WO2019061687A1 - 一种培育抗蚜烟草和小麦的方法和技术体系 - Google Patents

一种培育抗蚜烟草和小麦的方法和技术体系 Download PDF

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WO2019061687A1
WO2019061687A1 PCT/CN2017/109851 CN2017109851W WO2019061687A1 WO 2019061687 A1 WO2019061687 A1 WO 2019061687A1 CN 2017109851 W CN2017109851 W CN 2017109851W WO 2019061687 A1 WO2019061687 A1 WO 2019061687A1
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fragment
gene
plant
sequence
interference
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French (fr)
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夏兰琴
闫婷
孙永伟
杜文明
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中国农业科学院作物科学研究所
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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)
    • C12N15/8241—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
    • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—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 biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • 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
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present invention relates to a method and technical system for growing resistant tobacco and wheat.
  • Aphids are an important worldwide agricultural pest. There are hundreds of millions of direct economic losses caused by locusts worldwide each year. There are many types of mites, including more than 4,400 species of mites in about 10 families, which can cause harm during the whole growth period of plants. Aphid feeding crops can not only cause severe yield reduction but also a carrier of many viruses. Aphids use plant-sucking devices to absorb plant nutrients and affect the normal growth of plants; the secreted honeydew affects plant photosynthesis; many aphids are also spreaders of plant viruses, such as wheat yellow dwarf virus. Of the more than 300 insect viral mediators reported, more than 170 are aphids.
  • Maichang tube and peach aphid are the main pests of various economic crops, causing significant economic losses to agricultural production.
  • the annual area of wheat aphid damage in China can reach as high as 0.17 billion hectares, accounting for 62% of the total planted area of wheat.
  • Myzus persicae is a polyphagous pest.
  • RNAi technology has become one of the hotspots of crop insect-resistant genetic engineering.
  • the dsRNA of the corresponding insect-specific gene is expressed by the host plant, and the insects eat the plants and silence their corresponding genes to achieve the purpose of controlling pest damage.
  • the RNAi phenomenon is a double-stranded RNA (dsRNA) that enters the organism and is cleaved into 21-23 nt siRNA by Dicer.
  • the siRNA binds to the RNA-inducing silencing complex, binds to the target mRNA of the complementary sequence, and is recognized by Dicer. A decrease in the amount of gene expression.
  • the present invention firstly protects a method for preparing an anti-tuberculosis plant, comprising the steps of: inhibiting expression of a specific gene or a specific gene fragment in a starting plant to obtain an anti- ⁇ transgenic plant; the specific gene is represented by the sequence 1 having a sequence listing A gene of a nucleotide sequence; the specific gene fragment is shown as Sequence 1 of the Sequence Listing.
  • the method of "suppressing the expression of a specific gene or a specific gene fragment in a starting plant” is as follows: an interference vector or an interference fragment is introduced into the starting plant.
  • the interfering fragment has a coding gene for interfering RNA; the coding gene of the interfering RNA is composed of a fragment A, a spacer sequence and a fragment B; the fragment A is as shown in sequence 1 of the sequence listing; Fragment A is reverse complementary.
  • the coding gene of the interfering RNA is specifically shown as nucleotides 2161-2768 of SEQ ID NO: 4 of the Sequence Listing.
  • the interference fragment includes, in order from upstream to downstream, a promoter, a coding gene for interfering RNA, and a terminator.
  • the promoter may specifically be a Ubi promoter.
  • the terminator may specifically be a NOS terminator.
  • the interference fragment may specifically be represented by nucleotides 67-3044 in SEQ ID NO: 4 of the Sequence Listing.
  • the interference fragment can be specifically as shown in sequence 4 of the sequence listing.
  • the interfering vector has a coding gene for interfering RNA; the coding gene of the interfering RNA is composed of a fragment A, a spacer sequence and a fragment B; the fragment is as shown in sequence 1 of the sequence listing; Fragment A is reverse complementary.
  • the coding gene of the interfering RNA is specifically shown as nucleotides at nucleotide positions 626-12-333 of Sequence 3 of the Sequence Listing.
  • a specific expression cassette is included in the interference vector.
  • the specific expression cassette includes, in order from upstream to downstream, a promoter, a coding gene for interfering RNA, and a terminator.
  • the promoter may specifically be a 35S promoter, and the terminator may specifically be a NOS terminator.
  • the specific expression cassette is specifically shown as Sequence 3 of the Sequence Listing.
  • the interference vector is specifically a circular plasmid represented by SEQ ID NO: 2 of the Sequence Listing.
  • the anti-caries are anti-aphids.
  • the aphid may specifically be a peach aphid or a long tube.
  • the starting plant is a monocot or a dicot.
  • the starting plant may specifically be wheat (e.g., wheat variety Coonong 199) or tobacco (e.g., tobacco W38).
  • the present invention also protects a DNA molecule (interfering fragment) having a coding gene that interferes with RNA; the coding gene of the interfering RNA is composed of a fragment A, a spacer sequence and a fragment B; Sequence 1 of the Sequence Listing; said Fragment B is inversely complementary to said Fragment A.
  • the coding gene of the interfering RNA is specifically shown as nucleotides 2161-2768 of SEQ ID NO: 4 of the Sequence Listing.
  • the interference fragment includes, in order from upstream to downstream, a promoter, a coding gene for interfering RNA, and a terminator.
  • the promoter may specifically be a Ubi promoter.
  • the terminator may specifically be a NOS terminator.
  • the interference fragment may specifically be represented by nucleotides 67-3044 in SEQ ID NO: 4 of the Sequence Listing.
  • the interference fragment can be specifically as shown in sequence 4 of the sequence listing.
  • the present invention also contemplates a recombinant plasmid (interfering vector) having a coding gene that interferes with RNA; the coding gene of the interfering RNA is composed of a fragment A, a spacer sequence and a fragment B; the fragment is as shown in sequence 1 of the sequence listing The fragment B is inversely complementary to the fragment A.
  • the coding gene of the interfering RNA is specifically shown as nucleotides at nucleotide positions 626-12-333 of Sequence 3 of the Sequence Listing.
  • a specific expression cassette is included in the interference vector.
  • the specific expression cassette includes, in order from upstream to downstream, a promoter, a coding gene for interfering RNA, and a terminator.
  • the promoter may specifically be a 35S promoter, and the terminator may specifically be a NOS terminator.
  • the specific expression cassette is specifically shown as Sequence 3 of the Sequence Listing.
  • the interference vector is specifically a circular plasmid represented by SEQ ID NO: 2 of the Sequence Listing.
  • the invention also protects the use of the interference fragment or the interference vector in the cultivation of an anti- ⁇ transgenic plant.
  • the anti-caries are anti-aphids.
  • the aphid may specifically be a peach aphid or a long tube.
  • the plant is a monocot or a dicot.
  • the plant may specifically be wheat (e.g., wheat variety Coonong 199) or tobacco (e.g., tobacco W38).
  • the invention also contemplates a system for cultivating an anti- ⁇ transgenic plant, comprising the interfering fragment or the interfering vector.
  • the anti-caries are anti-aphids.
