Soybean NAC transcription factor gene and its application

Abstract

The present invention provides soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 and applications thereof, belonging to the field of genetic engineering technology. The soybean NAC transcription factor gene nucleotides include one or more of the following nucleotide sequences: (a) the nucleotide sequence shown in SEQIDNo.2; (b) the nucleotide sequence shown in SEQIDNo.4; (c) a nucleotide sequence encoding a protein having at least 95% homology with the amino acid encoded by the nucleotide sequence shown in SEQIDNo.2; (d) a nucleotide sequence encoding a protein having at least 95% homology with the amino acid encoded by the nucleotide sequence shown in SEQIDNo.4. The present invention has found that Glyma.02G109800 and Glyma.01G051300 can not only participate in regulating the root development of soybeans, but also participate in the response of soybeans to ABA and adverse stress. The present invention provides a theoretical basis for the study of plant root development and adverse stress.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a soybean NAC transcription factor gene and an application thereof. Background Art

[0002] Soil salinization is a global ecological problem. It is an important reason for limiting plant growth and development and causing crop yield reduction, which has a great negative impact on agricultural production. Soybean seeds are rich in protein, high-quality vegetable oil, minerals and vitamins, etc., and are an important edible economic crop. In 1984, Lauchli et al. classified soybeans as salt-sensitive sweet soil crops based on the classification of plant salt stress tolerance under high-salt conditions. Therefore, it is of great significance to conduct research on the molecular mechanism of soybean salt tolerance, identify related stress resistance genes and obtain plants with enhanced stress resistance.

[0003] Transcription factors can directly regulate the expression of stress resistance genes in plants and participate in a variety of physiological and biochemical processes, thereby improving the comprehensive stress resistance of plants. At present, it has been found that about 12% of the genes in the soybean genome are members of the transcription factor family, among which the NAC transcription factor family is a larger transcription factor family in plants. Studies in the model plant Arabidopsis have found that members of the NAC transcription factor family play an important role in abiotic stress response. There are 226 genes in soybean that are members of the NAC transcription factor family, but due to the large and complex soybean genome, research on NAC transcription factor family genes in salt stress response is very limited.

[0004] Adversity stress, including abiotic stress such as salinity, drought, and cold damage, and biotic stress such as pest and disease infection, affects the growth and development of plants and is one of the biggest constraints on agricultural and forestry production. Therefore, improving plant resistance to adversity through genetic modification is currently the most commonly used method in molecular breeding. Abscisic acid (ABA) is one of the most important plant hormones and is closely related to growth and development processes such as seed germination, root development, and plant senescence. More importantly, ABA plays a very important role in regulating plant adaptation to abiotic stress. Exogenous ABA treatment can improve plant resistance to abiotic stress, while abiotic stress such as salinity, drought, and cold damage can promote the synthesis of ABA, thereby activating the ABA signaling pathway, inducing the expression of ABA-responsive genes, and regulating plant stress resistance. However, many ABA-responsive genes are genes of unknown function.

[0005] So far, it has been found that transcription factors from multiple families, including MYB, NAC, bZIP, etc., are involved in ABA signal transduction, but usually overexpression of related transcription factor genes can enhance plant stress resistance, so they have potential application value in plant molecular breeding. However, overexpression of genes involves regulatory issues in transgenic plants, which seriously restricts the commercial value of known transcription factors involved in ABA signal transduction.

[0006] CRISPR / Cas9 technology is a new generation of gene editing technology developed in recent years. The principle of the function of this technology is: the CRISPR cluster is transcribed and processed to form a mature crRNA, which forms a complex with tracrRNA and Cas9, combines and matches the target DNA sequence and cuts the target DNA chain. When the organism repairs the DNA chain break, base insertion or deletion occurs. This insertion or deletion is mainly the insertion or deletion of the endogenous DNA sequence of the edited organism. Compared with zinc finger nuclease (ZFN) and transcription factor effector nuclease (TALEN), the CRISPR / Cas9 system has the advantages of low cost, high efficiency and simple operation. More importantly, non-transgenic mutants can be obtained through screening through CRISPR / Cas9 gene editing. Therefore, the application of CRISPR / Cas9 gene editing technology in plants is of great significance for basic plant research and crop improvement. However, the target genes that can be used for CRISPR / Cas9 gene editing to improve plant stress resistance are very limited. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a soybean NAC transcription factor gene and application thereof, wherein the soybean NAC transcription factor gene can be used to regulate root development and / or resist adverse stress.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a soybean NAC transcription factor gene, wherein the soybean NAC transcription factor gene nucleotide comprises one or more of the following sequences:

[0010] (a) the nucleotide sequence shown in SEQ ID No. 2;

[0011] (b) the nucleotide sequence shown in SEQ ID No.4;

[0012] (c) a nucleotide sequence encoding a protein having at least 95% homology with the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 2;

[0013] (d) A nucleotide sequence encoding a protein having at least 95% homology with the amino acids encoded by the nucleotide sequence shown in SEQ ID No.4.

[0014] The present invention also provides the use of the soybean NAC transcription factor gene in regulating root development and / or resisting adverse stress.

[0015] Preferably, the amino acid sequence of the soybean NAC transcription factor gene includes the amino acid sequence described in SEQ ID No.1 or the amino acid sequence described in SEQ ID No.3.

[0016] Preferably, the application is to knock out the soybean NAC transcription factor gene to obtain double mutant plants to promote root development and improve the ability to resist adverse stress.

[0017] Preferably, the adverse stress is abiotic stress, and the abiotic stress includes salt stress.

[0018] The present invention also provides a method for promoting plant root development and / or improving resistance to adverse stress, comprising the following steps:

[0019] The nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 4 were used to screen the target sequences to construct two sgRNAs for knocking out the soybean NAC transcription factor gene, and then a CRISPR / Cas9 gene editing vector containing the sgRNA was constructed, and the CRISPR / Cas9 gene editing vector was transformed into Agrobacterium tumefaciens. The Agrobacterium containing the CRISPR / Cas9 gene editing vector was then used to infect wild-type soybeans, and T1 transgenic seeds were harvested. After planting, purified double mutant plants without T-DNA / Cas9 were screened and identified.

[0020] Preferably, the gene target sequence of the sgRNA is shown as SEQ ID No.5 and SEQ ID No.6.

[0021] Preferably, the expression vector is pYLCRISPR / Cas9P ubi -B is the skeleton.

[0022] Preferably, the plant preferably comprises the Wm82 soybean variety.

[0023] Preferably, the Agrobacterium tumefaciens is Agrobacterium GV3101.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides the application of soybean NAC transcription factor genes in regulating root development and / or resisting adverse stress. The soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 of the present invention encode NAC transcription factors and belong to members of the NAC transcription factor family. The present invention reports the functions of soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 for the first time, enriching the functions of NAC transcription factor genes in soybean. The present invention has found that Glyma.02G109800 and Glyma.01G051300 can not only participate in regulating the root development of soybean, but also participate in the response of soybean to ABA and adverse stress. The present invention provides a theoretical basis for the study of adverse stress of plants.

[0026] The present invention constructs and separates double mutant plants without Cas9 by utilizing CRISPR / Cas9 gene editing, and the results show that the root systems of the double mutant plants are more robust and the resistance to salt stress is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The amino acid sequence alignment of soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300, where the same amino acids are represented in black, similar ones are represented in gray, and the underline represents the NAC domain;

[0028] Figure 2 The results of the response of soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 to ABA and salt stress;

[0029] Figure 3 Subcellular localization of proteins encoded by Glyma.02G109800 and Glyma.01G051300;

[0030] Figure 4 The transcriptional activity of proteins encoded by Glyma.02G109800 and Glyma.01G051300;

[0031] Figure 5In the figure, A is the location of the target sequences of Glyma.02G109800 and Glyma.01G051300, where the straight lines represent introns and the gray boxes represent exons, B is the editing mode of Glyma.02G109800 and Glyma.01G051300 in the double mutant, the underline is the PAM site, and the numbers represent their relative positions in the CDS, C is the amino acid sequence alignment of the proteins encoded by Glyma.02G109800 and Glyma.01G051300 in the double mutant, and the numbers represent their relative positions in the full-length amino acid sequence;

