Application of GmMYB14 protein in regulating plant salt tolerance and / or cyst nematode resistance

Abstract

The present invention discloses a GmMYB14 protein related to plant salt tolerance and / or cyst nematode resistance, and related biological materials and applications thereof. The GmMYB14 protein may specifically be the protein of any one of the following A1), A2) or A3): A1) a protein with an amino acid sequence of SEQ ID No.1 in the sequence listing; A2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1), having more than 75% identity with the protein shown in A1) and being related to plant salt tolerance and insect resistance; A3) a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2). The GmMYB14 protein and related biological materials can be used to regulate plant salt tolerance, insect resistance and yield increase.

Description

Technical Field

[0001] The present invention relates to the application of GmMYB14 protein in regulating plant salt tolerance and / or cyst nematode resistance in the field of biotechnology. Background Art

[0002] Soybean (Glycine max (L.) Merr.) is an important source of plant protein and oil globally. The fundamental way to promote soybean production is to increase soybean yield per unit area, enhance the stable yield performance of soybeans, and effectively utilize saline-alkali land. Soil salinization is also a worldwide problem. When the soil salt concentration exceeds 5 ds / m, the growth of soybeans is inhibited and the yield is significantly reduced. Due to the low organic matter content and poor soil fertility in saline-alkali soil, it is not easy to promote the growth of crops, and a large amount of saline-alkali land urgently needs to be utilized. In terms of the development of soybean industrial technology, there is also an urgent need to cultivate salt-tolerant soybean varieties, utilize saline-alkali land, and increase the total soybean production.

[0003] Soybean Cyst Nematode Soybean cyst nematode Ichinohe mainly harms the roots. The affected plants are underdeveloped and short. After being infected at the seedling stage, the cotyledons and true leaves turn yellow and the development is retarded; when adult plants are infected, the above-ground parts are dwarfed and wilted, with few or no pods formed, and in severe cases, the whole plant dies. The roots of diseased plants are underdeveloped, the lateral roots are significantly reduced, the fine roots increase, and the root nodules are scarce. The cyst nematode overwinters in the soil in cysts with eggs. The cyst has strong resistance to adverse environments. In the following spring, the second-stage larvae invade from the root hairs of the host young roots and develop into adults in the cortex of the soybean young roots. The female body gradually swells into a lemon shape with the formation of internal eggs, breaks through the surface layer and exposes outside the host body, and only adsorbs to the host root with the mouthparts. Soybean cyst nematode is widely distributed, seriously harmful, has a wide host range, multiple transmission routes, and a long survival time, and is a soil-borne disease that is extremely difficult to control. Applying disease-resistant varieties is an economical and effective measure to control soybean cyst nematode disease. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to regulate plant salt tolerance and cyst nematode resistance.

[0005] To solve the above technical problems, the present invention provides any one of the following applications of GmMYB14 protein or a substance that regulates the content or activity of the GmMYB14 protein:

[0006] U1 Application in regulating plant salt tolerance;

[0007] U2 Application in preparing a product for regulating plant salt tolerance;

[0008] U3 Application in regulating plant cyst nematode resistance;

[0009] U4 Application in preparing a product for regulating plant cyst nematode resistance;

[0010] Use of U5 in plant breeding;

[0011] The GmMYB14 protein is a protein as follows A1, A2 or A3:

[0012] A1. A protein with the amino acid sequence shown in SEQ ID No.1 in the sequence listing;

[0013] A2. A protein which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID No.1 in the sequence listing, has more than 80% identity with the protein shown in A1), and is related to plant salt tolerance and insect resistance;

[0014] A3. A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0015] In the above application, SEQ ID No.1 in the sequence listing consists of 291 amino acid residues.

[0016] In the above application, 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 the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0017] In the above application, the above-mentioned identity of more than 80% can be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0018] In the above application, the GmMYB14 protein can be derived from soybean.