  • the aphid may specifically be a peach aphid or a long tube.
  • the plant is a monocot or a dicot.
  • the plant may specifically be wheat (e.g., wheat variety Coonong 199) or tobacco (e.g., tobacco W38).
  • the present invention also protects a gene having the nucleotide sequence shown in SEQ ID NO: 1 of the Sequence Listing.
  • the present invention also protects a gene derived from aphid having the nucleotide sequence shown in SEQ ID NO: 1 of the Sequence Listing.
  • the aphid may specifically be a peach aphid or a long tube.
  • the invention also protects proteins encoded by the genes.
  • the invention also protects the use of the gene as a target for silencing in the cultivation of an amaranth plant.
  • the being silenced may specifically be to knock out all or part of the gene.
  • the being silenced may specifically inhibit the expression of the gene.
  • the anti-caries are anti-aphids.
  • the aphid may specifically be a peach aphid or a long tube.
  • the plant is a monocot or a dicot.
  • the plant may specifically be wheat (eg wheat variety) Coonong 199) or tobacco (eg tobacco W38).
  • the invention also protects the use of the protein as an inhibited target in the cultivation of an anthrax resistant plant.
  • the anti-caries are anti-aphids.
  • the inhibition may specifically be to reduce the level of the protein in the aphids.
  • the inhibition is such as to inhibit the activity of the protein in aphids.
  • the aphid may specifically be a peach aphid or a long tube.
  • the plant is a monocot or a dicot.
  • the plant may specifically be wheat (e.g., wheat variety Coonong 199) or tobacco (e.g., tobacco W38).
  • Figure 1 shows the results of PCR identification in Example 2.
  • Figure 2 is the result of Southern hybridization identification in Example 2.
  • Figure 3 is the result of Northern hybridization identification in Example 2.
  • Figure 4 is the result of mortality in Example 2.
  • Figure 5 is a photograph of two offspring of the same aphid in Example 2.
  • Fig. 6 is a graph showing the elapsed time and generational period of each stage of aphid development in Example 2.
  • Fig. 7 is a graph showing the results of the lifespan, the calving period, the length of life, the daily average yield, and the total calyx in Example 2.
  • Figure 8 is a graph showing the relative expression levels of target sequences in Myzus persicae in Example 2.
  • Fig. 9 is a graph showing the results of the lifespan, the calving period, and the length of life in Example 3.
  • Fig. 10 is a graph showing the results of daily average yield and total calcination in Example 3.
  • Figure 11 is the result of mortality in Example 3.
  • Fig. 12 is a graph showing the elapsed time of each stage of aphid development in Example 3, and the results of the current stage, the pre-calving period, and the period.
  • 1 year old period the period from the moment of birth to the time when the first layer of skin is removed.
  • 2nd infancy period the period from the first layer of skin peeling off to the time when the second layer of skin is removed.
  • 3rd infancy the period from the time when the second layer of skin is removed to the time when the third layer of skin is removed.
  • 4th infancy the period from the start of the third layer of skin to the loss of the fourth layer of skin. If the flood season: from the moment of birth to the fall of the fourth layer of skin The time period until then.
  • Pre-calving period the period from the time when the fourth layer of skin is removed to the time when the first head is produced. Duration: The period of time from birth to the first lice.
  • Length of life The period of time from birth to death.
  • Daily average yield The daily average yield of aphids during the period from the first aphid to the last aphid.
  • Total pupa The total number of calves produced throughout the life of the locust.
  • Agrobacterium tumefaciens AGL1:Y.He,HDJones,S.Chen,XMChen,DWWang,KXLi,DSWang and LQXia Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L.var.durum cv Stewart)with Improved efficiency. Journal of Experimental Botany, Vol. 61, No. 6, pp. 1567–1581, 2010.
  • Sitobion avenae (Fabricius): Abdellatef E., Will T., Koch A., Imani J., Vilcinskas A., Kogel KHSilencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae .Plant Biotechnology Journal, 2015, 13: 849-857.
  • Tobacco W38 (Nicotiana tabacum L.cv W38): Yu XD, Zhang YJ, Ma YZ, Xu ZS, Wang GP, Xia LQ Expressionion of an (E)- ⁇ -farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation. The Crop Journal, 2013, 1:50-60.
  • Kenong 199 Yamaong199: Yan Donghong, He Sha, Zhang Yan, Xia Lanqin. Optimization of main bombardment parameters of wheat transformed by gene gun. Acta Sinica, 2013, 1:60-67.
  • Protein A A new protein was found in the genus Aphididae, which was named Protein A.
  • Protein B A new protein was found from the peach aphid, which was named Protein B.
  • Both the gene encoding gene and the protein B encoding gene have the gene fragment described in SEQ ID NO: 1 of the Sequence Listing.
  • the recombinant plasmid RNAi pBI121-p35S-22544 (interference vector) was constructed. After sequencing, the recombinant plasmid RNAi pBI121-p35S-22544 was the circular plasmid shown in SEQ ID NO: 2 of the Sequence Listing. The recombinant plasmid RNAi pBI121-p35S-22544 has the expression cassette shown in SEQ ID NO: 3 of the Sequence Listing.
  • nucleotides 1-543 are the 35S promoter
  • nucleotides 626-12-33 are the genes encoding the interfering RNA
  • the nucleotide of -1233 is DNA fragment B
  • the DNA fragment A and the DNA fragment B are separated by a sequence
  • the DNA fragment A and the DNA fragment B are reverse-complementary
  • the nucleotides 1251-1509 are NOS terminators.
  • a double-stranded DNA molecule Ubi-22544-NOS (interference fragment) was prepared.
  • the DNA molecule Ubi-22544-NOS is shown in SEQ ID NO: 4 of the Sequence Listing.
  • nucleotides 67 to 2078 are Ubi promoters
  • nucleotides 2161-2768 are interfering RNA encoding genes
  • nucleotides 2161-2397 are DNA fragments A
  • 2532 - 2768 nucleotides are DNA fragment B
  • DNA fragment A and DNA fragment B are spacer sequences
  • DNA fragment A and DNA fragment B are reverse-complementary
  • nucleotides 2786-3044 are NOS terminators.
  • the recombinant plasmid RNAi pBI121-p35S-22544 was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium.
  • Disinfectant An aqueous solution containing 10% by volume of NaClO and 0.05% by volume of tween-20.
  • step 3 cut fresh leaves of the plant, rinse with 70% ethanol solution for 1 min, then disinfect with 10% (volume percent) NaClO aqueous solution for 10 min, remove the veins and leaf edges, and use the puncher for the rest.
  • the hole was obtained as a disc having a diameter of 1 cm 2 and was an explant.
  • step 4 Obtain the explant obtained in step 3. Place the paraxial surface on the pre-culture medium and incubate for 2 days.
  • Preculture medium MS solid medium containing 0.1 mg L -1 IAA and 1 mg L -1 6-BA.
  • step 4 the explants are taken, soaked in the Agrobacterium liquid obtained in the step 1, and gently shaken and infested for 3-5 minutes.
  • step 5 After completing step 5, the explants were taken, and the residual bacterial liquid was removed by a filter paper, and then placed on a preculture medium, and cultured in the dark for 2 days.
