CN108866081A - A kind of raising cadmium resistance and the gene of cadmium content and application thereof - Google Patents

A kind of raising cadmium resistance and the gene of cadmium content and application thereof Download PDF

Info

Publication number
CN108866081A
CN108866081A CN201810827621.5A CN201810827621A CN108866081A CN 108866081 A CN108866081 A CN 108866081A CN 201810827621 A CN201810827621 A CN 201810827621A CN 108866081 A CN108866081 A CN 108866081A
Authority
CN
China
Prior art keywords
cadmium
gene
sactp
resistance
content
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810827621.5A
Other languages
Chinese (zh)
Other versions
CN108866081B (en
Inventor
刘明英
贺雪莲
卓仁英
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Original Assignee
Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Institute of Subtropical Forestry of Chinese Academy of Forestry filed Critical Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Priority to CN201810827621.5A priority Critical patent/CN108866081B/en
Publication of CN108866081A publication Critical patent/CN108866081A/en
Application granted granted Critical
Publication of CN108866081B publication Critical patent/CN108866081B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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)
    • C12N15/8241—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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to gene engineering technology fields, relate more specifically to a kind of raising cadmium resistance and the gene of cadmium content and application thereof；The present invention tires out mine type Sedum alfredii Hance cDNA library by building cadmium ultraproduct, is screened out from itSaCTPGene, by the experimental verification of yeast contact plate, the gene can improve yeast cadmium resistance；It willSaCTPGene heterogenous expression in arabidopsis, the cadmium resistance and cadmium ion content of transgenic arabidopsis get a promotion；Gained of the inventionSaCTPGene to cultivate there is high cadmium resistance high cadmium content plant new germ plasm to provide genetic resources, and the plant biological that can be applied to heavy-metal contaminated soil repairs field.

