CN116769798A - Setaria viridis drought-resistant and salt-tolerant gene SvWRKY64 and application thereof - Google Patents
Setaria viridis drought-resistant and salt-tolerant gene SvWRKY64 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant biology, in particular to a drought-resistant and salt-resistant gene of green bristlegrassSvWRKY64And uses thereof, the drought-resistant and salt-tolerant geneSvWRKY64The nucleotide sequence of (2) is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 3. Drought-resistant and salt-tolerant gene of the inventionSvWRKY64Is the only WRKY transcription factor family which is screened from transcriptomes of 9 treatment stages of green bristlegrass drought and salt stress and can respond to adversity stress at high efficiency in each stress stageA member, thusSvWRKY64It is likely that a core gene of many members of the WRKY transcription factor family, responsive to various abiotic stresses, is in a central location in the regulatory network. Further understandingSvWRKY64The action mechanism of stress resistance function is exerted in the plant body, and the method has important theoretical and practical significance for cultivating novel stress resistance varieties of C4 crops such as millet, corn, sorghum and the like.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a drought-resistant and salt-resistant gene of green bristlegrassSvWRKY64And applications thereof.
Background
Green bristlegrassSetaria viridis) The green bristlegrass is a novel monocotyledonous, C4 transgenic mode plant which has a C4 photosynthesis system and is close to the relatives of millet, corn, sorghum, sugarcane, coix and important energy grass, and is an important C4 plant model; setaria viridis is an important cereal crop milletSetaria italica) The wild close relation of the setaria is monocotyledonous setaria gramineae, the nuclear types of the setaria is basically the same, the band types are similar, and the genome sequences are almost identical, so the setaria is an important model for researching the gene of the setaria, a mode for researching the influence of artificial selection on the evolution of crops is also researched, the current conversion efficiency of the setaria is extremely low, the setaria lays a foundation for quickly excavating excellent genes of the setaria, the research on the characteristic formation molecular mechanism of the setaria is performed on the setaria, corn, sorghum, sugarcane and the like of the same genus C4 plant, and especiallyHas important reference function on millet of the same genus green bristlegrass. The green bristlegrass has strong tolerance to abiotic stress and rich resistance genes, and is an important mode for researching stress reactions such as drought resistance, heat resistance, salt resistance and the like.
WRKY proteins are a class of important regulatory factors present in higher plants. At present, the function research of WRKY transcription factors in participation in plant stress response is more and more paid attention, the WRKY family is numerous in members, different members have respective functions and action mechanisms, the participating regulatory network is very complex, and at present, people have a certain knowledge on upstream regulatory genes and downstream influencing genes of certain family genes, but a plurality of regulatory nodes and regulatory modes are still blank. The research on the related functions and regulation modes of specific members of the WRKY family is imperfect, and the response mechanism of each member under various complex environmental conditions is unclear.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a drought-resistant salt-tolerant gene SvWRKY64 of green bristlegrass and application thereof, and discovers that the total number of WRKY transcription factor family members in the genome of the green bristlegrass is 109, and then the only WRKY transcription factor family member capable of efficiently responding to adverse stress in each stress stage is screened from the transcriptome of the green bristlegrass in 9 treatment stages of drought and salt stressSvWRKY64Transcription factor gene, experiment showsSvWRKY64In response to core genes under various abiotic stresses, in a central location of the regulatory network; research on action mechanism of acting stress resistance function in plant based on WRKY transcription factorSvWRKY64Has important theoretical and practical significance for cultivating new stress-resistant varieties of C4 crops such as millet, corn, sorghum and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a drought-resistant and salt-tolerant gene of green bristlegrassSvWRKY64The drought-resistant and salt-tolerant geneSvWRKY64The nucleotide sequence of (2) is shown as SEQ ID NO. 1.