[0019] In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant; further, the dicotyledonous plant can be a leguminous plant; furthermore, the leguminous plant can be a plant of the genus Glycine; specifically, the plant of the genus Glycine can be soybean.

[0020] In the above application, the substance for regulating the content or activity of the GmMYB14 protein may be a substance that performs at least one of the following six regulations: B1) regulation at the gene transcription level; B2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); B3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); B4) regulation of the translation of the gene; B5) regulation of the degradation of the mRNA of the gene; B6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0021] In the above application, the substance for regulating the content or activity of the GmMYB14 protein is a biological material related to the GmMYB14 protein; the biological material is any one of the following C1-C3:

[0022] C1. A nucleic acid molecule encoding the GmMYB14 protein;

[0023] C2. A nucleic acid molecule that enhances the expression of the GmMYB14 protein;

[0024] C3. An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in C1 or C2.

[0025] In the above application, the nucleic acid molecule described in C1 or C2 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.

[0026] In the above application, the nucleic acid molecule described in C1 may specifically be a nucleic acid molecule whose coding sequence of the coding strand is the 87th to 962nd positions of SEQ ID No.2 in the sequence listing.

[0027] In the above application, the substance for regulating the content or activity of the GmMYB14 protein may enhance the content or activity of the GmMYB14 protein, and specifically may be achieved by increasing the expression level of the coding gene of the GmMYB14 protein.

[0028] The present invention also provides a protein, which is the GmMYB14 protein and is a protein of any of the following A1, A2 or A3:

[0029] A1. A protein whose amino acid sequence is the amino acid sequence shown in SEQ ID No.1 in the sequence listing;

[0030] A2. A protein that has more than 80% identity with the protein shown in A1) and is related to the plant root angle, which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID No. 1 in the sequence listing;

[0031] A3. A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0032] The present invention also provides a biological material related to the GmMYB14 protein; the biological material is any one of the following C1-C3:

[0033] C1. A nucleic acid molecule encoding the GmMYB14 protein;

[0034] C2. A nucleic acid molecule that enhances the expression of the GmMYB14 protein;

[0035] C3. An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in C1 or C2.

[0036] To solve the above technical problems, the present invention provides a plant reagent for enhancing salt tolerance and / or resistance to cyst nematodes, and the active ingredient of the plant reagent is a substance that promotes the expression of the gene encoding the GmMYB14 protein and increases the abundance of the GmMYB14 protein. The active ingredient can specifically be the GmMYB14 protein and / or a biological material related to the GmMYB14 protein.

[0037] The active ingredient of the above plant reagent may further contain other biological components or / and non-biological components, and those skilled in the art can determine other active ingredients of the above plant reagent according to the salt tolerance and / or cyst nematode resistance effect of the plant.

[0038] To solve the above technical problems, the present invention also provides a method for improving the salt tolerance and / or resistance to cyst nematodes of plants, including the following steps: promoting the expression of the GmMYB14 protein in a recipient plant or increasing the abundance of the GmMYB14 protein to obtain a target plant with stronger salt tolerance and / or resistance to cyst nematodes than the recipient plant.

[0039] Among them, promoting the expression of the GmMYB14 protein in the recipient plant or increasing the abundance of the GmMYB14 protein can specifically be achieved by introducing the coding gene of the GmMYB14 protein into the recipient plant.

[0040] In this article, the plant can be any one of the following plants:

[0041] E1) A plant of the class Dicotyledoneae,

[0042] E2) Leguminous plants,

[0043] E3) Plants of the genus Glycine,

[0044] E4) Soybean.

[0045] In the present invention, the regulation may be up-regulation or enhancement or improvement.

[0046] In the present invention, the purposes of plant breeding may include cultivating plants with strong salt tolerance and / or strong resistance to cyst nematodes.