Description

A kind of raising cadmium resistance and the gene of cadmium content and application thereof
Technical field
The present invention relates to gene engineering technology field, relate more specifically to a kind of raising cadmium resistance and cadmium content gene and Its purposes.
Background technique
China is large agricultural country, soil be vertical agriculture this, as important natural environmental elements passively carry including Most of pollutant including heavy metal；The cultivated area of China's heavy metal pollution up to 20,000,000 hectares, is often only cadmium pollution Grain be up to 12,000,000 tons, caused by direct economic loss be more than 20,000,000,000 yuan.Heavy metal element it is difficult to degrade, easy enrichment and The transferability of food chain is brought to ecological environment and food safety and is seriously threatened, and is that human life and health and safety are " stealthy Killer ", the heavy metal pollutions such as " cadmium rice " food event takes place frequently in recent years, and painful disease is first, and cadmium pollution soil, which is administered to carve, not to be allowed It is slow.The functional study for carrying out the super accumulation type resistant gene in plant of cadmium illustrates the molecular mechanism of Plant Tolerance cadmium, cultivates cadmium resistance and turns Gene new germ plasm and improvement and reparation China's heavy-metal contaminated soil are of great significance.
Super accumulation type Sedum alfredii Hance is zinc/cadmium of China native country growth（Cd/Zn）Super accumulation type plant, on the ground part 9,000 μ g.g can be accumulated-1Cd and 29,000 μ g.g-1Zn is without being presented any signs of toxicity, great researching value；East The cadmium ion of high concentration is transported to aerial part by efficient root-stem transporting mechanism by southern red-spotted stonecrop, and is finally stored in mesophyll Parenchyma cell, stem marrow and cortical tissue in.
In order to parse super accumulation type Sedum alfredii Hance highly resistance and super accumulation ability, have researcher to participate in ion transport, chela The gene of the processes such as conjunction, oxidative damage reparation conducts a research；However, whether there is special cadmium in super accumulation type Sedum alfredii Hance Ion transport, removing toxic substances, response GAP-associated protein GAP are still unclear.It is existing research shows that super accumulation type Sedum alfredii HanceSaNramp6Gene can To improve the cadmium ion accumulation of plant, but excessively high cadmium ion accumulates so that the cadmium resistance of genetically modified plants reduces.Southeast scape It can not only promote the cadmium resistance of genetically modified plants with the presence or absence of a kind of gene in it but also cadmium content can be improved and need to conduct a research.
Summary of the invention
The present invention by building Sedum alfredii Hance cDNA library, by cadmium responsive type yeast screening assay cadmium resistant gene, for the first time from The gene with raising cadmium resistance and cadmium content function is had found in Sedum alfredii Hance cDNA library, and is named as Sedum alfrediiCd tolerance protein (SaCTP), it will be describedSaCTP geneIt is transferred to cadmium responsive type yeast mutants bacterium Strain（ycf1）In, it is substantially better than pair by yeast contact plate experiment discovery upgrowth situation of transgenic yeast on the culture medium containing cadmium According to group.It will be describedSaCTPGene heterogenous expression in arabidopsis, discovery heterogenous expression in arabidopsisSaCTPIt can be with after gene Significantly improve the cadmium resistance and cadmium ion uptake of transgenic arabidopsis.
An object of the present invention is to provide a kind of gene for improving cadmium resistance and cadmium content.
The second object of the present invention is to provide the transgenosis containing above-mentioned raising cadmium resistance and cadmium content related gene quasi- south Mustard plant.
The third object of the present invention, which is to provide, is improving plant containing above-mentioned raising cadmium resistance and Cd uptake amount related gene Cadmium resistance and cadmium content in terms of application.
In order to achieve the above object, the invention discloses a kind of raising cadmium resistance and the genes of cadmium contentSaCTP, the base CauseSaCTPSeparated from the Leaf cDNA Library of Sedum alfredii Hance to obtain, nucleotide sequence is as shown in SEQ.ID.No.1.
The present invention also provides the albumen of a kind of raising cadmium resistance and the coded by said gene of cadmium content, amino acid sequence is Shown in SEQ.ID.No.2.
The present invention also provides a kind of arabidopsis transgenic plants containing the gene for being improved cadmium resistance and cadmium content.