  • step 6 the explants are taken and placed on the induction medium for light-dark alternating culture (lighting for 16 hours/dark for 8 hours) until the bud length is 1-2 cm.
  • Induction medium MS solid medium containing 0.1 mg L -1 IAA, 1 mg L -1 6-BA, 160 mg -1 Timentin, 100 mg L -1 Kan.
  • step 7 transfer to rooting medium and culture until rooting to obtain regenerated plants (T 0 generation).
  • Rooting medium MS solid medium containing 100 mg L -1 Kan.
  • the genomic DNA of the T 0 generation regenerated plants was extracted, and PCR amplification was carried out using a primer pair consisting of 22544F and 22544R (the target sequence was 576 bp), followed by 1% agarose gel electrophoresis.
  • the recombinant plasmid RNAi pBI121-p35S-22544 was used as a positive control for genomic DNA of regenerated plants.
  • Genomic DNA of tobacco W38 was used as a negative control for genomic DNA of regenerated plants.
  • FIG. 1 M: 100 bp DNA Ladder; 1: positive control; 2: negative control; 3-6: different regenerated plants.
  • the results showed that the regenerated plants corresponding to lane 3 were negative for PCR and were non-transgenic plants; the regenerated plants corresponding to lanes 4 to 6 were positive by PCR and were transgenic plants.
  • the T 0 generation transgenic plants were taken, genomic DNA was extracted, and the restriction endonuclease Sac I was used for single digestion and Southern hybridization.
  • Southern hybridization probe preparation method using the recombinant plasmid RNAi pBI121-p35S-22544 as a template, using a primer pair consisting of P22544F and P22544R (target sequence is 459 bp) for PCR amplification, and then taking PCR amplification products, using digoxin mark.
  • the recombinant plasmid RNAi pBI121-p35S-22544 was used as a positive control for genomic DNA of transgenic plants.
  • Genomic DNA of tobacco W38 was used as a negative control for genomic DNA of transgenic plants.
  • P22544F 5'-ACCGTTTTTCATCATACGACCGCAT-3';
  • P22544R 5'-CCATGCCATTGTGCAGCTTACCTG-3'.
  • the T 0 generation transgenic plants were selfed to obtain progeny, which is a T 1 generation plant.
  • the T 1 generation plants were selfed to obtain progeny, which was a T 2 generation plant.
  • the T 2 generation plants were selfed to obtain progeny, which is a T 3 generation plant.
  • Each T 1 progenies of the individual, T 2 and T 3 generation were random sample generation plants can be identified by PCR, with the method steps 1.
  • T 0 generation transgenic plant if all T 1 , T 2 , and T 3 plants of the plant are positive for PCR, the plant and its progeny can be regarded as a homozygous transgenic line.
  • Tob8 strain Tob11 strain
  • Tob63 strain Three homozygous transgenic lines were obtained: Tob8 strain, Tob11 strain and Tob63 strain.
  • Plants to be tested Tob8 strain T 2 of progenies, Tob11 lines T 2 of progenies, Tob63 lines T 2 of progenies.
  • the mRNA of the plant to be tested is extracted and subjected to Northern hybridization.
  • Northern hybridization probe preparation method using the recombinant plasmid RNAi pBI121-p35S-22544 as a template, using a primer pair consisting of 22544T7F and 22544probeR (target sequence is 256 bp) for PCR amplification, and then taking PCR amplification products for in vitro reversal Record and then mark it with digoxin.
  • the mRNA of tobacco W38 was used as a negative control for the mRNA of the transgenic plants.
  • U6, 28s rRNA and 18s rRNA were used as internal parameters.
  • 22544T7F 5'- TAATACGACTCACTATAGGAAT CCATCTACTTGGATAAATCTCG-3';
  • 22544probeR 5'-CTCAAGCAGACTCAGGCCCCTCGTG-3'.
  • the T7 promoter sequence is underlined.
  • the empty vector differs only in the absence of nucleotides 626-12-3 in sequence 3 of the sequence listing.
  • the empty vector was replaced by the recombinant plasmid RNAi pBI121-p35S-22544, and the steps were followed in accordance with step two to obtain an empty vector.
  • Plants to be tested W38 tobacco (wild type), empty vector transfected lines T 2 of the generation plants T 2 of lines Tob8 generation plants T 2 of lines Tob11 generation plants lines T 2 progenies of Tob63.
  • the peach aphid of the first instar stage was inoculated on the plant to be tested and cultured at 19 ° C to 22 ° C.
  • the survival of the aphids was recorded daily.
  • the skin and the clams were carefully Remove from the blade.
  • Three sets of biological replicates were set for each line, and at least 20 aphids were inoculated on each plant as technical repeats.
  • the number of days from the inoculation of peach aphid was counted, and the mortality was counted every 3 days.
  • the mortality results from the 18th day of cultivation were shown in Fig. 4.
  • the mortality rate of aphids on the Tob8 plants was 88% on average
  • the mortality of aphids on the Tob11 plants was 65% on average
  • the mortality of aphids on the Tob63 plants was 79% on average.
  • the mortality rate of mites averages 10%.
  • the number of days from the inoculation of peach aphid was counted, and the adult life, puerperium, life duration, daily average yield and total calyx of the aphids were counted.
  • the results are shown in Fig. 7.
  • the average daily yield of aphids on the transgenic plants was significantly reduced, the total alfalfa production was significantly reduced, the lifespan of the alfalfa was significantly shortened, the calving period was significantly shortened, and the length of life was significantly reduced, resulting in a population of aphids.
  • the number is significantly reduced.
  • the average generation duration (T), intrinsic growth rate (r m ), proliferation rate limit ( ⁇ ), and net reproductive rate (R 0 ) index of the population were calculated to estimate the number of individuals and determine the death factor.
  • Net fertility rate: R 0 ⁇ Lx ⁇ m x .
  • the average generation period: T ⁇ x ⁇ Lx ⁇ m x / R 0 .
  • Intrinsic growth rate: r m LnR 0 /T.
  • Proliferation rate limit: ⁇ e rm .
  • Population doubling time: t Ln2 / r m .
  • x is the representative age (d)
  • Lx is the survival rate of the x phase
  • m x is the number of females per female.
  • the results are shown in Table 1.
  • the experimental data showed that there was no significant difference in the intrinsic growth rate, the proliferation rate limit, and the population doubling time of the aphids on the transgenic lines compared with the aphids on the wild type plants, indicating that when other species were excluded, at any specific temperature Under the combination of humidity, food quality, etc., the tested aphids on all lines have the same maximum growth rate and growth potential; compared with the aphids on wild-type plants, the net reproductive rate and average of the aphids on the transgenic lines The generational cycle is significantly reduced.
  • the plants to be tested were: W38 tobacco (wild type), T 2 generation plant of strain Tob8, T 2 generation plant of strain Tob11, T 2 generation plant of strain Tob63.
  • One-year-old peach aphid was inoculated on the plants to be tested, and cultured at 19 ° C to 22 ° C. When the development of the molting and calving were observed, the molting and the scorpion were carefully removed from the leaves. Three sets of biological replicates were set for each line, and at least 20 aphids were inoculated on each plant as technical repeats.
