CN113105534B

CN113105534B - Application of WRKY55 transcription factor in plant salt resistance

Info

Publication number: CN113105534B
Application number: CN202011528268.4A
Authority: CN
Inventors: 隋娜; 郑洪祥; 李思敏
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2023-05-30
Anticipated expiration: 2040-12-22
Also published as: CN113105534A

Abstract

The invention provides application of a WRKY55 transcription factor in plant salt resistance, and belongs to the technical field of biology. The invention reports a salt resistance negative regulation related protein WRKY55 and a coding gene thereof for the first time. According to the invention, the research shows that the expression quantity of the WRKY55 gene in sweet sorghum is obviously reduced under the condition of salt stress, the WRKY55 gene is introduced into arabidopsis, and the functional identification is carried out on the arabidopsis, so that the excessive expression of the WRKY55 is shown, the salt resistance of plants is reduced, and the negative regulation and control effect on the salt resistance of the plants are shown, so that a foundation is provided for cultivating resistant plants.

Description

Application of WRKY55 transcription factor in plant salt resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a WRKY55 transcription factor in plant salt resistance.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Soil salinization is a common problem worldwide. Currently, the global saline-alkali soil is wide in area and is 9.5xl0 ⁸ hm ² And there is an increasing trend. The inadequacy of irrigation measures in agricultural activities aggravates the generation of salinized soil, and the area is continually expanding. The salinized environment is harmful to most crops. Therefore, the saline soil is comprehensively treated to increase the cultivated area, and in addition, the plant can grow on the saline soil better by increasing the capability of resisting the saline environment, which is a great problem to be solved urgently for promoting the agricultural development.

The transcription factor is an important molecule in a regulatory network, and almost participates in all life processes of organisms through signal transduction and regulation of stress response genes, wherein the WRKY transcription factor is an important transcription factor discovered in recent years, is a super gene family and plays a key role in growth and physiological regulation of plants. WRKY transcription factors can specifically interact with cis-acting elements in eukaryotic gene promoters, are trans-acting factors playing an important role in the process, and promote or block the expression level of target genes to respond to various stress responses. However, the inventors found that the study is currently mainly conducted on a small number of model organisms such as Arabidopsis thaliana, etc., while the study on WRKY transcription factors in other plants is very small.

Disclosure of Invention

In order to overcome the technical problems, the invention provides application of the WRKY55 transcription factor in plant salt resistance. According to the invention, the research shows that under the condition of salt stress, the expression quantity of the WRKY55 gene in sweet sorghum is obviously reduced, the WRKY55 gene is introduced into arabidopsis, and the functional identification is carried out on the arabidopsis, so that the result shows that the WRKY55 gene plays a negative regulation role in the plant salt resistance process. Based on the above-described results, the present invention has been completed.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a protein designated SbWRKY55, being any one of the following a 1) to a 3):

a1 Protein shown in a sequence 1 in a sequence table;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 1 in the sequence table and has the same function and is derived from the sequence 1;

a3 Other genes code proteins which have more than 50% similarity with the amino acid sequence composition shown in the sequence 1 and have the activity of the protein shown in the sequence 1;

in the above a 2), the "substitution and/or deletion and/or addition of one or more amino acid residues" is substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the a 1) to a 3) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

In a second aspect of the invention, nucleic acid molecules encoding the above proteins are also within the scope of the invention.

The above-mentioned nucleic acid molecule may be a DNA molecule as described in any one of the following b 1) to b 4);

b1 The coding region is a DNA molecule shown as a sequence 2 in a sequence table;

b2 The nucleotide sequence is a DNA molecule shown as a sequence 2 in a sequence table;

b3 A DNA molecule which has 75% or more identity to the nucleotide sequence defined in b 1) or b 2) and which encodes said protein WRKY 55;

b4 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 1) or b 2) and which codes for said protein WRKY 55.

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, etc.

In addition, recombinant vectors, expression cassettes, transgenic cell lines, host bacteria or transgenic plants comprising nucleic acid molecules encoding the above protein SbWRKY55 are also within the scope of the invention. Wherein the host bacteria may be eukaryotic or prokaryotic such as, but not limited to, agrobacterium, yeast, E.coli, and the like.

The recombinant vector comprises inserting a nucleic acid molecule encoding the protein SbWRKY55 into an expression vector pROKII-GFP, and is named pROKII-GFP-WRKY55 for over-expression of the WRKY55 gene vector.

Primer pairs that amplify the full length of the nucleic acid molecule encoding the protein WRKY55 described above, or any fragment thereof, are also within the scope of the present invention. The primer comprises a sequence 3 and a sequence 4 in a sequence table.

The use of the above-described proteins, the above-described nucleic acid molecules or the above-described recombinant vectors, expression cassettes, transgenic cell lines, host bacteria or transgenic plants for regulating stress resistance of plants is also within the scope of the invention;

in the above application, the regulation of plant stress resistance may be decreasing plant stress resistance or increasing plant stress resistance; preferably to increase stress resistance of the plant. According to the invention, the research shows that the deletion or inhibition of the WRKY55 gene expression in the plant can improve the stress resistance of the plant, and the plant salt resistance is particularly improved.

It is emphasized that in the above application, the WRKY55 gene includes a gene having a high homology thereto, such as the AtWRKY55 gene in arabidopsis thaliana. The protein sequence homologous to the sweet sorghum WRKY55 amino acid sequence, the coding nucleotide sequence and the stress resistance application thereof are all within the protection scope of the invention.

In the above application, the stress resistance may be salt resistance.

In such applications, the plant may be dicotyledonous (e.g., arabidopsis, cotton, castor, pumpkin, peanut, cassava, morning glory, etc.), or monocotyledonous (e.g., sorghum, sweet sorghum, maize, rice, wheat, etc.).

In a fifth aspect of the present invention, there is provided a plant breeding method comprising knocking out or inhibiting the expression of the WRKY55 gene described above, and improving stress resistance of a plant.

It is emphasized that in the above method, the WRKY55 gene includes a gene having a high homology thereto, such as the AtWRKY55 gene in arabidopsis thaliana. The protein sequence homologous to the sweet sorghum WRKY55 amino acid sequence, the coding nucleotide sequence and the application of the coding nucleotide sequence in regulating and controlling plant stress resistance are all within the protection scope of the invention.

In the above method, the stress resistance may be salt resistance.

In the above method, the plant may be dicotyledonous plant (such as Arabidopsis, cotton, castor, pumpkin, peanut, cassava, morning glory, etc.), or monocotyledonous plant (such as sorghum, sweet sorghum, maize, rice, wheat, etc.).

The beneficial technical effects of the one or more technical schemes are as follows:

According to the technical scheme, a salt resistance negative regulation related protein WRKY55 and a coding gene thereof are reported for the first time, specifically, the technical scheme is that the WRKY55 gene is screened from sweet sorghum, the expression quantity of the WRKY55 gene is reduced under salt treatment conditions, the WRKY55 gene is introduced into arabidopsis, and functional identification is carried out on the Arabidopsis, so that the excessive expression of the WRKY55 gene leads to obvious increase of plant sodium ions and obvious reduction of potassium ions, biomass accumulation in plants, sodium-potassium ion steady state, ROS content and membrane lipid peroxidation process are influenced, and therefore, the salt resistance of the plants is reduced, and therefore, the negative regulation effect on the salt resistance of the plants is shown.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a graph showing the hydrophilicity analysis of sweet sorghum SbWRKY55 protein in example 1 of the present invention.

FIG. 2 shows the prediction of the signal peptide and transmembrane structure of SbWRKY55 protein in example 1 of the present invention; a, predicting a signal peptide; and B, predicting a transmembrane structure.

FIG. 3 is a functional domain of SbWRKY55 protein of example 1 of the present invention.

FIG. 4 shows the analysis of the homology of SbWRKY55 protein sequence in example 1 of the present invention.

FIG. 5 shows the expression level of SbWRKY55 at different NaCl concentrations in example 2 of the present invention.

FIG. 6 shows subcellular localization of SbWRKY55 protein in example 2 of the present invention.

FIG. 7 is a kana screening of Arabidopsis transformed seedlings in example 3 of the present invention.

FIG. 8 is a PCR identification of the genome of transgenic Arabidopsis lines in example 3 of the present invention.

FIG. 9 is a diagram of T in example 3 of the present invention ₃ Canada screening of transformed seedlings of Arabidopsis thaliana.

FIG. 10 is a PCR characterization of the genome of transgenic Arabidopsis lines in example 3 of the present invention.

FIG. 11 shows the relative expression levels of SbWRKY55 in different strains of transgenic Arabidopsis according to example 3 of the present invention.

FIG. 12 is a screen for homozygous mutants of different Arabidopsis T-DNA insertions in example 3 of the present invention; and (3) injection: lane 1, from left to right, is 2000marker; sample application sequence: even lanes are LBb1.3+RP and odd lanes are LP+RP.

FIG. 13 shows the relative expression levels of AtWRKY55 in the different Arabidopsis mutant lines of example 3 of the present invention.

FIG. 14 shows the phenotype of the individual strains of Arabidopsis thaliana after 7d treatment with NaCl at different concentrations in example 4 according to the invention.

FIG. 15 shows the effect of NaCl treatment at different concentrations on the germination rate of different Arabidopsis lines in example 4 according to the present invention.

FIG. 16 shows the effect of NaCl treatment at different concentrations on root length of different Arabidopsis lines in example 4 of the present invention.

FIG. 17 shows the effect of NaCl treatment on fresh and dry weight of seedlings of different Arabidopsis lines in example 4 of the present invention.

FIG. 18 shows the effect of NaCl treatment on MDA content in leaves of different Arabidopsis lines in example 4 of the present invention.

FIG. 19 is a graph showing the Na treatment of leaves of various Arabidopsis lines with NaCl treatment in example 4 of the present invention ⁺ Content, K ⁺ Content of Na ⁺ /K ⁺ Influence of the content.

FIG. 20 shows the expression level of an ion transport-related gene under salt stress in Arabidopsis roots in example 4 of the present invention; wherein a is SOS1, b is AtCNGC10, c is AtCNGC1, d is AtCNGC2, e is AtHKT1, and f is AtNHX1.

FIG. 21 shows a SbWRKY55 complementation of Arabidopsis mutant in example 4 of the present inventionSalt tolerance, wherein A is the relative expression amount of SbWRKY55 in different Arabidopsis lines, B is the influence of NaCl treatment with different concentrations on the fresh weight of seedlings of different Arabidopsis lines, C is the influence of NaCl treatment with different concentrations on the dry weight of seedlings of different Arabidopsis lines, D is the influence of NaCl treatment with different concentrations on the MDA content in leaves of different Arabidopsis lines, E is the influence of NaCl treatment with different concentrations on Na of different Arabidopsis lines ⁺ Effect of content, F is the effect of treatment with different concentrations of NaCl on different Arabidopsis lines K ⁺ Influence of the content.

FIG. 22 is a graph showing the effect of SbWRKY55 negative control on the salt resistance of sweet sorghum in example 4, wherein A is the effect of NaCl treatment at different concentrations on the growth of different sweet sorghum strains; b is the relative expression quantity of SbWRKY55 in different sweet sorghum strains, C is the influence of different concentration NaCl treatment on MDA content in leaves of different sweet sorghum strains, D is the influence of different concentration NaCl treatment on fresh weight of seedlings of different sweet sorghum strains, E is the influence of different concentration NaCl treatment on dry weight of seedlings of different sweet sorghum strains, F is the influence of different concentration NaCl treatment on Na of different sweet sorghum strains ⁺ Influence of the content, G is the effect of NaCl treatment at different concentrations on different sweet sorghum strains K ⁺ Influence of the content.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present disclosure, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1: bioinformatics analysis of sweet sorghum WRKY55 gene

1 test materials

Sweet sorghum (Sb 02G 011050), and the WRKY protein sequences of short anther wild rice (xm_ 015839135.1), brachypodium distachyon (xm_ 014901792.1), zucchini (xm_ 023659186.1), corn (xm_ 020540231.2), cassava (xm_ 021761582.1), anthurium (KY 597634.1), vitis vinifera (KY 411919.1), arabidopsis (AT 2G 40740) obtained by BLASTp on NCBI.

2 test method

Firstly, the primary structure and physicochemical properties of SbWRKY55 protein are analyzed by using software ExpASY. Detection of the signal peptide and prediction of the transmembrane region was performed using software Signal4.1 and TMHMM. SMART online soft can predict the structural domain of SbWRKY 55. Homologous sequences of SbWRKY55 are obtained from NCBI BLASTP on the net, and then the homologous sequences are subjected to protein aspect alignment by using software such as MegAlign and the like, and a evolutionary tree reflecting the relationship is constructed.

3 test results and analysis

3.1 Analysis of amino acid sequence and physicochemical Properties of SbWRKY55

MDAASRMTVLQNGVHDSYTWRKYGQKEILGARFPRSYYKCGRRPGCPAKKHVQQCDADPSKLEVTYLEAHTCDDPPPSSSHAVPDPTAGSDALLVPPVPTVPFPSAQCYGGGRPSPPPLPPYQVPYAATTLGSNVLTLTATGVLLPSASYDPVPDVTDCTPSLEQEQDHDLLHIPSPACSQSELLPMEAAKLSPHAHGLPLSLEHTLDCDFAVPEL(SEQ ID NO.1)

The SbWRKY55 has a gene registration number of Sb02g011050 (SEQ ID NO. 2), and the CDS sequence has 651 bases and can code 216 amino acids. Based on analysis of physicochemical properties, the isoelectric point (pI) of the sequence was found to be 5.21, indicating acidity. The molecular Formula (Formula) of SbWRKY55 protein is C1023H1582N274O318S11, and the relative molecular mass is 23kDa. The protein has 14 strong basic amino acids, such as Arg and Lys;24 strongly acidic amino acids, such as Asp, glu. In addition, the SbWRKY55 protein contains 62 and 59 hydrophobic amino acids and 59 polar amino acids, respectively. The protein has an instability index of greater than 40, 68.46, indicating protein instability. Average hydrophilicity (GRAVY): -0.364.

