICE1基因欧洲云杉的获得
克隆欧洲云杉PiaICE1基因cDNA，序列为SEQ ID NO：45，根据密码子的简并性特征，将PiaICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PiaICE1基因中的不同程度的甲基化位点。获得全部替换后PiaICE1基因序列为SEQ ID NO：86，命名为PiaICE1(DC)1；部分替换后PiaICE1基因序列为SEQ ID NO：127，命名为PiaICE1(DC)2。将PiaICE1(DC)1分成2段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGAGTTGGCCTGCGAGGGGTTGG-3’和5’-GGACTCGAACCTGCGAAGCTGGG-3’；PiaICE1(DC)2sgRNA序列为5’-GTTCAAGGCAATGCTGGACGCGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PiaICE1(DC)1、PiaICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
继代培养1个月的杉木试管苗，选取生理状态良好的茎尖，轻轻剥去针叶，切取靠近茎尖的长约0.5cm的茎段，在用农杆菌浸染之前先置于诱导培养基(DCR+6-BA 1.0mg/L+TDZ 0. 001mg/L+NAA 0.1mg/L，PH 5.8)上预培养3d。挑农杆菌EHA105单菌落，接种于5m1 YEB+5mg/L双丙氨磷的液体培养基中，27℃，220rpm振荡培养过夜。取0.5m1菌液转接入25m1 YEB+5mg/L双丙氨磷的液体培养基中扩大培养6h，至菌液OD600值为0.4时，5000rpm离心10min收集菌体，取等量液体诱导培养基悬浮菌体。将预培养后的茎段，浸入制备的浸染菌液，轻微振荡，浸泡15min，取出茎段后在无菌滤纸上吸去多余菌液，然后再放回诱导培养基上进行共培养(添加80uM AS)。黑暗条件下共培养一定时间后，茎段用液体诱导培养基洗涤3次以除去过多的农杆菌。并转接到初次筛选培养基(DCR+6-BA 1.0mg/L+TDZ 0.001mg/L+NAA 0.1mg/L+Cef 200mg/L+双丙氨磷5mg/L，PH 5.8)上培养。洗涤过的茎段转接到初次筛选培养基上培养约20d，待少数茎段开始长芽时转入二次筛选培养基(DCR+Cef 200mg/L+双丙氨磷5mg/L，PH 5.8)。在二次筛选培养基上25d继代一次。初次筛选培养基上共获得186个Km抗性芽，初次获得的Km抗性芽在二次筛选培养基上继代2次，在继代过程中大量芽白化，最终仍保持绿色的芽被认定为Km抗性芽。本研究共获得39个Km抗性芽。高约1-5cm的不定芽转接入生根培养基(1/4MS基本培养基附加IBA 0.2mg/L，NAA 0.1mg/L，蔗糖296，琼脂粉0.6％，pH 5.8)，接种7d后，不定芽基部开始长出根原基，25d后根原基可伸长到1-2cm。遗传转化分别获得了10、13株原位替换PiaICE1(DC)1、PiaICE1(DC)2基因抗性植株。与野生型欧洲云杉植株相比，其耐寒性分别提高了3.9℃和2.2℃。
实施例81.CRISPR/Cas9技术原位替换不同甲基化程度PsICE1基因甜杨的获得