  • the number of days from the inoculation of peach aphid The number of days from the inoculation of peach aphid, the aphid samples were taken every 3 days, total RNA was extracted and reverse transcribed into cDNA, and the primer pair consisting of My22544F and My22544R was used for qRT-PCR to detect the expression level of the target sequence of interfering RNA.
  • the internal reference gene is the Myzus Actin gene, and ActinF and ActinR constitute a primer pair for identifying the internal reference gene.
  • My22544F 5'-ACGTACCCGACTGTTTGGAA-3';
  • My22544R 5'-GTGGTTCAGGCTCACAACGA-3'.
  • the relative expression levels of target sequences in Myzus persicae are shown in Figure 8. After 6 days of culture, the relative expression levels of target genes in the aphids on the transgenic plants decreased to a very significant level compared to the aphids on the wild type plants. On day 12, the target gene in the aphids on the transgenic plants was almost completely silenced.
  • Aphids on wild-type plants After 6 days of culture, the target gene expression in aphids was highest; After 9 days of feeding, the aphids basically developed into 4th instar or adultery, and the expression level of the target gene fell back. After 12 days of culture, the aphids developed into adult mites, and the expression level of the target gene increased again.
  • Aphids on transgenic plants After 3 days of culture, the expression of target genes began to be lower than that of wild-type plants; after 6 days of culture, the expression of target genes was lower than that of wild-type plants, and the difference was extremely significant; after 9 days of culture After 12 days of culture, the expression level of the target gene was significantly lower than that of the wild type plant.
  • Induction medium MS + 1 mg / L VB 1 + 150 mg / L ASP + 2 mg / L 2, 4-D.
  • step 2 Take the DNA molecule Ubi-22544-NOS and use the PDS-1000/He gene gun of BIO-RAD to bombard the explants of step 1 (Psi 1100, 27.5 cm Hg column), and then transfer the explants. On a new induction medium, incubate at 22-25 ° C for 2-3 weeks in the dark.
  • step 2 After completing step 2, the explants are taken, and regeneration, screening and seedlings are sequentially carried out to obtain T 0 generation regenerated plants.
  • the genomic DNA of T 0 generation regenerated plants was extracted, and primers consisting of UbiF and AdhR were used for PCR amplification of primers consisting of A and AdhF and NosR. If primers were used, 524 bp amplification product was obtained and primer pair B was used. An amplification product of 541 bp was obtained, which was positive by PCR.
  • AdhR 5'-CAGATAAGCCGCCAAGAAGG-3'.
  • AdhF 5'-CCAAGGTATCTAATCAGCCATCC-3';
  • NosR 5'-TATAATTGCGGGACTCTAATC-3'.
  • the T 0 generation transgenic plants were selfed to obtain progeny, which is a T 1 generation plant.
  • the T 1 generation plants were selfed to obtain progeny, which was a T 2 generation plant.
  • the T 2 generation plants were selfed to obtain progeny, which is a T 3 generation plant.
  • Each T 1 progenies of the individual, T 2 and T 3 generation were random sample generation plants can be identified by PCR, with the method steps 1.
  • T 0 generation transgenic plant if all T 1 , T 2 , and T 3 plants of the plant are positive for PCR, the plant and its progeny can be regarded as a homozygous transgenic line.
  • the plants to be tested were: wheat variety KONIN 199 (wild type), 234-1 strain T 3 generation plant, 297-3 strain T 3 generation plant, and 309-8 strain T 3 generation plant.
  • the plants to be tested were taken, and the leaves were clamped with a positive cube eco-box (2.5 ⁇ 2.5 ⁇ 2.5 cm), and the larvae of the larvae of the larvae inoculated within 24 hours (inoculation amount of 15 plants/plants) were cultured.
  • the culture conditions were: 18-20 ° C for 16 hours (400 W sodium lamp, light intensity of 750 ⁇ Es -1 m -2 ) / 10-14 ° C dark for 8 hours, humidity of 50-70%. From the beginning of the inoculation, observe twice a day to record the growth and development of the long tuber (the cockroach, the larvae and the dead cockroaches are removed at any time until the death of the cockroach). 2-10 sets of biological replicates were set for each line.
  • the following biological parameters of the wheat long tube mites are counted: mortality (lethality rate), developmental duration of nymphs of various ages, nymphal period, pre-calving period, duration, sputum life, puerperium (production) ⁇ Duration), length of life, average daily production and total output.
  • the results of Chengyu's life, puerperium and life duration are shown in Figure 9.
  • the results of daily average yield and total yield are shown in Figure 10.
  • the results of the mortality rate are shown in Figure 11.
  • the results of the developmental period, the nymphal period, the pre-calving period and the duration of the nymphs of each age are shown in Fig. 12.
  • the mortality of aphids on transgenic wheat lines was significantly increased, and the daily average yield and total alfalfa production were significantly reduced. If the flood season, duration, calving period and life time were significantly shortened, the yield was significantly reduced. The previous period was significantly extended. The results showed that transgenic wheat had significant anti-caries results.
  • the inventors of the present invention isolated the aphid-specific target gene 22544 from the intestinal tract of aphids, and the preliminary in vitro feeding results indicated that it is a better RNAi target gene.
  • the target gene 22544 was introduced as an object of inhibition expression, and the interference fragment or the interference vector was introduced, and both the transgenic tobacco and the transgenic wheat showed a good anti-sputum phenotype.
  • the invention provides a reference for excavating and identifying novel anti-caries genes, and improving the anti-caries properties of tobacco, wheat and other crops by plant-mediated RNAi genetic engineering breeding.
  • the invention has great application and promotion value for breeding anti-tubing transgenic crops.

