CN103588867B - Soybean transcription factor GmMYB174a, and coding gene and applications thereof - Google Patents

Abstract

The invention discloses a soybean transcription factor GmMYB174a and its coding gene and application. The present invention provides a protein, which is the following (a) or (b): (a) a protein composed of the amino acid sequence shown in sequence 1 in the sequence listing; (b) the amino acid sequence shown in sequence 1 in the sequence listing A protein derived from Sequence 1 that has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant stress tolerance. The experiment of the present invention proves that the present invention discovers a GmMYB174a gene whose expression in soybean is induced by high salt, drought, low temperature and plant hormone ABA treatment. The present invention is of great value for cultivating new varieties of crops and forests and grasses resistant to abiotic stress (salt tolerance), and can be used for the cultivation and identification of stress-tolerant plant varieties required for agriculture, animal husbandry and ecological environment management. The improvement of crop yield in high-saline soil is of great significance.

Description

Soybean transcription factor GmMYB174a and its coding gene and application

technical field

The invention relates to the field of biotechnology, in particular to a soybean transcription factor GmMYB174a and its coding gene and application.

Background technique

Changes in physical and chemical factors in the environment, such as drought, salinity, low temperature, etc., have an important impact on the growth and development of plants. In severe cases, they will cause large-scale crop yield reduction. Cultivating stress-tolerant crops is one of the main goals of planting. At present, genetic engineering breeding has become one of the important methods to enhance crop stress tolerance. Higher plant cells have multiple pathways to respond to various stresses in the environment, and transcription factors play a role in regulating the expression of stress tolerance-related effector genes. Many types of transcription factors have been found in plants related to plant stress tolerance, for example: DREB in EREBP/AP2, bZIP, MYB, WRKY and so on.

The MYB transcription factor family refers to a class of transcription factors containing the MYB domain. The MYB domain is a peptide segment of about 51-53 amino acids, including a series of highly conserved amino acid residues and spacer sequences.

Since the ZmMYBC1 gene related to pigment synthesis in maize was cloned in 1987, MYB genes with different functions have been isolated from many plants. Most of the MYB proteins in plants contain only two MYB domains. They are a large family of transcription factors involved in many biological processes, such as the regulation of secondary metabolism, control of cell differentiation, response to hormone stimulation and external environmental stress, and resistance to pathogenic bacteria. infringement. MYB proteins containing one MYB domain and MYB proteins containing three MYB domains have also been found in plants. They may respectively play an important role in maintaining the integrity of chromosome structure, regulating gene transcription, participating in the control of cell cycle and regulating cell differentiation.

MYB is one of the largest transcription factor families in plants. Studies have shown that MYB is involved in the response to abiotic stress. In the abiotic stress regulatory signaling pathway, the MYB gene was induced by ABA and not induced by ABA or even suppressed expression, indicating that the MYB transcription factor may participate in the ABA regulatory pathway and also participate in the non-ABA regulatory pathway in response to abiotic stress. Atmyb2, a member of Arabidopsis MYB family, is rapidly induced by drought stress, and its expression is also induced by high salt and exogenous ABA. Overexpression can enhance the drought tolerance of transgenic plants; the mutant plant hos10-1 of Arabidopsis MYB member HOS10 has a sharp decrease in adaptability to chilling injury, and is highly sensitive to dehydration and NaCl treatment; the content of ABA increases under dehydration stress conditions. This suggests that HOS10 is essential for chilling adaptation and affects plant tolerance to dehydration stress by controlling ABA biosynthesis. The following genes are not induced by ABA. Arabidopsis thaliana HPPBF-1 is a single MYB transcription factor gene of MYB type induced by salt; rice Osmyb4 is induced by low temperature, and overexpression in Arabidopsis can increase the chilling injury and Freeze damage resistance. After classifying 1500 salt-responsive genes in Arabidopsis, only four MYB genes were found to be induced by NaCl.

Contents of the invention

One object of the present invention is to provide a soybean transcription factor GmMYB174a and its coding gene.

The present invention provides a seed protein named GmMYB174a, which is derived from soybean (Glycine max (L.) Merrill), and is as follows (a) or (b): (a) consists of the amino acid sequence shown in sequence 1 in the sequence listing (b) The amino acid sequence shown in Sequence 1 in the Sequence Listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues, and a protein derived from Sequence 1 that is related to plant stress tolerance.