[0032] Figure 6 The effect of NaCl on Wm82 wild soybean and double mutant soybean plants. A is the root length of wild type and double mutant soybean plants before and after NaCl treatment, scale 5 cm, B is the initial root length statistics of wild type soybean and double mutant soybean plants, C is the root length inhibition rate of wild type soybean and double mutant soybean plants after NaCl treatment. DETAILED DESCRIPTION

[0033] The present invention provides two highly homologous soybean NAC transcription factor genes, wherein the soybean NAC transcription factor gene nucleotides include one or more of the following nucleotide sequences:

[0034] (a) the nucleotide sequence shown in SEQ ID No. 2;

[0035] (b) the nucleotide sequence shown in SEQ ID No.4;

[0036] (c) a nucleotide sequence encoding a protein having at least 95% homology with the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 2;

[0037] (d) A nucleotide sequence encoding a protein having at least 95% homology with the amino acids encoded by the nucleotide sequence shown in SEQ ID No.4.

[0038] The present invention also provides the use of the two highly homologous soybean NAC transcription factor genes in regulating root development and / or resisting adverse stress.

[0039] In the present invention, the soybean NAC transcription factor gene of the present invention preferably includes Glyma.02G109800 and Glyma.01G051300. As a preferred embodiment, the amino acid sequence encoded by the soybean NAC transcription factor gene Glyma.02G109800 is shown in SEQ ID No. 1, and the amino acid sequence encoded by the soybean NAC transcription factor gene Glyma.01G051300 is shown in SEQ ID No. 3. The present invention has no particular limitation on the method for obtaining the Glyma.02G109800 and Glyma.01G051300 genes, and methods known in the art can be used.

[0040] In the present invention, as a preferred embodiment, the application is to obtain double mutant plants using soybean NAC transcription factor gene to promote root development and improve resistance to adverse stress. The adverse stress is preferably salt stress. The double mutant plants of the present invention tolerate NaCl concentrations ≤ 250 mM. In the present invention, the preparation method of the double mutant plants preferably includes the following steps: using the nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 4 to screen the target sequence to construct two sgRNAs targeting the knockout of soybean NAC transcription factor gene, and then constructing a CRISPR / Cas9 gene editing vector containing the sgRNA, transforming the above CRISPR / Cas9 gene editing vector into Agrobacterium tumefaciens, and then infecting wild-type soybeans with the Agrobacterium containing the CRISPR / Cas9 gene editing vector, harvesting T1 transgenic seeds, and screening and identifying after planting to obtain purified double mutant plants without T-DNA / Cas9.

[0041] In the present invention, the target sequences are screened by using the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.4 to construct two sgRNAs for targeting the knockout of soybean NAC transcription factor genes. The present invention selects two targets to construct two sgRNAs, namely sgRNA1 and sgRNA2, that simultaneously target the Glyma.02G109800 and Glyma.01G051300 genes. The target gene sequence of the sgRNA1 is preferably as shown in SEQ ID No.5, and the target gene sequence of the sgRNA2 is preferably as shown in SEQ ID No.6. The results show that only the target sequence of sgRNA1 is edited. In the present invention, a CRISPR / Cas9 gene editing vector containing the sgRNA is constructed, and the vector is preferably pYLCRISPR / Cas9P ubi-B is the skeleton. In the present invention, the above-mentioned CRISPR / Ca9 gene editing vector is transformed into Agrobacterium tumefaciens, and the Agrobacterium tumefaciens is preferably GV3101 Agrobacterium, and the transformation method is preferably heat shock, such as heat shock at 42°C for 90s. In the present invention, the Agrobacterium containing the CRISPR / Cas9 gene editing vector is then used to infect wild-type soybeans, and the plants preferably include the Wm82 soybean variety. The present invention has no special limitations on the Glyma.02G109800 and Glyma.01G051300 gene target sequences and the construction method of the CRISPR / Cas9 gene editing vector, and methods known in the art can be used.

