Disclosure of Invention
The invention provides an application of GhPSKR gene in regulating and controlling plant salt tolerance in order to develop a new application of GhPSKR gene in cotton stress resistance. In order to achieve the above purpose, the present invention adopts the following technical scheme.
The first aspect of the invention provides an application of GhPSKR gene in regulating and controlling salt tolerance of plants, wherein the nucleotide sequence of GhPSKR gene is shown as SEQ ID NO. 1.
The invention firstly reveals the forward regulation and control function of GhPSKR genes in the salt tolerance of cotton, and provides excellent regulation and control targets and theoretical guidance for the salt tolerance cultivation of cotton. The invention provides an application of GhPSKR gene in regulating and controlling plant salt tolerance, and develops a new application of GhPSKR gene in cotton stress resistance.
Preferably, the amino acid sequence of the GhPSKR gene expressed protein is shown as SEQ ID NO. 2.
Preferably, the GhPSKR gene in the plant is overexpressed by genetic engineering methods to improve the salt tolerance of the plant.
Preferably, the genetic engineering method is to construct a recombinant expression vector by using the GhPSKR gene and introduce the recombinant expression vector into plants so as to improve the salt tolerance of the plants.
Preferably, the method of introducing the recombinant expression vector into a plant comprises direct DNA transformation by any one of Ti plasmid, ri plasmid and plant viral vector, or by any one of microinjection, electric conduction and Agrobacterium mediation. The invention clones cotton GhPSKR1 gene, constructs GhPSKR1 over-expressed transgenic arabidopsis plant by using transgenic technology and constructs GhPSKR silencing plant by using VIGS technology, and performs functional verification on GhPSKR1, which confirms that GhPSKR1 can improve salt tolerance of plant. The method is favorable for deeply researching the response mechanism of the plant to the salt stress signal, lays a good molecular foundation for effectively improving the salt tolerance of the plant, and has great value for exploring the signal regulation network of the plant under the salt stress.
In a second aspect, the present invention provides a recombinant expression vector comprising the GhPSKR gene.
Preferably, the recombinant expression vector is obtained by inserting the DNA fragment of GhPSKR gene into Sma I and Kpn I cleavage sites of super1300-GFP vector.
Wherein, reverse transcription is carried out on RNA of the plant to obtain template DNA, and PCR amplification is carried out on the basis of the template DNA through primer pairs shown as SEQ ID NO.3 and SEQ ID NO.4, thereby obtaining the DNA fragment of GhPSKR gene.
The primer in the primer pair shown in SEQ ID NO.3 is an upstream primer F1, and the nucleotide sequence of the upstream primer F1 is as follows:
5’-gtaccagattacgctcatatgATGGGGGCTCAAGGCTGCTG-3’。
the primer in the primer pair shown in SEQ ID NO.4 is a downstream primer R1, and the nucleotide sequence of the primer is as follows:
5’-actggcctccatggccatatgTCAAACACTAGATAACGATGTC-3’。
Further, the construction method of the recombinant expression vector containing GhPSKR gene comprises the following steps:
The plasmid PGADT-GhPSKR with correct sequence was used as template for amplification.
The amplified product is connected with a binary plant transformation vector super1300-GFP vector which is subjected to double digestion by Sma I and Kpn I, transformed and extracted to verify correct positive cloning plasmid, namely a recombinant expression vector 35S:: ghPSKR1-GFP.
The method comprises the steps of designing forward primers for amplifying the whole coding region of the GhPSKR gene according to the multiple cloning site of a plant binary transformation vector super1300-GFP and the coding region sequence of the GhPSKR gene, wherein the forward primers comprise an upstream primer F1 and a reverse primer and a downstream primer R1, and a 35S: ghPSKR1-GFP recombinant expression vector is obtained, and the specific construction method is as follows:
the GhPSKR gene is used as a template, forward primers and reverse primers are used for carrying out PCR amplification to obtain a product containing the GhPSKR gene, smaI and KpnI are used for respectively carrying out enzyme digestion on the product containing the GhPSKR gene and the super1300 to obtain an enzyme digestion product and a carrier framework respectively and recovering the enzyme digestion product and the carrier framework, and a recombinant expression vector 35S obtained by connecting the enzyme digestion product and the carrier framework is GhPSKR-GFP, namely, a vector obtained by replacing a DNA fragment between SmaI and KpnI enzyme digestion sites of the super1300 with the GhPSKR gene as shown in SEQ ID NO.1 and keeping other sequences of the super1300 unchanged.
Wherein, the plant binary transformation vector super1300-GFP is a recombinant expression vector connected with GFP gene, and the recombinant expression vector is recorded in the doctor's thesis of the metabolic characteristics of the dodder in the in vitro growth process and the research of the agrobacterium mis gene in the genome, zhang Yuexia, chinese university of agriculture, 2020, and the public can obtain from the Li Zhaohu subject group of Chinese university of agriculture after the agreement of the authors, so as to repeat the experiment in the invention, and the experiment can not be used as other application, and is called as the super1300 in the invention.
The structure of the recombinant expression vector 35S GhPSKR-GFP is shown in FIG. 3.
In a third aspect the invention provides a method of improving salt tolerance in a plant by over-expressing the GhPSKR gene in the plant or increasing the activity of the protein expressed by the GhPSKR gene in the plant or increasing the content of the protein expressed by the GhPSKR gene in the plant to improve salt tolerance in the plant.
In the above method, the improvement of the expression level of the gene of the protein GhPSKR1 and/or the activity of the protein GhPSKR in the target plant and/or the content of the protein GhPSKR1 is to overexpress the protein GhPSKR in the plant.