Sequence 1 in the above sequence listing consists of 361 amino acid residues.

The protein in (a) or (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the protein in (b) above can be deleted by deleting one or several amino acid residue codons from the DNA sequence shown in Sequence 1 in the Sequence Listing, and/or performing missense mutations of one or several base pairs , and/or connect the coding sequence of the tag shown in Table 2 at its 5' end and/or 3' end.

Some of the substitutions, substitutions and/or additions of one or several amino acid residues in the amino acid sequences of the above proteins are caused by naturally occurring variations, and some are caused by artificial mutagenesis.

The gene encoding the above-mentioned protein is also within the protection scope of the present invention.

The above-mentioned gene is a DNA molecule as follows (1) or (2) or (3):

(1) The DNA molecule shown in sequence 2 in the sequence listing;

(2) A DNA molecule that hybridizes to the DNA sequence defined in (1) under stringent conditions and encodes a plant stress tolerance-related protein;

(3) At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the DNA sequence defined in (1) , a DNA molecule having at least 98% or at least 99% homology and encoding a plant stress tolerance-related protein.

The above sequence listing sequence 2 consists of 1086 nucleotides, and the coding region is 1-1086 nucleotides from the 5' end.

The above-mentioned stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1mM EDTA, rinsing at 50°C, 2×SSC, 0.1% SDS; Can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; can also be: 50°C, in 7% SDS , 0.5M NaPO ₄ and 1mM EDTA mixed solution, rinse at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, 7% SDS, 0.5M NaPO ₄ and 1mM EDTA mixed solution Hybridization at 50°C, rinsing in 0.1×SSC, 0.1% SDS; can also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, at 65°C, 0.1×SSC, Rinse in 0.1% SDS; alternatively: hybridize in 6×SSC, 0.5% SDS solution at 65°C, then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS .

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above genes all belong to the protection scope of the present invention.

The above-mentioned recombinant vector is a recombinant vector for expressing the above-mentioned protein by inserting the coding gene of the above-mentioned protein into the expression vector; the above-mentioned recombinant vector can be used as the starting vector, and the sequence in the sequence list is inserted between the multiple cloning sites XbaI and KpnI of pPROKII The recombinant plasmid pPROKII-GmMYB174a obtained from the 1-1086 deoxynucleotides at the 5' end of 1.

The above-mentioned plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal can direct polyA to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions.

When using GmMYB174a to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, maize ubiquitin promoter (Ubiquitin)), or tissue-specific expression promoters (such as seed-specific expression promoters), which can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must The same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical resistance marker genes (such as herbicide resistance genes), etc.

A pair of primers for amplifying the full length of the above-mentioned genes or any fragment thereof also falls within the protection scope of the present invention.

The nucleotide sequence of one primer in the above primer pair is sequence 3 in the sequence listing, and the nucleotide sequence of the other primer in the primer pair is sequence 4 in the sequence listing.

The application of the above-mentioned proteins, above-mentioned genes or above-mentioned recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in cultivating stress-tolerant plants is also within the protection scope of the present invention.

In the above application, the stress tolerance is drought tolerance and/or salt tolerance;

The above application is specifically to introduce the gene into the target plant through the recombinant vector to obtain the transgenic hairy root, the growth rate of the transgenic hairy root is greater than that of the empty vector hairy root; wherein, the empty vector hairy root The hairy root is the empty vector hairy root obtained by transferring pROKⅡ into the target plant.

Described plant is dicotyledonous plant or monocotyledonous plant; Described dicotyledonous plant is preferably leguminous plant, as soybean, lotus root, alfalfa, peanut, mung bean, kidney bean, kidney bean etc.; Or other dicotyledonous plant, as rape, Cucumber, tomato, cabbage, tobacco, eggplant, etc.; said monocotyledonous plants are preferably rice, wheat, corn, lawn grass, etc.; they can also be trees, such as poplar, acacia, pagoda tree, pumice, etc.

Transformed cells, tissues or plants as described above are understood to include not only the end products of the transformation process, but also transgenic progeny thereof.