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Abstract

提供了一种培育抗蚜烟草和小麦的方法。所述方法包括：抑制出发植物中特异基因或特异基因片段的表达，得到抗蚜转基因植物。所述特异基因片段是从蚜虫肠道中分离出蚜虫特异的靶标基因22544，核苷酸序列如序列1所示，所述特异基因为具有序列1所示核苷酸序列的基因。还提供了一种用于培育抗蚜转基因植物的系统，包括抑制出发植物中特异基因或特异基因片段的表达的干扰片段或干扰载体。将靶标基因22544作为被抑制表达的对象，导入干扰片段或干扰载体，获得的转基因植物表现出良好的抗蚜表型。

Description

一种培育抗蚜烟草和小麦的方法和技术体系 技术领域
本发明涉及一种培育抗蚜烟草和小麦的方法和技术体系。
背景技术
蚜虫是一种重要的世界性农业害虫。世界范围内每年因蚜虫造成的直接经济损失数以亿计。蚜虫种类繁多，包括约10个科4400多种蚜虫，在植物整各个生育期都可造成危害。蚜虫取食作物不仅可造成严重减产而且还是许多病毒的传播载体。蚜虫通过刺吸式口器吸食植物营养，影响植株正常生长；分泌的蜜露影响植物光合作用；许多蚜虫还是植物病毒的传播者，如小麦黄矮病毒。在报道的300多种昆虫病毒传播介体中，170多种为蚜虫。其中，麦长管蚜和桃蚜是多种经济作物的主要害虫，对农业生产造成了重大的经济损失。据统计，中国每年小麦蚜虫危害面积可高达0.17亿公顷，占小麦总种植面积的62％。桃蚜是广食性害虫，寄主约有350多种，主要危害烟草、桃、李、梅、梨等果树和白菜、萝卜、辣椒、菠菜等蔬菜，造成了很大的经济损失。近年来，随着大量氮肥的使用及CO2浓度的升高，耕作制度变化等因素，使蚜虫的繁殖能力和适应性显著增强，其危害日趋严重。目前蚜虫防治以喷洒农药为主，但过多使用农药不仅造成环境污染，而且易使蚜虫产生抗药性，另外农药的使用对蚜虫天敌也可造成危害。培育抗蚜虫小麦和蔬菜品种是防止蚜虫危害的最有效途径，但由于现有种质资源中缺乏有效的抗蚜基因，抗性机制尚不明确，常规育种难以奏效。挖掘和利用新型抗蚜基因并通过基因工程培育作物抗蚜新种质具有重要意义。
植物介导的RNAi技术已成为农作物抗虫基因工程的热点之一，通过寄主植物表达相应昆虫特异基因的dsRNA，昆虫取食植物后沉默其相应的基因从而达到控制害虫危害的目的。RNAi现象其作用过程是双链RNA(dsRNA)进入生物体内，被Dicer酶切割成21-23nt的siRNA，siRNA与RNA诱导沉默复合物结合，与互补序列的靶mRNA结合，被Dicer识别，造成靶基因表达量的下降。近年来，利用dsRNA体外饲喂或注射来筛选RNA靶标基因，导致靶标基因表达和沉默，已经广泛应用于昆虫生长发育关键基因的鉴定和功能分析。孟山都公司通过植物介导的昆虫肠道特异基因RNAi技术成功获得了抗玉米 根螟的转基因玉米，有效缓解了长期应用Bt转基因玉米诱发昆虫产生抗性等问题，目前已完成生产性试验。
发明公开
本发明的目的是提供一种培育抗蚜烟草和小麦的方法和技术体系。
本发明首先保护一种制备抗蚜转基因植物的方法，包括如下步骤：抑制出发植物中特异基因或特异基因片段的表达，得到抗蚜转基因植物；所述特异基因为具有序列表的序列1所示核苷酸序列的基因；所述特异基因片段如序列表的序列1所示。
所述“抑制出发植物中特异基因或特异基因片段的表达”的实现方法如下：在所述出发植物中导入干扰载体或干扰片段。
所述干扰片段中具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。所述干扰RNA的编码基因具体如序列表的序列4第2161-2768位核苷酸所示。所述干扰片段自上游至下游依次包括：启动子、干扰RNA的编码基因和终止子。所述启动子具体可为Ubi启动子。所述终止子具体可为NOS终止子。所述干扰片段具体可如序列表的序列4中第67-3044位核苷酸所示。所述干扰片段具体可如序列表的序列4所示。
所述干扰载体中具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。所述干扰RNA的编码基因具体如序列表的序列3第626-1233位核苷酸位核苷酸所示。所述干扰载体中包括特异表达盒。所述特异表达盒自上游至下游依次包括：启动子、干扰RNA的编码基因和终止子。所述启动子具体可为35S启动子，所述终止子具体可为NOS终止子。所述特异表达盒具体如序列表的序列3所示。所述干扰载体具体为序列表的序列2所示的环形质粒。
所述抗蚜为抗蚜虫。所述蚜虫具体可为桃蚜或麦长管蚜。
所述出发植物为单子叶植物或双子叶植物。所述出发植物具体可为小麦(例如小麦品种科农199)或烟草(例如烟草W38)。
本发明还保护一种DNA分子(干扰片段)，具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如 序列表的序列1所示；所述片段乙与所述片段甲反向互补。所述干扰RNA的编码基因具体如序列表的序列4第2161-2768位核苷酸所示。所述干扰片段自上游至下游依次包括：启动子、干扰RNA的编码基因和终止子。所述启动子具体可为Ubi启动子。所述终止子具体可为NOS终止子。所述干扰片段具体可如序列表的序列4中第67-3044位核苷酸所示。所述干扰片段具体可如序列表的序列4所示。
本发明还保护一种重组质粒(干扰载体)，具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。所述干扰RNA的编码基因具体如序列表的序列3第626-1233位核苷酸位核苷酸所示。所述干扰载体中包括特异表达盒。所述特异表达盒自上游至下游依次包括：启动子、干扰RNA的编码基因和终止子。所述启动子具体可为35S启动子，所述终止子具体可为NOS终止子。所述特异表达盒具体如序列表的序列3所示。所述干扰载体具体为序列表的序列2所示的环形质粒。
本发明还保护所述干扰片段或所述干扰载体在培育抗蚜转基因植物中的应用。所述抗蚜为抗蚜虫。所述蚜虫具体可为桃蚜或麦长管蚜。所述植物为单子叶植物或双子叶植物。所述植物具体可为小麦(例如小麦品种科农199)或烟草(例如烟草W38)。
本发明还保护一种用于培育抗蚜转基因植物的系统，包括所述干扰片段或所述干扰载体。所述抗蚜为抗蚜虫。所述蚜虫具体可为桃蚜或麦长管蚜。所述植物为单子叶植物或双子叶植物。所述植物具体可为小麦(例如小麦品种科农199)或烟草(例如烟草W38)。
本发明还保护具有序列表的序列1所示核苷酸序列的基因。
本发明还保护来源于蚜虫的具有序列表的序列1所示核苷酸序列的基因。所述蚜虫具体可为桃蚜或麦长管蚜。
本发明还保护所述基因编码的蛋白质。
本发明还保护所述基因作为被沉默的靶标在培育抗蚜植物中的应用。所述被沉默具体可为将所述基因全部或部分敲除。所述被沉默具体可为抑制所述基因的表达。所述抗蚜为抗蚜虫。所述蚜虫具体可为桃蚜或麦长管蚜。所述植物为单子叶植物或双子叶植物。所述植物具体可为小麦(例如小麦品种 科农199)或烟草(例如烟草W38)。