[0047] In the present invention, by cloning the soybean GmMYB14 gene, constructing a binary expression vector driven by the CaMV35S promoter for the target gene GmMYB14 transforming soybean, transgenic GmMYB14 soybean plants are obtained. Through the functional analysis of wild-type and transgenic GmMYB14 soybean plants, it is found that compared with the wild-type, the salt tolerance and the effect against cyst nematodes of transgenic GmMYB14 soybean plants are significantly increased. It shows that GmMYB14 the gene is an important candidate gene for improving plant stress resistance and insect resistance and has potential breeding value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 shows the RNA expression level results of GmMYB14 transgenic soybean lines OX1 and OX9 in Example 1 of the present invention. Among them, WT is wild-type soybean (negative control), and both OX1 and OX9 are T1-generation transgenic GmMYB14 gene soybean positive lines. In the figure, *** represents the result of significant difference analysis with P < 0.001.

[0049] Figure 2 shows the phenotypic diagrams of GmMYB14 transgenic soybean lines OX1 and OX9 under high salt stress in Example 1 of the present invention. Among them, WT is wild-type soybean (negative control), and both OX1 and OX9 are T1-generation transgenic GmMYB14 gene soybean positive lines.

[0050] Figure 3 shows the sodium ion content, sodium-potassium ratio, hydrogen peroxide content and malondialdehyde content of GmMYB14 transgenic soybean lines OX1 and OX9 under normal and high salt conditions in Example 1 of the present invention. Among them, WT is wild-type soybean (negative control), and both OX1 (OX-1) and OX9 (OX-9) are T1-generation transgenic GmMYB14 gene soybean positive lines. In the figure, ns represents no significant difference, ** represents the result of significant difference analysis with P < 0.01, and *** represents the result of significant difference analysis with P < 0.001.

[0051] Figure 4 Figure 1 is a graph showing the resistance of GmMYB14 transgenic soybean lines OX1 and OX9 after inoculation with cyst nematodes in Example 1 of the present invention. GmMYB14 Genetic soybean positive lines. The unit of the vertical axis is bars. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0053] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0055] The entry vector pGWC in the following examples is a product of Invitrogen. The literature describing the vector is ChenQJ, Zhou HM, Chen J, Wang XC. Using a modified TA cloning method to create entry clones. Anal Biochem. 2006, 358(1): 120-125.

[0056] The vector pB2GW7 in the following examples is a product of the NTCC Type Culture Collection and contains the CaMV35S promoter.

[0057] The soybean variety Tianlong No. 1 in the following examples passed the national crop variety approval in 2008, with the approval number of Guoshendou 2008023. It is a well-known public soybean variety and can be purchased commercially.

[0058] Example 1: Application of GmMYB14 in regulating soybean salt tolerance

[0059] one, GmMYB14 Gene cloning

[0060] Using soybean cDNA as template, PCR amplification GmMYB14 The coding region sequence (876 bp, the nucleotide sequence is shown in 87-962 positions of SEQ ID No. 2, and the encoded amino acid sequence is shown in SEQ ID No. 1), the PCR amplification product is recovered and purified, and constructed into the entry vector pGWC. The specific steps are as follows:

[0061] 1. Primer design

[0062] According to the soybean database (https: / / phytozome.jgi.doe.gov / pz / portal.html#!info?alias=Org_Gmax) GmMYB14 Design primers based on the gene sequence to amplify the full-length sequence of the coding region. The primer pair consists of upstream primer F1 and downstream primer R, and the specific sequence is as follows:

[0063] F1: 5'-ATGGGGAGAGCTCCATGCTG-3';

[0064] R: 5'-CAAACAACAGAAGAACAACGAC-3'.

[0065] 2. PCR amplification of target fragment

[0066] TRIZOL was used to extract RNA from soybean Tianlong No. 1, reverse transcribed into cDNA, and PCR amplified using the cDNA as a template and the primers designed in step 1 to obtain the target gene fragment. The PCR reaction system is shown in Table 1. The PCR reaction procedure is shown in Table 2.