"Polynucleotide", "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "open reading frame (ORF)" and the like mentioned in the present invention include single-stranded or double-stranded DNA and RNA Molecules, which may contain one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences including exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

The gene of the present invention can be introduced into the host in the following way: the gene of the present invention is inserted into the expression cassette, and then the expression cassette is introduced into the host through a plant expression vector, a non-pathogenic self-replicating virus or Agrobacterium. The expression vector carrying the gene of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conduction, and Agrobacterium-mediated.

The plants into which the gene of the present invention has been transferred can propagate the gene in the species, and can also transfer the gene into other varieties of the same species, especially commercial varieties, using conventional breeding techniques.

The gene of the present invention can be introduced into the host in the following way: the gene of the present invention is inserted into the expression cassette, and then the expression cassette is introduced into the host through a plant expression vector, a non-pathogenic self-replicating virus or Agrobacterium. The expression vector carrying the gene of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conduction, and Agrobacterium-mediated.

The gene of the present invention can be modified as follows on the basis of sequence 2, and then introduced into the host to achieve better expression effect:

1) In order to express the nucleotide sequence of the present invention in transgenic plants, the nucleotide sequence of the present invention can be modified and optimized according to actual needs. For example, according to the codons preferred by recipient plants, the codons thereof can be changed to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequence of the present invention. Moreover, in the optimization process, it is best to keep a certain GC content in the optimized coding sequence, so as to best realize the high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, preferably more than 45%. , more preferably more than 50%, most preferably more than about 60%.

2) For efficient initiation of translation, the gene sequence adjacent to the initial methionine can be modified. For example, modifications are made using sequences known to be effective in plants.

3) Linking the gene of the present invention with various plant-expressed promoters to facilitate its expression in plants. Such promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters. The choice of promoter will vary with expression time and space requirements, and also depends on the target species. For example, a tissue- or organ-specific expression promoter depends on at what stage of development the receptor is desired. Although many promoters derived from dicots have been shown to be functional in monocots and vice versa, ideally dicot promoters are selected for expression in dicots, monocots Promoters are used for expression in monocots.

Preferred constitutive promoters include the CaMV 35S and 19S promoters. The promoter may also be a promoter derived from several actin genes expressed in most cell types. Another preferred constitutive promoter is the ubiquitin promoter. The above-mentioned promoters can also be promoters that direct expression in roots, pith, leaves or pollen, that is, tissue-specific promoters. Cotton ribulose bisphosphate carboxylase-oxygenase promoter (US patent US 6,040,504), rice sucrose synthase promoter (US patent US 5,604,121), night scent yellow leaf curl virus promoter (WO 01/73087) .

The chemically inducible promoter may be the Rab29A promoter (US Patent US 5,614,395).

4) Linking the gene of the present invention with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention. For example, tml from CaMV, E9 from rbcS. Any available terminator known to function in plants may be linked to the gene of the present invention.

5) Enhancer sequences can be introduced into the gene of the present invention, such as intron sequences (eg derived from Adhl and bronze) and viral leader sequences (eg derived from TMV, MCMV and AMV).

In practice, the gene of the present invention can also be targeted to cells. It can be realized by utilizing existing technologies in the art. For example, the target gene sequence derived from the target organelle is fused with the gene sequence of the present invention, and then introduced into the plant cell to achieve localization.

The starting vector among the above recombinant vectors can be selected according to the transformation technique used and the characteristics of the target plant species. The above-mentioned selection can be reflected in the selection of the resistance marker in the vector. For some target species, different antibiotic or herbicide selectable markers may be preferred. Selectable markers commonly used in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics, the bar gene, which confers resistance to the herbicide phosphinothricin, hph, which confers resistance to the antibiotic hygromycin gene, and the dhfr gene that confers resistance to metharexate, the EPSPS gene that confers resistance to glyphosate, and the mannose-6-phosphate isomerase gene that confers the ability to metabolize mannose.