The present invention also provides the genes of a kind of raising cadmium resistance and cadmium content in cadmium resistance and the cadmium content side for improving plant The application in face.
Compared with prior art, the beneficial effects of the invention are as follows:
（1）The present invention provides a kind of with the plant code gene for improving cadmium resistance and cadmium contentSaCTP, the gene is at present still Have no relevant report research, which is derived from the Cd stress screening after Sedum alfredii Hance cDNA library yeast heterogenous expression；It is logical Cross buildingSaCTPYeast expression carrier, it is anti-to can verify that gene of the present invention can improve yeast cadmium using the experiment of yeast contact plate Property.
（2）The present invention willSaCTPIt is quasi- to find that the gene can significantly improve transgenosis for gene heterogenous expression in arabidopsis The cadmium resistance and cadmium ion content of southern mustard seedling and seedling, compared with common arabidopsis, the biomass of seedling is improved after Cd stress 35-50%, seedling root long improve 75-90%, and the cadmium ion content of seedling improves 20-60%.
（3）It is of the present inventionSaCTPGene to cultivate there is high cadmium resistance high cadmium content plant new germ plasm to provide gene Resource can be applied in the soil monitoring and improvement in cadmium pollution area, in research plant biological recovery technique, improve a soil huge sum of money Belong to pollution situation etc. to play an important role.
Detailed description of the invention
Fig. 1 is that the mixing plasmid Electroporation of the tired mine type Sedum alfredii Hance cDNA library of cadmium ultraproduct enters yeast mutantsycf1Containing 15 μM of L afterwards-1 CdCl2SD-U culture medium on bacterium colony growing state；
Fig. 2 is in the present inventionSaCTPThe yeast colony PCR of gene expands electrophoretogram；
Fig. 3 is T-easy-SaCTPPCR amplification electropherogram in Escherichia coli；
Fig. 4 is after transgenic arabidopsis T3 is cultivated ten days on 1/2MS solid medium for seed and wildtype Arabidopsis thaliana seed Phenotype comparison diagram；Wherein, Fig. 4 A is the control group without cadmium, and Fig. 4 B is to contain 100 uM CdCl2Stress group；
Fig. 5 is after transgenic arabidopsis T3 is cultivated ten days on 1/2MS solid medium for seed and wildtype Arabidopsis thaliana seed Root long and mean fresh comparison diagram；Wherein, Fig. 5 A is the root long comparison diagram after cultivating ten days, and Fig. 5 B is that 5 seedling are average fresh Weight comparison diagram；
Fig. 6 is wild-type Arabidopsis plants and overexpression after transplantingSaCTPTransgenic Arabidopsis plants phenotype comparison diagram；Its Middle Fig. 6 A is without CdCl2The Arabidopsis plant of Stress treatment, as control；Fig. 6 B is 0.5 mM CdCl2At stress 2 weeks Arabidopsis plant；
Fig. 7 is the metal cumulant comparison diagram of transgenic Arabidopsis plants and wild-type Arabidopsis plants.
Specific embodiment
With reference to the accompanying drawing, the present invention is further illustrated, these embodiments are merely to illustrate the present invention rather than limitation The scope of the invention；The reagent and biomaterial commercially obtain unless otherwise specified.
Experimental material and reagent：
（1）Bacterial strain and carrier：Escherichia coli/yeast shuttle vector pYES2.0G, Escherichia coli/yeast shuttle expression vector PYES2-DEST, Agrobacterium EHA105 are purchased from Invitrogen company.Bacillus coli DH 5 alpha is century biotechnology purchased from health Co., Ltd.T-easy carrier is purchased from Promega company.Cadmium responsive type yeast mutants bacterial strain（ycf1）Purchased from Euroscarf Company.
（2）Enzyme and kit：Total RNA Purification Kit plant tissue total RNA extraction reagent box is purchased from Norgen company.SMARTTMCDNA library constructs kit and is purchased from Clontech company.DNA gel QIAquick Gel Extraction Kit, plasmid DNA Mini Kit is purchased from Axygen biotech company.Restriction enzymesfi I Purchased from New England Biolabs company.
（3）Reaction substrate：SMART™ IV Oligonucleotide, CDS III/3' PCR Primer,5' PCR Primer is purchased from Clontech company.
Embodiment 1
Cadmium ultraproduct tires out the building of mine type Sedum alfredii Hance cDNA library
(1）Mine type Sedum alfredii Hance is tired out with the cadmium ultraproduct filtered out（The ancient Pb-Zn ore district in Quzhou City of Zhejiang Province one is picked up from, after It transplants to this laboratory）Rooted Cuttings are material, through 400 μM of L-1Caddy（CdCl2）It is total with plant tissue after Stress treatment RNA extracts kit (Total RNA Purification Kit) extracts the Sedum alfredii Hance blade total serum IgE under Cd stress, passes through Purifying mRNA is obtained by filtration in few (dT)-fibre columns；
(2）The acquisition of full-length cDNA
The synthesis of cDNA chain uses SMARTTMCDNA library constructs kit and completes；