Further, the drought-resistant salt-tolerant geneSvWRKY64CDS sequence shown in SEQ ID NO. 2。
The invention also provides a drought-resistant and salt-tolerant geneSvWRKY64The amino acid sequence of the coded protein SvWRKY64 is shown as SEQ ID NO. 3.
The invention also provides a method for obtaining the drought-resistant and salt-resistant gene of the green bristlegrassSvWRKY64The primer pair is as follows:
the invention also provides a method for obtaining drought-resistant and salt-resistant gene by using the primer pairSvWRKY64A method of CDS sequence comprising the steps of:
cDNA of green bristlegrass is used as a template and the green bristlegrass drought-resistant and salt-resistant gene is usedSvWRKY64PCR amplification and purification are carried out to obtain drought-resistant salt-tolerant geneSvWRKY64CDS sequences.
The invention also provides an over-expression vector, which is pZmUbi-GUS (ZmUbis-HPH)SvWRKY64Contains the drought-resistant and salt-resistant geneSvWRKY64Expression vector of CDS sequence.
The invention also provides a plant gene editing vectorpRLG103- SvWRKY64Comprises drought-resistant and salt-resistant genesSvWRKY64 Target site knockout primers designed for the nucleotide sequence of (2) pairs, as follows:
the invention also provides a host cell containing the over-expression vector or the plant gene editing vector, and the host cell is agrobacterium AGL1.
Use of one of the following in stress tolerance regulation in drought and salt stress conditioned plants, comprising:
(1) The drought-resistant and salt-tolerant gene of the green bristlegrassSvWRKY64；
(2) The above-mentioned over-expression vector;
(3) The plant gene editing vector;
(4) The host cell described above;
the plant is green bristlegrass or millet or corn or sorghum.
The application of one of the following items in the cultivation of new stress-resistant varieties comprises:
(1) The drought-resistant and salt-tolerant gene of the green bristlegrassSvWRKY64；
(2) The above-mentioned over-expression vector;
(3) The plant gene editing vector;
(4) The host cell described above;
the plant is green bristlegrass or millet or corn or sorghum.
The invention has the beneficial effects that:
1. the WRKY transcription factor family in the green bristlegrass genome has 109 members in total, and the drought-resistant and salt-tolerant gene of the inventionSvWRKY64The single WRKY transcription factor family member which can respond to adversity stress in each stress stage is screened from transcriptomes of 9 processing stages of drought and salt stress of green bristlegrass, so that SvWRKY64 can be a core gene responding to various abiotic stresses for a plurality of WRKY transcription factor families of family members and is positioned in a central position of a regulatory network. The invention further knows the action mechanism of the WRKY transcription factor playing stress-resistant function in the plant body, and has important theoretical and practical significance for cultivating novel stress-resistant varieties of C4 crops such as millet, corn, sorghum and the like.
2. The drought-resistant and salt-tolerant gene SvWRKY64 belongs to a WRKY transcription factor family of a tropical mode plant green bristlegrass, the green bristlegrass belongs to a monocotyledonous plant of the Gramineae, the green bristlegrass is often distributed in tropical and subtropical areas, sorghum, sugarcane, corn, millet, coix, huang Mao, cogongrass and the like belong to the green bristlegrass, and the plant has typical characteristics of tropical plants. The green bristlegrass is a novel transgenic mode plant, has the advantages of short growth cycle, short stature, easy planting, easy transformation, small genome, diploid, capability of producing a large amount of inbred line seeds and the like as dicotyledonous mode plant Arabidopsis thaliana, and is an excellent monocotyledonous mode plant; because of its origin in the tropics, it has a C4 photosynthesis system, close to the relatives of millet, corn, sorghum, sugarcane, coix and important energy grass, it is an important C4 plant model; the green bristlegrass is a wild close relation of millet (Setaria algorithm), the nuclear types of the green bristlegrass and the Setaria algorithm are basically the same, the band types of the green bristlegrass and the Setaria algorithm are similar, the genome sizes of the green bristlegrass and the Setaria algorithm are 510 and Mb, and the genome sequences of the green bristlegrass are almost identical, so that the green bristlegrass is also an important model for researching the gene of the millet, and is also a mode for researching the influence of artificial selection on the evolution of crops; the green bristlegrass has strong tolerance to abiotic stress and rich resistance genes, and is an important mode for researching stress reactions such as drought resistance, heat resistance, salt resistance and the like; the setaria is similar to the important energy crops such as switchgrass, miscanthus and the like in morphology, and is an important mode plant for researching the energy crops.