[0067] Table 1

[0068]

[0069] Table 2

[0070]

[0071] 3. Recovery of target fragments

[0072] The target gene fragment was recovered by 0.8% agarose gel electrophoresis and purified using a gel recovery kit from Novozymes Biotech to obtain a target fragment recovery product. For detailed operation steps, please refer to the product manual.

[0073] 4. Entry vector digestion, T addition, and ligation

[0074] The entry vector pGWC was linearized by AhdI digestion, and the digestion product was subjected to T reaction at 72°C for 2 hours. Then the target fragment recovery product was connected to the vector pGWC, and the connection reaction system was shown in Table 3. The connection was carried out at 16°C overnight.

[0075] Table 3

[0076]

[0077] 5. Identification of positive clones

[0078] Transform 10 μL of the ligation product into Escherichia coli DH5α competent cells, then evenly spread them on an LB solid culture plate containing chloramphenicol, and incubate them upside down at 37 °C for 16 hours. Pick monoclonal colonies on the transformation plate, and perform PCR amplification according to the PCR reaction system and reaction program described in Table 1 and Table 2. A positive clone is detected when the target fragment of 876 bp is contained. Select 2 - 3 positive clones and send them to Tsingke Biological Company for sequencing, and obtain positive clones containing the gene with the nucleotide sequence from position 87 to 962 of SEQ ID No. 2 GmMYB14 gene, which contains the recombinant plasmid pGWC-GmMYB14 (containing the gene with the nucleotide sequence from position 87 to 962 of SEQ ID No. 2 GmMYB14 gene)

[0079] II. Construction of Recombinant Vectors and Recombinant Bacteria

[0080] Use the pB2GW7 vector (containing the CaMV35S promoter) to construct a binary expression vector driven by the CaMV35S promoter for gene expression, and transform it into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens containing the binary expression vector. The specific steps are as follows: GmMYB14 1. Extract the pB2GW7 vector plasmid from Escherichia coli

[0081] 2. Extract the recombinant plasmid pGWC-GmMYB14 from the TA positive clone obtained in step 1

[0082] 3. Carry out an LR reaction on the two vector plasmids pB2GW7 and pGWC-GmMYB14 obtained in the above steps 1 and 2 under the catalysis of LR ligase (Invitrogen, 1179OX19), and react overnight at 25 °C to obtain a ligation product, completing the construction of the plant binary vector pB2GW7-

[0083] driven by the CaMV35S promoter for gene expression. The ligation reaction system is shown in Table 4 GmMYB14 gene expression. The ligation reaction system is shown in Table 4 GmMYB14 The construction of pB2GW7-

[0084] Table 4

[0085]

[0086] pB2GW7- GmMYB14 contains GmMYB14 gene (the nucleotide sequence is from position 87 to 962 of SEQ ID No. 2), and the promoter driving GmMYB14 gene expression is the CaMV35S promoter. The amino acid sequence of the expressed protein is as shown in SEQ ID No. 1

[0087] 4. Add 5 μL of the ligation product (pB2GW7-GmMYB14 Transform competent Escherichia coli DH162 cells, and then evenly spread them on an LB solid culture plate containing spectinomycin. Incubate the plate upside down at 37 °C for 16 hours.

[0088] 5. Pick monoclonal colonies on the transformation plate for colony PCR. The primer pair consists of the upstream primer F2 on the vector and the downstream primer R of the target gene GmMYB14. The specific sequences are as follows:

[0089] F2: 5’-CATTTGGAGAGGACTCCGG-3’;

[0090] R: 5’-CAAACAACAGAAGAACAACGAC-3’.

[0091] Detect the target gene fragment (with a size of 1050 bp, including a 950-bp target gene amplification fragment (shown at positions 87 - 1036 of SEQ ID No.2) + about 100 bp of a sequence on the vector), and send the positive clones that amplify the target fragment to the company for sequencing.