[0042] The present invention utilizes CRISPR / Cas9 gene editing technology for gene editing, which can effectively shorten the process and time of obtaining homozygous double mutant plants, and the operation is simple and fast. The results show that the double mutant plants obtained by the present invention can promote root development and improve resistance to adverse stress, especially resistance to salt stress.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] In order to explore the role of soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 in soybean ABA and salt stress responses, transcriptome sequencing was first performed to screen out the genes Glyma.02G109800 and Glyma.01G051300 that were regulated by both ABA and salt stress, and then RT-PCR was used to determine their responses to ABA and salt stress.

[0046] Cultivation and growth of soybean:

[0047] Williams82 (Wm82) wild-type soybean seeds for ABA treatment and salt tolerance assays, ABA treatment and salt tolerance gene expression were grown in Petri dishes or plant growth bags on filter paper surfaces moistened with 1 / 2 MS liquid medium in a growth chamber. The growth conditions in the growth chamber were 25°C, long-day conditions (16 h light / 8 h dark), and a light intensity of ~600 μmol m -2 s -1 .

[0048] Two-week-old soybean seedlings were treated with 100 μM ABA solution, methanol control group (0 μM ABA), water control group (0 mM NaCl) and 200 mM NaCl solution in the dark for 4 hours. The roots and leaves of the seedlings were immediately frozen in liquid nitrogen and stored at -80°C. After RNA was extracted, part of the samples were sent for transcriptome sequencing, and part was reverse transcribed into cDNA for RT-PCR detection. The expression level of GmEF-1α was used as an internal reference.

[0049] The full-length amino acid sequences of the proteins encoded by soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 were compared using BioEdit software. Figure 1 .

[0050] Depend on Figure 1 It can be seen that Glyma.02G109800 and Glyma.01G051300 encode NAC transcription factors, and the amino acid sequences of the proteins encoded by Glyma.02G109800 and Glyma.01G051300 are highly similar, with an amino acid sequence similarity of 99%.

[0051] Among them, the amino acid sequence of the protein encoded by the soybean NAC transcription factor gene Glyma.02G109800 is:

[0052] MENRT SSVLPPGFRFHPTDEELIVYYLCNQATSRPCPASIIPEVDIYKFDPWELPEKTDFGEKEWYFF SPRERKYPNGVRPNRATVSGYWKATGTDKAIYSGSKHVGVKKALVFYKGKPPKGLKTDWIMHEYRLIGSRRQANRQ VGSMRLDDWVLCRIYKK KNIGKSMEAKEEYPIAQINLTPANNDSEQEMVKFPRTSSLTHLLEMDYLGPISHILSDATYNSTFDFQINTANGGIDPFVKPQPVEIPYAADSGKYQVKQNSTINPTIFVNQVYYQRG (SEQ ID No. 1), wherein the underlined portion is the NAC domain.

[0053] The nucleotide sequence of soybean NAC transcription factor gene Glyma.02G109800 is:

[0054] ATGGAGAACAGAACAAGCTCTGTACTCCCACCTGGCTTCAGGTTTCATCCCACTGATGAAGAACTAATTGTTTACTATCTTTGCAACCAAGCCACATCAAGACCATGCCCTGCATCAATCATCCCAGAAGTGGATATCTATAAGTTTGATCCATGGGAATTACCTGAAAAAACAGACTTTGGAGAAAAGGAGTGGTACTTCTTTAGTCCACGAGAGAGGAAGTATCCAAATGGGGTGAGGCCTAATAGAGCAACCGTGTCTGGGTATTGGAAGGCCACTGGCACAGATAAAGCAATCTATAGTGGGTCTAAGCATGTAGGGGTGAAGAAGGCTTTGGTTTTTTATAAGGGTAAGCCACCAAAGGGTCTCAAGACAGATTGGATTATGCATGAGTATAGATTAATTGGATCAAGAAGACAAGCCAATAGACAAGTTGGATCCATGAGGCTAGACGACTGGGTCTTGTGTAGGATCTACAAGAAGAAGAACATTGGAAAGTCAATGGAGGCAAAAGAGGAATACCCAATAGCCCAAATCAATCTTACACCAGCAAACAATGACAGTGAACAAGAAATGGTGAAATTTCCAAGGACTAGTTCCCTTACTCATCTTTTGGAAATGGATTACTTGGGTCCAATATCACATATATTATCTGATGCCACATACAATTCAACCTTTGATTTTCAAATTAACACTGCCAATGGTGGAATAGACCCGTTCGTAAAACCACAGCCGGTTGAAATCCCTTATGCAGCAGATTCAGGGAAGTACCAAGTGAAACAAAATAGCACCATCAACCCCACCATATTTGTGAACCAAGTGTATTATCAAAGAGGATAA(SEQ ID No.2)。