Preferably, the GhPSKR gene in the plant is over-expressed in an agrobacterium-mediated manner, and the plant is transformed into the GhPSKR gene to improve the salt tolerance of the plant.
Preferably, the plant is a monocot or dicot. More preferably, the plant is a cruciferous and/or malvaceae plant. More preferably, the plant is a cotton and/or arabidopsis plant. Most preferably, the plant is arabidopsis thaliana and/or cotton.
In a fourth aspect, the present invention provides a method for breeding a salt tolerant plant, wherein the GhPSKR gene in the plant is overexpressed to obtain a salt tolerant transgenic plant.
Wherein the plant is M1) or M2) or M3) or M4) or M5).
M1) monocotyledonous or dicotyledonous plants.
M2) cruciferae.
M3) arabidopsis thaliana.
M4) cotton plants.
M5) cotton.
In a fifth aspect, the present invention provides a protein, named cotton polypeptide receptor GhPSKR, ghPSKR1 protein, derived from cotton, which is any one of the following:
a1 A protein comprising or being the amino acid sequence of SEQ ID NO. 2.
A2 A fusion protein obtained by connecting a tag to the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 2.
A3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2, has more than 90 percent of identity with the protein shown in A1) and has the same function.
The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.
Among the above proteins, the protein tag refers to a polypeptide or protein which is fusion expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag.
In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, perresidue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.
In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
The biological material related to GhPSKR protein also belongs to the protection scope of the invention, and the invention also provides a new application of the biological material related to GhPSKR protein.
The invention relates to an application of a biological material related to GhPSKR protein in regulating plant stress resistance, wherein the biological material is any one of the following:
c1 Nucleic acid molecules encoding GhPSKR proteins.
C2 An expression cassette comprising the nucleic acid molecule of C1).
C3 A recombinant expression vector comprising the nucleic acid molecule of C1) or a recombinant expression vector comprising the expression cassette of C2).
C4 A recombinant microorganism comprising the nucleic acid molecule of C1), or a recombinant microorganism comprising the expression cassette of C2), or a recombinant microorganism comprising the recombinant expression vector of C3).
C5 A transgenic plant cell line comprising the nucleic acid molecule of C1), or a transgenic plant cell line comprising the expression cassette of C2), or a transgenic plant cell line comprising the recombinant expression vector of C3).
C6 A transgenic plant tissue comprising the nucleic acid molecule of C1), or a transgenic plant tissue comprising the expression cassette of C2), or a transgenic plant tissue comprising the recombinant expression vector of C3).
C7 A transgenic plant organ containing the nucleic acid molecule of C1), or a transgenic plant organ containing the expression cassette of C2), or a transgenic plant organ containing the recombinant expression vector of C3).
C8 A transgenic plant containing the nucleic acid molecule of C1), or a transgenic plant containing the expression cassette of C2), or a transgenic plant containing the recombinant expression vector of C3).
C9 A tissue culture produced by regenerable cells of the transgenic plant of C8).
C10 Protoplasts produced from the tissue culture of C9).
C11 A recombinant expression vector or a recombinant microorganism which suppresses the expression level of the GhPSKR protein gene and/or suppresses the activity of the GhPSKR protein and/or reduces the GhPSKR protein content.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA. The nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
In the above biological material, the nucleic acid molecule of C1) is any one of the following:
B1 A DNA molecule shown in SEQ ID NO. 1.
B2 The coding sequence is a DNA molecule shown as SEQ ID NO. 1.
B3 A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined under B1) or B2) and which codes for a GhPSKR protein.
The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each.
In the above biological material, the expression cassette of C2) refers to DNA capable of expressing GhPSKR a protein in a host cell, and the DNA may include not only a promoter for initiating transcription of GhPSKR1 gene but also a terminator for terminating transcription of GhPSKR.
Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, promoters of the GhPSKR gene itself, constitutive promoters, tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus, wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) PlantPhysiol:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible by jasmonic acid a ester), heat shock promoter (U.S. Pat. No. 5,187,267), tetracycline inducible promoter (U.S. Pat. No. 5,057,422), seed specific promoters such as the millet seed specific promoter pF128 (Chinese patent CN 101063139B), seed storage protein specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beach et al (1985) EMBO J.4:3047-3053) which may be used alone or in combination with other plant promoters, all references cited herein are incorporated by reference in their entirety, suitable transcriptional terminators include, but are not limited to, the GhPSKR Gene own terminator, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tmL terminator, the pea rbcS E9 terminator and the lipo and octopine synthase terminators (see, e.g., ohber et al (1985; 262: fig. 262; 1987; 125; 1997; see also, gfan et al, 1997; 1987; 1981; 1997; see also, gene 125; 1981; mol, et al, gduct, 1997; 1981), 5:141, mogen et al (1991) PLANT CELL,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) nucleic acids Res.17:7891; joshi et al (1987) nucleic acids Res., 15:9627).
In the above biological material, the recombinant expression vector of C3) may contain a DNA molecule shown in SEQ ID NO.1 for encoding protein GhPSKR 1. Recombinant expression vectors containing the GhPSKR protein gene or the protein GhPSKR gene expression cassette can be constructed using existing plant expression vectors. The plant expression vector can be a Gateway system vector or a binary expression vector, etc., such as super1300、pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2300、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa or pCAMBIA1391-Xb. When GhPSKR1 is used to construct a recombinant expression vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters, and in addition, when the plant expression vector is constructed using the gene of the present invention, enhancers including translation enhancers or transcription enhancers may be used, and these enhancer regions may be ATG initiation codons or adjacent region initiation codons and the like, but must be the same as the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.
In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants.