[0092] 6. Extract plasmids from the positive clones with correct sequencing, and transform Agrobacterium tumefaciens EHA105 by the freeze-thaw method to obtain Agrobacterium tumefaciens EHA105 containing pB2GW7- GmMYB14 named EHA105- GmMYB14 for soybean transformation.

[0093] III. Obtaining Transgenic Soybean Plants GmMYB14 1. Using the Agrobacterium-mediated method, with the cotyledon node as the explant, transform Glycine max cv. Tianlong No. 1 with the recombinant Agrobacterium tumefaciens EHA105-

[0094] constructed in step 2 to complete soybean genetic transformation. The specific transformation method refers to the method in the literature "Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K (2006) Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep. 25(3):206 - 213." to obtain T0 generation transgenic soybean seedlings. GmMYB14 2. Detection of Bar protein in transgenic soybean seedlings by strip

[0095] using a strip

[0096] The glufosinate resistance of T0 transgenic seedlings was detected using a Bar protein test strip. Seedlings with two bands on the test strip were preliminarily determined to be transformed seedlings with glufosinate resistance, and further PCR detection was carried out.

[0097] 3. PCR Detection of Transgenic Soybean Plants

[0098] Leaves of T0 transgenic seedlings were taken, and leaf DNA was extracted using the CTAB method. PCR amplification was carried out using the following primers to obtain PCR products. The primer pair consisted of the above upstream primer F2 and downstream primer R.

[0099] The PCR reaction system and reaction program were referred to Table 1 and Table 2.

[0100] The PCR products were subjected to agarose gel electrophoresis. The target band of 876 bp appeared in transgenic positive plants, and the gene band did not appear in non-transgenic plants, proving that the target fragment had been integrated into the plant genome.

[0101] 4. Obtaining of T1 Homozygous Transgenic Soybean Lines

[0102] For T0 positive transgenic soybean plants, seeds were harvested from each single plant. The offspring plants were detected using the Bar protein test strip and PCR technology. The detection methods were the same as those in Step 2 and Step 3 to obtain T1 segregating lines. Herbicides were applied to the leaves for identification. Seeds were separately harvested from single plants resistant to herbicides to obtain T2. T2 was continuously planted. If all the offspring were resistant to herbicides, the T1 seeds were homozygous. Two T1 positive transgenic soybean homozygous lines were selected and named OX1 and OX9 for GmMYB14 gene function analysis.

[0103] IV. GmMYB14 Expression Analysis of Transgenic Soybean Lines OX1 and OX9

[0104] 1. Five healthy (plump grains and smooth surfaces without disease spots) seeds of wild-type soybeans (Tianlong No. 1) and transgenic soybeans (T1 positive transgenic soybean homozygous lines OX1 and OX9) were picked and sown in nutrient soil (the ratio of soil to vermiculite was 3:1), under long-day conditions (16 h light / 8 h dark), and cultured at 28 °C for 12 days.

[0105] 2. Thin the seedlings, and leave 2 wild-type or transgenic soybean plants with consistent growth in each pot, and continue to culture.

[0106] 3. After the first trifoliate leaf unfolded, take the middle small leaf and put it into a liquid nitrogen quick-freezing tube containing steel beads.

[0107] 4. Grind the leaves until they are completely ground at low temperature and with vibration. Add 1 ml of trizol extraction solution. After vigorously shaking the homogenized sample, let it stand at room temperature for 5 min. Add 0.2 ml of chloroform, shake vigorously for 15 s, let it stand at room temperature for 2 - 3 min, centrifuge at 4°C and 12,000 rpm for 10 min. Transfer the upper aqueous layer to a clean centrifuge tube and add an equal volume of isopropanol. Invert and mix well, then let it stand at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Add 75% ethanol to wash the precipitate, centrifuge for another 3 min, discard the supernatant, air-dry, and add 30 μl of RNase-free water to fully dissolve the RNA.