[0055] The amino acid sequence of the protein encoded by the soybean NAC transcription factor gene Glyma.01G051300 is as follows:

[0056] MENRT SSVLPPGFRFHPTDEELIVYYLCNQASSRPCPASIIPEVDIYKFDPWELPDKTDFGEKEWYFF SPRERKYPNGVRPNRATVSGYWKATGTDKAIYSGSKHVGVKKALVFYKGKPPKGLKTDWIMHEYRLIGSRRQANRQ VGSMRLDDWVLCRIYKKKNIGKSMEAKEDYPIAQINLTPANNNSEQELVKFPRTSSLTHLLEMDYLGPISHILPDASYNSTFDFQINTANGGIDPFVKPQLVEIPYATDSGKYQVKQNSTINPTIFVNQVYDQRG (SEQ ID No. 3), wherein the underlined portion is the NAC domain.

[0057] The nucleotide sequence of soybean NAC transcription factor gene Glyma.01G051300 is:

[0058] ATGGAGAATAGAACAAGCTCTGTACTCCCACCTGGCTTCAGGTTTCACCCCACTGATGAGGAACTAATTGTGTACTACCTTTGCAACCAAGCCTCATCAAGGCCATGCCCTGCATCAATCATTCCAGAAGTGGATATCTATAAGTTTGATCCATGGGAATTACCTGATAAAACAGACTTTGGAGAAAAGGAGTGGTACTTCTTTAGCCCACGAGAGAGGAAGTATCCAAATGGGGTGAGGCCTAATAGAGCAACCGTGTCTGGGTATTGGAAGGCCACTGGGACAGATAAAGCAATCTATAGTGGGTCTAAGCATGTAGGGGTGAAGAAGGCTTTGGTTTTTTACAAGGGTAAGCCACCAAAGGGTCTCAAGACAGATTGGATTATGCATGAGTATAGATTAATTGGATCAAGAAGACAAGCCAATAGACAAGTTGGATCCATGAGGCTAGATGACTGGGTCTTGTGTAGGATTTACAAGAAGAAGAATATTGGAAAATCAATGGAGGCAAAAGAGGACTACCCAATAGCCCAAATCAACCTTACACCAGCAAACAATAACAGTGAACAAGAATTGGTGAAATTTCCAAGGACTAGTTCCCTTACTCATCTTTTGGAAATGGATTACTTGGGTCCAATATCACATATATTACCTGATGCCTCATACAATTCAACCTTTGATTTTCAAATTAACACTGCCAATGGTGGAATAGACCCGTTCGTAAAACCACAGCTGGTTGAAATCCCTTATGCAACAGATTCAGGGAAGTACCAGGTGAAACAGAATAGCACCATCAACCCCACCATATTTGTGAACCAAGTGTATGATCAAAGAGGATAA(SEQ ID No.4).

[0059] It can be seen from Figure 2 that the expression of soybean NAC transcription factor genes Glyma.02G109800 and Glyma.01G051300 is induced by ABA but inhibited by NaCl.