In a specific embodiment of the invention, the recombinant expression vector C3) is a 35S-GhPSKR-GFP recombinant expression vector, wherein the 35S-GhPSKR-GFP recombinant expression vector is obtained by replacing a DNA fragment between SmaI and KpnI cleavage sites of the super1300 with a GhPSKR1 gene of SEQ ID NO.1 and keeping other sequences of the super1300 unchanged.
In the above biological material, C11) the recombinant expression vector is a recombinant expression vector pYL-GhPSKR 1, the pYL-GhPSKR 1 is a recombinant expression vector obtained by replacing the DNA fragment between EcoR I and BamH I cleavage sites of the pYL156 vector with the DNA molecule shown in positions 1-311 of SEQ ID NO.1, and keeping other sequences of the pYL156 vector unchanged.
In the above biological material, the recombinant microorganism may be yeast, bacteria, algae and fungi, and the bacteria may be agrobacterium GV3101.
In the above biological material, the transgenic plant organ may be the root, stem, leaf, flower, fruit and seed of the transgenic plant.
In the above biological material, the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
In the above biological materials, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.
The invention further provides a product for regulating plant stress resistance, which contains GhPSKR protein or related biological materials.
The use of the GhPSKR protein or the biological material related to the GhPSKR protein in any of the following is also within the scope of the present invention:
D1 Use in the cultivation of transgenic plants with increased stress resistance.
D2 Use of the same for the production of transgenic plant products with increased stress resistance.
D3 For the cultivation of stress-reduced gene-silenced plants.
D4 The use of the composition for preparing a gene silencing plant product with reduced stress resistance.
D5 Use in plant breeding.
Among the above applications, the plant breeding may specifically be performed by crossing plants containing GhPSKR protein or biological material related thereto (for example, the gene GhPSKR1 encoding GhPSKR protein) with other plants.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides an application of GhPSKR gene in regulating and controlling salt tolerance of plants. The research result of the invention proves that GhPSKR gene silencing plants (especially cotton) have more obvious growth inhibition under the salt stress treatment, more obvious salt damage characteristics, increased accumulation of sodium ions in leaves and roots and more sensitivity to salt stress, and compared with wild type arabidopsis thaliana, the root length of GhPSKR gene overexpression transgenic homozygous plants (especially arabidopsis thaliana) under the salt stress treatment is obviously prolonged, and has good stress resistance, thereby showing that GhPSKR1 gene plays a positive regulation role in regulating and controlling plant salt stress. The invention firstly reveals the forward regulation and control function of GhPSKR in the salt tolerance of cotton, and provides excellent regulation and control targets and theoretical guidance for the salt tolerance cultivation of cotton. The invention provides an application of GhPSKR gene in regulating and controlling plant salt tolerance, and develops a new application of GhPSKR gene in cotton stress resistance.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Virus-induced gene silencing is widely used for identifying plant genome functions as an effective reverse genetics technology, can successfully silence endogenous genes at different parts of plants, and provides a practical means for researching gene functions in different growth periods. The present examples provide GhPSKR1 gene silencing plants by virus-induced gene silencing methods to demonstrate the function of the gene. Wherein, the English name of Virus-induced GENE SILENCING and VIGS of Virus-induced gene silencing.
The invention proves that GhPSKR < 1 > silences cotton plants, has more obvious growth inhibition under salt stress treatment, more obvious salt damage characteristics, increased accumulation of sodium ions in leaves and roots and more sensitivity to salt stress, and compared with wild type arabidopsis thaliana, the root length of GhPSKR < 1 > over-expression transgenic homozygous arabidopsis thaliana plants under salt stress treatment is obviously prolonged, and has good stress resistance, thereby showing that GhPSKR < 1 > genes play a positive regulation role in regulating and controlling plant salt stress.
For this purpose, the embodiment of the first aspect of the invention proposes the use of GhPSKR gene for regulating the salt tolerance of plants,
EXAMPLE 1 discovery, cloning, positioning of the GhPSKR1 Gene and construction of the recombinant vector for over-expression of GhPSKR1
1.1 GhPSKR1 discovery of Gene
In this example, the genes related to the salt of the VIGS cDNA library were screened and retrieved using the cotton database. A protein is obtained from cotton variety Guoxin No. 3 and named GhPSKR protein, and the amino acid sequence of GhPSKR protein is shown as SEQ ID NO. 2:
MGAQGCWAMVIILGLLLQPQFLSSQNLTCNQKDWTALRYFMGNLTKTLEGWTTNSSADCCDWEGITCDPPSAGRVIKLELPNKRLSGKLSDSLYGLDQLKTLNLSHNFLKDSLPLSLFHMPNLEQLDLSYNDFSGLIPESINLPSIRSLNISANSLKGSLPSHICVNSTGIGFLSLAVNYFSGNISPGLGKCSSLEELYLGTNQLTGVVTEDIFELQNLRRLGLQDNNLGGKLSPGIANLSNLVRLDISSNNFSGEIPDVFSELKRFQYLIANSNNFSGGIPSSLSNSPTVSLLNLRNNSLEGSIYLNCSAMVTLNSLDLATNKFTGPVPDNLPLCRQLQDVNLARNNFSGQIPESFKQFHSLSYLSLSNSSLHNLSSALQILQQCRNLTALVLTLNFPVETLPDDPNLHFEKLKVLVIASCQLRGSIPQWLSKISSLQLLDLSWNHLNGALPPWLGSYRDLFYLDLSNNSFTGEIPKSLTELPSLIHGNISLEEPSPDFPFFMKRNESARGLQYNQIWSFPPTLELGHNFLSGPVWPEFGNLKKLIVFDLKFNNLSGPIPENLSEMSSLEILDLSHNDLTGTIPPSLESLSFLSIFNVAYNRLYGKIPSGGQFQTFPNSSFEGNNLCGDHRFSCQDNTSEHVSPKRSRRNKDIIIGMVVGIIFGTALLIGLMYLFVLRTHKRNEVDPEKEEPDTNDKNLEELSSRLVVLFQNWESYKELCIDDLLESTNNFDQANIIGCGGFGLVYRGTLPDGRKVAIKRLSGDCGQMDREFRAEVEALSRAQHPNLVHLQGYCMHKNDRLLIYSYMENGSLDYWLHEKVDGSSLLGWETRLKIAQGAARGLAYLHQSCEPHILHRDIKSSNILLDENFKAHLADFGLARLILPYDTHVTTDLVGTLGYIPPEYGQASVATYKGDVYSFGVVLLELLTGKRPMDMCKPKGTRDLISWVIRMKMENKESEVFDPFIYGKQHDKEMLRVLEIACLCLNESPKIRPTTQQLVYWLDKVTSLSSV.