本发明还保护所述蛋白质作为被抑制的靶标在培育抗蚜植物中的应用。所述抗蚜为抗蚜虫。所述被抑制具体可为降低所述蛋白质在蚜虫中的水平。所述被抑制具有可为抑制所述蛋白质在蚜虫的活性。所述蚜虫具体可为桃蚜或麦长管蚜。所述植物为单子叶植物或双子叶植物。所述植物具体可为小麦(例如小麦品种科农199)或烟草(例如烟草W38)。
附图说明
图1为实施例2中PCR鉴定结果。
图2为实施例2中Southern杂交鉴定的结果。
图3为实施例2中Northern杂交鉴定的结果。
图4为实施例2中死亡率的结果。
图5为实施例2中同一蚜虫的两个后代的照片。
图6为实施例2中蚜虫发育每一龄期的历经时间及世代历期的结果。
图7为实施例2中成蚜寿命、产蚜期、生活时长、日均产蚜量和总产蚜量的结果。
图8为实施例2中桃蚜中靶序列的相对表达水平的结果。
图9为实施例3中成蚜寿命、产蚜期、生活时长的结果。
图10为实施例3中日均产蚜量和总产蚜量的结果。
图11为实施例3中死亡率的结果。
图12为实施例3中蚜虫发育每一龄期的历经时间以及若蚜期、产蚜前期、历期的结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
1龄若蚜期：从出生一刻开始到褪掉第一层皮为止的时间段。2龄若蚜期：从褪掉第一层皮开始到褪掉第二层皮为止的时间段。3龄若蚜期：从褪掉第二层皮开始到褪掉第三层皮为止的时间段。4龄若蚜期：从褪掉第三层皮开始到褪掉第四层皮为止的时间段。若蚜期：从出生一刻开始到褪掉第四层皮 为止的时间段。产蚜前期：从褪掉第四层皮开始到产生第一头子蚜之前的时间段。历期：蚜虫从出生到产生第一头子蚜的时间段。生活时长：蚜虫从出生到死亡的时间段。日均产蚜量：蚜虫在开始生第一头蚜虫到生最后一头蚜虫期间的日均产蚜量。总产蚜量：蚜虫整个生命过程中产蚜总数。
根癌农杆菌AGL1：Y.He,H.D.Jones,S.Chen,X.M.Chen,D.W.Wang,K.X.Li,D.S.Wang and L.Q.Xia Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L.var.durum cv Stewart)with improved efficiency.Journal of Experimental Botany,Vol.61,No.6,pp.1567–1581,2010。
桃蚜，Myzus persicae (Sulzer)：Christiaens O.,Swevers L.,Smagghe G.DsRNA degradation in the pea aphid (Acyrthosiphon pisum) associated with lack of response in RNAi feeding and injection assay.Peptides,2014,53:307-314.。
麦长管蚜，Sitobion avenae (Fabricius)：Abdellatef E.,Will T.,Koch A.,Imani J.,Vilcinskas A.,Kogel K.H.Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae.Plant Biotechnology Journal,2015,13:849-857.。
烟草W38(Nicotiana tabacum L.cv W38)：Yu X.D.,Zhang Y.J.,Ma Y.Z.,Xu Z.S.,Wang G.P.,Xia L.Q.Expression of an (E)-β-farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation.The Crop Journal,2013,1:50-60.。
小麦品种科农199(Kenong199)：闵东红,何莎,张彦,夏兰琴.基因枪转化小麦主要轰击参数的优化.作物学报,2013,1:60-67。
实施例1、基因片段的发现以及干扰载体/干扰片段的构建
一、功能蛋白及其编码基因的发现
从麦长管蚜中发现一个新蛋白，将其命名为蛋白甲。从桃蚜中发现一个新蛋白，将其命名为蛋白乙。蛋白甲的编码基因和蛋白乙的编码基因中均具有序列表的序列1所述的基因片段。
二、干扰载体和干扰片段的构建
构建重组质粒RNAi pBI121-p35S-22544(干扰载体)。经测序，重组质粒RNAi pBI121-p35S-22544为序列表的序列2所示的环形质粒。重组质粒RNAi pBI121-p35S-22544中具有序列表的序列3所示的表达盒。序列表的序列3中，第1-543位核苷酸为35S启动子，第626-1233位核苷酸为干扰RNA的编码基因(第626-862位核苷酸为DNA片段甲，第997-1233位核苷酸为DNA片段乙，DNA片段甲和DNA片段乙之间为间隔序列，DNA片段甲和DNA片段乙反向互补)，第1251-1509位核苷酸为NOS终止子。
制备双链的DNA分子Ubi-22544-NOS(干扰片段)。DNA分子Ubi-22544-NOS如序列表的序列4所示。序列表的序列4中，第67-2078位核苷酸为Ubi启动子，第2161-2768位核苷酸为干扰RNA的编码基因(第2161-2397位核苷酸为DNA片段甲，第2532-2768位核苷酸为DNA片段乙，DNA片段甲和DNA片段乙之间为间隔序列，DNA片段甲和DNA片段乙反向互补)，第2786-3044位核苷酸为NOS终止子。
实施例2、转基因烟草的制备和鉴定
一、转基因烟草的制备
1、将重组质粒RNAi pBI121-p35S-22544导入根癌农杆菌AGL1，得到重组农杆菌。用LB培养基悬浮重组农杆菌，得到OD600nm＝1.0的农杆菌菌液。
2、取烟草W38种子，用70％酒精水溶液灭菌1min，然后用消毒液消毒20min，然后用无菌水漂洗5次，然后置于MS固体培养基上培养至植株长出4-5片叶子。
消毒液：含10％(体积百分含量)NaClO和0.05％(体积百分含量)tween-20的水溶液。
3、完成步骤2后，剪取植株的新鲜叶子，用70％乙醇水溶液漂洗1min，然后用10％(体积百分含量)NaClO水溶液消毒10min，切除叶脉和叶片边缘，剩余部分用打孔器打孔，得到直径1cm2大小的圆片，即为外植体。
4、取步骤3得到的外植体，近轴面向上置于预培养培养基上，光照培养2天。
预培养培养基：含0.1mg L-1IAA和1mg L-1 6-BA的MS固体培养基。
5、完成步骤4后，取外植体，浸泡于步骤1得到的农杆菌菌液中，轻轻晃动，侵染3-5min。
6、完成步骤5后，取外植体，用滤纸吸除残留菌液，然后置于预培养培养基上，避光培养2天。
7、完成步骤6后，取外植体，置于诱导培养基上进行光暗交替培养(光照16小时/黑暗8小时)，直至芽长为1-2cm。
诱导培养基：含0.1mg L-1IAA、1mg L-1 6-BA、160mg-1 Timentin、100mg L-1Kan的MS固体培养基。
8、完成步骤7后，转移到生根培养基上培养至生根，获得再生植株(T0代)。
生根培养基：含100mg L-1Kan的MS固体培养基。
二、分子鉴定
1、PCR鉴定