[0060] In order to study the subcellular localization of proteins encoded by Glyma.02G109800 and Glyma.01G051300, pUC19-GFP-Glyma.02G109800 and pUC19-GFP-Glyma.01G051300 vectors were constructed: the target RT-PCR fragments of Glyma.02G109800 and Glyma.01G051300 digested with NdeⅠ and SacⅠ were purified and ligated into the pUC19-GFP vector digested with the same enzymes. After transformation into Escherichia coli DH5α, single clones were picked, shaken and small-extracted plasmids were respectively extracted, and the restriction enzyme digestion detection of pUC19-GFP-Glyma.02G109800 and pUC19-GFP-Glyma.01G051300 vector plasmid DNA was performed. The sizes were consistent with expectations, and the expression vectors were successfully constructed.

[0061] The constructed pUC19-GFP-Glyma.02G109800 and pUC19-GFP-Glyma.01G051300 plasmid DNAs were co-transfected with pUC19-NLS-RFP nuclear localization vector plasmid DNA into Arabidopsis protoplasts, respectively. After transfection, the cells were cultured in the dark at room temperature for 20 to 22 hours, and the GFP and RFP fluorescence were observed under a fluorescence microscope.

[0062] Depend on Figure 3 It can be seen that the green fluorescence signal is concentrated in the cell nucleus, indicating that the proteins encoded by Glyma.02G109800 and Glyma.01G051300 are both localized in the cell nucleus.

[0063] After confirming that the proteins encoded by Glyma.02G109800 and Glyma.01G051300 were both localized in the nucleus, we further tested whether the proteins encoded by Glyma.02G109800 and Glyma.01G051300 had transcriptional activity. The reporter gene pUC19-Gal4:GUS and the effector gene pUC19-GD-Glyma.02G109800 or pUC19-GD-Glyma.01G051300 plasmids were co-transfected into Arabidopsis protoplasts, and cultured in the dark at room temperature for 20-22h after transfection. The GUS enzyme activity was determined by a microplate reader, and the pUC19-GD co-transfection was used as a control. The data represent the mean ± standard deviation of three replicates.

[0064] Depend on Figure 4 It can be seen that the GUS activity of the pUC19-GD-Glyma.02G109800 or pUC19-GD-Glyma.01G051300 co-transfection group increased significantly, indicating that the proteins encoded by Glyma.02G109800 and Glyma.01G051300 have transcriptional activation activity.

[0065] Example 2

[0066] Obtaining the Glyma.02G109800 and Glyma.01G051300 gene-edited double mutant described in Example 1

[0067] (1) The exon nucleotide sequences of Glyma.02G109800 and Glyma.01G051300 were used for target sequence analysis (https: / / cctop.cos.uni-heidelberg.de / species.html), and target sequences that simultaneously target two genes were selected. Two sets of target site sequences that simultaneously target two genes were determined in the DNA sequences of Glyma.02G109800 and Glyma.01G051300, and used to construct sgRNA1 and sgRNA2, respectively. The specific target sequences constructed into sgRNA are shown in Table 1. sgRNA1 and sgRNA2 were cloned into pYLCRISPR / Cas9P ubi -B vector, a gene editing vector simultaneously targeting Glyma.02G109800 and Glyma.01G051300 was constructed.

[0068] Table 1 Target sequences in sgRNA

[0069]

[0070] (2) pYLCRISPR / Cas9P targeting both Glyma.02G109800 and pYLCRISPR / Cas9 ubi -B gene editing vector was transformed into GV3101 Agrobacterium competent cells, infected with Wm82 wild-type soybean, obtained positive plants, extracted leaf DNA for PCR amplification, sequenced to detect gene editing, and harvested T1 generation seeds. The T1 generation seeds were planted in the soil, the DNA of the seedlings was extracted and PCR tested, and plants without Cas9 were screened. The gene editing was then sequenced to detect the situation, and Cas9-free homozygous double mutants of Glyma.02G109800 and Glyma.01G051300 genes were obtained. Finally, two strains of Cas9-free homozygous mutants were obtained, double mutant-c1 or double mutant-c2 (double mutant-c1 and double mutant-c2).

[0071] The location of the Glyma.02G109800 and Glyma.01G051300 target sequences in the double mutant and their editing patterns, e.g. Figure 5 shown.