the coding gene of GhPSKR protein is named GhPSKR gene, and the nucleotide of the open reading frame is shown as SEQ ID NO. 1:
ATGGGGGCTCAAGGCTGCTGGGCTATGGTTATTATTCTTGGGTTGTTGTTGCAACCTCAGTTTCTCAGCTCTCAAAACCTGACATGCAATCAAAAGGACTGGACTGCTCTCCGGTATTTCATGGGCAATTTGACAAAAACACTTGAAGGTTGGACGACAAATTCCTCTGCTGATTGTTGTGACTGGGAAGGCATAACCTGTGATCCTCCATCTGCTGGTAGAGTAATCAAGTTGGAACTGCCCAATAAAAGGTTATCAGGCAAATTATCTGATTCTTTGTATGGTTTGGATCAGCTTAAAACTCTCAATCTTTCCCACAATTTCCTTAAAGATTCATTGCCACTTTCTTTGTTTCATATGCCGAATTTAGAGCAACTTGACTTGAGCTATAATGACTTCTCTGGACTAATCCCAGAAAGCATCAATCTGCCTTCAATTCGAAGTCTTAACATCTCTGCCAATTCCTTGAAGGGTTCCCTTCCTTCCCATATATGTGTTAACTCCACTGGAATTGGGTTCCTTAGTTTGGCAGTGAACTATTTCTCTGGTAATATTTCACCAGGGCTTGGAAAATGTTCTTCCTTGGAGGAACTGTATCTTGGCACAAATCAACTCACTGGTGTGGTGACTGAAGACATCTTTGAGCTTCAAAATTTGAGACGTCTGGGGCTTCAAGATAACAACTTGGGTGGGAAACTTAGTCCTGGTATTGCTAATCTCTCTAATCTTGTTCGTTTAGATATTTCTTCAAATAATTTTTCTGGAGAGATCCCGGATGTGTTTAGTGAGCTAAAGAGATTTCAGTATCTTATAGCAAATTCAAATAACTTTAGTGGTGGAATTCCCAGTTCACTGTCCAATTCTCCAACCGTTAGTTTGCTGAATTTGAGGAATAATTCATTGGAGGGTTCTATATATCTTAATTGCTCTGCTATGGTTACTTTGAATTCTCTGGATTTGGCTACCAATAAGTTCACTGGGCCTGTGCCTGATAATCTTCCCTTATGCAGACAATTGCAAGATGTCAACCTTGCCCGCAATAATTTCAGTGGCCAAATCCCTGAGAGCTTCAAACAATTCCATAGCCTCTCTTATCTTTCCCTCTCGAATTCGAGCCTCCATAATCTTTCCTCTGCTCTTCAAATTCTGCAGCAGTGTAGGAACCTAACTGCTTTGGTTCTCACCTTGAATTTCCCTGTTGAAACATTACCTGATGATCCTAATCTGCACTTTGAAAAGTTGAAGGTTCTTGTTATTGCAAGCTGTCAACTTAGAGGTTCAATTCCCCAATGGTTGAGCAAGATTTCTTCATTGCAGTTGTTGGATTTGTCATGGAACCATCTGAATGGAGCACTTCCACCATGGTTGGGAAGCTATAGGGATCTGTTTTACTTGGACTTATCGAACAATTCGTTTACTGGAGAGATCCCTAAGAGTTTAACTGAATTACCGAGCCTCATTCATGGAAATATCTCACTGGAGGAACCTTCACCAGATTTCCCTTTTTTCATGAAAAGGAATGAAAGTGCGAGGGGATTGCAGTATAATCAAATTTGGAGTTTTCCACCAACACTTGAACTTGGTCACAACTTTCTTAGTGGACCAGTTTGGCCTGAGTTCGGGAATCTGAAAAAGCTTATTGTTTTTGATTTGAAATTCAATAATCTCTCTGGACCGATTCCGGAAAATTTGTCTGAGATGAGCAGCTTGGAGATTCTGGATCTATCACATAATGACTTAACTGGGACCATACCACCTTCACTGGAAAGTCTCAGTTTTCTGTCTATCTTTAATGTTGCCTACAATCGACTTTATGGGAAGATTCCTTCTGGAGGTCAATTTCAGACATTCCCGAATTCAAGCTTTGAAGGAAACAATCTTTGTGGTGATCATCGGTTCAGTTGTCAAGATAATACAAGTGAACATGTGTCTCCAAAAAGATCAAGAAGGAACAAAGATATCATCATTGGAATGGTTGTTGGAATCATATTTGGGACAGCCCTTTTGATTGGCCTTATGTATTTGTTTGTTCTGCGTACACATAAACGCAACGAGGTCGATCCCGAGAAAGAGGAGCCTGACACCAATGATAAAAATTTAGAAGAACTCAGTTCAAGGCTAGTGGTGCTCTTCCAAAATTGGGAATCCTACAAGGAGCTTTGCATTGATGACCTTTTGGAATCAACCAACAATTTTGACCAAGCAAATATCATCGGTTGTGGTGGTTTTGGTCTGGTTTATAGAGGCACTCTCCCAGATGGTCGTAAGGTTGCCATTAAACGGCTTTCTGGTGATTGTGGTCAGATGGATAGAGAATTCCGTGCTGAAGTTGAAGCACTTTCAAGAGCTCAGCATCCAAATCTTGTCCATCTACAAGGATATTGCATGCATAAAAATGATAGGCTCTTAATATATTCTTACATGGAGAATGGTAGCTTGGACTATTGGTTGCATGAGAAGGTTGATGGATCATCCTTGCTTGGTTGGGAAACAAGGCTGAAAATTGCACAAGGGGCAGCTAGGGGTCTAGCTTATTTGCACCAGTCATGTGAGCCTCATATTCTTCACAGAGATATAAAGTCAAGTAACATTCTTTTAGATGAGAATTTTAAAGCTCACTTGGCCGATTTTGGTCTTGCAAGGCTCATACTTCCCTATGATACCCATGTCACCACTGATCTGGTAGGAACATTAGGCTACATTCCTCCTGAGTACGGACAAGCTTCAGTTGCAACCTATAAAGGTGATGTGTACAGTTTCGGGGTAGTGCTTTTGGAGCTTCTGACCGGGAAAAGGCCCATGGATATGTGCAAGCCAAAAGGTACCCGGGATTTGATCTCTTGGGTGATTCGCATGAAGATGGAAAACAAGGAGAGTGAGGTTTTTGACCCGTTTATCTATGGCAAGCAGCATGATAAGGAAATGTTGCGGGTTCTTGAAATCGCATGCCTTTGTTTAAACGAAAGCCCTAAAATTAGGCCTACAACGCAGCAGCTAGTTTATTGGCTTGACAAAGTGACATCGTTATCTAGTGTTTGA.
the screening method is described in 'ViGS technology for research on the function of stress-resistant genes of cotton, li Fangjun, university of agriculture in China, 2014'.