The synthesis of the first chain of cDNA：Using purifying mRNA is template obtained in above-mentioned steps (1), SMART IV (sequence is as shown in SEQ.ID.No.3 by Oligonucleotide (10 μM)：5'- AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 ') and CDS III/3'PCR Primer (10 μM), sequence Column are as shown in SEQ.ID.No.4：5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3’(N = A, G, C, or T; N-1 =A, G, or C) it is used as primer, synthesis the first chain of cDNA is loaded according to kit specification；
The synthesis of the second chain of cDNA：Using the first chain of cDNA as template, (sequence is such as by CDS III/3'PCR Primer Shown in SEQ.ID.No.4) and 5'PCR Primer (sequence is as shown in SEQ.ID.No.5：5'- AAGCAGTGGTATCAACGCAGAGT-3 ') it is used as primer, buffer is sequentially added according to kit specification（Buffer）, DNTP Mix, Polymerase Mix and distilled water, synthesis obtains full-length cDNA in PCR instrument.
PCR response procedures：95℃→1min；（95℃→15sec；68℃→6min）× 20 circulations.
(3）Construction cDNA library
Withsfi IEnzyme is respectively to Escherichia coli/yeast shuttle vector pYES2.0G and above-mentioned steps（2）Obtained full-length cDNA Digestion is carried out, digestion products are observed by agarose gel electrophoresis and is greater than the item of 500bp with DNA gel QIAquick Gel Extraction Kit recycling Band；
By Escherichia coli/yeast shuttle vector pYES2.0G digestion recovery product and the full-length cDNA digestion products for being greater than 500 bp In 16 DEG C of connections overnight；Connection product is transferred in bacillus coli DH 5 alpha by Electroporation conversion, Electroporation product, which is applied to, to be contained Have and be inverted culture overnight for 37 DEG C on the LB culture medium of amicillin resistance, selects the monoclonal grown at random respectively with T7（Sequence Column are as shown in SEQ.ID.No.6：5'-TAATACGACTCACTATAGGG-3'）, pYES2-R（Sequence such as SEQ.ID.No.7 institute Show：5'-TCG GTT AGA GCG GAT GTG-3'）Primer detection carries out PCR identification.
Embodiment 2
The screening and identification for the transformant of resistance to cadmium
（1）Extract the mixing plasmid that cadmium ultraproduct obtained in embodiment 1 tires out mine type Sedum alfredii Hance cDNA library, Electroporation cadmium Responsive type yeast mutants（ycf1）：
CDNA library mixing plasmid is extracted using Plasmid DNA Mini Kit, extremely by the mixing plasmid Electroporation of extractionycf1In bacterial strain, Electroporation applied voltage 1.5V；Converted product coated plate is to containing 15 μM of L-1 CdCl2SG-U plate on； It is cultivated 3 days in 28 DEG C of inversions, yeast colony as shown in Figure 1 is uniform in size, well-grown.
（2）It carries out TA clone and is sequenced to obtain Insert Fragment sequence information：
Respectively to step in embodiment 2（1）Obtained yeast colony carries out PCR identification, is separated and is seen with agarose gel electrophoresis It examinesSaCTPGene target fragment band, PCR the primer are T7（As shown in SEQ.ID.No.6）And pYES2-R（Such as Shown in SEQ.ID.No.7）.As shown in Fig. 2, M is DNA molecular amount standard in figure；1 represents using T7 as primer detectionSaCTPGene Yeast colony PCR product, 2 represent using pYES2-R as primer detectionSaCTPThe PCR product of the yeast colony of gene；It uses DNA gel QIAquick Gel Extraction Kit recyclingSaCTPThe PCR band of gene yeast conversion daughter colony.PCR is connected by TA clone technology to return Product and T-easy carrier are received, is transferred to bacillus coli DH 5 alpha, and LB training of the coated plate extremely containing amicillin resistance by heat shock method It supports and is incubated overnight on base in 37 DEG C of inversions.Picking monoclonal carries out T-easy-SaCTPPCR is detected in Escherichia coli, electrophoresis knot For fruit as shown in figure 3, M is DNA molecular weight standard in figure, it is primer detection T-easy- that 1-3, which respectively represents M13F/M13R,SaCTP PCR product in Escherichia coli.
Bacterium solution is sent into the sequencing of Shanghai Sheng Gong bioengineering Co., Ltd, obtains the sequence in sequence table by sequence assembly SEQ.ID.No.1.It is obtained according to sequencingSaCTPThe open reading frame of gene（ORF）, extrapolate the amino of gene coded protein Acid sequence SEQ.ID.No.2.PCR and sequencing primer are T-easy carrier universal primer M13-F（As shown in SEQ.ID.No.8： 5'-TGTAAAACGACGGCCAGT-3'）And M13-R（As shown in SEQ.ID.No.9：5'-CAGGAAACAGCTATGACC-3'）.
Embodiment 3
Yeast contact plate experimental verificationSaCTPGene cadmium resistance
It is obtained according to sequencingSaCTPThe open reading frame of gene（ORF）Sequence separately designs primer NG3-F1（Such as Shown in SEQ.ID.No.10：atggcttccggcacgttctt）And NG3-R1（As shown in SEQ.ID.No.11： tcaagcgacttgaattgtatc）, clone and obtain from cDNA librarySaCTPGene coded sequence；
It will by Gateway technologySaCTPGene is inserted into Escherichia coli/yeast shuttle expression vector pYES2-DEST and is containedSaCTPThe expression vector pYES2- of gene SaCTP。