[0108] 5. After reverse transcription of 1 μg of RNA, perform fluorescence quantitative analysis on the RNA expression levels of transgenic soybean lines OX1 and OX9. The results show that, compared with the wild-type control WT, the RNA expression levels of transgenic soybean lines OX1 and OX9 are significantly increased ( Figure 1 ).

[0109] V. Salt tolerance function analysis of genes in transgenic soybeans GmMYB14 Gene

[0110] 1. Select 6 healthy (full and smooth seeds without disease spots) seeds each of wild-type soybeans (Tianlong No. 1) and transgenic soybeans (T1 generation positive transgenic soybean homozygous lines OX1 and OX9), and sow them in nutrient soil (the ratio of soil to vermiculite is 1:1), under long-day conditions (14 h light / 10 h dark), and culture at 25°C for 12 days.

[0111] 2. Thin the seedlings, leaving 4 wild-type or transgenic soybean plants with consistent growth in each pot, and continue the culture.

[0112] 3. After the first trifoliate leaf unfolds, perform saline irrigation treatment. The saline solution is a solution prepared with sodium chloride as the solute and a nutrient solution containing green manure as the solvent; the content of sodium chloride in the saline solution is 200 mM; for the nutrient solution containing green manure, the green manure used is the full water-soluble fertilizer of the Hua Wu Que series of Shanghai Yongtong Ecological Engineering Co., Ltd., and its content in the nutrient solution is 2.4 g / L. Irrigate 2 L of saline solution each time, irrigate saline solution once every 3 days. After 3 treatments, perform rehydration (irrigate the nutrient solution containing green manure) for about 5 - 7 days. After the wild-type shows salt damage symptoms, take pictures and record (see Figure 2 ).

[0113] The results show that the bottom of the wild-type plants withers and dies, and the top leaves are significantly yellowed. The leaves of the transgenic soybean lines are slightly yellowed, and most of the leaves can still grow normally.

[0114] 4. Take the root tissues of wild-type soybeans, overexpressing OX1 and OX9 soybeans, quickly freeze them in liquid nitrogen, and measure the sodium ion content, sodium-potassium ratio, hydrogen peroxide content, and malondialdehyde content. The results show that the sodium ion content, sodium-potassium ratio, hydrogen peroxide content, and malondialdehyde content of the overexpressing transgenic soybean lines are all reduced, further proving that the salt tolerance of transgenic soybeans OX1 and OX9 is enhanced (see Figure 3 ).

[0115] VI. Functional analysis of GmMYB14 genes against soybean cyst nematode in transgenic soybeans