The cotton variety Guoxin No. 3 is shown in detail in Guoxin No. 3 insect-resistant cotton cultivation technology Feng Sulian, hebei agriculture, 2011,06.
1.2 Cloning of GhPSKR Gene
In this example, the GhPSKR gene was clonally amplified by reverse transcription PCR, confirmed by electrophoresis, and the obtained clone was further sequenced.
(1) Cotton leaf RNA was extracted using the ideley kit, and first strand cDNA was synthesized using the M-mLV reverse transcription kit, and the resulting first strand cDNA was used as a template for amplifying the full length of the GhPSKR1 gene.
Wherein, the ideley kit is purchased from the beijing ideley biotechnology company. M-mLV reverse transcription kit was purchased from Takara company.
(2) Designing two specific primers, namely an upstream primer F1 and a downstream primer R1, and carrying out PCR amplification to obtain a PCR product.
Wherein the use of North-AfricanThe Max Super-Fidelity DNAPolymerase kit amplifies the full length of GhPSKR gene.
The nucleotide sequence of the upstream primer F1 is shown as SEQ ID NO. 3:
5’-gtaccagattacgctcatatgATGGGGGCTCAAGGCTGCTG-3’。
the nucleotide sequence of the downstream primer R1 is shown as SEQ ID NO. 4:
5’-actggcctccatggccatatgTCAAACACTAGATAACGATGTC-3’。
the PCR amplification reaction system is shown in Table 1:
TABLE 1 PCR reaction system
The PCR amplification reaction procedure is shown in Table 2:
TABLE 2PCR amplification procedure
| Temperature/°c | Time of | Number of cycles |
| 95 | 3 Minutes | 1 Time |
| 95 | 15 Seconds | 35 Times |
| 52 | 15 Seconds | 35 Times |
| 72 | 40 Seconds | 35 Times |
| 72 | For 5 minutes | 1 Time |
(3) The PCR amplification reaction products were electrophoresed on a 1.5% agarose gel.
(4) And cutting off a target band after electrophoresis under an ultraviolet lamp, and recovering and purifying by using an agarose gel DNA recovery kit. Wherein, agarose gel DNA recovery kit is purchased from Biotechnology Co.
(5) The recovered fragment was recombined with pGADT7 vector, and the recombination reaction system was reacted at 37℃for 30min as shown in Table 3.
TABLE 3 ligation reaction System
(6) Mu.L of the ligation product was used to transform E.coli DH 5. Alpha. By heat shock, and positive clones were selected from LB solid plates containing 50mg/L ampicillin. The method of thermal shock is described in "J. Sambrook, et al, huang Peitang, et al, guidelines for molecular cloning experiments (third edition), science Press, 2002 edition).
(7) 5 Clones were picked and sent to Beijing qingke sequencing company for sequencing to obtain the required full-length gene CDS, and GhPSKR genes were obtained.
The agarose gel electrophoresis results are shown in FIG. 1.
Sequencing results show that the full length of the GhPSKR gene sequence is 3039bp, and the complete ORF reading frame of 1012 amino acids is encoded.
1.4 Subcellular localization of GhPSKR gene
Subcellular localization of GhPSKR protein was studied using tobacco epidermal cells.
Agrobacterium expressing the super 100-GhPSKR-GFP plasmid was cultured overnight in 10ml of YEP liquid medium containing 50mg/ml Kan and 25mg/ml Gen. The supernatant was removed by centrifugation at 6000rpm for 10min and resuspended to od=0.6 with a resuspension. The resuspended bacterial liquid is injected into tobacco leaves. Two days later, the injected tobacco leaves were observed under a laser confocal microscope, and the excitation light of GFP was 488nm.
FIG. 3 is a chart showing subcellular localization of GhPSKR protein according to the example of the present invention. It is shown that GhPSKR a is localized on the cell membrane.
EXAMPLE 2 VIGS method to obtain GhPSKR 1-silenced plants and phenotypic Studies under salt stress
In the embodiment, a VIGS-GhPSKR1 silencing vector is constructed by a VIGS method, ghPSKR silencing plants are further obtained by using the VIGS-GhPSKR1 silencing vector, and the phenotype of the plants under salt stress is studied.
2.1 Construction of the VIGS-GhPSKR1 silencing vector
(1) Total RNA from leaves of cotton variety "Guoxin No. 3" was extracted and reverse transcribed into cDNA according to the procedure in example 1.
(2) And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and using the following primer pair to obtain a PCR amplification product.
The nucleotide sequence of the primer F1 is shown in SEQ ID NO. 5:
5'-gtgagtaaggttaccgaattcTGTGGTCAGATGGATAGAGA-3'. Wherein. The underlined cleavage site for EcoR I in the sequence.
The nucleotide sequence of the primer R1 is shown in SEQ ID NO. 6:
5'-gagacgcgtgagctcggtaccAAGAATGTTACTTGACTTTA-3'. Wherein. The underlined BamHI cleavage sites in the sequence.
(3) And (3) carrying out double digestion on the PCR amplification product obtained in the step (2) by using restriction endo EcoRI and BamHI, and recovering the digested product.
(4) The pYL vector was digested simultaneously with restriction enzymes EcoRI and BamHI, and the vector backbone was recovered. Among them, pYL156,156 vector is also called pTRV2: RNA2 vector, which is described in non-patent document "Gao X,2013,Functional genomic analysis ofcotton genes with agrobacterium-mediated virus-induced gene silencing.".
(5) And (3) connecting the enzyme digestion product of the step (3) and the vector skeleton of the step (4) to obtain the recombinant plasmid pYL156,156-GhPSKR 1, wherein the connecting system is referred to as the steps in the examples.
(6) Sequencing verification was performed on recombinant vector pYL-GhPSKR 1.
Sequencing results show that the recombinant plasmid pYL-GhPSKR is a vector obtained by replacing a DNA fragment between EcoR I and BamHI cleavage sites of the pYL156 vector with a part GhPSKR gene fragment shown in positions 1-300 of SEQ ID NO.1 and keeping other sequences of the pYL156 vector unchanged, namely a VIGS-GhPSKR1 silencing vector.
FIG. 5 (A) is a graph showing the results of identifying the gene silencing efficiency of GhPSKR gene in cotton according to the example of the present invention.
As can be seen from FIG. 5, the GhPSKR gene was indeed silenced in the VIGS-GhPSKR1 plants compared to the VIGS-GFP plants.
2.1 Acquisition of VIGS-GhPSKR1 silenced plants
(1) The pYL156-GhPSKR1, pYL156-GFP, pTRV-RNA1 and pYL156-GhCLA1 are respectively subjected to electric shock transformation to obtain agrobacterium GV3101, and recombinant bacteria pYL156-GhPSKR1/GV3101, recombinant bacteria pYL156-GFP/GV3101, recombinant bacteria pTRV1/GV3101 and recombinant bacteria pYL156-GhCLA1/GV3101 are obtained.
Wherein pYL156-GhPSKR1 is the silencing vector of VIGS-GhPSKR1 constructed in step 2.1 in this example.
PYL156-GFP, pTRV1 (pTRV-RNA 1) and pYL-GhCLA 1 are described in non-patent document "Gao X,2013,Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.".
(2) The recombinant bacteria were shake-cultured in LB liquid medium containing antibiotics at 28℃respectively.
Wherein the formula of the LB liquid medium containing antibiotics comprises 50 mug/mL kanamycin, 25 mug/mL gentamycin, 10mM MES with pH of 5.6 and 20 mug acetosyringone, and the solvent is water.
(3) Recombinant bacteria were resuspended in VIGS solution and the bacterial concentration was adjusted to OD600 =1.5.
Wherein the formula of the VIGS solution is 10mM MES, 10mM MgCl2 and 200 mu M acetosyringone with pH of 5.6, and the solvent is water
(4) Recombinant bacteria pYL to GhPSKR1/GV3101, pYL156-GFP/GV3101 and pYL to GhCLA1/GV3101 with specific concentration are mixed with the bacterial liquid of recombinant bacteria pTRV1/GV3101 according to the volume ratio of 1:1 to obtain a mixed liquid 1, a mixed liquid 2 and a mixed liquid 3.
(5) Mix 1, mix 2 and mix 3 were filled with the lower surfaces of different cotton "Xin-test 17" cotyledons with a 1mL needle-less syringe and cultured for 2 weeks to obtain VIGS-GhPSKR1 silenced plants, VIGS-GFP control plants and VIGS-GhCLA indicator plants, respectively.
(6) After VIGS-GhCLA1 of the injection mixed solution 3 indicates that the plant has albino phenotype for about two weeks, respectively extracting RNA of leaf parts from the plants of the injection mixed solution 1 and the mixed solution 2, carrying out reverse transcription on cDNA, carrying out gene silencing efficiency analysis by fluorescent real-time quantitative PCR, and calculating the relative expression quantity by adopting a method of 2-ΔΔCt.