ATGGATGCTGCATCCCGGATGACAGTGCTACAGAATGGAGTTCATGATTCGTACACATGGAGGAAATACGGGCAGAAGGAAATTCTGGGCGCCAGATTTCCAAGGAGTTACTACAAATGTGGCCGCCGGCCGGGCTGCCCCGCGAAGAAGCACGTGCAGCAATGCGACGCGGATCCGTCCAAGCTGGAGGTCACCTACTTGGAGGCACACACGTGCGATGATCCACCACCGTCGTCGTCCCATGCTGTTCCAGATCCGACGGCCGGCTCCGACGCTCTGCTCGTACCACCAGTCCCGACCGTTCCGTTTCCATCAGCTCAGTGCTACGGCGGCGGCCGACCGTCGCCGCCGCCGCTGCCGCCGTACCAGGTGCCGTACGCCGCGACGACGCTCGGCTCCAACGTCCTGACGCTGACGGCCACCGGTGTTCTTCTGCCTAGTGCAAGCTACGACCCTGTTCCGGATGTCACGGACTGCACGCCGTCGTTGGAGCAGGAGCAAGACCATGATCTGCTTCACATACCTTCGCCGGCTTGTTCACAGTCAGAGCTGCTGCCGATGGAGGCTGCCAAGCTTTCACCGCACGCGCACGGGCTGCCTCTGTCGTTGGAGCACACGCTGGATTGCGACTTTGCTGTACCCGAGCTTTAA(SEQ ID NO.2)。

3.2 Hydrophilicity and hydrophobicity analysis of SbWRKY55 amino acid sequence

According to the analysis of the hydrophilicity and hydrophobicity of the target protein by software, in FIG. 1, the hydrophilicity of the SbWRKY55 protein is predicted, the greater the score of the amino acid is, the stronger the hydrophobicity is, and from the distribution perspective, the hydrophilic region of the SbWRKY55 protein is far greater than the hydrophobic region, and the score of the amino acid is negative, so that the SbWRKY55 is hydrophilic.

3.3 SbWRKY55 signal peptide and transmembrane structure prediction and analysis

From the analysis of FIG. 2, the SbWRKY55 protein is devoid of signal peptide. It has no transmembrane region, so the SbWRKY55 protein has no transmembrane helix region and is a mature protein secreted outside the cell.

3.4 Conserved domain of SbWRKY55 protein

As can be seen from the analysis of FIG. 3, sbWRKY55 has a typical WRKY domain between amino acids 14-74, with WRKYGQK at the N-terminus and C-terminal zinc finger structure C ₂ HC, family classification is class III.

3.5 Homology analysis of SbWRKY55 protein and proteins of other species

By comparison of SbWRKY55 protein with homologous WRKY proteins of other species, they were found to be highly homologous within about 60 amino acids, which are conserved, and to have both the core sequences WRKYGQK and the C2HC zinc finger structure, belonging to class III of the WRKY family classification (FIG. 4). As can be seen from the evolutionary tree of FIG. 4, the closest relationship with sweet sorghum is wild rice, and the relationship with Arabidopsis is far away.

EXAMPLE 2 cloning of sweet sorghum WRKY55 Gene, vector construction and subcellular localization

1 test materials and Material handling

Sweet sorghum salt-tolerant high sugar-content inbred line M-81E seed (benefit of Shandong national academy of sciences), tobacco (benthos) seed, escherichia coli strain (DH 5 alpha), agrobacterium strain (GV 3101), cloning vector pEASY-Blunt3 simple, pROKII-GFP expression vector driven by CaMV35S promoter, and the like.

Selecting sweet sorghum seeds with similar sizes and plump seeds, and placing the sweet sorghum seeds in a mesh bag for washing for 12 hours in running water. The sand is washed by tap water and then is packaged into flower pots. The seeds were then planted uniformly in sand, 8 per pot, approximately 2cm deep, without too shallow a depth to prevent flushing out during watering. Tap water is poured once a day until water can flow out from the holes at the bottom of the flowerpot, and the tap water is poured thoroughly. 1/2Hoagland nutrient solution is poured every day after seedling emergence. When the seedlings developed the third true leaves, 4 seedlings with balanced vigour were selected per pot. And then irrigating with complete Hoagland nutrient solution, and obtaining the full length of SbWRKY55 gene from the root material-extracting RNA when the plant grows to the 4-leaf stage. When a part of sorghum seedlings grow to three leaves and one heart, the sorghum seedlings are treated with Hoagland nutrient solution containing 0, 50, 100 and 150mM NaCl respectively, roots of the sorghum seedlings are obtained after 48 hours, and the expression quantity of SbWRKY55 genes is verified.

2 test method

2.1 extraction of RNA

RNA was extracted using RNAprep Pure plant Total RNA extraction kit (TIANGEN).

2.2 cDNA Synthesis

The extracted RNA was reverse transcribed into cDNA using the reverse transcription kit (Code: FSQ-101) for Vaccinium.

2.3 analysis of expression of the sweet sorghum WRKY55 Gene under salt treatment at different concentrations

Finding the sequence of the WRKY55 gene of sweet sorghum according to the gene number from NCBI website, designing quantitative primers by using Beacon Designer 7 software according to the cDNA sequence, wherein the Sb beta-actin gene is selected as an internal reference primer, and the primer sequence is as follows:

Sb actin-S:TGGCATCTCTCAGCACATTC(SEQ ID NO.5)

Sb actin-A:AATGGCTCTCTCGGCTTGC(SEQ ID NO.6)

W55-S:ATGACAGTGCTACAGAAT(SEQ ID NO.7)

W55-A:TAGTAACTCCTTGGAAATCT(SEQ ID NO.8)

preparing a fluorescent quantitative PCR reaction system.

2.4 cloning of full Length of sweet sorghum WRKY55 Gene

Primers were designed based on the CDS sequence of the WRKY55 gene. And (3) finding out the restriction enzyme sites on the gene sequences, selecting restriction enzyme sites which are not arranged on one gene sequence and are easy to restriction enzyme on the expression vector according to the restriction enzyme sites on the pROKII-GFP of the connected expression vector, and adding the base sequences of the corresponding restriction enzyme sites to the 5' ends of the forward and reverse primers according to the direction on the vector. The 5' end of the primer is respectively added with enzyme cutting sites XbaI and KpnI, and the specific primers are as follows:

w55-5:5’-gctctagaatggatgctgcatcccggatg-3’(SEQ ID NO.3)

w55-3:5’-ggggtaccaagctcgggtacagcaaagtc-3’(SEQ ID NO.4)

the cDNA sequence of the sweet sorghum WRKY55 obtained by reverse transcription is used as a template, and the designed w55-5 and w55-3 primers are used for amplification. The PCR reaction product thus obtained was subjected to agarose gel electrophoresis to determine whether or not the desired target band was present. And if so, performing cutting adhesive recovery so as to perform subsequent tests. And (3) connecting and transforming the target fragment and pEASY-Blunt3 Vector, carrying out colony PCR by using a screened positive clone single colony as a template and using a primer for amplifying the WRKY55 target fragment, preserving strain in a bacterial solution with glycerol after sequencing correctly, extracting plasmids, and extracting plasmids by using a plasmid small extraction kit (TIANprep Mini Plasmid Kit).

2.5 construction of expression vectors

The expression vector used in this experiment was pROKII-GFP. And selecting two enzyme cutting sites of XbaI and KpnI, respectively carrying out double enzyme cutting on the extracted plasmid and the expression vector, connecting a target fragment WRKY55 with an expression vector pROKII-GFP to obtain pROKII-SbWRKY55-GFP connection product, and converting the pROKII-SbWRKY55-GFP connection product into escherichia coli (DH 5 alpha). Since the expression vector has kana resistance, 50mg/mL kana antibiotic was added to the medium to screen successfully transformed E.coli. After culturing, the single colony positive to colibacillus is selected for PCR verification. And (3) performing agarose gel electrophoresis on the obtained PCR product, then performing liquid culture on positive single colonies of the target fragment, and finally sending the obtained bacterial liquid to sequencing to verify whether the bacterial liquid is the target gene. The bacterial liquid with correct sequencing is preserved with glycerine first, then expression vector plasmid is extracted, and the extracted plasmid is preserved in-20 deg.c refrigerator.