[0116] Select healthy (plump grains and smooth surfaces without disease spots) seeds of wild-type soybeans (Tianlong No. 1), wild-type soybeans (Tianlong No. 1), and transgenic soybeans (T2 generation positive transgenic soybean homozygous lines OX1 and OX9) with plump and uniform sizes, sow them in the greenhouse, and conduct resistance identification of soybean cyst nematode. The soil property is sandy soil. At the peak of cyst formation, completely remove the soybean roots, wrap the cyst roots in a self-sealing bag, place them at room temperature for 48 hours, then wash the cysts into water, stir evenly, and pass through a set of sieves with 20 meshes on top and 60 meshes on the bottom (pore sizes are 850 μm and 250 μm respectively). Use a wash bottle to wash the cysts on the 60-mesh sieve into a 50-ml centrifuge tube. Store the cysts at 12 °C. After 40 days, the cysts are completely browned, and take pictures and count. The results show that the number of cysts of transgenic soybean lines OX1 and OX9 (6.9 ± 5.3 cysts / plant and 10 ± 4.8 cysts / plant respectively) is significantly lower than that of the control (14.4 ± 9.6 cysts / plant), indicating that GmMYB14 the overexpression of the Figure 4 gene improves the resistance of soybeans to soybean cyst nematode (see ). Use the EXCEL statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and the T-test is used. *, *** represent significant differences of P < 0.05 and P < 0.001 respectively. The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the following appended claims. Sequence Listing <110> Oil Crops Research Institute, Chinese Academy of Agricultural Sciences <120> Application of GmMYB14 Protein in Regulating Plant Salt Tolerance and / or Resistance to Soybean Cyst Nematode <130> GNCSY221122 <160> 2 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 291 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Arg Ala Pro Cys Cys Glu Lys Met Gly Leu Lys Arg Gly Pro 1 5 10 15 Trp Thr Pro Glu Glu Asp Gln Ile Leu Ile Asn Tyr Ile Asn Thr Tyr 20 25 30 Gly His Ala Asn Trp Arg Ala Leu Pro Lys Leu Ala Gly Leu Leu Arg 35 40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp 50 55 60 Ile Lys Arg Gly Asn Phe Thr Arg Glu Glu Glu Asp Thr Ile Ile Ser 65 70 75 80 Leu His Glu Met Leu Gly Asn Arg Trp Ser Ala Ile Ala Ala Arg Leu 85 90 95 Ser Gly Arg Thr Asp Asn Glu Ile Lys Asn Val Trp His Thr His Leu 100 105 110 Lys Lys Arg Leu Pro Gln Asn Tyr Gln Gln Ser His His Thr Lys Lys 115 120 125 Arg Ser Lys Lys Gln Pro Pro Lys Leu Asp Ala Asp Ala Ser Lys Ser 130 135 140 Asn Gln Asp Ala Lys Leu Glu Gln Gln Asp Pro Val Asn Ile His Gly 145 150 155 160 Ser Asn Ser Glu Asp Met Met Met Pro Leu Ser Pro Pro His Cys Ser 165 170 175 Ser Asp Met Ser Ser Ser Leu Thr Thr Ser Asp Asn Asn Ser Asn Val 180 185 190 Asn Ile Ile Asn Asn Asn His Asp Met Ser Leu Asn Val Asn Asp Tyr 195 200 205 Asp Ile Asp Thr Pro Glu Asn Asn Leu Ala Leu Asp Glu Asp Phe Trp 210 215 220 Ser Glu Val Leu Ser Ser Asp Asn Ser Gly Val Thr Ser Gly Phe Pro 225 230 235 240 Thr Leu Asp Tyr Asp Gln Phe Gln Pro Met Ser Pro Leu Val Thr Glu 245 250 255 Glu Gly Val Leu Ile Asp Cys Ser Ser Ser Thr Cys Asp Asp Gly Met 260 265 270 Asp Phe Trp Cys Asn Val Tyr Ser Arg Ala Glu Glu Phe Thr Gln Leu 275 280 285 Leu Glu Leu 290 <210> 2 <211> 1196 <212> DNA <213> Artificial Sequence <400> 2 aagagagagc tagcagtgca agctttcaac atccaaaaca acagagagtt tttggcttta 60 gaagctgaag gaatatagtt atcacaatgg ggagagctcc atgctgtgag aaaatggggt 120 tgaagagagg gccatggact ccagaagaag atcaaattct catcaattac atcaacactt 180 atggccatgc taattggcgt gcactgccca aactagctgg gctattaagg tgtggaaaga 240 gttgtagact ccggtggata aattatctga gaccggacat caaacggggc aactttacca 300 gagaagaaga ggacactata atcagtttgc atgaaatgtt gggaaacaga tggtcggcta 360 ttgcggcaag gttgtcaggg agaacagaca acgagataaa aaacgtgtgg cacacccact 420 tgaagaagag gctgccacaa aattaccaac aaagccatca cactaaaaaa cgaagcaaaa 480 aacaaccacc aaagttggat gcggacgcct ccaaatccaa ccaagacgcc aaactagaac 540 aacaagaccc cgttaatatt catggatcga actctgagga catgatgatg ccactatctc 600 ctcctcattg ttctagcgac atgtcctcct ccctcaccac cagtgacaac aatagtaacg 660 ctcctcattg ttctagcgac atgtcctcct ccctcaccac cagtgacaac aatagtaacg 660 ttaatattat taataataat catgatatgt ccttaaatgt taatgattat gatattgaca 720 ttaatattat taataataat catgatatgt ccttaaatgt taatgattat gatattgaca 720 ccccagagaa taatcttgca ttggacgagg atttctggtc ggaagttttg tcatctgata 780 ccccagagaa taatcttgca ttggacgagg atttctggtc ggaagttttg tcatctgata 780 attccggcgt gacaagcgga tttccgaccc ttgactatga tcagtttcaa ccaatgtctc 840 attccggcgt gacaagcgga tttccgaccc ttgactatga tcagtttcaa ccaatgtctc 840 ctctagtgac agaagaaggg gtccttatag attgttcgtc gagtacgtgc gacgatggca 900 ctctagtgac agaagaaggg gtccttatag attgttcgtc gagtacgtgc gacgatggca 900 tggatttttg gtgcaatgtt tactcgagag ctgaggaatt tacccagtta ctggaattgt 960 tggatttttg gtgcaatgtt tactcgagag ctgaggaatt tacccagtta ctggaattgt 960 gatgttagct attgttgttg ttcttaaata gagaccaagt ccttcattca acatgtcgtt 1020 gatgttagct attgttgttg ttcttaaata gagaccaagt ccttcattca acatgtcgtt 1020 gttcttctgt tgtttggact tgaattaatt cccttgttag aaattaataa atcgtgtaca 1080 gttcttctgt tgtttggact tgaattaatt cccttgttag aaattaataa atcgtgtaca 1080 ttgatgattg ttactggacg tcattatgtt tttaccaaaa gcgtcatttg tagcaaaaca 1140 ttgatgattg ttactggacg tcattatgtt tttaccaaaa gcgtcatttg tagcaaaaca 1140 ttgttccata atcctcatga gtgttgaaat aataatacta tgttccttta tataga 1196 ttgttccata atcctcatga gtgttgaaat aataatacta tgttccttta tataga 1196