The primer pairs and PCR procedure used are as follows:
The nucleotide of the primer F2 is shown as SEQ ID NO. 7:
5’-AAGATGTCAACCTTGCCCG-3’。
the nucleotide of the primer R2 is shown as SEQ ID NO. 8:
5’-GGTGAGAACCAAAGCAGTTAGG-3’。
PCR procedure 94℃denaturation 30s, 94℃denaturation 5s, 60℃annealing 35s, 40 cycles.
Wherein, the cotton Actin9 gene is used as a control, and the primer pair for identifying the cotton Actin9 gene is as follows:
the nucleotide of the primer F3 is shown as SEQ ID NO. 9:
5’-GCCTTGGACTATGAGCAGGA-3’。
the nucleotide of the primer R3 is shown as SEQ ID NO. 10:
5’-AAGAGATGGCTGGAAGAGGA-3’。
FIG. 5 (A) is a graph showing the results of identifying the gene silencing efficiency of GhPSKR gene in cotton according to the example of the present invention.
The results of panel (A) in FIG. 5 show that the GhPSKR gene expression level of the plants injected with mixture 1 was significantly lower than that of the plants injected with mixture 2, i.e., the VIGS-GhPSKR1 silenced plants with the GhPSKR gene silenced by the above method and the VIGS-GFP control plants were successfully obtained.
Wherein, the mixed solution 1 contains pYL156-GhPSKR1/GV3101 and pTRV1/GV3101 bacterial solutions.
Mixed solution 2 contained pYL-GFP/GV 3101 and pTRV1/GV3101 bacterial solutions.
2.3 Phenotypic Studies of VIGS-GhPSKR1 silenced plants under salt stress
(1) The VIGS-GhPSKR 1-silenced plants obtained in step 2.2 in this example and VIGS-GFP control plants were subjected to 250mM NaCl stress treatment and observed for growth phenotype.
The stress treatment method is that when cotton grows to a three-leaf period, the VIGS-GhPSKR1 silent plant and the VIGS-GFP control plant are treated with 250mM NaCl.
(2) And 2. Carrying out 250mM NaCl stress treatment on the VIGS-GhPSKR1 silent plant and the VIGS-GFP control plant obtained in the step 2, taking the leaf and root system of the plant, and detecting the sodium ion content of the leaf and root system of the plant.
FIG. 5 (B) is a graph showing the results of the growth phenotype of the stress resistance verification of GhPSKR gene-silenced plants according to the examples of the present invention. The results show that under salt stress, compared with a VIGS-GFP control plant, the VIGS-GhPSKR1 silent plant has more obvious plant growth inhibition, smaller leaves, more obvious salt damage characteristics such as wilting and curling at the edges of the leaves and more sensitive to salt stress, and in a control group which is not treated by NaCl, the VIGS-GhPSKR1 silent plant and the VIGS-GFP control plant have no difference in growth characteristics.
FIG. 6 is a graph showing sodium ion content of leaves and root systems of GhPSKR gene-silenced plants according to an example of the present invention. The results indicate that VIGS-GhPSKR1 silenced plants have increased sodium accumulation in leaves and roots compared to VIGS-GFP control plants.
In summary, the expression level of GhPSKR gene is obviously reduced by utilizing the VIGS gene silencing technology in cotton, the gene silencing efficiency reaches 73.9%, compared with a VIGS-GFP control plant, the growth level of the VIGS-GhPSKR silencing plant is obviously inhibited under salt stress treatment, the salt damage characteristic is more obvious, the accumulation of sodium ions in leaves and roots is increased, and the plant is more sensitive to salt stress.
The experimental result shows that GhPSKR gene plays a positive regulation role in cotton salt stress and the like.
Example 3 construction of GhPSKR1 overexpression vector, acquisition of overexpressed transgenic plants and phenotypic Studies under salt stress
3.1 GhPSKR1 over-expression vector construction
(1) PCR amplification was performed using GhPSKR gene as template and forward and reverse primers to obtain a product containing GhPSKR gene, see example 1. Wherein, the forward primer is the upstream primer F1, and the reverse primer is the downstream primer R1.
(2) The product containing GhPSKR gene and super1300-GFP vector were digested with Sma I and Kpn I, respectively, to obtain digested product and vector frame, and recovered.
(3) GhPSKR1-GFP recombinant vector, ghPSKR1 over-expression vector, was ligated to the vector frame to give a recombinant expression vector 35S, ligation was described in example 1.
A recombinant expression vector 35S of GhPSKR-GFP was obtained by replacing the DNA fragment between Sma I and Kpn I cleavage sites of super1300 with GhPSKR gene of SEQ ID NO.1, and keeping the other sequences of super1300 unchanged. Wherein, the structure schematic diagram of the recombinant expression vector 35S GhPSKR-GFP is shown in figure 3.
3.2 GhPSKR1 acquisition of transgenic plants overexpressing
(1) The recombinant expression vector 35S obtained in the step 3.1 is introduced into an agrobacterium strain GV3101 by GhPSKR-GFP to obtain recombinant agrobacterium.
(2) The recombinant Agrobacterium obtained in (1) was shake-cultured in LB liquid medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL gentamicin at 28℃for 24 hours.
(3) The recombinant agrobacterium was collected and resuspended in a resuspension solution at 4000rpm for 10min, the concentration of the resuspension solution was adjusted to OD600 = 0.8. Wherein, the formulation of the heavy suspension is 50mM MES with pH of 5.6 and 5wt% sucrose, and the solvent is water.
(4) Adding silwetL-77 with the concentration of 500 μl/L to the bacterial suspension, dipping the inflorescence of the arabidopsis, wrapping the arabidopsis with a black plastic bag to keep humidity, horizontally placing, dark culturing at 20 ℃ for 24 hours, removing the plastic bag, recovering illumination, culturing plants to be firm according to a conventional method, and harvesting mature T0 generation seeds.
Wherein silwetL-77 are available from Beijing Cool Lei Bo technology Co., ltd., product number CS9791.
(5) T0 -generation seeds were cultured using 1/2MS medium containing 50mg/L hygromycin and positive plants were selected therefrom. Wherein, the positive plants are expressed as true She Jiankang which is dark green, and the roots are elongated into the culture medium.
(6) The obtained positive plants were subjected to selfing to obtain T1 -generation seeds, and then T1 -generation seeds were cultured with a 1/2MS medium containing 25mg/L hygromycin and positive plants were selected from the seeds. Wherein, the screening standard is that the proportion of positive plants is more than 3:1.
(7) Selfing the positive plants obtained in the step (6) to obtain T2 -generation seeds, culturing the T2 -generation seeds by using a 1/2MS culture medium containing 25mg/L hygromycin, and selecting positive plants from the seeds. Wherein, the screening standard is that all the strains are positive plants.
(8) And (3) carrying out selfing on the positive plant obtained in the step (7) to obtain T3 generation seeds, and cultivating T3 generation seeds to obtain T3 generation transgenic GhPSKR Arabidopsis plants.
(9) Leaf samples are taken by a puncher when the T3 generation GhPSKR1 generation Arabidopsis plants grow to the fourth week, leaf tissues are ground by liquid nitrogen, SDS plant total protein extracting solution (250 mM Tris-HCl pH 6.8,4% SDS,40% glycerol, 0.1% bromophenol blue and 4% beta-mercaptoethanol) is added, the mixture is uniformly mixed and denatured for 10 minutes at more than 95 ℃, and the supernatant is centrifugally taken to obtain denatured total protein, and SDS-PAGE detection is carried out.
Wherein, the formula of the SDS plant total protein extract comprises 250mM Tris-HCl with pH of 6.8, 4wt% SDS, 40wt% glycerol, 0.1wt% bromophenol blue and 4wt% beta-mercaptoethanol.
Transgenic GhPSKR A1 Arabidopsis plants, ghPSKR A1, overexpress transgenic Arabidopsis.
FIG. 7 is a graph showing the Western Blot identification of GhPSKR A1 over-expressed transgenic Arabidopsis homozygous lines described in the examples of the present invention. The results show that GhPSKR a transgenic arabidopsis homozygous strain OE1 and GhPSKR a transgenic arabidopsis homozygous strain OE2 were successfully obtained by this example.
Phenotypic study of 3.3GhPSKR1 overexpressing transgenic plants under salt stress
(1) The GhPSKR1 over-expression transgenic Arabidopsis homozygous strain OE1, ghPSKR1 over-expression transgenic Arabidopsis homozygous strain OE2 obtained in the 3.2 step, wild type Arabidopsis (WT) seeds were spring-treated at 4℃for 72 hours, and then transferred into a 20℃greenhouse, and cultured in a 16h light/8 h dark culture chamber with a light intensity of 60. Mu. Mol/m2/s and a humidity of 65.+ -.5% for 4 days.
(2) Arabidopsis seedlings were then transferred to medium containing 0mM or 100mM NaCl for continued growth for 8 days, and their growth phenotype was observed and root length changes were counted.
FIG. 8 is a graph showing the comparison of the growth conditions of GhPSKR A transgenic homozygous Arabidopsis thaliana and wild Arabidopsis thaliana under salt stress conditions, according to the example of the present invention, wherein the plants from left to right in FIG. 8 are wild Arabidopsis thaliana (WT) and GhPSKR A transgenic homozygous Arabidopsis thaliana OE1 and GhPSKR A transgenic homozygous Arabidopsis thaliana OE2, respectively.
FIG. 9 is a graph showing root length statistics of GhPSKR over-expressed transgenic Arabidopsis homozygous lines and wild type Arabidopsis under salt stress conditions as described in the examples of the present invention.
The results showed that there was no significant difference in root length between wild-type arabidopsis thaliana (WT) and GhPSKR a1 over-expressed transgenic arabidopsis thaliana homozygous strain OE1, ghPSKR a1 over-expressed transgenic arabidopsis thaliana homozygous strain OE2 on MS medium as normal control. And when subjected to salt stress, the root length of GhPSKR1 over-expression transgenic arabidopsis thaliana homozygous strain OE1 and GhPSKR1 over-expression transgenic homozygous strain OE2 is obviously prolonged compared with that of wild type arabidopsis thaliana (WT). This shows that GhPSKR1 over-expressed transgenic arabidopsis homozygous strain OE1, ghPSKR1 over-expressed transgenic arabidopsis homozygous strain OE2 has good stress resistance. The GhPSKR gene plays a positive regulation role in regulating plant salt stress. GhPSKR1 gene can be applied to improving the salt tolerance of plants.
The invention clones cotton GhPSKR gene, constructs GhPSKR1 over-expressed transgenic arabidopsis plant by using transgenic technology and constructs GhPSKR silencing plant by using VIGS technology, performs functional verification on GhPSKR gene, and confirms GhPSKR1 gene can improve salt tolerance of plant, is beneficial to deeper research on response mechanism of plant to salt stress signal, lays good molecular foundation for effectively improving salt tolerance of plant, and has important value for exploring signal regulation network of plant under salt stress.
The invention firstly reveals the forward regulation and control function of GhPSKR genes in the salt tolerance of cotton, and provides excellent regulation and control targets and theoretical guidance for the salt tolerance cultivation of cotton. The invention provides an application of GhPSKR gene in regulating and controlling plant salt tolerance, and develops a new application of GhPSKR gene in cotton stress resistance.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and in order to prevent redundancy, the present invention describes a preferred embodiment.
While the preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts, all such variations and modifications are within the scope of the invention.