提取T0代再生植株的基因组DNA，采用22544F和22544R组成的引物对(靶序列为576bp)进行PCR扩增，然后进行1％琼脂糖凝胶电泳。采用重组质粒RNAi pBI121-p35S-22544作为再生植株的基因组DNA的阳性对照。采用烟草W38的基因组DNA作为再生植株的基因组DNA的阴性对照。
22544F：5’-TTCCATTGCCCAGCTATCTGTCACTTT-3’；
22544R：5’-TGTGAAACGGTGCATTATGATGGTGA-3’。
PCR鉴定结果见图1。图1中：M：100 bp DNA Ladder；1：阳性对照；2：阴性对照；3-6：不同的再生植株。结果表明：泳道3对应的再生植株PCR鉴定为阴性，为非转基因植株；泳道4至6对应的再生植株PCR鉴定为阳性，为转基因植株。
2、Southern杂交
取T0代转基因植株，提取基因组DNA，用限制性内切酶Sac I进行单酶切后进行Southern杂交。Southern杂交的探针的制备方法：以重组质粒RNAi pBI121-p35S-22544为模板，用P22544F和P22544R组成的引物对(靶序列为459bp)进行PCR扩增，然后取PCR扩增产物，用地高辛标记。采用重组质粒RNAi pBI121-p35S-22544作为转基因植株的基因组DNA的阳性对照。采用烟草W38的基因组DNA作为转基因植株的基因组DNA的阴性对照。
P22544F:5′-ACCGTTTTTCATCATACGACCGCAT-3′；
P22544R:5′-CCATGCCATTGTGCAGCTTACCTG-3′。
Southern 杂交鉴定结果见图2。图2中：P：阳性对照；CK：阴性对照；8：Tob8植株；11：Tob11植株；63：Tob63植株。结果表明，Tob8植株、Tob11植株和Tob63植株中导入的外源片段的拷贝数依次为3个拷贝、4个拷贝和3个拷贝，外源片段在植物基因组中成功整合。
3、获得纯合转基因株系
将T0代转基因植株自交获得后代，即为T1代植株。将T1代植株自交获得后代，即为T2代植株。将T2代植株自交获得后代，即为T3代植株。分别对各个T1代植株、T2代植株以及随机抽样的T3代植株进行PCR鉴定，方法同步骤1。
对于某一T0代转基因植株来说，如果该植株的所有T1代、T2代、T3代植株均为PCR鉴定阳性，该植株及其后代可视为一个纯合的转基因株系。
得到了三个纯合的转基因株系：Tob8株系、Tob11株系和Tob63株系。
4、Northern杂交
待测植株为：Tob8株系的T2代植株、Tob11株系的T2代植株、Tob63株系的T2代植株。提取待测植株的mRNA，进行Northern杂交。Northern杂交的探针的制备方法：以重组质粒RNAi pBI121-p35S-22544为模板，用22544T7F和22544probeR组成的引物对(靶序列为256bp)进行PCR扩增，然后取PCR扩增产物进行体外反转录，然后用进行地高辛标记。采用烟草W38的mRNA作为转基因植株的mRNA的阴性对照。采用U6、28s rRNA和18s rRNA为内参。
22544T7F：5′-TAATACGACTCACTATAGGAATCCATCTACTTGGATAAATCTCG-3′；
22544probeR：5′-CTCAAGCAGACTCAGGCCCCTCGTG-3′。
22544T7F中，下划线标注T7启动子序列。
Northern杂交的结果见图3。图3中：CK：阴性对照；8：Tob8株系；11：Tob11株系；63：Tob63株系。结果表明：外源片段在转基因植株的转录水平有表达。
三、转空载体烟草的制备
与重组质粒RNAi pBI121-p35S-22544相比，空载体的差异仅在于缺少了序列表的序列3中第626-1233位核苷酸。将空载体代替重组质粒RNAi pBI121-p35S-22544，依次按照步骤一并参照步骤二进行操作，得到转空载体 株系。
四、抗蚜效果鉴定
待测植株为：W38烟草(野生型)、转空载体株系的T2代植株、株系Tob8的T2代植株、株系Tob11的T2代植株、株系Tob63的T2代植株。
取1龄若蚜期的桃蚜，接种到待测植株上，19℃-22℃培养，每日观察记录蚜虫的存活情况，在观察到蜕皮发育和产蚜时，将蜕皮和若蚜小心的从叶片上移除。每个株系设置3组生物学重复，每植株上至少接种20只蚜虫作为技术重复。
从接种桃蚜开始计天数，每3天统计死亡率，培养至第18天的死亡率结果见图4。培养18天后，株系Tob8植株上蚜虫的死亡率平均为88％，株系Tob11植株上蚜虫的死亡率平均为65％，株系Tob63植株上蚜虫的死亡率平均为79％，野生型植株上蚜虫的死亡率平均为10％。
观察蚜虫及其后代的生长发育状况。转基因株系植株上一些新生蚜虫出现发育不正常现象，正常新生蚜虫会在出生后出于本能将蜷缩在一起的肢体展开，然后开始自由取食活动，但畸形发育的蚜虫似丧失了肢体展开的能力，最终蜷缩死亡。同一蚜虫的两个后代的照片见图5，其中1个后代正常发育，另一个后代畸形发育(无法展开肢体，最终虫体颜色变黄变暗，蜷缩死亡)。
从接种桃蚜开始计天数，统计蚜虫发育每一龄期的历经时间及世代历期，结果见图6。野生型植株上的蚜虫与各转基因株系植株上的蚜虫之间无显著性差异，说明在植株中导入重组质粒RNAi pBI121-p35S-22544，没有影响到蚜虫的发育历期。
从接种桃蚜开始计天数，统计蚜虫的成蚜寿命、产蚜期、生活时长、日均产蚜量和总产蚜量，结果见图7。与野生型植株上的蚜虫相比，转基因植株上的蚜虫日均产蚜量显著降低，总产蚜量显著降低，成蚜寿命显著缩短，产蚜期显著缩短，生活时长显著减少，使蚜虫种群数量明显降低。计算种群的平均世代历期(T)、内禀增长率(rm)、增殖率极限(λ)及净生殖率(R0)指数，以此进行个体数的估算和死亡因子的确定。净生殖率：R0＝∑Lx·mx。平均世代周期：T＝∑x·Lx·mx/R0。内禀增长率：rm＝LnR0/T。增殖率极限：λ＝erm。种群加倍时间：t＝Ln2/rm。x为代表性年龄(d)，Lx为x期的存活率，mx为每雌产雌数。结果见表1。实验数据显示：与野 生型植株上的蚜虫相比，转基因株系上的蚜虫的内禀增长率、增殖率极限、种群加倍时间均无显著性差异,说明当排除其他物种，在任一特定的温度、湿度、食物质量等的组合下，所有株系上的测试蚜虫均有同等的最大增长率及增长潜能；与野生型植株上的蚜虫相比，转基因株系上的蚜虫的净生殖率和平均世代周期显著减小。
转空载体植株的各个结果数据，均与野生型植株无显著差异。
表1
Figure PCTCN2017109851-appb-000001
五、干扰RNA对靶序列的沉默效果
待测植株为：W38烟草(野生型)、株系Tob8的T2代植株、株系Tob11的T2代植株、株系Tob63的T2代植株。
取1龄桃蚜，接种到待测植株上，19℃-22℃培养，在观察到蜕皮发育和产蚜时，将蜕皮和若蚜小心的从叶片上移除。每个株系设置3组生物学重复，每植株上至少接种20只蚜虫作为技术重复。
从接种桃蚜开始计天数，每3天取蚜虫样本，提取总RNA并反转录为cDNA,采用My22544F和My22544R组成的引物对进行qRT-PCR，检测干扰RNA的靶序列的表达水平。内参基因为桃蚜Actin基因，ActinF和ActinR组成用于鉴定内参基因的引物对。