Claims

1. Any of the following applications of the GmMYB14 protein or a substance that increases the content of the GmMYB14 protein: Use in enhancing the resistance of plants to soybean cyst nematodes; enhancing the resistance of plants to soybean cyst nematodes means increasing the content of the GmMYB14 protein to enhance the resistance of plants to soybean cyst nematodes; Use in preparing a product for enhancing the resistance of plants to soybean cyst nematodes; The GmMYB14 protein is a protein of any of the following A1 or A2: A1. A protein with an amino acid sequence shown in SEQ ID No.1 in the sequence listing; A2. A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1; The GmMYB14 protein is derived from soybean; The substance that increases the content of the GmMYB14 protein is a biological material related to the GmMYB14 protein; the biological material is any one of the following C1 - C2: C1. A nucleic acid molecule encoding the protein; C2. An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in C1; The plant is soybean.

2. The application according to claim 1, characterized in that: The coding sequence of the nucleic acid molecule described in C1 as the coding strand is the nucleic acid molecule at positions 87-962 of SEQ ID No.2 in the sequence listing.

3. A method for enhancing the resistance of plants to soybean cyst nematodes, characterized in that: It includes the following steps: promoting the expression of the GmMYB14 protein described in claim 1 in the recipient plant or increasing the abundance of the GmMYB14 protein described in claim 1 to obtain a target plant with stronger cyst nematode resistance than the recipient plant, and the plant is soybean.

4. The method according to claim 3, characterized in that: The promotion of the expression of the GmMYB14 protein described in claim 1 in the recipient plant or the increase in the abundance of the GmMYB14 protein described in claim 1 is achieved by introducing the coding gene of the GmMYB14 protein described in claim 1 into the recipient plant.

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