2.6 transformation of Agrobacterium with expression vectors

mu.L of pROKAI-SbWRKY 55-GFP ligation product and 100. Mu.L of Agrobacterium competent cells GV3101 were mixed, transformed and cultured, and positive single colonies of Agrobacterium competent cells GV3101 were selected for PCR verification, and the specific method was the same as above. And (3) performing agarose gel electrophoresis on the obtained PCR product, then performing liquid culture on positive single colonies of the target fragment, and finally sending the obtained bacterial liquid to sequencing to verify whether the bacterial liquid is the target gene.

2.7 Subcellular localization analysis of SbWRKY55

The test adopts a tobacco transient transformation method, and agrobacterium tumefaciens bacteria liquid with a target gene is injected into tobacco, so that the positioning condition of the target gene is observed, and specific steps refer to a test operation method of Wu Yingjie and the like (2010).

3 test results and analysis

3.1 expression level of the sweet sorghum WRKY55 Gene under salt treatment at different concentrations

Based on the transcriptome results, it was found that the expression level of M81-E WRKY55 gene of sorgo was significantly reduced after 48 hours of 150mM NaCl treatment, and for this purpose, the expression level of SbWRKY55 was measured after 48 hours by treating sorgo with 0, 50, 100, 150mM NaCl to measure the expression level of SbWRKY55 under salt treatment. As shown in FIG. 5, the expression level of the sweet sorghum WRKY55 was not significantly different from that of the 0mM NaCl treatment. The expression level of SbWRKY55 was significantly decreased when the treatment with 100mM NaCl was started, and the expression level of SbWRKY55 was the lowest when the NaCl concentration was 150 mM.

3.2 cloning of the sweet sorghum WRKY55 Gene

The SbWRKY55 gene is amplified by using a W55-5 and W55-3 primer, the size of target bands is 664bp respectively, the target bands are connected with a cloning vector pEASY-Blunt3 after gel recovery through electrophoresis detection, and then the connection products are transformed into escherichia coli DH5a for overnight culture. The following day, single colonies were picked and streaked while performing colony PCR. The PCR product was then run to see if the target band was present. Inoculating the positive single colony into a liquid LB culture medium for culture, and sending the cultured escherichia coli bacterial liquid to Boshang company for sequencing. And (5) preserving the strain in the corresponding bacterial liquid with correct sequencing, and extracting plasmids for the next test.

3.3 construction of expression vectors

The cloning vector plasmid with correct sequence and the empty expression vector pROKII-GFP are subjected to double digestion, and digestion sites are XbaI and KpnI respectively. Comparing the double digested expression vector with the non-digested expression vector, comparing the bands above the lanes, namely that the bands are cut, recovering the target gene fragment and the cut vector fragment, connecting the target gene fragment and the cut vector fragment, transferring the target gene fragment to escherichia coli competent DH5a, and performing colony PCR verification to obtain positive colonies. The plasmid of the E.coli strain, in which the SbWRKY55 gene and pROKII-GFP were successfully ligated, was subjected to extraction and sequencing. And (3) transferring the expression vector plasmid which is sequenced successfully into agrobacterium competent GV3101, and performing colony PCR verification to obtain a positive single colony.

3.4 Subcellular localization analysis of SbWRKY55

The target fragment is connected with pROKII-GFP expression vector to construct GFP fusion protein, so that we use tobacco transient transformation method to make subcellular localization on SbWRKY55 expression. As a result, as shown in FIG. 6, it was found that the green fluorescent signal of the tobacco hypoepidermic cells transformed with pROKII-GFP empty vector was observed under a microscope in almost whole cells such as nucleus and cell membrane, and in cytoplasm, but not so much. The expression vector with the target gene is transferred into tobacco, and the green fluorescent signal of the lower epidermal cell is almost distributed in the cell nucleus. Transcription factors generally play a regulatory role in the nucleus, so it is preliminarily ascertained that the SbWRKY55 gene is expressed in the nucleus and is a nuclear transcription factor.

Example 3: obtaining of Arabidopsis SbWRKY55 overexpression and AtWRKY55 mutant Strain

1 test materials

Columbia ecotype (Col-0) Arabidopsis plants Arabidopsis WRKY55 (AT 2G 40740) mutants purchased from the Tair website were WRKY55-1 (SALK_ 084192), WRKY55-2 (SALK_ 021677), WRKY55-3 (SALK_ 121437), WRKY55-4 (SALK_ 084288), WRKY55-5 (SALK_082916), WRKY55-6 (CS 838667), respectively.

2 test method

2.1 acquisition of the Arabidopsis SbWRKY55 overexpressing line

After the agrobacteria with pROKII-GFP-SbWRKY 55 are used for impregnating the inflorescence of the Arabidopsis, the black plastic film is used for covering, the dark culture is carried out for about 24 hours, and then the black preservative film is uncovered for normal culture. And (3) carrying out secondary infection after about 7d, and placing the infected plants in an artificial climate incubator (22-16 h of illumination/18-8 h of darkness) for continuous culture. After about 24 hours, the black plastic film was removed and the culture was continued. After about one week, the dip dyeing is continued once, this time being about 20-30 s. After three continuous dip-dyeing, no new inflorescences are generated basically, and the culture is continued in an incubator until the arabidopsis seeds are ripe, and the seeds are collected for later-period transformation seedling screening.

1/2MS culture medium is prepared, and 1mL of culture medium is used before the plate is poured on an ultra clean bench: the kanavidines were added in a proportion of 1. Mu.L 50mg/L kanavidines. Counting the infected seeds as T ₀ Instead, the seeds are placed in an EP tube of 1.5mL, 75% alcohol is added for 3-4 min, and the total three times are performed. Then the ultra-clean workbench is sterilized for three times by using 95 percent alcohol, and is washed for 3 to 4 times by using sterile water. A few 0.1% agar was added to the centrifuge tube to suspend the seeds and to enable the seeds to be immobilized on the medium. On-culture with a pipetteOn-demand T on nutrient medium in large quantity ₀ Seed generation. After sealing the membrane, the culture dish is placed in spring flowers at 4 ℃ for 3d, then is taken out and vertically placed in a tissue culture chamber for culture, and the temperature is about 25 ℃. We continued to screen each well grown viable seedling as a single transgenic independent line. Transferring the surviving seedlings to nutrient soil, putting the seedlings into a artificial climate incubator (22-16 h illumination/18-8 h darkness), marking, and continuing culturing until the seeds are mature. The single seed harvest of each seedling is T ₁ And (3) replacing. The received seeds were allowed to air dry for approximately two weeks to allow for post maturation. Culture medium was also prepared, and kana was added and the same strain was placed in each dish. T (T) ₁ Seed generations have begun to segregate for homozygosity and heterozygosity. We until essentially well grown seedlings were screened on 1/2MS medium supplemented with kana antibiotics.