The experiment of the present invention proves that the present invention discovers a GmMYB174a gene whose expression in soybean is induced by high salt, drought, low temperature and plant hormone ABA treatment. The GmMYB174a gene was introduced into the root of soybean Kefeng No. 1, and the hairy root transfected with GmMYB174a gene was obtained. The phenotypic analysis of the hairy roots after salt and drought stress respectively proved that the growth state of the hairy roots of the transgenic GmMYB174a gene was significantly better than that of the control (transfer of empty vector hairy roots), indicating that the hairy roots of the transgenic GmMYB174a gene provided by the present invention Shaped roots can significantly improve the salt/drought tolerance of plants. The invention is of great value for cultivating stress-tolerant plant varieties, especially for cultivating new varieties such as crops and forest grasses resistant to abiotic stress (salt tolerance), and can be used for the development of stress-tolerant plant varieties required for agriculture, animal husbandry and ecological environment management. Breeding and identification are important for improving crop yields in arid/high salinity soils.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Description of drawings

Figure 1 is the expression characteristics of GmMYB174a gene under various treatments

Figure 2 is a schematic diagram of the plant expression vector pPROKII-GmMYB174a

Figure 3 is the growth of GmMYB174a overexpression hairy root under normal conditions

Figure 4 is the analysis of the salt tolerance of transgenic GmMYB174a hairy roots

Fig. 5 is the drought tolerance analysis of transgenic GmMYB174a gene hairy roots

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

% in the following examples, unless otherwise specified, are mass percentages. In the quantitative experiments in the following examples, three repeated experiments were set up, and the data were the mean value or mean ± standard deviation of the three repeated experiments.

All plant material was grown at 22°C with 16h/8h per day light (light/dark).

Glycine max L.Merr.Kefeng 1 (Glycine max L.Merr.Kefeng 1) was recorded in W.K.Zhang, Y.J.Wang, G.Z.Luo, J.S.Zhang, C.Y.He, X.L.Wu, J.Y.Gai, S.Y.Chen, QTL mapping of tenagronomic traits on the soybean( Glycine max L.Merr.) genetic map and theirassociation with EST markers, Theor.Appl.Genet, 2004, 108:1131-1139; also recorded in Wang Xiuqiang, Gai Junyi, Yu Deyue, broad-spectrum Kangyuan Kefeng No. 1 on soybean Genetic research on the resistance of virulent mosaic virus strain group SC-8, Soybean Science, Issue 03, 2003; the public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences;

pROKII vector (binary expression vector) is described in D.C.Baulcombe, G.R.Saunders, M.W.Bevan, M.A.Mayo and B.D.Harrison, Expression of biologically active viral satelliteRNA from the nuclear genome of transformed plants. Nature 321(1986), pp.446–449 The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Agrobacterium rhizogenes K599 is recorded in Attila Kereszt, et al., Agrobacterium rhizogenes-mediated transformation of soybean to study of root biology, Nature Protocols, 2007, 2(4), 549-552), the public can get from Professor Peter M Gressnon, The University of Queensland, St Lucia, Queensland 4072, Australia, or the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Professor Peter M Gressnon (written consent).

Arabidopsis ecotype Colombia (col-0): Seeds were purchased from Arabidopsis Biological Resource Center (ABRC).

Cloning and expression of embodiment 1, soybean transcription factor GmMYB174a gene

In the study of soybean transcription factor MYB family in response to high salt, drought and low temperature stress, it was found that a gene was induced by the above treatment, and it was named GmMYB174a; the details are as follows:

1. Cloning of soybean transcription factor GmMYB174a gene

The 3-week-old seedlings of Soybean Kefeng No. 1 were treated with 200mM NaCl for 6 hours, 2 grams of fresh leaves were taken, ground in liquid nitrogen, suspended in 4mol/L guanidine sulfhydric acid aqueous solution, the mixture was extracted with acidic phenol and chloroform, and put on Total RNA was obtained by adding absolute ethanol to the supernatant. The extracted total RNA was reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed with Primer-F and Primer-R to obtain PCR amplification products. The PCR amplification product was detected by 1% agarose gel electrophoresis, and a band with a molecular weight of about 1Kb was obtained, and the fragment was recovered with an agarose gel recovery kit (TIANGEN).

Primer-F: 5'-CCC TCTAGA ATGGCGATTCAAGATCAAAATG-3' (underlined XbaⅠ enzyme recognition site) (SEQ ID NO: 3);

Primer-R: 5'-CCC GGTACC CTACAAGGAAAGATGGATGCAT-3' (underlined KpnI enzyme recognition site) (SEQ ID NO: 4).