Drawings
FIG. 1 is a schematic view ofSvWRKY64Expression profiles under drought and salt stress conditions;
FIG. 2 is a schematic view ofSvWRKY64Deducing amino acid sequence and other plantsWRKYPhylogenetic analysis of the amino acid sequence of the gene;
FIG. 3 is a diagram of pZmUbi-GUS (ZmUbis-HPH) plasmid vector
FIG. 4 is a diagram of pRLG103 plasmid vector
FIGS. 5-10 are phenotypic diagrams of SvWRKY64 over-expression lines and gene editing lines under drought and salt stress;
in the figures, the first behavior ME34 in FIGS. 5-7 is the phenotype under untreated, drought stress treatment and salt stress treatment,
the second, third and fourth lines in FIG. 5 are phenotypes of SvWRKY64 overexpressing line 3-1 without stress treatment, drought and salt stress treatment, respectively;
the second, third and fourth lines in FIG. 6 are phenotypes of SvWRKY64 overexpressing lines 3-3, respectively, without stress, drought and salt stress treatments;
the second and third lines in FIG. 7 are phenotypes of SvWRKY64 overexpressing lines 5-4, 5-3, 5-2, respectively, without stress and drought stress treatment;
the first, second and third rows in fig. 8 are phenotypes of SvWRKY64 gene editing line 521 under untreated, drought stress treatment and salt stress treatment, respectively;
the first, second and third rows in fig. 9 are phenotypes of SvWRKY64 gene editing line 528 under untreated, drought stress treatment and salt stress treatment, respectively;
the first, second and third rows in fig. 10 are phenotypes of SvWRKY64 gene editing line 586 under untreated, drought stress treatment and salt stress treatment, respectively;
FIG. 11 is a view showing the behavior of wild-type and genetically modified green bristlegrass lines;
in the figure, A is a comparison graph of growth conditions of wild type and gene editing strains under untreated conditions,
the first strain on the left is a wild type strain, and the five strains on the right are different strains of a gene editing strain;
b is a comparison graph of growth conditions of wild type and gene editing strains under drought stress treatment conditions,
the first strain on the left is a wild type strain, and the five strains on the right are different strains of a gene editing strain;
c is a comparison graph of growth conditions of wild type strain and gene editing strain under the condition of salt stress treatment,
the first strain on the left is a wild-type strain, and the five strains on the right are different strains of the gene editing strain.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
Example 1 drought and salt tolerance gene of Setaria viridisSvWRKY64Screening of (C)
1. Study of drought resistance and salt tolerance of seed in germination period
1.1 Material
The plant material is the green bristlegrass a10 line, which is supplied by Thomas p. Brutnell laboratories of us Donald Danforth Plant Science Center;
1.2 Method of
a. Mannitol and sodium chloride hypertonic culture medium with different concentration gradients
Preparing a basic culture medium: 1/2MS medium, 10 g sucrose, pH5.8, gelzan plant gel 4 g,
drought stress culture medium preparation of different mannitol: mannitol is added into the basic culture medium to simulate drought stress, the concentration gradient is 0, 100, 200, 300 and 400 mmol/L, and the culture medium is sterilized at 120 ℃ for 20 min and then poured into a dish for standby.