After the resistant seedlings screened above were cultured for about two weeks, the leaves were taken to extract DNA, and PCR amplification was performed using 35s, W55-3 primers. After amplification, 5. Mu.L of the PCR product was subjected to 1% agarose gel electrophoresis to detect the presence or absence of a correct target band.

And (3) carrying out semi-quantitative expression analysis detection and real-time fluorescence quantitative analysis detection on the SbWRKY55 genes of different Arabidopsis overexpression lines.

2.2 screening of the Arabidopsis WRKY55 homozygous mutant

Mutants purchased on the Tair website are mostly heterozygotes, and further screening is needed to obtain homozygotes. The corresponding mutant of the Arabidopsis WRKY55 is T-DNA insertion type, and insertion sites are all on the promoter. Screening and identification are carried out by adopting a two-primer method, namely, different specific primers on genes and a primer LBb1.3 on T-DNA are designed according to different T-DNA insertion positions, so as to screen homozygous mutants. Different mutant numbers are input from a T-DNA Primer Design (http:// signal. Salk. Edu/tdnaprimers.2. Html) website, corresponding primers are obtained to extract genome DNA, and mutant homozygotes are screened.

3 test results and analysis

3.1 acquisition of the Arabidopsis SbWRKY55 overexpressing line

3.1.1 Carna screening of overexpressed lines and identification at the DNA level

After about 7 days, as indicated by the arrow in fig. 7, the cotyledons grown from the germinated seeds were found to be mostly yellow, the roots were also extremely short, and finally almost wilting and dying. Only leaves of a few seedlings in each dish are green, roots are relatively long, the seedlings grow well, the seedlings are preliminarily determined to be transgenic seedlings, tens of positive seedlings are obtained through screening, DNA is extracted, and the seedlings have bright target bands after PCR verification (figure 8). Each surviving individual seedling was used as a line and seeds were harvested individually. After the T3 generation was found by Cana screening and PCR identification, the growth of several strains, S2, S4, S5, S6, S10, S12, S15, S51, S53 (FIG. 9), were stable. Almost all can survive after being screened by kana, the cotyledons are fresh green, and the root system is longer. The target bands were all verified by PCR (FIG. 10), and were essentially determined to be homozygous over-expressed lines.

3.1.2 semi-quantitative expression analysis and detection of different overexpressing lines

After DNA level verification, 9 strains of SbWRKY55 over-expression Arabidopsis strains S2, S4, S5, S6, S10, S12, S15, S51 and S53 are selected. In order to verify the expression level of the SbWRKY55 genes in their respective bodies, specific primers of the Arabidopsis Actin2 genes are used as internal references, the internal references are adjusted to be consistent, and the gene loading amount of the internal references is used as a template amount to perform specific PCR amplification of the SbWRKY 55. The stripe brightness of different Arabidopsis over-expression lines is not uniform, which indicates that the expression amount of the gene in each line is different. The SbWRKY55 transgenic Arabidopsis numbers S6, S15 and S51 have higher expression level under the same condition, and S10, S12 and S53 times.

3.1.3 real-time PCR analysis of different overexpression lines of Arabidopsis thaliana

From FIG. 11, it can be seen that the transcription level of SbWRKY55 of the overexpressed Arabidopsis line is significantly increased compared with the wild-type, so that it can be fully demonstrated that SbWRKY55 has been successfully transferred into the wild-type Arabidopsis plant, and that the SbWRKY55 gene is overexpressed in the transgenic plant. Second, the level of SbWRKY55 transcription in S6, S15, S51 is relatively high, while S10, S12, S53 times, which is also consistent with semi-quantitative results.

3.2 screening of the Arabidopsis WRKY55 homozygous mutant

3.2.1 identification of homozygous mutants of Arabidopsis thaliana

DNA of different mutants is used as a template, the primers LBb1 and RP have bands during amplification, and no bands during amplification of LP and RP are homozygous mutants. We screened three homozygous mutant lines wrky55-1, wrky55-2, wrky55-3 of arabidopsis thaliana, and then we identified the next generation of these three mutant lines, all homozygous.

3.2.3 semi-quantitative expression analysis of different mutants of Arabidopsis thaliana

Three homozygous mutants of the Arabidopsis WRKY55 are screened, and in order to detect the expression quantity condition of the AtWRKY55 gene in mutants, the semi-quantitative condition of the AtWRKY55 gene in different mutant strains is firstly carried out. Firstly, regulating the expression quantity of the internal reference gene Actin2 in the three mutants to be consistent, judging according to the electrophoresis result of PCR products, and when the basic brightness of the strips is consistent, respectively amplifying AtWRKY55 by using the template quantity corresponding to each strain at the moment. As a result, it was found that AtWRKY55 was expressed in very low amounts in each mutant line, and in particular, in wrky55-1 and wrky55-2 mutants, was hardly expressed.

3.2.3 real-time PCR analysis of different mutants of Arabidopsis thaliana

FIG. 13 shows that the transcript levels of AtWRKY55 in the Arabidopsis mutant lines were significantly reduced compared to the wild type, especially the transcript levels of wry 55-1 and wrky55-2 were very low and almost none, similar to the semi-quantitative results above.

Example 4: functional analysis of SbWRKY55 gene under salt stress

3T 3 generation SbWRKY55 over-expression lines (S6, S15 and S51) with high relative expression quantity and good growth vigor of SbWRKY55 and 2 Arabidopsis T-DNA insertion mutation homozygotes wry 55-1 and wrky55-2 with extremely low expression quantity are selected for test.

1 test materials and treatments

1.1 test materials

Seeds of Arabidopsis thaliana Columbia ecology (Col-0), S6, S15 and S51 screened out in the above examples, and seeds of wrky55-1, wrky55-2.

1.2 Material handling

First, 1/2MS medium was prepared, and 0, 50, 100, 150mM NaCl was added to the medium. Autoclaving at 121deg.C for 15min. After the removal, the culture medium was removed and poured into a large dish of 13 cm. Times.13 cm until the culture medium was not hot. After sterilization, the seeds were sown on demand in 1/2MS medium containing different salt concentrations, 6 lines, WT, S6, S15, S51, wrky55-1, wrky55-2, respectively, were placed one row. Sealing the sealing film, and then placing the sealing film in a refrigerator at 4 ℃ for vernalization for 3d. After vernalization, the dishes were placed vertically in a tissue culture chamber (25 ℃, light intensity 4000Lx, light 16h, dark 8 h) for cultivation.

Further, WT, S6, S15, S51, wrky55-1, wrky55-2 were ordered in 1/2MS medium without salt. After vernalization for 3d in a refrigerator at 4 ℃, the petri dish is vertically placed in a tissue culture room for culture. When four true leaves are grown, they are transferred to nutrient soil and placed in an incubator for cultivation. After two weeks, seedlings of each strain of Arabidopsis thaliana were treated with 1/2Hoagland nutrient solution containing 0mM and 100mM NaCl, respectively. And (5) taking materials to measure various physiological indexes after 10d of seedling treatment, and taking materials to extract RNA after 48h of treatment for real-time PCR verification.

1.3 data processing

Data analysis was performed using biological software such as Excel, imageJ, SPSS 17.0.0 and SigmaPlote 10.0.

2 test method

2.1 Germination test of Arabidopsis wild type, overexpressed and mutant plants under NaCl treatment

2.1.1 determination of seed germination Rate and Main root Length

Sterilized WT, S6, S15, S51, wrky55-1, wrky55-2 seeds were sown in rows in 1/2MS medium containing 0mM, 50mM, 100mM and 150mM NaCl, and after vernalization for 3d, placed in a tissue culture room for culture, and after 24 hours germination rates were counted. Germination rate= (number of germinated seeds +.total number of seeds) ×100%. The main root length measurement was performed after 7d of growth.