pYES2- SaCTPYeast expression carrier plasmid and pYES2-DEST empty carrier plasmid difference Electroporation are quick to cadmium Sense type yeast mutantsycf1In bacterial strain, by the yeast transformant of acquisition and empty carrier in SD-U plate streaking culture 3 days.
Picking individual colonies, shaken cultivation to OD600 successively dilute 10 times and are being free of cadmium respectively, are containing 15 μM when being 1.0 L-1With 30 μM of L-1 CdCl2SG-U plate on carry out contact plate experiment, verify resistance to cadmium function.As a result, it has been found that cadmium is not added On culture medium, the bacterium colony size and number of yeast is almost the same, illustrate containing empty expression vector andSaCTPExpression vector Yeast growing way is almost the same, and upgrowth situation is also more consistent；In 15 μM of L-1With 30 μM of L-1When Cadmium treated, two kinds of ferment Female growth is by a degree of inhibition, but unloaded yeast transformant is than expressionSaCTPThe yeast transformant of gene is suppressed Situation processed is serious.The result shows that under the conditions of Cadmium treated,SaCTPGene can significantly improve cadmium responsive type yeast mutantsycf1Resistance to cadmium, it was demonstrated thatSaCTPThe heterogenous expression of gene can play its effect for improving cadmium resistance and cadmium ion content.
Embodiment 4：
Transgenic arabidopsis seedling Cd stress experimental verificationSaCTPGene heterogenous expression improves arabidopsis cadmium resistance
Design forward primer（Primer sequence is as shown in SEQ.ID.No.12：caccatggcttccggcacgttctt）With reversely draw Object（Primer sequence is as shown in SEQ.ID.No.13：tcaagcgacttgaattgtatc）, PCR amplifies cDNA segmentSaCTP Gene whole coding sequence.It will using Gateway methodSaCTPGene is inserted into plant expression vector pH2GW7.0, is containedSaCTPThe expression vector pH2GW7.0- of geneSaCTP。
By pH2GW7.0-SaCTPIt is transferred to Agrobacterium EHA105 in electric shock method, obtains Agrobacterium engineering cell system；Using Inflorescence infestation method carries out transformation of Arabidopsis thaliana, and the arabidopsis after infecting is lain low and is put into the plastic tub being protected from light, after spraying a small amount of water Preservative film is covered to prevent water point evaporation, newspaper after cultivating 16-24 h, is thrown off in preservative film upper cover to keep dark surrounds Preservative film, and arabidopsis is taken out, continue to be placed in arabidopsis room and cultivate；Seed is collected after arabidopsis is mature, in 4 DEG C after drying It saves backup.
T0 after drying is being passed through into 75% ethanol disinfection for seed on superclean bench, is equably being sprinkling upon mould containing tide Element（Hyg）20 mg·L-11/2 MS solid medium on；Culture dish, is then transferred to quasi- by dark culture 2 days under 4 DEG C of environment It cultivates, cultivates one week or so in southern mustard room；Then battalion will can be transferred to the Arabidopsis plant with Hyg resistance of normal growth It supports in soil, and is cultivated in arabidopsis room.After PCR and RT-qPCR detection, the high positive plant of expression quantity is chosen as super ExpressionSaCTPTransgenic Arabidopsis plants strain, by be commissioned to train support until harvest T3 for homozygote seed and in -4 DEG C save, use Biological function analysis later.
By the overexpression of acquisitionSaCTPTransgenic arabidopsis T3 for seed（OE1, OE2 and OE3）And wildtype Arabidopsis thaliana Seed（WT）Containing 100 uM CdCl with sterilizing toothpick sowing21/2MS solid medium on, while will be grown in and be free of The overexpression of the 1/2MS solid medium of cadmiumSaCTPTransgenic arabidopsis T3 for seed（OE1, OE2 and OE3）And wild type Arabidopsis seed（WT）It as control, is observed after 10 days, as shown in figure 4, being grown in the 1/2MS solid medium without cadmium Wildtype Arabidopsis thaliana and overexpressionSaCTPThe equal normal growth of transgenic arabidopsis, do not have notable difference, and containing 100 uM CdCl21/2MS solid medium on wild-type Arabidopsis plants growth be obviously suppressed.
As shown in Figure 5A, under non-stress state, Arabidopsis plant root long in 4.5 cm or so, is coerced in heavy metal cadmium Afterwards, wild-type Arabidopsis plants root long is in 0.8 cm or so, and overexpressesSaCTPTransgenic arabidopsis root long although growth Also it is suppressed, but its rhizome length is considerably longer than wildtype Arabidopsis thaliana, the statistics root of tri- strains of OE-1, OE-2, OE-3 Long average value is 1.47 cm, 1.55 cm, 1.40 cm.As shown in Figure 5 B, overexpression is also indicated that by the measurement of biomassSaCTPTransgenic arabidopsis Cd stress after three strains 5 seedling mean freshs be 17.87 mg, 18.72 mg, 16.04 mg, and wild type is 12.23 mg, is significantly higher than WT strain.Seedling stress experiment shows transgenic arabidopsis Seedling is higher than wild type to the tolerance of Cd stress,SaCTPOverexpression can significantly improve the cadmium resistance of transgenic arabidopsis.