MS sodium chloride hypertonic culture medium preparation: sodium chloride is respectively added into the basic culture medium for salt stress, the concentration gradients are 0, 40, 80, 120 and 160 mmol/L5 gradients, and the culture medium is sterilized at 120 ℃ for 20 min and then poured into a dish for standby.
b. Aseptic treatment and inoculation of seeds:
selecting dry mature seeds of the green bristlegrass subjected to dormancy treatment, removing seed coats, filling about 1 g seeds into a 14 mL centrifuge tube, adding 10 mL sodium hypochlorite solution with the effective concentration of about 1% -3%, adding 50 mu L of Tween 20 solution, and sterilizing for about 10 min; after washing with sterile water several times, seeds were inoculated onto hypertonic media containing mannitol and sodium chloride in different concentration gradients with the shoots facing upwards, approximately 20 seeds were inoculated per dish of media, 3 dishes (3 replicates) were inoculated per gradient, and the whole plant was subjected to transcriptome sequencing analysis after 7 d days at 24℃in light/dark culture (12,000 lx,16 h/8 h,50% -60% humidity).
1.3 Conclusion(s)
Transcriptome analysis of 9 treatments for green bristlegrass drought stress (total 4 stress gradients) and salt stress (total 4 stress gradients) plus control showed a total of 40 differentially expressed genes in total for both drought stress and salt stress 9 treatments. WhileSvWRKY64The transcription factor gene is one of 40 differential genes closely related to drought stress and salt stress obtained by transcriptome analysis, and then the gene is subjected to the following stepsSvWRKY64Fluorescence quantitative verification was performed (FIG. 1), consistent with the analysis results of the transcriptome. It can be seen from the figure that under drought and salt stress conditionsSvWRKY64The gene obviously down-regulates expression, and gradually down-regulates with the deepening of the stress degree, and the down-regulating degree is larger under the salt stress condition than under the drought stress condition;from this, it is speculated that the SvWRKY64 gene is sensitive to abiotic stress, is a gene closely related to drought and salt stress, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1:
atgtcaacgcggctcaactccgcgaggaagtcgcagccgccgcgctcctccgacaggagggacgctgcgatccaggagctcaggaggggcacccagctggcagatctgctcaggaagcaggtgaagctcatcccggagccaaaccgccgtgacgctgcagtggccaacgtgggcgagatatccatggccatggagtcgtcgctcaacatcctccagtatgagatagagcatccctcctcccccgaggtcggcacggtggacatggctgcgcacgctggctactcctccgatggaggcaccggggaaagaaatcgtgccgttccccgtaccagaagggtgaggcaccggcgaggcaggcatggagttgagctcccaatgtgagtcctacaaacctgtgatttttgttgcgtcttgcatgctacagtgagggtgttaaaacagatatatagcctgcaacctgaataaaactatgggaatctcatgttacaaagattcagatctctgcatatctcatttgtgtttaagggagcaaattactgctgcttactttcttactggattagttaacttgctttagaggcatgaagaaaaatggattggaaagttagtttatggctagagctttccagatctacattaccaatgaaagggaaaagaaaagaagatcaggtgtttctggatcttgttttatccaagttcatctcgggtagcatatatatgatgtacatgtgtgctttccttctcgcgtaggtgcatatctgggatctatatttcatggttggatagaaagtttgtgcaaggtcatgcggtgaaattacattcagtttctcttccaccttgttattcaggaaggagatactgactgaggcaccagaaaacgatcgtttccactggaggaaatatggtgagaagaccatcctcaatgctgaatatccaaggtatgcatctttaatggagttccatgcatcatccactccccaagtcactttctctccagatttctcactagctaatataaatcatgaatgttcatccgattcctccttgcaatcaatagtgaggaaacctttttctgtaaaaaaattacagctttacagaacataaatagttcatagtactacttatatatattctataaaaagtatgagtttcatggatcataacctcctctcggcatttaatatgcaatttatgatttgtagtgctagacccatttgggtattagcttgattagcttcctctagcagcttcaattccttccgctcacaccccttgttgtggggggtttggcgtttagcttgcagttcttgcgtgcttttttttgcttgcttcactaaaaagtcagatccaacagccgcttattcagtcaggggaggtagccgctgcttcctcaaggataacccaccttgtggagcagttggatccggcttgtaaactgaattcatgctgaaactaaaggcccttcttgttccagcttgttaactgtgtagtattccagtttgcaaattgtaacaacctgcaagctgcaatccacagcagctgaaataaaaaaggcctaagatccatcaagttgtgcaatatattttcaattatatatttctagttgtgcctgttgatttctgctaatatattactattattatcttctatactccttcaatttcaaattataggtcgttttgacttttttagatacatagattttcctatatatgtagacataatctatatttcagtgcatagcaaaaagtatgtatctagaagaatgaaaaatatctacaattttgatggagagagtaaaaaaaaaagcgcgtagaaaataaaggggctagtatggtatgcattatgttctttcttgaaattaaattagataaaaatttatatatataattgaatttgtggatggaccgtacttcatacttgattcagcggcacggaaaaacgaaaatggatgctaagcatatccagaagttgtttttggctatttgactgcatgtggtaatatgcatgaagatttgacctaagttcaaacatttaaattttcgatagaaaatggatatgctaggcatattcatgtacaaagaaaacaagatattttctctttctcttatcagcaaacaagcgctcacctccacatgtacatgaaatgtcgttatccacctgccaataatttacttctagcttagtatgatttcactccatacattgtttaggggacattccatactttaattttctcaatggataaaaagttatgcttgttctagatttatgttcataccttcggttatcaatttgcaggttatactacaaatgtggttacagcgatgaccacaagtgtccagcaaagaaatacgtgcagcagcaaagcaacagcggccatccacgtttcatggtcaccctgatcaacgagcatacgtgcgaagctttgttcccggatgagcccacctcaagcagcagtagtgcttcgcaggttctcgacttcacaaaggcatcgctttctcctccattgatggccgccgctgcctcagggtcgttgaagaaagaggaagaagacagcatgtctgtgtgtatgcacagctactcgtatgatgaatacttgtcttcttcgttcccaacgatgtcgccagatggagatcaggtccaattttctccggggcccgggtggtagcattctaataaatgagttagttagatacacaaacctgtttaattatttcttgttttagcagctagtcgaaccataaataatctagcgacgactcatgcaccgcaagagtttcctcgttattagcaccaagtatgcatgatatgcatggtgttaattacactttcctgttctatcaacggtttattagatggatatggtttctctttggacgtatacggagtagcatttcattgcacattgctccatgcaaaatgaccttttgtatttgatgaacccatatgtctatagcaggatca；
the CDS sequence of the drought-resistant and salt-tolerant gene SvWRKY64 is shown in SEQ ID NO: 2:
atgtcaacgcggctcaactccgcgaggaagtcgcagccgccgcgctcctccgacaggagggacgctgcgatccaggagctcaggaggggcacccagctggcagatctgctcaggaagcaggtgaagctcatcccggagccaaaccgccgtgacgctgcagtggccaacgtgggcgagatatccatggccatggagtcgtcgctcaacatcctccagtatgagatagagcatccctcctcccccgaggtcggcacggtggacatggctgcgcacgctggctactcctccgatggaggcaccggggaaagaaatcgtgccgttccccgtaccagaagggtgaggcaccggcgaggcaggcatggagttgagctcccaatgaaggagatactgactgaggcaccagaaaacgatcgtttccactggaggaaatatggtgagaagaccatcctcaatgctgaatatccaaggttatactacaaatgtggttacagcgatgaccacaagtgtccagcaaagaaatacgtgcagcagcaaagcaacagcggccatccacgtttcatggtcaccctgatcaacgagcatacgtgcgaagctttgttcccggatgagcccacctcaagcagcagtagtgcttcgcaggttctcgacttcacaaaggcatcgctttctcctccattgatggccgccgctgcctcagggtcgttgaagaaagaggaagaagacagcatgtctgtgtgtatgcacagctactcgtatgatgaatacttgtcttcttcgttcccaacgatgtcgccagatggagatcaggtccaattttctccggggcccgggtggtag
the amino acid sequence of the drought-resistant and salt-tolerant gene SvWRKY64 is protein SvWRKY64 shown in SEQ ID NO: 3:
MSTRLNSARKSQPPRSSDRRDAAIQELRRGTQLADLLRKQVKLIPEPNRRDAAVANVGEISMAMESSLNILQYEIEHPSSPEVGTVDMAAHAGYSSDGGTGERNRAVPRTRRVRHRRGRHGVELPMKEILTEAPENDRFHWRKYGEKTILNAEYPRLYYKCGYSDDHKCPAKKYVQQQSNSGHPRFMVTLINEHTCEALFPDEPTSSSSSASQVLDFTKASLSPPLMAAAASGSLKKEEEDSMSVCMHSYSYDEYLSSSFPTMSPDGDQVQFSPGPGW*
drought-resistant and salt-resistant gene of green bristlegrassSvWRKY64The amino acid sequences of (a) and other species homologous sequences are used for constructing a phylogenetic tree (figure 2) by using an NJ method, and phylogenetic analysis shows that:
green bristlegrassSetaria viridis) And millet (millet)Setaria italica) Recently, the green bristlegrass has many similarities with grains of food crops from the appearance. In particular, both seedlings, are difficult to distinguish. By means of research of molecular biology and cytogenetics, chromosomes are almost identical, gene level proves that the green bristlegrass is the wild ancestor of millet, the karyotypes of the green bristlegrass and the wild ancestor are basically identical, the karyotypes of the green bristlegrass and the wild ancestor are similar, the genome sizes are 510 and Mb, and the genome sequences are almost identical. SvWRKY64 has 98.2% sequence similarity to the amino acid sequence of SiWRKY 63. And important energy crops of switchgrassPanicum virgatum) The parent source relation is relatively close to that of C4 plant sorghumSorghum bicolor) And cornZea mays) Is closely related to wheatTriticum dicoccoides) Radix seu herba HeterophyllaeBrachypodium distachyon) And the like are somewhat distant. The analysis of the relationship shows that the green bristlegrass lays a foundation for rapid excavation of excellent genes of the millet, and the research on the molecular mechanism of the characteristic formation of the green bristlegrass has important reference effects on the millet, corn, sorghum, sugarcane and the like of the same genus C4 plant, especially on the millet of the same genus green bristlegrass.
EXAMPLE 2 construction of the overexpression vector
Extracting RNA of wild green bristlegrass ME34, reverse transcribing into cDNA, and extractingSvWRKY64The CDS region of the gene is deleted for stop codon, inThe two ends of the gene are respectively added with enzyme cutting sitesSpeIAndKpnIthe following primers were designed and amplified using cDNA as a templateSvWRKY64，Introduction of cloning vector pMD ® In 18T, cloning vector containing target gene is then carried outSvWRKY64-pMD ® Enzymes for 18T and pZmUbi-GUS (ZmUbis-HPH)SpeIAndKpnIthe target fragment is connected to the digested pZmUbi-GUS (ZmUbis-HPH) vector to obtain the over-expression vector pZmUbi-GUS (ZmUbis-HPH)SvWRKY64(FIG. 3).
Example 3 construction of plant Gene editing vector:
analysis of green bristlegrass Using an on-line CRISPR target analysis website (http:// crispor. Tefor. Net)SvWRKY64The target point of the exon of the gene is selected to be suitable according to the specificity, editing efficiency and off-target rate of the target point, and two target points are selected at the front 300 bp of the first exon. Target site knockout primers were designed based on the target sequences (table 1).