2.2 Seedling stage test of Arabidopsis wild type, over-expressed, mutant plants under NaCl treatment

2.2.1 determination of the biomass

Seedling plants of the Arabidopsis wild-type, over-expressed, mutant lines were treated with 0, 100mM NaCl for 10d, respectively. The seedlings were then carefully removed from the nutrient soil, the material rinsed clean with deionized water, and the water on the plant surface was wiped dry. The Fresh weight (Fresh weight) of the plants was weighed, then placed in an oven at 70 ℃ until oven dried, and Dry weight was weighed. Each treatment was repeated 6 times.

2.2.2 determination of the extent of lipid peroxidation (MDA content) of the Membrane

The MDA content is measured by the method of Lin Zhifang et al (1984).

2.2.3 Na ⁺ 、K ⁺ Determination of ion content

WT, S6, S15, S51, wrky55-1, wrky55-2 plants 10d were treated with 0 and 100mM NaCl and the material was harvested. 0.1g of leaf was weighed separately and three replicates were taken per treatment. Put into a test tube, add 5mL ddH ₂ O, sealing the test tube mouth by using a sealing film. Boiling in boiling pot for 2 hr, filtering, and constant volume reaching 10mL. The group treated with 100mM NaCl was treated with ddH because of the high ion content ₂ O was diluted 4-fold. Then, na of each strain was measured by each treatment with a flame spectrophotometer ⁺ 、K ⁺ The content is as follows.

2.2.4 real-time PCR analysis of genes related to ion transport

In long-term evolution, plants have formed a range of mechanisms to cope with sodium ions in the soil environment. These mechanisms require multiple channel/transporter involvement. Cyclic nucleotide-gated channels (CNGCs are regulated by the involvement of cAMP and cGMP AtCNGC1 is able to participate in Na ⁺ And K ⁺ AtCNGC2 is capable of mediating K ⁺ Is not capable of mediating Na ⁺ Is capable of transporting K by AtCNGC10 ⁺ (Apse2007)。SOS1(salt overly sensitive 1，Na ⁺ /H ⁺ Antiport protein) located on plasma membrane, capable of regulating root cell Na ⁺ Is arranged outside the furnace; arabidopsis AtHKT1 transported Na only ⁺ The method comprises the steps of carrying out a first treatment on the surface of the NHX1 is Na localized on the vacuole membrane ⁺ /H ⁺ Transport body capable of transporting Na ⁺ . We haveThese genes were selected, their expression levels under salt stress were detected, and their relationship with the WRKY55 gene was analyzed. We selected the SbWRKY55 overexpressing line S15 and the Arabidopsis WRKY55 mutant WRKY55-1, 2 lines and WT for testing. The expression levels of these genes in WT and these 2 lines under salt stress were examined. cDNA sequences of the genes were found from the tair website, corresponding primers were designed, RNA was extracted, cDNA was synthesized, and Real-time PCR was performed.

3 test results and analysis

3.1 Experiments of Arabidopsis wild type, over-expressed and mutant plants in germination period under NaCl treatment

3.1.1 phenotyping of wild-type, overexpressed and mutant plants under salt treatment

As shown in fig. 14, the phenotype showed a significant difference after 7 days of treatment with different salt concentrations. With increasing salt concentration, the growth of each strain of Arabidopsis is significantly inhibited. In particular, at 150mM NaCl treatment, all strains showed little cotyledon growth or little yellowing, even without emergence directly. The growth vigor of the respective lines of Arabidopsis thaliana was substantially uniform with no significant difference upon treatment with 0mM NaCl. When treated with 50mM NaCl, the phenotypes of the individual lines started to differ, but not very much. When treated at 100mM NaCl, there was a significant difference in vigour between the individual lines, especially the overexpressed lines S6, S15, S51 had small and yellow cotyledons, even no cotyledons; the roots of the over-expressed lines were significantly shorter relative to the wild type and mutant.

3.1.2 Effect of salt treatment on Arabidopsis seed germination Rate

There was no difference in germination rate of seeds when treated with 0mM NaCl. As the salt concentration increases, the seed germination rate of each strain decreases significantly. When treated with 50 and 100mM NaCl, the germination rate of the mutant strain was found to be significantly higher than that of the wild-type Arabidopsis thaliana, while that of the overexpressing strain was significantly lower than that of the wild-type; in the 150mM NaCl culture medium, the germination rate of each strain of Arabidopsis thaliana is very low. This suggests that during germination the mutant strain has a stronger resistance to salt than the wild type, whereas the overexpressed strain has a weaker resistance to salt (FIG. 15).

3.1.3 Effect of salt treatment on Arabidopsis Main root Length

Root length of each strain was measured after 7d and found that there was no significant difference in root length of the wild type, over-expressed and mutant strains of Arabidopsis thaliana upon treatment with 0mM NaCl. As the salt concentration increases, the root length of each line decreases significantly. When treated with 50, 100mM NaCl, significant differences in root length occurred for each strain of Arabidopsis thaliana, whereas the root length of the mutant was significantly greater than that of the wild type and that of the overexpressed strain was significantly less than that of the wild type. At 150mM NaCl treatment, the root length of each strain was very short, and in particular, the root length of the overexpressing strain S6, S15 was almost 0, and no growth was observed (FIG. 16).

3.2 Seedling stage experiments of Arabidopsis wild type, overexpressed and mutant plants under NaCl treatment

3.2.1 Effect of salt treatment on Arabidopsis seedling biomass

FIG. 17 shows that the dry fresh weight of each strain of Arabidopsis was significantly reduced under salt treatment, but the reduction of the overexpressed strain was significantly higher than that of the wild-type strain, and the reduction of the mutant strain was significantly lower than that of the wild-type strain; the fresh weights of WT, S6, S15, S51, wrky55-1 and wrky55-2 were reduced by 37.89%, 48.13%, 59.11%, 47.77%, 20.64%, 24.55%, respectively, and the dry weights were reduced by 43.09%, 57.45%, 60.1%, 54.01%, 29.32%, 32.64%, respectively. These changes in biomass data indicate that salt treatment inhibited biomass accumulation to a significantly lower extent than the wild type strain, while over-expressed strains were inhibited to a significantly higher extent than the wild type strain.

3.2.2 influence of salt on MDA content in Arabidopsis leaves

As seen in FIG. 18, the MDA content of each strain of Arabidopsis thaliana was increased to a different extent under the treatment of 100mM NaCl. However, the over-expressed strain has a higher MDA content than the wild-type strain, whereas the mutant strain has a lower MDA content and a significant difference. The MDA content of the Arabidopsis overexpression lines S6, S15 and S51 is 1.3 times, 1.34 times and 1.28 times that of the wild Arabidopsis respectively, the MDA contents of the mutant lines wrky55-1 and wrky55-2 are 0.84 times and 0.91 times that of the wild Arabidopsis respectively, which shows that the overexpression lines have higher oxidation degree than the wild type membrane lipid and the membrane lipid oxidation degree of the mutant lines is lower.

3.2.3 salt treatment of Na in Arabidopsis leaves ⁺ 、K ⁺ Influence of the content

After 10d treatment of Arabidopsis thaliana with 0mM NaCl and 100mM NaCl, respectively, leaves of each strain of Arabidopsis thaliana were taken and Na was measured ⁺ And K ⁺ Is contained in the composition. As can be seen from FIG. 19, na of each strain of Arabidopsis thaliana was treated with 0mM NaCl ⁺ There was no difference in content, and the content was very low. However, when treated with 100mM NaCl, arabidopsis WT overexpressed lines S6, S15, S51 and the mutant wrky55-1, wrky55-2 Na ⁺ The content is obviously increased by 20 times, 21 times, 25.07 times, 25 times, 15.64 times and 18.42 times respectively; it can be seen that the Na of the overexpressed strain ⁺ The content is significantly higher than that of wild type, and the Na of mutant ⁺ The content is significantly smaller than that of the wild type. K in Arabidopsis lines at 0mM NaCl treatment ⁺ The content is not obviously different. But at 100mM NaCl, K ⁺ The content is obviously reduced, and the WT, S6, S15, S51, wrky55-1 and wrky55-2 are respectively reduced by 63 percent, 73.74 percent, 76.29 percent, 71.72 percent, 55.45 percent and 58 percent; it can be seen that the K of the overexpressing strain ⁺ The level of decrease was significantly higher than in the wild type, whereas the level of decrease was less for the mutant than in the wild type. Na of the respective lines of Arabidopsis thaliana under salt treatment ⁺ /K ⁺ The compound is obviously increased by 55.34 times, 80.71 times, 107.74 times, 88.97 times, 35.10 times and 43.33 times respectively; na over-expressing lines S6, S15, S51, especially S15 ⁺ /K ⁺ Significantly higher than wild type and mutant Na ⁺ /K ⁺ Lowest.

3.2.4 real-time PCR analysis of genes related to ion transport

There have been many studies in which some CNGCs may be involved in the salt reaction of plants. Under 0mM NaCl treatment, the expression level of AtCNGC10 in the roots of the WT and wrky55-1 strains is obviously different, and the relative expression level in the over-expression strain S15 is highest and is obviously higher than that of the WT; whereas the expression level at wrky55-1 was significantly lower than that of the wild type. When treated with 100mM NaCl, the expression levels of AtCNGC10 at WT, S15 and wrky55-1 were significantly reduced by 54.3%,46.5% and 56.5%, respectively.

There was no significant difference in the expression levels of AtCNGC1 in WT, S15, wrky55-1, both in 0 and 100mM NaCl treatments. However, the expression level of AtCNGC1 was increased in all three lines under NaCl treatment, 1.2-fold, 1.3-fold and 1.2-fold, respectively, when not treated with salt.

Under 0mM NaCl treatment, the difference in the expression level of AtCNGC2 in WT, S15, wrky55-1 roots was not significant. However, under 100mM NaCl treatment, the expression level of AtCNGC2 in roots was decreased, but there was no significant difference in expression level between the individual lines.

AtHKT1 at Na ⁺ Plays an important role in transportation of Na ⁺ The root is transported back, the damage to the overground part is avoided, and the method plays an important role in responding to salt stress. Under 0mM NaCl treatment, there was a clear difference in the expression level of AtHKT1 in WT, S15, wrky55-1, and the expression level was the highest in the overexpressed strain S15, and the expression level was the lowest in the mutant wrky 55-1. When treated with 100mM NaCl, the expression level of AtHKT1 at WT, S15 was significantly increased, and there was no significant difference. However, in mutant wrky55-1, the expression level of AtHKT1 was very low, both at 0 and 100mM NaCl, and significantly smaller than that of WT and S15.

There was little difference in the expression levels of AtNHX1 at WT, S15, wrky55-1 under 0 and 100mM NaCl treatments. The expression level of AtNHX1 was significantly increased in salt treatment compared to 0mM NaCl treatment, and there was no significant difference between the individual lines.

3.3 SbWRKY55 complements the salt tolerance of Arabidopsis mutant

To explore the role of SbWRKY55 in salt resistance, we generated a SbWRKY55 complementation line in the mutant background. RT-PCR verifies that the expression quantity of SbWRKY55 in the COM1 and COM2 strains is higher than that of the wild type strain. Afterwards we performed salt tolerance tests on COM1, COM2 homozygous lines. It was found that the anaplerotic lines were almost identical to the wild type, regardless of biomass accumulation, MDA content, or sodium potassium ion content.

3.4 SbWRKY55 negative regulation sweet sorghum salt resistance

To further investigate the role of SbWRKY55 in salt resistance, sweet sorghum SbWRKY55 over-expressed lines under the control of the CaMV 35S promoter were generated. The relative expression level of SbWRKY55 in each strain was examined by RT-PCR, and the results showed that the expression level of SbWRKY55 in OE1, OE2 and OE3 strains was higher than that in M-81E. Afterwards we performed salt tolerance tests on homozygous sweet sorghum over-expression lines. The over-expression strain shows the symptoms of slow growth, weak growth vigor, yellowing or even purple leaf, reduced root system, purple and the like after the sand-cultured sweet sorghum grown under normal laboratory conditions is treated by 0mM NaCl. Next, we studied the biological processes affected by salt stress. For biomass accumulation, OE strain biomass accumulated less than control. For membrane lipid peroxidation, we compared the root MDA content. The MDA content of the over-expressed strain is significantly increased after salt treatment compared to the wild type. Next, we studied the sodium potassium ion content in each strain under salt stress. Under salt stress, the sodium ions of the over-expression strain are obviously increased, and the potassium ion content is obviously reduced. Therefore we believe that SbWRKY55 after overexpression in sweet sorghum also affects biomass accumulation in plants, sodium potassium ion homeostasis, ROS content and membrane lipid peroxidation.

EXAMPLE 5 analysis of expression control of SbWRKY55 Gene

There is growing evidence that WRKY proteins function by forming protein complexes with other interactors. To find potential chaperones for the WRKY55 protein, we used a yeast two hybrid system. WRKY55 was first fused to the BD domain of the pGBKT7 vector as a bait. After the yeast system demonstrated that the gene had no self-activating activity, yeast cells were co-transformed with pGBKT7-WRKY55 using a cDNA library comprising a prey protein fused to GAL 4-AD. The results show that the binding of SbFYVE to SbWRKY55 is stable at all times. By NCBI alignment, sbFYVE and Arabidopsis AT3G43230 are homologous, and each has a typical FYVE domain, belonging to FYVE type zinc finger proteins, so we have once named SbFYVE. To confirm their interaction in yeast, a yeast two-hybrid experiment was performed with Y2H competence co-transformed with the prey plasmid and the decoy plasmid. As shown, sbWRKY55 has a strong interaction with SbFYVE.

To determine whether these interactions are also present in plant cells, we subsequently employed a two-molecule fluorescence complementation (BiFC) system. The cDNA of SbFYVE was fused to the N-terminal region of Yellow Fluorescent Protein (YFP) to give SbFYVE-N-YFP vector. The cDNA of SbWRKY55 is fused to the C-terminal region of Yellow Fluorescent Protein (YFP) to SbWRKY55-C-YFP. Mixing the two agrobacteria, and injecting the mixed solution into tobacco leaves by using a needle tube. In parallel, empty vector was combined with each fusion construct and co-injected into tobacco lamina. After 2 days of incubation, YFP signal was observed by fluorescence microscopy. The co-transformed samples showed YFP fluorescence in the nuclei with interaction, whereas none of the control samples gave any YFP signal.