My22544F：5′-ACGTACCCGACTGTTTGGAA-3′；
My22544R:5′-GTGGTTCAGGCTCACAACGA-3′。
ActinF：5′-CGGTTCAAAAACCCAAACCAG-3′；
ActinR：5′-TGGTGATGATTCCCGTGTTC-3′。
桃蚜中靶序列的相对表达水平见图8。培养6天后，与野生型植株上的蚜虫相比，转基因植株上的蚜虫中的靶基因的相对表达水平下降,达到极显著水平。第12天时，转基因植株上的蚜虫中的靶基因几乎完全被沉默。
野生型植株上的蚜虫：培养6天后，蚜虫中的靶基因的表达量最高；培 养9天后，蚜虫基本发育为4龄若蚜或成蚜前期，靶基因的表达量回落；培养12天后，蚜虫发育为成蚜，靶基因的表达量再次升高。转基因植株上的蚜虫：培养3天后，靶基因的表达量开始低于野生型植株上的蚜虫；培养6天后，靶基因的表达量低于野生型植株上的蚜虫，差异极显著；培养9天后和培养12天后，靶基因的表达量均显著低于野生型植株上的蚜虫。
实施例3、转基因小麦的制备和鉴定
一、转基因小麦的制备
1、取小麦品种科农199授粉后第14天的幼胚，作为外植体接种于诱导培养基，22-25℃条件下暗培养1-2天。
诱导培养基：MS+1mg/L VB1+150mg/L ASP+2mg/L 2,4-D。
2、取DNA分子Ubi-22544-NOS，采用BIO-RAD公司的PDS-1000/He基因枪对完成步骤1的外植体进行轰击(Psi 1100，27.5cm Hg柱)，然后将外植体转移到新的诱导培养基上，22-25℃黑暗培养2-3周。
3、完成步骤2后，取所述外植体，依次进行再生、筛选和壮苗，得到T0代再生植株。
二、分子鉴定
1、PCR鉴定
提取T0代再生植株的基因组DNA，分别采用UbiF和AdhR组成的引物对甲和AdhF和NosR组成的引物对乙进行PCR扩增，如果采用引物对甲得到524bp的扩增产物且采用引物对乙得到541bp的扩增产物，PCR鉴定为阳性。
UbiF：5’-TTTAGCCCTGCCTTCATACG-3’；
AdhR：5’-CAGATAAGCCGCCAAGAAGG-3’。
AdhF：5’-CCAAGGTATCTAATCAGCCATCC-3’；
NosR：5’-TATAATTGCGGGACTCTAATC-3’。
2、获得纯合转基因株系
将T0代转基因植株自交获得后代，即为T1代植株。将T1代植株自交获得后代，即为T2代植株。将T2代植株自交获得后代，即为T3代植株。分别对各个T1代植株、T2代植株以及随机抽样的T3代植株进行PCR鉴定，方法同步骤1。
对于某一T0代转基因植株来说，如果该植株的所有T1代、T2代、T3代植株均为PCR鉴定阳性，该植株及其后代可视为一个纯合的转基因株系。
得到了三个纯合的转基因株系：234-1株系、297-3株系和309-8株系。
三、抗蚜效果鉴定
待测植株为：小麦品种科农199(野生型)、234-1株系的T3代植株、297-3株系的T3代植株、309-8株系的T3代植株。
取待测植株，用正立方体生态盒(2.5×2.5×2.5cm)将叶片其夹住，接种24h内的麦长管蚜初产若蚜(接种量为15只/株)，进行培养。培养条件为：18-20℃光照16小时(400W钠灯，光强为750μEs-1m-2)/10-14℃黑暗8小时，湿度为50-70％。从接种开始，每天观察两次，记载麦长管蚜的生长发育情况(期间随时移去蚜蜕、仔蚜和死蚜，直到成蚜死亡为止)。每个株系设置2-10组生物学重复。
根据调查结果，统计麦长管蚜的如下生物学参数：死亡率(致死率)、各龄若虫的发育历期、若蚜期、产蚜前期、历期、成蚜寿命、产蚜期(产蚜时长)、生活时长、日均产蚜量和总产蚜量。
成蚜寿命、产蚜期、生活时长的结果见图9。日均产蚜量和总产蚜量的结果见图10。死亡率的结果见图11。各龄若虫的发育历期、若蚜期、产蚜前期、历期的结果见图12。与野生型小麦相比，转基因小麦株系上的蚜虫的死亡率显著增高，日均产蚜量和总产蚜量显著降低，若蚜期、历期、产蚜期和生活时长显著缩短，产蚜前期显著延长。结果表明，转基因小麦具有明显的抗蚜结果。
工业应用
本发明的发明人从蚜虫肠道中分离出蚜虫特异的靶标基因22544，前期体外饲喂结果表明，它是一个较好的RNAi靶标基因。将靶标基因22544作为被抑制表达的对象，导入干扰片段或干扰载体，转基因烟草和转基因小麦均表现出良好的抗蚜表型。本发明为挖掘和鉴定新型抗蚜基因,并通过植物介导的RNAi基因工程育种提高烟草、小麦等农作物的抗蚜特性提供了参考。本发明对于培育抗蚜转基因作物具有重大的应用推广价值。

Claims (16)

  1. 一种制备抗蚜转基因植物的方法，包括如下步骤：抑制出发植物中特异基因或特异基因片段的表达，得到抗蚜转基因植物；所述特异基因为具有序列表的序列1所示核苷酸序列的基因；所述特异基因片段如序列表的序列1所示。
  2. 如权利要求1所述的方法，其特征在于：所述“抑制出发植物中特异基因或特异基因片段的表达”的实现方法如下：在所述出发植物中导入干扰载体或干扰片段。
  3. 如权利要求2所述的方法，其特征在于：所述干扰片段为具有干扰RNA的编码基因的DNA分子；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。
  4. 如权利要求2所述的方法，其特征在于：所述干扰载体为具有干扰RNA的编码基因的重组质粒；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。
  5. 如权利要求1至4中任一所述的方法，其特征在于：所述抗蚜为抗蚜虫；所述蚜虫为桃蚜或麦长管蚜。
  6. 一种DNA分子，具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。
  7. 一种重组质粒，具有干扰RNA的编码基因；所述干扰RNA的编码基因由片段甲、间隔序列和片段乙组成；所述片段甲如序列表的序列1所示；所述片段乙与所述片段甲反向互补。
  8. 干扰片段在培育抗蚜转植物中的应用；所述干扰片段为权利要求6所述DNA分子。
  9. 干扰载体在培育抗蚜转植物中的应用；所述干扰载体为权利要求7所述的重组质粒。
  10. 一种用于培育抗蚜转基因植物的系统，包括干扰片段；所述干扰片段为权利要求6所述DNA分子。
  11. 一种用于培育抗蚜转基因植物的系统，包括干扰载体；所述干扰载体为权利要求7所述的重组质粒。
  12. 具有序列表的序列1所示核苷酸序列的基因。
  13. 来源于蚜虫的具有序列表的序列1所示核苷酸序列的基因。
  14. 权利要求12或权利要求13所述基因编码的蛋白质。
  15. 权利要求12或权利要求13所述基因作为被沉默的靶标在培育抗蚜植物中的应用。
  16. 权利要求13所述蛋白质作为被抑制的靶标在培育抗蚜植物中的应用。
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