PCR amplification system (50 μl): cDNA 1 μl, 10× buffer 5 μl, dNTP (10 mM) 1 μl, Primer-F 1 μl, Primer-R 1 μl, Pfu DNA Polymerase (TaKaRa) 1 μl, ddH ₂ O 40 μl. The amplification conditions are: 94°C for 3 minutes; 30 cycles of 94°C for 30s, 50°C for 30s, and 72°C for 30s; 72°C for 10m.

The recovered fragment (PCR product) was ligated with pGEM-T Easy (Promega), and the ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were screened according to the carbenicillin resistance marker on the pGEM-T Easy vector to obtain the recovered Fragments of recombinant plasmids. Using the T7 and SP6 promoter sequences on the recombinant plasmid vector as primers to determine its nucleotide sequence, the sequencing results show that the PCR product has the nucleotides shown in sequence 2 in the sequence table, and the gene of the PCR product is named GmMYB174a, the coding region of the gene is 1-1086 nucleotides from the 5' end of sequence 2 in the sequence listing, and the protein encoded by the gene is named GmMYB174a, and the amino acid sequence of the protein is shown in sequence 1 in the sequence listing. Sequence 2 in the sequence listing consists of 1086 nucleotides, and sequence 1 in the sequence listing consists of 361 amino acid residues.

Sequence 2 in the sequence listing can also be artificially synthesized, and PCR is performed with Primer-F and Primer-R to obtain the same PCR product.

2. The expression of soybean transcription factor GmMYB174a gene is induced by abiotic stress

The 3-week-old seedlings of Soybean Kefeng No. 1 were subjected to the following stress treatments:

1) Salt stress treatment (200mM NaCl treatment): move the seedlings into 200mM NaCl aqueous solution, room temperature, about 25°C;

2) Osmotic stress treatment (drought treatment): carefully absorb water from the roots of the seedlings, place them on filter paper and expose them to air at room temperature (25°C);

3) Low temperature stress treatment (low temperature treatment at 4°C): immerse the seedlings in pre-cooled water and place them in a refrigerator at 4°C;

4) Abscisic acid (ABA) treatment (100 μM ABA treatment), immerse the seedling roots in 100 μM ABA aqueous solution, room temperature, about 25°C.

5) CK treatment: Seedlings without any treatment are recorded as CK;

6) Water treatment: immerse the seedlings in distilled water at a temperature of about 25°C;

Collect 1 g of fresh leaves at 1, 3, 6, and 12 hours for each treatment, grind them in liquid nitrogen (collect the leaves directly in the CK group), suspend them in 4 mol/L guanidine thiohydrogen aqueous solution, and extract the mixture with acidic phenol and chloroform , adding absolute ethanol to the supernatant to obtain total RNA. The expression characteristics of GmMYB174a gene in the above treatments were analyzed by quantitative PCR, and the primers were Primer-F and Primer-R. The soybean GmTubulin gene was used as an internal standard, and the primers used were Primer-TF and Primer-TR. The value obtained by Q-PCR is the expression level of the gene relative to GmTubulin. RT-PCR needs to adjust the amount of template cDNA to make the expression of GmTubulin consistent, and then perform gene amplification. The experiment was repeated three times, and the results were average ± standard deviation.

Primer-F: 5'-ATGGCGATTCAAGATCAAAATG-3'

Primer-R: 5'-CTACAAGGAAAGATGGATGCAT-3'

Primer-TF: 5'-AACTCCATTTCGTCCATTCCTTC-3'

Primer-TR: 5'-TTGAGTGGATTCCCAACAACG-3'

The results are shown in Figure 1, A is drought treatment; B is 200mM NaCl treatment; C is 4°C low temperature treatment; D is 100μM ABA treatment; it can be seen from the figure that GmMYB174a gene has the same effect on low temperature, drought, high salinity and ABA treatment. There is a response, but the pattern of the response is different. Except for ABA treatment, the expression of GmMYB174a was significantly induced at 1 hour of the above treatments, and the expression of GmMYB174a continued to increase up to 12 hours in the drought treatment, and rose to the highest at 3 hours in the high-salt and low-temperature treatments, and then decreased, and the high-salt treatment It will be the lowest at 12 o'clock, and it will rise again after 12 hours of low temperature treatment. When ABA was treated for 1 hour, it decreased slightly, increased significantly at 3 hours, and decreased again at 6 hours.