We have examined the expression of this gene in sweet sorghum immediately and found that overexpression of SbWRKY55 results in an increase in SbFYVE expression under salt stress conditions. These results indicate that WRKY55 and SbFYVE co-localize and interact in the plant cell nucleus.

To determine how WRKY55 is involved in regulating plant salt tolerance, we studied transcriptional changes in wild-type, over-expressed and mutant strains before and after salt treatment. We used 48h NaCl treatment in RNA sequence experiments and performed three replicates. After obtaining transcriptome sequencing data, analysis found that 459 DEGs in wild type, 976 DEGs in over-expressed strain, 679 DEGs in mutant after salt treatment, we intersected these DGE to obtain 391 DGEs, followed by treatment of these 391 DEGs. An analysis was performed of KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology Consortium), respectively. GO analysis of these genes indicated that these differential genes are involved in biological processes (Biological Process), molecular functions (Molecular Function) and cellular components (Cellular Component). KEGG enrichment analysis indicated that these differential genes were primarily metabolized with phenylalanine. Among the 391 differential genes, genes related to salt resistance are screened for expression level analysis. After that, we found homologous genes in sweet sorghum and analyzed the promoters of these genes, we speculated that the genes with W-box in the promoters might be potential targets for SbWRKY 55.

Based on the transcriptome data described above, we further explored the interaction of SbWRKY55 with downstream target genes. To verify whether SbWRKY55 directly regulated these genes in vitro, we performed a yeast single hybridization assay. Through single impurity experiments, the SbWRKY55 has obvious interaction with SbCHI12 and SbBGLU 22. Yeast single hybridization analysis showed that LacZ gene expression was activated and positive colonies were blue on SD/-Trp-Ura+X-a-gal medium only when pLacZi-SbCHI12, pLacZi-SbBGLU22 were co-transferred with GAD-SbWRKY55, respectively, to EGY48 yeast. In contrast, yeast cells with the GAD-SbWRKY55 and pLacZi, and GAD and pLacZi-SbCHI12/SbBGLU22 control combinations did not turn blue even after prolonged incubation. These results confirm that SbWRKY55 specifically binds to W-box sequences in sbbchi 12, sbBGLU22 promoters.

To investigate whether SbWRKY55 directly regulated these genes in vivo, we performed luciferase complementation imaging analysis. The analysis results show that when LUC-SbCHI12, LUC-SbBGLU220 co-rotates with pMWB110-SbWRKY55, respectively, a strong fluorescent signal is observed in the in vivo imager. Next, we examined the expression of both genes in each strain of sweet sorghum, and found that under salt stress conditions, the expression of both genes was reduced. This suggests that SbWRKY55 can interact directly with SbCHI12, sbBGLU22 in sweet sorghum to modulate salt tolerance in plants.

Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.

SEQUENCE LISTING

<110> Shandong university of teachers and students

Application of <120> WRKY55 transcription factor in plant salt resistance

<130>

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 216

<212> PRT

<213> SbWRKY55 protein

<400> 1

Met Asp Ala Ala Ser Arg Met Thr Val Leu Gln Asn Gly Val His Asp

1 5 10 15

Ser Tyr Thr Trp Arg Lys Tyr Gly Gln Lys Glu Ile Leu Gly Ala Arg

20 25 30

Phe Pro Arg Ser Tyr Tyr Lys Cys Gly Arg Arg Pro Gly Cys Pro Ala

35 40 45

Lys Lys His Val Gln Gln Cys Asp Ala Asp Pro Ser Lys Leu Glu Val

50 55 60

Thr Tyr Leu Glu Ala His Thr Cys Asp Asp Pro Pro Pro Ser Ser Ser

65 70 75 80

His Ala Val Pro Asp Pro Thr Ala Gly Ser Asp Ala Leu Leu Val Pro

85 90 95

Pro Val Pro Thr Val Pro Phe Pro Ser Ala Gln Cys Tyr Gly Gly Gly

100 105 110

Arg Pro Ser Pro Pro Pro Leu Pro Pro Tyr Gln Val Pro Tyr Ala Ala

115 120 125

Thr Thr Leu Gly Ser Asn Val Leu Thr Leu Thr Ala Thr Gly Val Leu

130 135 140

Leu Pro Ser Ala Ser Tyr Asp Pro Val Pro Asp Val Thr Asp Cys Thr

145 150 155 160

Pro Ser Leu Glu Gln Glu Gln Asp His Asp Leu Leu His Ile Pro Ser

165 170 175

Pro Ala Cys Ser Gln Ser Glu Leu Leu Pro Met Glu Ala Ala Lys Leu

180 185 190

Ser Pro His Ala His Gly Leu Pro Leu Ser Leu Glu His Thr Leu Asp

195 200 205

Cys Asp Phe Ala Val Pro Glu Leu

210 215

<210> 2

<211> 651

<212> DNA

<213> SbWRKY55 Gene sequence

<400> 2

atggatgctg catcccggat gacagtgcta cagaatggag ttcatgattc gtacacatgg 60

aggaaatacg ggcagaagga aattctgggc gccagatttc caaggagtta ctacaaatgt 120

ggccgccggc cgggctgccc cgcgaagaag cacgtgcagc aatgcgacgc ggatccgtcc 180

aagctggagg tcacctactt ggaggcacac acgtgcgatg atccaccacc gtcgtcgtcc 240

catgctgttc cagatccgac ggccggctcc gacgctctgc tcgtaccacc agtcccgacc 300

gttccgtttc catcagctca gtgctacggc ggcggccgac cgtcgccgcc gccgctgccg 360

ccgtaccagg tgccgtacgc cgcgacgacg ctcggctcca acgtcctgac gctgacggcc 420

accggtgttc ttctgcctag tgcaagctac gaccctgttc cggatgtcac ggactgcacg 480

ccgtcgttgg agcaggagca agaccatgat ctgcttcaca taccttcgcc ggcttgttca 540

cagtcagagc tgctgccgat ggaggctgcc aagctttcac cgcacgcgca cgggctgcct 600

ctgtcgttgg agcacacgct ggattgcgac tttgctgtac ccgagcttta a 651

<210> 3

<211> 29

<212> DNA

<213> artificial sequence

<400> 3

gctctagaat ggatgctgca tcccggatg 29

<210> 4

<211> 29

<212> DNA

<213> artificial sequence

<400> 4

ggggtaccaa gctcgggtac agcaaagtc 29

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

tggcatctct cagcacattc 20

<210> 6

<211> 19

<212> DNA

<213> artificial sequence

<400> 6

aatggctctc tcggcttgc 19

<210> 7

<211> 18

<212> DNA

<213> artificial sequence

<400> 7

atgacagtgc tacagaat 18

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

tagtaactcc ttggaaatct 20

Claims

1. Application of proteins, nucleic acid molecules, recombinant vectors, expression cassettes, transgenic cell lines or host bacteria in regulating and controlling plant salt resistance;

wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1;

the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2;

the recombinant vector, the expression cassette, the transgenic cell line and the host bacteria contain the nucleic acid molecules;

the plant is sweet sorghum or arabidopsis.

2. A method of plant breeding, the method comprising: the method comprises knocking out or inhibiting expression of nucleic acid molecules, and improving salt resistance of plants; the plant is sweet sorghum or arabidopsis;

wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.