TABLE 1 Setaria viridis LinneSvWRKY64Target site knockout primer for gene editing
Remarks: the underlined sequence is the editing site recognition sequence.
Double-stranded primers are subjected to double-stranded primer, double-stranded fragments of two targets are respectively subjected to enzyme digestion by endonucleases BsmBI and BsaI and then are respectively connected into intermediate vectors pJG310 and pJG338, and a connection product is converted into escherichia coli DH5 alpha, and monoclonal sequencing verification is selected; finally, pJG310 and pJG338 vectors connected with the correct target sequences and pJG471 vectors containing cas9 enzyme are connected into a skeleton vector pRLG103 (figure 4) together after being subjected to AarI digestion, and the connection products are transformed into escherichia coli DH5 alpha, and single clone is selected for bacterial liquid PCR identification and sequence determination, and positive clones are screened. Finally, the plant gene editing vector containing 2 editing sites is constructedpRLG103-SvWRKY64。
Example 4
1. Transforming C4 mode plant green bristlegrass:
the green bristlegrass ME34 strain is used as plant material, which is provided by Thomas P.Brutnell laboratories of America Donald Danforth Plant Science Center. Mature seeds of the ME34 strain subjected to dormancy treatment are peeled and germinated, and the embryogenic callus induced is used as a transformation explant material. The seed is subjected to aseptic treatment and embryogenic callus induction, and then the seed is subjected to the treatment with a strain containing an overexpression vector pZmUbi-GUS (ZmUbis-HPH)SvWRKY64Agrobacterium containing the plant gene editing vector pRLG103-SvWRKY 64. And screening transformants by using hygromycin in the green bristlegrass transformation to obtain a SvWRKY64 gene overexpression strain and a pRLG103-SvWRKY64 gene editing strain.
2. Stress treatment methods for wild type green bristlegrass (ME 34), svWRKY64 gene overexpression strain and pRLG103-SvWRKY64 gene editing strain:
seeds of wild type green bristlegrass (ME 34), svWRKY64 gene overexpression line and gene editing line were sown on filter paper containing 100 mmol/L mannitol and 40 mmol/L sodium chloride respectively, grown for 7 days, and culture conditions: 24. culturing at a temperature of 12,000lx, 16 hours/8 hours and humidity of 50% -60% in a light/dark manner, and observing the properties.
The green bristlegrass ME34 and the gene editing strain which grow in the seedling raising pot for 1 month are subjected to continuous 7-day drought treatment without watering, and the plant characters are observed after 7 days. Brisk green bristlegrass ME34 growing in a seedling raising basin for 1 month and a gene editing strain are irrigated with 350 mmol/L sodium chloride for salt stress treatment, and the plant properties are observed after 7 days. Culturing conditions of green bristlegrass in the seedling raising basin: 30. culturing for 16 h by illumination at the temperature of 22 ℃, culturing for 8 h in a dark state at the temperature of 22 ℃, wherein the illumination intensity is 12 lx and the humidity is 50% -60%.
3. Conclusion(s)
(1) By growing wild-type ME34 of green bristlegrass for one week,SvWRKY64over-expression linesSvWRKY64The gene editing transgenic lines were observed under drought and salt stress conditions (FIGS. 5-10) and found to be transformedSvWRKY64The over-expressed strain of the gene becomes drought-tolerantSalt stress is insensitive and phenotypes under drought and salt stress are significantly better than wild-type and gene-editing strains. WhileSvWRKY64The growth vigor of the gene editing strain under drought and salt stress is obviously weaker than that of a wild type strain, the germination rate is lower, and the plant is also dwarf. By observing the characters of wild type and gene editing transgene strain grown for one month under drought and salt stress conditions, the gene is found to be transferredSvWRKY64The stress resistance of the green bristlegrass of the gene editing vector was significantly poorer than that of the wild type under stress conditions (FIG. 11).