克隆甜杨PsICE1基因cDNA，序列为SEQ ID NO：46，根据密码子的简并性特征，将PsICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PsICE1基因中的不同程度的甲基化位点。获得全部替换后PsICE1基因序列为SEQ ID NO：87，命名为PsICE1(DC)1；部分替换后PsICE1基因序列为SEQ ID NO：128，命名为PsICE1(DC)2。将PsICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段500bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GGTTTCTTCCAACCCTTCAAAGG-3’，5’-GGTCGTGGTGAGGATCAAAAGGG-3’和5’-GCAGTCCTACTGGATTCAGCTGG-3’；PsICE1(DC)2sgRNA序列为5’-GCAGCTAGAGGAAAGAACTTGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PsICE1(DC)1、PsICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取保存于-70℃的农杆菌GV3101菌株，冰上融化，取适量的菌液涂于含有相应抗生素的LB培养基平板上，28℃倒置培养至单菌落产生，2d左右。从平板中挑取单菌落，接种到附加spc 100mg/L，GM 50mg/L的5ml LB液体培养基中，28℃，220rpm振荡培养24h，再从中取出0.4ml菌液转接到20ml含相应抗生素的LB培养基中，培养6h左右至OD600值为0.5，菌液在5000离心l0min收集菌体，然后用等体积的1/2MS液体培养基重悬菌体，稀释菌液至OD600值为0.5，用于侵染。加入20uM乙酰丁香酮，25℃温和振荡(90rpm)菌液30min；挑选生长势基本一致(4-6周)的甜杨组培苗，取2，3节茎段，修剪掉叶片和腋芽，用刀片迅速将其切为1-1.5m的茎段，转入制备好的浸染液中，25℃温和振荡(250ml三角瓶，90rpm)30min后取出茎段，用滤纸吸干残余菌液，转入不含抗生素的筛选培养基上暗培养；共培养的培养基为分化培养基，共培养采用暗培养的方法。共培养2d后，将茎段放到筛选培养基(愈伤诱导培养基+cef200mg/L+5mg/L双丙氨磷，PH 5.6)上进行筛选培养，15-20d后在茎段两端边缘便会长出绿色的抗性愈伤。待筛选培养的茎段完全长出小芽以后，将其移到芽伸长培养基(MS+6-BA 0.2mg/L+TDZ 0.01mg/L+cef 200mg/L+5mg/L双丙氨磷，PH 5.6)上，芽伸长培养基含有较低的激素浓度，有利于抗性芽的快速生长。当芽长至1-2cm的时候，转至生根培养基(1/2MS+cef 200mg/L+5mg/L双丙氨磷，PH 5.6)进行诱导生根，生根后经过一段时间的培养，即可形成完整的转基因植株。本次遗传转化分别获得了20、15株原位替换PsICE1(DC)1、PsICE1(DC)2基因抗性植株。与野生型甜杨植株相比，其耐寒性分别提高了3.6℃和2.1℃。
实施例82.CRISPR/Cas9技术原位替换不同甲基化程度PtICE1基因杨的获得
克隆杨PtICE1基因cDNA，序列为SEQ ID NO：47，根据密码子的简并性特征，将PtICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PtICE1基因中的不同程度的甲基化位点。获得全部替换后PtICE1基因序列为SEQ ID NO：88，命名为PtICE1(DC)1；部分替换后PtICE1基因序列为SEQ ID NO：129，命名为PtICE1(DC)2。将PtICE1(DC)1分成3段进行CRISPR/Cas9载体构建和原位替换，每段400bp左右，通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，序列分别为5’-GTTTCATCTCTGTTAAACAGGGG-3’，5’-GATGTTAGCTTTGATGGGTCGGG-3’和5’-GCTTCCAGCTAAGAATTTGATGG-3’；PtICE1(DC)2sgRNA序列为5’-GTTTCATCTCTGTTAAACAGGGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PtICE1(DC)1、PtICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、 AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