[0072] Depend on Figure 5The results showed that in the double mutants, only the target sequence sgRNA1 was edited, and the Glyma.02G109800 gene had one editing mode, and the Glyma.02G109800 nucleotide sequence in the double mutant-c1 or double mutant-c2 had a base deletion at 299bp, resulting in several amino acids in the Glyma.02G109800 encoding amino acid sequence changing and terminating prematurely. The Glyma.01G051300 gene had two editing modes, and the Glyma.01G051300 nucleotide sequence in the double mutant-c1 had two base deletions at 298bp and 299bp, resulting in the Glyma.01G051300 encoding amino acid sequence terminating prematurely. The double mutant -c2 had a four-base deletion in the Glyma.01G051300 nucleotide sequence at 297-300 bp, which resulted in several amino acid changes in the amino acid sequence encoded by Glyma.01G051300 and premature termination.

[0073] Therefore, the present invention successfully obtained homozygous double mutants of Glyma.02G109800 and Glyma.01G051300 genes, namely double mutant-c1 and double mutant-c2.

[0074] Example 3

[0075] Glyma.02G109800 and Glyma.01G051300 regulate plant responses to stress

[0076] In order to explore the role of Glyma.02G109800 and Glyma.01G051300 in soybean response to adverse stress, further evidence was obtained by detecting Glyma.02G109800 and Glyma.01G05130 in regulating soybean response to NaCl.

[0077] The seeds of the wild type Wm82 and the double mutant-c1 and double mutant-c2 in Example 2 obtained at the same time under the same growth conditions were collected, and after disinfection, the seeds were planted in a plant growth bag containing 1 / 2MS liquid culture medium to cultivate seedlings. The seedlings were transferred to a new plant growth bag containing 1 / 2MS liquid culture medium (control group) and 1 / 2MS culture medium containing 200mM NaCl (experimental group), and continued to be cultivated. The root lengths of the plants in the control group and the experimental group were observed and counted. The results are as follows: Figure 6 shown.

[0078] Depend on Figure 6The results showed that in the control group, that is, in the absence of salt stress, the double mutant had a significantly shorter taproot length than the wild type, but had well-developed lateral roots and a more robust root system. In the experimental group, that is, in the presence of salt stress, the double mutant still had a shorter taproot than the wild type and had well-developed lateral roots and a robust root system, but the degree of root length inhibition was significantly lower than that of the wild type. Therefore, the double mutant of the present invention has a more robust root system and significantly improves tolerance to NaCl.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of soybean NAC transcription factor gene in regulating soybean root development and / or salt stress, characterized in that: The nucleotide sequences of the soybean NAC transcription factor gene are shown in SEQ ID No. 2 and SEQ ID No. 4; The application is to knock out the soybean NAC transcription factor gene to obtain double mutant plants, so as to promote root development and improve the ability to resist salt stress.

2. The use according to claim 1, characterized in that: The amino acid sequences of the soybean NAC transcription factor gene are shown in SEQ ID No.1 and SEQ ID No.

3.

3. A method for promoting soybean root development and / or improving salt stress resistance, characterized in that: The steps include: The nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 4 were used to screen the target sequences to construct two sgRNAs for knocking out the soybean NAC transcription factor gene, and then a CRISPR / Cas9 gene editing vector containing the sgRNA was constructed, and the CRISPR / Cas9 gene editing vector was transformed into Agrobacterium tumefaciens. The Agrobacterium containing the CRISPR / Cas9 gene editing vector was then used to infect wild-type soybeans, and T1 transgenic seeds were harvested. After planting, homozygous double mutant plants without T-DNA / Cas9 were screened and identified.

4. The method according to claim 3, characterized in that The nucleotide sequences of the gene target sequences of the two sgRNAs are shown in SEQ ID No.5 and SEQ ID No.

6.

5. The method according to claim 3, characterized in that: The vector is pYLCRISPR / Cas9P ubi -B For the skeleton.

6. The method according to claim 3, characterized in that The soybeans are of the Wm82 soybean variety.

7. The method according to claim 3, characterized in that The Agrobacterium tumefaciens is Agrobacterium GV3101.

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