The wildtype Arabidopsis thaliana WT and overexpression cultivated according to the method described aboveSaCTPTransgenic arabidopsis strain OE1, OE2 and OE3 30 days or so after transplanting, chooses the consistent wild type of growth conditions and transgenic arabidopsis starts with containing 0.5 mM CdCl2Aqueous solution irrigate arabidopsis, irrigated after antecedent soil moisture with same solution, processing the time be 2 weeks, with not Through CdCl2The arabidopsis of Stress treatment is as control.As shown in fig. 6, to wild type and the quasi- south of transgenosis during Cd stress 2 weeks The phenotypic difference of mustard plant carries out it has been observed that after by Cd stress, and wild type and transgenic Arabidopsis plants can Growth, it is less obvious to coerce initial phenotypic difference；But with the extension of time, the color of the blade of wildtype Arabidopsis thaliana starts to become It is deep, purple is gradually become, and there is faint yellow in the foreign steamer blade periphery of transgenic arabidopsis, blade purpleization is unobvious.Therefore Speculate that wildtype Arabidopsis thaliana is poor to the patience of cadmium from phenotype.
The wild type of different disposal time and the ground position of transgenic Arabidopsis plants are separated and obtain, it is clear with distilled water It washes three times；By all samples put into 105 DEG C of baking ovens after dry 30 min 80 DEG C until sample thoroughly drying to constant weight.High-content Cadmium element using Flame Atomic Absorption Spectrophotometry measure, and the cadmium element of low content using graphite electrode atomic absorption spectrometry survey It is fixed.The cadmium content in plant tissue is calculated by upper machine concentration, sample weighting amount and constant volume.As a result as shown in fig. 7, not by cadmium When processing, cadmium content is substantially not detectable in wild type and transgenic arabidopsis；After 0.5mM Cd handles 2 w, wild type is quasi- The cadmium content of southern mustard strain is 50.17 μ g/g FW, and the cadmium content of three transgenic lines is respectively 82.56 μ g/g FW, 71.96 μ g/g FW, 60.33 μ g/g FW, the results showed that, overexpressionSaCTPTransgenic arabidopsis pair can be improved after gene The content of cadmium and maintain more normal growth conditions.
SaCTPGene coded sequence is as shown in SEQ.ID.No.1：
ATGGCTTCCGGCACGTTCTTCTATGCAGAGTGCGATGATCGTTGCACTCTTGTATCAAAATCGCCTTGTAAAG AGTCTTGCTTTATGTGCGAGAAGCCGCTTGGATTCAGCGCTGATATATTCATGTACATGGGGAATACACCGTTTTGT AGCACGGAGTGTAGACAAGAGCAGATTGAAATGGACGATGCGGAGGAGAGGAGGAAGAGGAAGAAATCCGCGCTGAG GGCTAAGGCGGAGACAGCAAGATCGACGGCCGGGGGTAAATCGGTCAGGACGGATACAATTCAAGTCGCTTGA。
SaCTPAmino acid sequence is as shown in SEQ.ID.No.2：
MASGTFFYAECDDRCTLVSKSPCKESCFMCEKPLGFSADIFMYMGNTPFCSTECRQEQIEMDDAEERRKRKKS ALRAKAETARSTAGGKSVRTDTIQVA。
Sequence table
<110>Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences
<120>A kind of raising cadmium resistance and the gene of cadmium content and application thereof
<160> 13
<170> SIPOSequenceListing 1.0
<210> 14
<211> 300
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
atggcttccg gcacgttctt ctatgcagag tgcgatgatc gttgcactct tgtatcaaaa 60
tcgccttgta aagagtcttg ctttatgtgc gagaagccgc ttggattcag cgctgatata 120
ttcatgtaca tggggaatac accgttttgt agcacggagt gtagacaaga gcagattgaa 180
atggacgatg cggaggagag gaggaagagg aagaaatccg cgctgagggc taaggcggag 240
acagcaagat cgacggccgg gggtaaatcg gtcaggacgg atacaattca agtcgcttga 300
<210> 14
<211> 99
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 14
Met Ala Ser Gly Thr Phe Phe Tyr Ala Glu Cys Asp Asp Arg Cys Thr
1 5 10 15
Leu Val Ser Lys Ser Pro Cys Lys Glu Ser Cys Phe Met Cys Glu Lys
20 25 30
Pro Leu Gly Phe Ser Ala Asp Ile Phe Met Tyr Met Gly Asn Thr Pro
35 40 45
Phe Cys Ser Thr Glu Cys Arg Gln Glu Gln Ile Glu Met Asp Asp Ala
50 55 60
Glu Glu Arg Arg Lys Arg Lys Lys Ser Ala Leu Arg Ala Lys Ala Glu
65 70 75 80
Thr Ala Arg Ser Thr Ala Gly Gly Lys Ser Val Arg Thr Asp Thr Ile
85 90 95
Gln Val Ala
<210> 3
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aagcagtggt atcaacgcag agtggccatt acggccggg 39
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
attctagagg ccgaggcggc cgacatg 27
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
aagcagtggt atcaacgcag agt 23
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
taatacgact cactataggg 20
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tcggttagag cggatgtg 18
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
tgtaaaacga cggccagt 18
<210> 9
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
caggaaacag ctatgacc 18
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
atggcttccg gcacgttctt 20
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
tcaagcgact tgaattgtat c 21
<210> 12
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
caccatggct tccggcacgt tctt 24
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tcaagcgact tgaattgtat c 21