(2) The invention willSvWRKY64The gene is transferred into the wild ME34 of the green bristlegrass to obtain the green bristlegrass strain with drought resistance and salt tolerance, and the genetic relationship analysis is combined,SvWRKY64the gene has important theoretical and practical significance for cultivating novel stress-resistant varieties of C4 crops such as millet, corn, sorghum and the like.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. Setaria viridis drought-resistant and salt-resistant geneSvWRKY64The method is characterized in that: the drought-resistant and salt-resistant geneSvWRKY64The nucleotide sequence of (2) is shown as SEQ ID NO. 1.
2. The drought-resistant salt-tolerant gene of green bristlegrass according to claim 1SvWRKY64The method is characterized in that: the drought-resistant and salt-resistant geneSvWRKY64The CDS sequence is shown as SEQ ID NO. 2.
3. Drought-resistant salt-tolerant gene according to claim 1 or claim 2SvWRKY64The amino acid sequence of the coded protein SvWRKY64 is shown as SEQ ID NO. 3.
4. Drought-resistant and salt-tolerant gene for obtaining green bristlegrass according to claim 2SvWRKY64Is characterized in that: the primer pair is as follows:
SvWRKY64-F： GGACTAGTATGTCAACGCGGCTCAACTC，
SvWRKY64-R： GGGGTACCCCACCCGGGCC。
5. drought-resistant salt-tolerant gene obtained by using primer pair as claimed in claim 4SvWRKY64A method of CDS sequence, characterized by: the method comprises the following steps:
cDNA of green bristlegrass is used as a template and the green bristlegrass drought-resistant and salt-resistant gene is usedSvWRKY64PCR amplification and purification are carried out to obtain drought-resistant salt-tolerant geneSvWRKY64CDS sequences.
6. An over-expression vector, characterized in that: the overexpression vector pZmUbi-GUS (ZmUbis-HPH)SvWRKY64A method of controlling drought and salt tolerance comprising the drought and salt tolerance gene according to claim 2SvWRKY64Expression vector of CDS sequence.
7. A plant gene editing vector, characterized in that: the plant gene editing vectorpRLG103-SvWRKY64Comprises drought-resistant and salt-resistant genesSvWRKY64 Target site knockout primers designed for the nucleotide sequence of (2) pairs, as follows:
SvWRKY64-S1F： ACTTGGATCGCAGCGTCCCTCCTGTG，
SvWRKY64-S1R： AAAACACAGGAGGGACGCTGCGATCC；
SvWRKY64-S2F： GAAGACATGGATATCTCGCCCACGTG，
SvWRKY64-S2R： AAAACACGTGGGCGAGATATCCATGT。
8. a host cell comprising the overexpression vector of claim 6 or comprising the plant gene-editing vector of claim 7, characterized in that: the host cell is agrobacterium AGL1.
9. The application of one of the following in plant stress resistance regulation under drought and salt stress conditions is characterized in that: it comprises the following steps:
(1) The drought-resistant salt-tolerant gene of green bristlegrass according to claim 1 or 2SvWRKY64；
(2) The overexpression vector of claim 6;
(3) The plant gene editing vector of claim 7;
(4) The host cell of claim 8;
wherein the plant is green bristlegrass or millet or corn or sorghum.
10. The application of one of the following items in the cultivation of new stress-resistant varieties is characterized in that: it comprises the following steps:
(1) The drought-resistant salt-tolerant gene of green bristlegrass according to claim 1 or 2SvWRKY64；
(2) The overexpression vector of claim 6;
(3) The plant gene editing vector of claim 7;
(4) The host cell of claim 8.
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| US20200239901A1 (en) * | 2019-01-28 | 2020-07-30 | Ut-Battelle, Llc | Genes for enhancing salt and drought tolerance in plants and methods of use |
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| CN110066327A (en) * | 2019-04-30 | 2019-07-30 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | Application of the protein TaWRKY13 in regulation stress resistance of plant |
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