杨遗传转化实验操作方法同甜杨遗传转化实验(实施例39)。遗传转化实验中，共获得原位替换杨PtICE1(DC)1、PtICE1(DC)2基因植株18和22株。与野生杨植株相比，其耐寒性分别提高了3.7℃和2.2℃。
实施例83.CRISPR/Cas9技术原位替穿叶眼子菜不同甲基化程度PopICE1基因拟南芥的获得
克隆穿叶眼子菜PopICE1基因cDNA，序列为SEQ ID NO：48，根据密码子的简并性特征，将PopICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PopICE1基因中的不同程度的甲基化位点。获得全部替换后PopICE1基因序列为SEQ ID NO：89，命名为PopICE1(DC)1；部分替换后PopICE1基因序列为SEQ ID NO：130，命名为PopICE1(DC)2。通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，PopICE1(DC)1和PopICE1(DC)2基因sgRNA序列均为5’-GCTCCTATTTCGATGGGGTT NGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PtICE1(DC)1、PtICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
取保存的根癌农杆菌GV3101在已制备好的YEB固体培养基平板上画线，并在28℃条件下暗培养1-2d后，用接种环挑取单菌落接种于5ml YEB液体培养基中，于28℃、220rpm振荡培养24h后，在无菌条件下吸取1ml菌液到YEB液体培养基中，于28℃、220rpm振荡培养4-6h后，在600nm处测得菌液OD值为0.6-0.8左右即可用于拟南芥侵染。将盛花期拟南芥的花表面部分浸泡在农杆菌悬浮液(OD＝0.8)中5分钟，同时轻轻旋转。将浸染后植株套袋保持高度的湿润状态，暗室培养24小时。种子成熟，角果自然开裂后可以收种子。收集到的拟南芥种子于4℃春化2天，种子消毒后在含有5mg/L双丙氨磷的平板上进行培养。成功转入重组质粒的种子能够在抗性培养及上正常生长出4片以上真叶。非转基因种子不能正常生长，仅能长出2片子叶，根的生长也受到严重抑制。将筛选到的植株移植到蛭石：泥炭土：珍珠岩为9：3：1的基质中，覆盖薄膜2-3天。遗传转化分别获得了14、19、17株原位替换PopICE1、PopICE1(DC)1、PopICE1(DC)2基因抗性植株。与野生型拟南芥植株Col-0相比，其耐寒性分别提高了0.8℃、2.0℃和1.5℃。
实施例84.CRISPR/Cas9技术原位替竹叶眼子菜不同甲基化程度PwICE1基因拟南芥的获得
克隆竹叶眼子菜PwICE1基因cDNA，序列为SEQ ID NO：49，根据密码子的简并性特征，将PwICE1基因中理论上可甲基化的位点进行替换，根据目的性状要求，定量替换PwICE1基因中的不同程度的甲基化位点。获得全部替换后PwICE1基因序列为SEQ ID NO：90，命名为PwICE1(DC)1；部分替换后PwICE1基因序列为SEQ ID NO：131，命名为PwICE1(DC)2。通过人工设计具有引导作用的sgRNA，sgRNA的长度为20nt，PwICE1(DC)1和PwICE1(DC)2基因序列均为5’-GCTCCTATTTCGATGGGGTT NGG-3’，引导Cas9蛋白对DNA的定点切割。启动子选择YAO启动子，通过限制性内切酶BsaI、KpnI切割载体pHEE401E和不同甲基化程度的PtICE1(DC)1、PtICE1(DC)2基因，将目的基因连入zCas9序列后面；在Bar基因两端添加SacII、AsiSI酶切位点，通过SacII、AsiSI酶切pHEE401E和Bar基因，将Bar基因连入pHEE401E作为抗性筛选基因，构建CRISPR/Cas9表达载体。