Claims (4)

1. a kind of gene for improving cadmium resistance and cadmium content, which is characterized in that the gene for improving cadmium resistance and cadmium content from It separates and obtains in the Leaf cDNA Library of Sedum alfredii Hance, nucleotide sequence is as shown in SEQ.ID.No.1.
2. a kind of albumen of the coded by said gene as described in claim 1 for improving cadmium resistance and cadmium content, which is characterized in that its Amino acid sequence is shown in SEQIDNo.2.
3. a kind of arabidopsis transgenic plant of the gene containing raising cadmium resistance described in claim 1 and cadmium content.
4. application of the gene as described in claim 1 in terms of the cadmium resistance and cadmium content for improving plant.
CN201810827621.5A 2018-07-25 2018-07-25 Gene for improving cadmium resistance and cadmium content and application thereof Active CN108866081B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810827621.5A CN108866081B (en) 2018-07-25 2018-07-25 Gene for improving cadmium resistance and cadmium content and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810827621.5A CN108866081B (en) 2018-07-25 2018-07-25 Gene for improving cadmium resistance and cadmium content and application thereof

Publications (2)

Publication Number Publication Date
CN108866081A true CN108866081A (en) 2018-11-23
CN108866081B CN108866081B (en) 2021-04-02

Family

ID=64304902

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810827621.5A Active CN108866081B (en) 2018-07-25 2018-07-25 Gene for improving cadmium resistance and cadmium content and application thereof

Country Status (1)