Embodiment 2, application of soybean transcription factor GmMYB174a gene

1. Obtaining GmMYB174a hairy roots

1. Construction of recombinant expression vector pPROKII-GmMYB174a

Use restriction endonucleases XbaI and KpnI to double digest the PCR product obtained by using Primer-F and Primer-R as primers in Example 1, recover the digested product, and use the digested product with the plant expression vector pROKII through the same digestion The resulting vector backbones are ligated to obtain ligation products. The ligation product was transformed into Escherichia coli to obtain a transformant. The plasmid of the transformant was extracted and sent for sequencing. The plasmid was a vector obtained by inserting sequence 2 in the sequence table between the XbaI and KpnI restriction sites of pROKII. The vector was named pROKII-GmMYB174a, and the sequence in the sequence table was Sequence 2 is located behind the CaMV 35S promoter. The schematic diagram of the structure of the recombinant expression vector pROKⅡ-GmMYB174a is shown in Fig. 2 .

2. Obtaining GmMYB174a-transformed hairy roots (overexpressing GmMYB174a gene hairy roots)

1) The recombinant expression vector pROKⅡ-GmMYB174a obtained in the above 1 was introduced into Agrobacterium rhizogenes K599 by electric shock method to obtain recombinant Agrobacterium.

The plasmid of the recombinant Agrobacterium was extracted and verified by sequencing. The result was that the plasmid was pROKⅡ-GmMYB174a, and the recombinant Agrobacterium containing the plasmid was named K599/pROKⅡ-GmMYB174a.

2) Inoculate recombinant Agrobacterium K599/pROKⅡ-GmMYB174a seedlings containing two true leaves for 6 days with a syringe to inoculate soybean Kefeng 1 seedlings, and grow under moisture: 16 hours of light, temperature 25°C, humidity 50%. After 2 weeks, hairy roots grow and become transformed hairy roots. A total of 32 transgenic GmMYB174a hairy root systems were obtained. When the hairy roots grow to 4-6 cm, further transgenic identification and stress tolerance testing can be carried out. In the same way, the empty vector pROKⅡ was transformed into soybean Kefeng 1 seedlings, and 27 hairy roots transformed with the empty vector were obtained as experimental controls.

3) Total RNA was extracted from the hairy roots transfected with GmMYB174a numbered 1-32 and the hairy roots transformed with empty vectors numbered 1-27, and reverse-transcribed into cDNA. Using cDNA as a template, the expression of GmMYB174a gene was analyzed with Primer-F and Primer-R. The Real-Time PCR reaction uses TOYOBO's RealTime PCRMaster Mix kit and operates according to the instructions. The primers used for detection of GmMYB174a gene expression were Primer-F and Primer-R; soybean GmTubulin gene was used as internal standard, and the primers used were Primer-TF and Primer-TR. The experiment was repeated three times, and the results were average ± standard deviation.

Primer-F: 5'-ATGGCGATTCAAGATCAAAATG-3'

Primer-R: 5'-CTACAAGGAAAGATGGATGCAT-3'

Primer-TF: 5'-AACTCCATTTCGTCCATTCCTTC-3'

Primer-TR: 5'-TTGAGTGGATTCCCAACAACG-3'

The average relative expression level of GmMYB174a in molecular identification of GmMYB174a-transformed hairy roots numbered 1-15 and empty vector hairy roots numbered 1-15 is shown in Figure 4A, wherein, OE is GmMYB174a-transformed hairy roots, The control is the hairy root of the empty vector. It can be seen from the figure that the relative expression level of GmMYB174a in the hairy root of the transgenic GmMYB174a is 3.65 ± 0.3; the relative expression level of GmMYB174a detected in the hairy root of the empty vector is the The expression of GmMYB174a was 0.50 ± 0.15.

The average relative expression level of GmMYB174a in the molecular identification of the GmMYB174a-transformed hairy root numbered 16-32 and the empty vector hairy root numbered 16-27 is shown in Figure 5A, wherein, OE is the GmMYB174a-transformed hairy root, The control is the hairy root of the empty vector. It can be seen from the figure that the relative expression level of GmMYB174a in the hairy root of the transgenic GmMYB174a is 3.35±0.25; the relative expression of GmMYB174a in the hairy root of the empty vector is 0.40±0.2.

The above results indicated that the expression level of GmMYB174a in the GmMYB174a gene-transformed root system was much higher than that in the empty vector-transferred root system.

2. Identification of Drought Tolerance, Salt Tolerance and Low Temperature Tolerance of Transgenic GmMYB174a Hairy Root System

The experimental samples were as follows: the hairy roots transformed with empty vector were recorded as control; the hairy roots transformed with GmMYB174a were recorded as OE.

There was no significant difference in hairy root growth between the control and OE cultured in water for 10 days, with lengths of 8.1 ± 3.9 cm and 7.8 ± 3.7 cm, see Figure 3.

1. Identification of salt tolerance

Ten hairy roots of transgenic GmMYB174a (OE) and empty vector (control) were treated with 100mM NaCl aqueous solution for 3 days, that is, immersed in 100mM NaCl aqueous solution at 25° for 3 days. Each strain has 10 individual plants.

After 3 days of treatment, take pictures and observe the results, as shown in Figure 4B, the phenotypes of the empty vector hairy roots and GmMYB174a hairy roots treated with 100mM NaCl for 3 days, it can be seen that under the treatment of 100mM NaCl, the hairy roots of the two There was a significant difference in root growth rate.

The hairy root length was measured, the experiment was repeated three times, and the results were average ± standard deviation, as follows: the lengths of the GmMYB174a gene-transformed hairy root and the empty carrier hairy root were 4.3 ± 2.8 cm and 4.5 cm, respectively, after being treated with 100 mM NaCl aqueous solution. ±2.7cm, there was no significant difference between the two; the hairy roots of transgenic GmMYB174a gene and transgenic vector were 6.2±2.3cm and 5.2±2.1cm after treatment with 100mM NaCl aqueous solution;

Hairy root growth rate = (root length after treatment - root length before treatment) / root length before treatment

The growth rate was plotted as shown in Fig. 4C, the length growth rate of hairy roots transfected with GmMYB174a gene was 42±28% after being treated with 100mM NaCl aqueous solution. However, the growth rate of the hairy root length of the empty carrier treated with 100mM NaCl aqueous solution was 16±35%, and there was a significant difference between the two.

2. Drought tolerance identification:

Transgenic GmMYB174a gene hairy roots (OE) and empty carrier hairy roots (control) were immersed in 1% (volume percentage) PEG (polyethylene glycol 6000) at 25° for 3 days. Each strain has 10 individual plants.

After 3 days of treatment, take photos and observe. The results are shown in Figure 5B. The phenotypes of the empty vector hairy roots and GmMYB174a hairy roots treated with 1% PEG for 3 days can be seen. There were significant differences in growth status.

The length of the hairy root was measured, the experiment was repeated three times, and the results were average ± standard deviation, as follows: the lengths of the hairy root of the transgenic GmMYB174a gene and the transgenic vector hairy root were 4.6 ± 2.1 cm and 4.1 cm respectively after 1% PEG treatment. ±2.3cm; the lengths of the GmMYB174a gene-transformed hairy roots and the empty vector-transferred hairy roots after treatment with 1% PEG were 6.4±2.0cm and 5.7±2.4cm, respectively;

Hairy root growth rate = (root length after treatment - root length before treatment) / root length before treatment

The growth rate was plotted as shown in Figure 5C. When the PEG concentration was 1%, the growth rate of the hairy root (control) transfected with the empty vector was about 3923%, while that of the transgenic GmMYB174a hairy root (OE) was about 63± twenty one%.

The growth rate of the hairy root of transgenic GmMYB174a gene was slightly different from the growth rate of the transgenic vector hairy root.

Claims (3)

1. Application of GmMYB174a protein in cultivating stress-tolerant plants; The amino acid sequence of the GmMYB174a protein is sequence 1 in the sequence listing; The stress tolerance is drought tolerance and/or salt tolerance; The plant is soybean. 2. Application of GmMYB174a protein coding gene in cultivating stress-tolerant plants; The nucleotide sequence of the GmMYB174a protein coding gene is sequence 2 in the sequence listing; The stress tolerance is drought tolerance and/or salt tolerance; The plant is soybean. 3. The application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the GmMYB174a protein coding gene in cultivating stress-tolerant plants; The nucleotide sequence of the GmMYB174a protein coding gene is sequence 2 in the sequence listing; The stress tolerance is drought tolerance and/or salt tolerance; The plant is soybean.