竹叶眼子菜不同甲基化程度PwICE1基因遗传转化拟南芥实验操作方法同实施例47。遗传转化实验中，共获得原位替换竹叶眼子菜PwICE1、PwICE1(DC) 1、PwICE1(DC) 2基因植株18、15和19株。与野生型拟南芥植株Col-0相比，其耐寒性分别提高了0.7℃、1.9℃和1.2℃。
显然，本发明不限于以上实施例，还可以有许多变形。与上述实施例水稻、玉米、小麦和大麦同属禾本科作物中的高粱、甘蔗等，与烟草、茄、番茄同属茄科的辣椒、马铃薯等，与白菜、油菜、萝卜同属十字花科的其他青菜等，热带作物橡胶、木薯、龙眼、可可以及其他热带作物如木瓜等。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (14)

  1. 一个表观遗传操作植物表型可塑性性状的方法,其特征在于通过简并密码子定量替换目的基因的可甲基化位点，设计合成基因，进行转基因操作；或利用CRISPR/Cas9编辑技术构建植物表达载体进行原位在体基因简并密码子定量替换可甲基化位点后，定量调控外源或在体目的基因在受体中的甲基化程度和基因表达水平，达到定量调控植物的可塑性性状。
  2. 根据权利要求1所述的方法,其特征在于通过简并密码子定量替换目的基因的可甲基化胞嘧啶位点后设计合成基因，并进行转基因遗传操作，定量调控外源目的基因在受体中的甲基化程度和基因表达水平，达到定量调控植物的可塑性性状。
  3. 根据权利要求1所述的方法,其特征在于利用CRISPR/Cas9技术构建植物表达载体进行原位在体基因简并密码子定量替换可甲基化胞嘧啶位点后，定量调控在体目的基因的甲基化程度和基因表达水平，达到定量调控植物的可塑性性状。
  4. 根据权利要求1-3中任一项所述的方法,其特征在于所述的可塑性性状为耐寒性。
  5. 根据权利要求4所述的方法，其特征在于所述的目的基因为ICE1基因。
  6. 根据权利要求4所述的方法，其特征在于所述的ICE1基因选自紫茎泽兰AaICE1基因，木薯MeICE1基因，猕猴桃AcICE1基因，野生蕉MaICE1基因，芭蕉MabICE1基因，可可TcICE1基因，枳CtICE1基因，葡萄VvICE1基因，苹果MdICE1基因，芥CbICE1基因，茄子ScICE1基因，番茄SlICE1基因，桃PpICE1基因，玉米ZmICE1基因，棉花GhICE1基因，花生AhICE1基因，芥菜BjICE1基因，油菜BrICE1基因，菊花CdICE1基因，小盐芥ThICE1基因，野胡萝卜DcICE1基因，大豆GmICE1基因，萝卜RsICE1基因，小麦TaICE1基因，大麦HvICE1基因，橡胶树HbICE1基因，胡桃JrICE1基因，莴苣LsICE1基因，水稻OsICE1基因，蝴蝶兰PaICE1基因，梅花PmICE1基因，茶CsICE1基因，拟南芥AtICE1基因，芜菁BrrICE1基因，菘蓝ItICE1基因，赤桉EcICE1基因，麻风树JcICE1基因，欧洲云杉PiaICE1基因，甜杨PsICE1基因，杨PtICE1基因，穿叶眼子菜PopICE1基因，竹叶眼子菜PwICE1基因；其中，紫茎泽兰AaICE1基因核苷酸序列为SEQ ID NO：1，木薯MeICE1基因核苷酸序列为SEQ ID NO：9，猕猴桃AcICE1基因核苷酸序 列为SEQ ID NO：10，野生蕉MaICE1基因核苷酸序列为SEQ ID NO：11，芭蕉MabICE1基因核苷酸序列为SEQ ID NO：12，可可TcICE1基因核苷酸序列为SEQ ID NO：13，枳CtICE1基因核苷酸序列为SEQ ID NO：14，葡萄VvICE1基因核苷酸序列为SEQ ID NO：15，苹果MdICE1基因核苷酸序列为SEQ ID NO：16，芥CbICE1基因核苷酸序列为SEQ ID NO：17，茄子ScICE1基因核苷酸序列为SEQ ID NO：18，番茄SlICE1基因核苷酸序列为SEQ ID NO：19，桃PpICE1基因核苷酸序列为SEQ ID NO：20，玉米ZmICE1基因核苷酸序列为SEQ ID NO：21，棉花GhICE1基因核苷酸序列为SEQ ID NO：22，花生AhICE1基因核苷酸序列为SEQ ID NO：23，芥菜BjICE1基因核苷酸序列为SEQ ID NO：24，油菜BrICE1基因核苷酸序列为SEQ ID NO：25，菊花CdICE1基因核苷酸序列为SEQ ID NO：26，小盐芥ThICE1基因核苷酸序列为SEQ ID NO：27，野胡萝卜DcICE1基因核苷酸序列为SEQ ID NO：28，大豆GmICE1基因核苷酸序列为SEQ ID NO：29，萝卜RsICE1基因核苷酸序列为SEQ ID NO：30，小麦TaICE1基因核苷酸序列为SEQ ID NO：31，大麦HvICE1基因核苷酸序列为SEQ ID NO：32，橡胶树HbICE1基因核苷酸序列为SEQ ID NO：33，胡桃JrICE1基因核苷酸序列为SEQ ID NO：34，莴苣LsICE1基因核苷酸序列为SEQ ID NO：35，水稻OsICE1基因核苷酸序列为SEQ ID NO：36，蝴蝶兰PaICE1基因核苷酸序列为SEQ ID NO：37，梅花PmICE1基因核苷酸序列为SEQ ID NO：38，茶CsICE1基因核苷酸序列为SEQ ID NO：39，拟南芥AtICE1基因核苷酸序列为SEQ ID NO：40，芜菁BrrICE1基因核苷酸序列为SEQ ID NO：41，菘蓝ItICE1基因核苷酸序列为SEQ ID NO：42，赤桉EcICE1基因核苷酸序列为SEQ ID NO：43，麻风树JcICE1基因核苷酸序列为SEQ ID NO：44，欧洲云杉PiaICE1基因核苷酸序列为SEQ ID NO：45，甜杨PsICE1基因核苷酸序列为SEQ ID NO：46，杨PtICE1基因核苷酸序列为SEQ ID NO：47，穿叶眼子菜PopICE1基因核苷酸序列为SEQ ID NO：48，竹叶眼子菜PwICE1基因核苷酸序列为SEQ ID NO：49。
  7. 根据权利要求6所述的方法，其特征在于所述的通过简并密码子全部替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：3，木薯：SEQ ID NO：50，猕猴桃：SEQ ID NO：51，野生蕉：SEQ ID NO：52，芭蕉：SEQ ID NO：53，可可：SEQ ID NO：54，枳：SEQ ID NO：55，葡萄：SEQ ID NO：56，苹果：SEQ ID NO：57，芥：SEQ ID NO：58，茄子：SEQ ID NO：59，番茄：SEQ  ID NO：60，桃：SEQ ID NO：61，玉米：SEQ ID NO：62，棉花：SEQ ID NO：63，花生：SEQ ID NO：64，芥菜：SEQ ID NO：65，油菜：SEQ ID NO：66，菊花：SEQ ID NO：67，小盐芥：SEQ ID NO：68，野胡萝卜：SEQ ID NO：69，大豆：SEQ ID NO：70，萝卜：SEQ ID NO：71，小麦：SEQ ID NO：72，大麦：SEQ ID NO：73，橡胶树：SEQ ID NO：74，胡桃：SEQ ID NO：75，莴苣：SEQ ID NO：76，水稻：SEQ ID NO：77，蝴蝶兰：SEQ ID NO：78，梅花：SEQ ID NO：79，茶：SEQ ID NO：80，拟南芥：SEQ ID NO：81，芜菁：SEQ ID NO：82，菘蓝：SEQ ID NO：83，赤桉：SEQ ID NO：84，麻风树：SEQ ID NO：85，欧洲云杉：SEQ ID NO：86，甜杨：SEQ ID NO：87，杨：SEQ ID NO：88，穿叶眼子菜：SEQ ID NO：89，竹叶眼子菜：SEQ ID NO：90；通过简并密码子部分替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：4，木薯：SEQ ID NO：91，猕猴桃：SEQ ID NO：92，野生蕉：SEQ ID NO：93，芭蕉：SEQ ID NO：94，可可：SEQ ID NO：95，枳：SEQ ID NO：96，葡萄：SEQ ID NO：97，苹果：SEQ ID NO：98，芥：SEQ ID NO：99，茄子：SEQ ID NO：100，番茄：SEQ ID NO：101，桃：SEQ ID NO：102，玉米：SEQ ID NO：103，棉花：SEQ ID NO：104，花生：SEQ ID NO：105，芥菜：SEQ ID NO：106，油菜：SEQ ID NO：107，菊花：SEQ ID NO：108，小盐芥：SEQ ID NO：109，野胡萝卜：SEQ ID NO：110，大豆：SEQ ID NO：111，萝卜：SEQ ID NO：112，小麦：SEQ ID NO：113，大麦：SEQ ID NO：114，橡胶树：SEQ ID NO：115，胡桃：SEQ ID NO：116，莴苣：SEQ ID NO：117，水稻：SEQ ID NO：118，蝴蝶兰：SEQ ID NO：119，梅花：SEQ ID NO：120，茶：SEQ ID NO：121，拟南芥：SEQ ID NO：122，芜菁：SEQ ID NO：123，菘蓝：SEQ ID NO：124，赤桉：SEQ ID NO：125，麻风树：SEQ ID NO：126，欧洲云杉：SEQ ID NO：127，甜杨：SEQ ID NO：128，杨：SEQ ID NO：129，穿叶眼子菜：SEQ ID NO：130，竹叶眼子菜：SEQ ID NO：131。
  8. 根据权利要求6所述的方法，其特征在于合成或CRISPR/Cas9原位进行简并密码子全部或部分替换可甲基化的胞嘧啶后的AaICE1基因，将其转化到水稻、木薯、香蕉、番茄、可可、橡胶、柠檬、甜橙、柑橘、甘蔗、番木瓜、枇杷、荔枝、龙眼、芒果、马铃薯、辣椒、高粱、葡萄、苹果、芥、茄子、猕猴桃、桃、玉米、棉花、花生、芥菜、油菜、菊花、胡萝卜、大豆、萝卜、小麦、大麦、胡桃、莴苣、大花蕙兰、蝴蝶兰、腊梅、茶、赤桉植株中，获得抗寒性增强的转基 因植株；所述的全部替换可甲基化的胞嘧啶后的AaICE1基因序列为SEQ ID NO：3，部分替换可甲基化的胞嘧啶后的AaICE1基因序列为SEQ ID NO：4。
  9. 一种耐寒基因，其特征在于选自全部或部分替换可甲基化的胞嘧啶后的ICE1基因。
  10. 根据权利要求9所述的基因，其特征在于野生型ICE1基因选自紫茎泽兰AaICE1基因，木薯MeICE1基因，猕猴桃AcICE1基因，野生蕉MaICE1基因，芭蕉MabICE1基因，可可TcICE1基因，枳CtICE1基因，葡萄VvICE1基因，苹果MdICE1基因，芥CbICE1基因，茄子ScICE1基因，番茄SlICE1基因，桃PpICE1基因，玉米ZmICE1基因，棉花GhICE1基因，花生AhICE1基因，芥菜BjICE1基因，油菜BrICE1基因，菊花CdICE1基因，小盐芥ThICE1基因，野胡萝卜DcICE1基因，大豆GmICE1基因，萝卜RsICE1基因，小麦TaICE1基因，大麦HvICE1基因，橡胶树HbICE1基因，胡桃JrICE1基因，莴苣LsICE1基因，水稻OsICE1基因，蝴蝶兰PaICE1基因，梅花PmICE1基因，茶CsICE1基因，拟南芥AtICE1基因，芜菁BrrICE1基因，菘蓝ItICE1基因，赤桉EcICE1基因，麻风树JcICE1基因，欧洲云杉PiaICE1基因，甜杨PsICE1基因，杨PtICE1基因，穿叶眼子菜PopICE1基因，竹叶眼子菜PwICE1基因；其中，紫茎泽兰AaICE1基因核苷酸序列为SEQ ID NO：1，木薯MeICE1基因核苷酸序列为SEQ ID NO：9，猕猴桃AcICE1基因核苷酸序列为SEQ ID NO：10，野生蕉MaICE1基因核苷酸序列为SEQ ID NO：11，芭蕉MabICE1基因核苷酸序列为SEQ ID NO：12，可可TcICE1基因核苷酸序列为SEQ ID NO：13，枳CtICE1基因核苷酸序列为SEQ ID NO：14，葡萄VvICE1基因核苷酸序列为SEQ ID NO：15，苹果MdICE1基因核苷酸序列为SEQ ID NO：16，芥CbICE1基因核苷酸序列为SEQ ID NO：17，茄子ScICE1基因核苷酸序列为SEQ ID NO：18，番茄SlICE1基因核苷酸序列为SEQ ID NO：19，桃PpICE1基因核苷酸序列为SEQ ID NO：20，玉米ZmICE1基因核苷酸序列为SEQ ID NO：21，棉花GhICE1基因核苷酸序列为SEQ ID NO：22，花生AhICE1基因核苷酸序列为SEQ ID NO：23，芥菜BjICE1基因核苷酸序列为SEQ ID NO：24，油菜BrICE1基因核苷酸序列为SEQ ID NO：25，菊花CdICE1基因核苷酸序列为SEQ ID NO：26，小盐芥ThICE1基因核苷酸序列为SEQ ID NO：27，野胡萝卜DcICE1基因核苷酸序列为SEQ ID NO：28，大豆GmICE1基因核苷酸序列为SEQ ID NO：29，萝卜RsICE1基因核苷酸序 列为SEQ ID NO：30，小麦TaICE1基因核苷酸序列为SEQ ID NO：31，大麦HvICE1基因核苷酸序列为SEQ ID NO：32，橡胶树HbICE1基因核苷酸序列为SEQ ID NO：33，胡桃JrICE1基因核苷酸序列为SEQ ID NO：34，莴苣LsICE1基因核苷酸序列为SEQ ID NO：35，水稻OsICE1基因核苷酸序列为SEQ ID NO：36，蝴蝶兰PaICE1基因核苷酸序列为SEQ ID NO：37，梅花PmICE1基因核苷酸序列为SEQ ID NO：38，茶CsICE1基因核苷酸序列为SEQ ID NO：39，拟南芥AtICE1基因核苷酸序列为SEQ ID NO：40，芜菁BrrICE1基因核苷酸序列为SEQ ID NO：41，菘蓝ItICE1基因核苷酸序列为SEQ ID NO：42，赤桉EcICE1基因核苷酸序列为SEQ ID NO：43，麻风树JcICE1基因核苷酸序列为SEQ ID NO：44，欧洲云杉PiaICE1基因核苷酸序列为SEQ ID NO：45，甜杨PsICE1基因核苷酸序列为SEQ ID NO：46，杨PtICE1基因核苷酸序列为SEQ ID NO：47，穿叶眼子菜PopICE1基因核苷酸序列为SEQ ID NO：48，竹叶眼子菜PwICE1基因核苷酸序列为SEQ ID NO：49；
    所述的通过简并密码子全部替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：3，木薯：SEQ ID NO：50，猕猴桃：SEQ ID NO：51，野生蕉：SEQ ID NO：52，芭蕉：SEQ ID NO：53，可可：SEQ ID NO：54，枳：SEQ ID NO：55，葡萄：SEQ ID NO：56，苹果：SEQ ID NO：57，芥：SEQ ID NO：58，茄子：SEQ ID NO：59，番茄：SEQ ID NO：60，桃：SEQ ID NO：61，玉米：SEQ ID NO：62，棉花：SEQ ID NO：63，花生：SEQ ID NO：64，芥菜：SEQ ID NO：65，油菜：SEQ ID NO：66，菊花：SEQ ID NO：67，小盐芥：SEQ ID NO：68，野胡萝卜：SEQ ID NO：69，大豆：SEQ ID NO：70，萝卜：SEQ ID NO：71，小麦：SEQ ID NO：72，大麦：SEQ ID NO：73，橡胶树：SEQ ID NO：74，胡桃：SEQ ID NO：75，莴苣：SEQ ID NO：76，水稻：SEQ ID NO：77，蝴蝶兰：SEQ ID NO：78，梅花：SEQ ID NO：79，茶：SEQ ID NO：80，拟南芥：SEQ ID NO：81，芜菁：SEQ ID NO：82，菘蓝：SEQ ID NO：83，赤桉：SEQ ID NO：84，麻风树：SEQ ID NO：85，欧洲云杉：SEQ ID NO：86，甜杨：SEQ ID NO：87，杨：SEQ ID NO：88，穿叶眼子菜：SEQ ID NO：89，竹叶眼子菜：SEQ ID NO：90；
    通过简并密码子部分替换可甲基化的胞嘧啶后的ICE1基因核苷酸序列分别为紫茎泽兰：SEQ ID NO：4，木薯：SEQ ID NO：91，猕猴桃：SEQ ID NO：92，野生蕉：SEQ ID NO：93，芭蕉：SEQ ID NO：94，可可：SEQ ID NO：95，枳：SEQ ID NO： 96，葡萄：SEQ ID NO：97，苹果：SEQ ID NO：98，芥：SEQ ID NO：99，茄子：SEQ ID NO：100，番茄：SEQ ID NO：101，桃：SEQ ID NO：102，玉米：SEQ ID NO：103，棉花：SEQ ID NO：104，花生：SEQ ID NO：105，芥菜：SEQ ID NO：106，油菜：SEQ ID NO：107，菊花：SEQ ID NO：108，小盐芥：SEQ ID NO：109，野胡萝卜：SEQ ID NO：110，大豆：SEQ ID NO：111，萝卜：SEQ ID NO：112，小麦：SEQ ID NO：113，大麦：SEQ ID NO：114，橡胶树：SEQ ID NO：115，胡桃：SEQ ID NO：116，莴苣：SEQ ID NO：117，水稻：SEQ ID NO：118，蝴蝶兰：SEQ ID NO：119，梅花：SEQ ID NO：120，茶：SEQ ID NO：121，拟南芥：SEQ ID NO：122，芜菁：SEQ ID NO：123，菘蓝：SEQ ID NO：124，赤桉：SEQ ID NO：125，麻风树：SEQ ID NO：126，欧洲云杉：SEQ ID NO：127，甜杨：SEQ ID NO：128，杨：SEQ ID NO：129，穿叶眼子菜：SEQ ID NO：130，竹叶眼子菜：SEQ ID NO：131。
  11. 包含权利要求10所述的全部或部分替换可甲基化的胞嘧啶后的ICE1基因的表达载体。
  12. 权利要求10所述的ICE1基因在培育耐寒植物方面的应用。
  13. 根据权利要求12所述的应用，其特征在于：包括以下步骤：
    (1)利用DNA重组技术，构建含有所述ICE1基因的植物转化质粒；
    (2)将步骤(1)中构建的植物转化质粒通过基因枪或农杆菌浸染方法或花粉介导法等导入植物组织，或通过CRISPR/Cas9技术进行受体植株原位替换，获得含有不同甲基化程度ICE1基因的转基因或基因编辑的植物。
  14. 根据权利要求12或13所述的应用，其特征在于所述的植物选自水稻、木薯、香蕉、番茄、可可、橡胶、柠檬、甜橙、柑橘、甘蔗、番木瓜、枇杷、荔枝、龙眼、芒果、马铃薯、辣椒、高粱、葡萄、苹果、芥、茄子、猕猴桃、桃、玉米、棉花、花生、芥菜、油菜、菊花、胡萝卜、大豆、萝卜、小麦、大麦、胡桃、莴苣、大花蕙兰、蝴蝶兰、腊梅、茶、赤桉。
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