Country Link
CN (1) CN108866081B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113046362A (en) * 2021-03-17 2021-06-29 浙江中医药大学 Gene for improving cadmium resistance cadmium content and relieving cadmium stress DNA damage and application thereof
CN116970616A (en) * 2023-09-25 2023-10-31 烟台大学 Application of ArWz-4 gene of red stripe Mao Fudan soft-shelled turtles in cadmium pollution monitoring
CN117106791A (en) * 2023-10-23 2023-11-24 烟台大学 Gene and application thereof in cadmium pollution monitoring

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274121A (en) * 2015-12-04 2016-01-27 中国林业科学研究院亚热带林业研究所 Gene capable of promoting cadmium accumulation and application of gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274121A (en) * 2015-12-04 2016-01-27 中国林业科学研究院亚热带林业研究所 Gene capable of promoting cadmium accumulation and application of gene

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
D.M. DENG ET AL.: "Zinc and cadmium accumulation and tolerance in populations of Sedum alfredii", 《ENVIRONMENTAL POLLUTION》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113046362A (en) * 2021-03-17 2021-06-29 浙江中医药大学 Gene for improving cadmium resistance cadmium content and relieving cadmium stress DNA damage and application thereof
CN113046362B (en) * 2021-03-17 2023-02-17 浙江中医药大学 Gene for improving cadmium resistance cadmium content and relieving cadmium stress DNA damage and application thereof
CN116970616A (en) * 2023-09-25 2023-10-31 烟台大学 Application of ArWz-4 gene of red stripe Mao Fudan soft-shelled turtles in cadmium pollution monitoring
CN117106791A (en) * 2023-10-23 2023-11-24 烟台大学 Gene and application thereof in cadmium pollution monitoring

Also Published As

Publication number Publication date
CN108866081B (en) 2021-04-02

Similar Documents

Publication Publication Date Title
CN101802190A (en) Method for improving stress resistance in plants and materials therefor
CN105755036A (en) Plants Having Enhanced Yield-related Traits And Method For Making The Same
CN108866081A (en) A kind of raising cadmium resistance and the gene of cadmium content and application thereof
CN111527101A (en) Photosynthetic organism gene regulation for improved growth
CN110468150B (en) Application of RGS1 gene as negative regulatory factor in improving tomato bacterial leaf spot resistance in low-irradiation environment
CN109666681A (en) Plant drought, salt tolerant protein EeCIPK26 and its encoding gene and application
CN102002101A (en) Plant root development related protein ZmNR1 and coding gene thereof
CN109971763A (en) Florescence control gene C MP1 and relevant carrier and its application
CN108997487A (en) Application of the resistance relevant protein Z76 in regulation stress resistance of plant
CN107326035B (en) Deubiquitinating enzyme gene UBP5 for regulating rice grain shape and leaf color and application thereof
CN106749580B (en) Plant salt tolerance GAP-associated protein GAP TaPUB15-D and its encoding gene and application
CN106432449B (en) Drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and application
CN115851821B (en) Application of BBX16 gene in improving plant salt tolerance
CN103172716B (en) Heat-resistant plant gene and application thereof
CN108864264B (en) Corn OXS2a gene, and encoding protein and application thereof
CN107267525B (en) Application of panax notoginseng polygalacturonase inhibitor protein gene PnPGIP
CN113234731B (en) GmARF16 gene for coding soybean ARF transcription factor and application thereof
CN112679591B (en) Application of substance for inhibiting OaGS3 gene expression in regulating and controlling length of tetraploid wild rice grains
CN114657188A (en) Gene PK1 for regulating and controlling rice cadmium accumulation, protein and application thereof
CN107266542A (en) Thick boisiana IpLEA genes and its encoding proteins and application
CN108034662A (en) The breeding method of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_06025 genes in stripe rust prevention
CN114736907A (en) Waxy synthesis gene PsFAR of Hibiscus solenopsis Lecanicillium and application thereof
CN104805062B (en) A kind of resistant gene in plant and its application
CN106811448B (en) Cotton tyrosine phosphatase GhPTP1 and its encoding gene and application
CN111560055A (en) Application of rice gene OsLAT3 in regulation of absorption and accumulation of diquat

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant