CN108409846B - Soybean salt tolerance related MYB transcription factor and coding gene and application thereof - Google Patents

Abstract

The invention relates to a soybean salt tolerance related MYB transcription factor, and a coding gene and application thereof, and belongs to the field of plant genetic engineering. The base sequence of the soybean MYB transcription factor gene is SEQ ID N0.1, and the amino acid sequence is SEQ ID N0.2. The invention clones a soybean GmMYB68 transcription factor gene related to salt tolerance, analyzes the expression mode and drought resistance of the gene in wild type and transgenic soybeans, and has important significance for cultivating salt-resistant soybean varieties.

Description

Soybean salt tolerance related MYB transcription factor and coding gene and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a soybean salt tolerance related transcription factor MYB, and a coding gene and application thereof.

Background

Soybeans (Glycine max L.) provide vegetable proteins and fats, and are also used as industrial raw materials and animal feeds. On one hand, China is the largest world imported soybean, and on the other hand, biotic and abiotic stresses including diseases and insect pests, low temperature, drought and soil salinization cause serious threats to domestic soybean production. Therefore, it is urgent to enhance the adversity defense ability and to breed germplasm resources with better stress resistance in agronomic characters. Although the traditional breeding method achieves a large amount of results, the research is limited by the long breeding period, the complex stress-resistant mechanism and the lack of excellent germplasm resources. The modern molecular biology technology and the traditional breeding mode are combined to research the stress resistance mechanism of the plants, and the method becomes a necessary choice for broad masses of breeding workers.

The abiotic stress network of plants is extremely complex and contains a large number of transcriptional controls at the gene level. The plant can regulate physiological development by regulating and controlling the transcription of related stress genes. Environmental stimuli such as ion stress, pathogenic bacteria, water, temperature and the like which affect the signal conduction of plants, in order to avoid the damage of adversity stress, the plants have a set of self-regulation mechanisms to avoid the adverse environmental stresses such as low temperature, drought, high salinity and the like. MYB gene is an important member in transcription factor family, participates in plant growth metabolism and environmental factor response, and has important regulation effect on physiological and biochemical mechanism establishment in various aspects of plants. The N-terminal of the amino acid sequence of the MYB transcription factor in the plant is provided with 1-3 helix-turn-helix conformation sequences (R1, R2 and R3) which are composed of 51-53 amino acids, namely MYB structural domains. The role of the hydrophobic core is mainly derived from the 3 conserved tryptophan residues in the MYB binding domain, which play a major role in maintaining the helix-turn-helix conformation. Most MYB proteins are transcriptional activators, can activate the expression of target genes, and also have a small part of inhibitory effect on the expression of the target genes to play a role in negative regulation. The transcription factor is mainly combined with a target sequence specifically, so that the transcription factor plays a role in regulating and controlling downstream genes. Romero and Urao et al showed that MYB transcription factors bind to MYBSI (conserved sequence T/CAACG/TGA/C/TA/C/T), MYBS II (conserved sequence TAACTAAC), CNGTTR, GKTWGTTR, GKTWGGTR (N is A, G, C or T; K, G or T; R, A or G; W, A or T), TAACTG and other elements.

The gene function expression network of eukaryote is very complex, and relates to a plurality of modification, translation and transcription processes, wherein the regulation and control on the transcription level are particularly key, and the gene function expression network has important influence on the growth and development of plants, stress reaction, signal conduction, disease resistance and the like.

A large number of MYB transcription factors can exist in both monocotyledons and dicotyledons, and as MYB plays an important role in stress, other members in a soybean MYB family are subjected to functional identification, so that effective gene resources can be provided for enhancing the stress tolerance quality of soybeans by adopting a plant gene breeding means and creating excellent stress-resistant materials.

Disclosure of Invention

The invention provides a soybean salt tolerance related MYB transcription factor, and a coding gene and application thereof.

The salt tolerance related MYB transcription factor provided by the invention is derived from a soybean variety Jilin 32 (given by a health researcher at the academy of agricultural sciences of Jilin province), and is named GmMYB 68.

The base sequence of the soybean MYB transcription factor gene is SEQ ID N0.1.

The amino acid sequence of the protein coded by the soybean MYB transcription factor gene is SEQ ID N0.2.

The sequence SEQ ID N0.1 consists of 780 bases; SEQ ID N0.2 consists of 259 amino acid residues, containing two SANT domains, the MYB domain: is located at amino acids 4-53 and 56-104 and belongs to a typical R2R3-MYB transcription factor.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the coding gene of the GmMYB68 provided by the invention into a plant cell, so that the transgenic plant with salt resistance is obtained. When a plant expression vector is constructed using the gene of the present invention, any one of an enhancer promoter and an inducible promoter may be added in front of the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, vectors to be used may be processed, for example, by adding a plant selectable marker (GUS gene, luciferase gene, etc.) or an antibiotic marker having resistance (gentamicin, kanamycin, etc.). The expression vector carrying the GmMYB68 of the present invention can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant tissues into plants. The host to be transformed may be either a monocotyledonous plant or a dicotyledonous plant.

The invention clones a soybean GmMYB68 transcription factor gene related to salt tolerance, analyzes the expression mode and drought resistance of the gene in wild type and transgenic soybeans, and has important significance for cultivating salt-resistant soybean varieties.

Drawings

Fig. 1 is a graph of the PCR amplification result of the GmMYB68 gene, where M: DNA Marker (DL 2000); 1,2,3,4: performing reverse transcription amplification on the GmMYB 68;

FIG. 2 is a diagram of stress-induced expression pattern of the GmMYB68 gene;

FIG. 3 is a diagram of the tissue expression pattern of the GmMYB68 gene;

FIG. 4 is a schematic diagram of a plant expression vector structure;

fig. 5.1 is a herbicide (glyphosate) smear screen plot in the identification results of part of T2 transgenic soybean for the GmMYB68 gene, where: the arrow marks the position where the herbicide is applied, after the herbicide is applied, the wild plants wither and turn yellow, and the transgenic plants have no obvious change;

fig. 5.2 is a Bar gene detection map of the GmMYB68 gene in the results of the identification of a partial T2 generation transgenic soybean in which: m:

DNA Marker (DL 2000); 1-22: pTF101.1-GmMYB68 transgenic plant; +: recombinant plasmids; -: negative control (template water);

fig. 5.3 is a Southern blot hybridization analysis plot of the GmMYB68 gene in the identification of partial T2 generations of transgenic soybeans, M:

DNA Marker; 1,2,3,4: carrying out Southern blot identification on T2 transgenic plants digested by Hind III; +: recombinant plasmids; -: negative control (wild type plants);

FIG. 6.1 is a salt tolerance identification chart of transgenic plants of GmMYB68, which is the results of salt and alkali treatment of wild soybean and transgenic soybean of T3 generation, wherein WT is wild soybean and OE is transgenic soybean of T3 generation;

FIG. 6.2 is a graph of germination rate determination for GmMYB68 transgenic soybeans, where WT is wild-type soybean, OE is T3 transgenic soybean,

FIG. 7 is a graph of the change of soluble sugar content (A) and proline content (B) of GmMYB68 transgenic plants under saline-alkali stress, Wild-type: wild type soybean; GmMYB 68-OE: the accumulation of soluble sugar and proline in the GmMYB68 transgenic plant is related to the stress reaction in the plant, the content of the soluble sugar and the proline in the wild type and the transgenic plant are obviously increased after the saline-alkali stress treatment, wherein the increase amplitude of the GmMYB68 transgenic plant is obviously increased compared with that of the wild type;

FIG. 8 is a fluorescent real-time quantitative PCR result chart of GmMYB68 stress-resistance-associated genes in T3 transgenic soybeans, wherein: a: an ABA signaling pathway key regulatory factor GmABI 5; b: molecular response regulation gene DREB2 for high salt, drought and low temperature stress of plants; c: na (Na)⁺the/H transporter gene GmNHX 1; d: phenylalanine ammonia lyase, a key enzyme of the phenylpropane metabolic pathway; e: a gene GmNPR1-1 related to the response of the disease course of the plant; f: the gene GmNPR1-2 related to the disease course response of the plant, and the 5 genes are important genes related to the stress resistance of the plant which are proved in related literatures.

Detailed Description

Example 1: cloning and sequence analysis of GmMYB68 gene

Extraction of soybean RNA and cDNA Synthesis: with reference to the whole body of gold

Plant RNA Kit instruction for extracting total RNA from soybean Jilin 32 leaves

First-Strand cDNA Synthesis Super Mix (purchased from gold, Kagaku) to reverse transcription, Synthesis of cDNA;

sequencing the resulting sequences (20d, 30d, 50d) using the immature embryo expression profile of soybean variety Jilin 32, using NCBI sequence analysis tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) Two unknown cDNA sequences with high homology with MYB transcription factors are obtained through analysis, primers are designed by utilizing Primer5 software, and immature embryo cDNA is used as a template for PCR amplification. The reaction system is as follows:

the PCR procedure was:

carrying out electrophoresis detection on the PCR product on 1% agarose gel, and shooting and storing by an ultraviolet gel imager;

the amplified fragment was recovered from the kit by using agarose gel

After recovery, the Quick Gel Extraction Kit (gold Kasei) was ligated with the cloning vector pMD18-T (available from TaKaRa) in the following reaction system:

t vector 0.5ul

PCR recovery of 2ul product

Solution I 2.5ul

After overnight ligation at 16 ℃, escherichia coli competence was transformed and sent to Huada gene for sequencing, and the sequence was verified to be correct.

PCR amplification is carried out to obtain a sequence containing an open reading frame of the GmMYB68 gene, and 259 amino acids are coded. The GmMYB68 protein was predicted to have a molecular weight of 145.636kDa and an isoelectric point pI of 5.0. Amino acid structure the protein is predicted to contain two SANT domains, the MYB domain, located at amino acids 4-53, 56-104, which are typical of R2R3-MYB transcription factors. .

Example 2: expression pattern of GmMYB68 gene under stress induction

When the plant grows to the four-leaf stage, the seedlings are transferred to Hogland nutrient solution to be cultured for 3d and then treated as follows:

salt stress treatment: placing soybean seedlings in Hogland nutrient solution containing 200mM NaCl;

low-temperature treatment: placing soybean seedlings in a light incubator at 4 ℃;

drought treatment: placing soybean seedlings in Hogland nutrient solution containing 20% PEG 8000;

and (3) mechanical injury treatment: slightly scratching 3-5 wounds on soybean seedling leaves by using a sterilized dissecting blade;

ABA treatment: spraying soybean seedlings with ABA containing 200 uM;

sampling the plant materials after the treatment for 0h, 1h, 2h, 5h, 10h and 24h respectively, and quickly placing the plant materials in liquid nitrogen to store at-80 ℃ for later use;

total RNA extraction and cDNA Synthesis methods in the same manner as in example 1, real-time fluorescent quantitative PCR primers (F: 5'-TCGGTCTGGGAAATCGTG-3'; R: 5'-CTCACCACTCGCAGCATCT-3') were designed based on the cDNA sequence of GmMYB 68; using soybean constitutive expression gene GmACTII (GeneBank accession number: U60500) (F: 5'-GAGCTATGAATTGCCTGATGG-3'; R: 5'-CGTTTCATGAATTCCAGTAGC-3') as an internal reference gene, and using ABI PRISM7500 real-time quantitative PCR instrument and cDNA of each tissue part and each stress treatment sampling point of soybean as a template to perform fluorescence real-time quantitative PCR; the specific steps refer to Tip Green qPCR Supermix instructions for operation;

reaction system:

the PCR procedure was:

94℃ 30s

55℃ 5s

72℃ 34s

40 cycles, using 2^-△△CTThe method analyzes data, determines the relative expression quantity of genes, sets 3 technical repeats for each sampling point, and sets 3 biological repeats for the test.

Table 1 relative expression level of GmMYB68 gene under stress treatment;

the result shows that the GmMYB68 gene has an up-regulation expression trend under various stress treatments. After the salt stress treatment is carried out for 5h, the expression level of GmMYB68 reaches the highest level, and the overall expression level is higher than that of other stress treatments, so that the GmMYB68 is deduced to be induced and expressed by various adversity stresses, wherein the expression response to the salt stress is the most obvious.

Example 3: expression pattern of GmMYB68 gene in soybean tissue

Total RNAs of roots, stems, flowers, leaves, cotyledonary nodes, hypocotyls and 20-day embryos of soybean Jilin 32 were extracted and reverse-transcribed into cDNAs in the same manner as in example 1. The procedure of real-time fluorescent quantitative PCR using cDNA of the above tissue as a template was the same as in example 2.

Table 2 expression levels of the GmMYB68 gene in different tissues of soybean:

stem of a tree	Leaf of Chinese character	Flower (A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A. B. A	Immature embryo	Cotyledon node	Hypocotyl
						1.413027	1.512265	24.98434	4.243418	2.049935	13.57257

GmMYB68 is expressed in all tissue parts of soybean, wherein the expression level of flowers, hypocotyls and immature embryos is the highest, and the expression level of other parts is low although the expression level is also high; the expression level in roots is minimal.

Example 4: construction of plant expression vector of GmMYB68 gene and genetic transformation of soybean cotyledonary node

Taking overnight culture liquid pMD18T-GmMYB68 to extract a plasmid as a template, adding XbaI and SacI enzyme cutting sites into upstream and downstream primers, (F: 5'-TGCTCTAGACAAAGGACATGGATCGGATAAAAGG-3', R: 5'-CGAGCTCTTATTCAACCCTACCAATTCCCATCC-3') to perform PCR amplification, and performing restriction enzyme cutting reaction on an amplification product and a plant expression vector pTF101.1-35S respectively, wherein the reaction system refers to the reagent specification of NEB company:

and (3) carrying out enzyme digestion for 2h at 37 ℃, carrying out agarose gel electrophoresis detection on the enzyme digestion product, and recovering the target fragment. And connecting the PCR fragment after enzyme digestion with a vector by adopting T4 ligase, and after the sequencing identification is correct, naming the recombinant plasmid with the correct sequencing as pTF101.1-GmMYB68, transforming agrobacterium EHA101 (purchased from Biovector), and storing the strain for later use.

Referring to the method of Guodong et al, the Agrobacterium-mediated transformation method was used to transform soybean cotyledonary node tissues. Taking the whole mature soybean seeds with smooth surface, no scab, no mould and no crack, putting the seeds into a closed container, and fumigating and sterilizing the seeds for 10 to 12 hours by using chlorine generated by 100ml of NaClO and 3.5ml of concentrated hydrochloric acid. After blowing off residual chlorine gas on an ultra-clean workbench, inoculating the sterilized soybean seeds into a germination culture medium through an umbilicus for about 16h overnight, cutting two cotyledons along the central axis by using a sterile scalpel, removing primary leaf buds, and making scratches with the length of about 3mm at the joints of the cotyledons and the hypocotyls. Placing the prepared explants into a sterile dish filled with engineering bacteria infection liquid, and infecting about 80 explants per 70ml of bacterial liquid, wherein the sterile explant can be infected when infectedSealing the culture dish, placing on a shaking table, and lightly shaking at 100rpm for 20-30min to make the explant fully contact with the invasion solution; and (3) paving a layer of sterile filter paper on the co-culture medium, placing infected cotyledon nodes on the filter paper with the paraxial surface upward, and carrying out dark culture at 24 +/-1 ℃ for 4 days at about 8 explants in each dish. After the co-culture for 4 days, cutting off the extended hypocotyl of the cotyledonary node, keeping about 3mm, and obliquely inserting the cotyledonary node explant with 45-degree paraxial surface upwards into a differentiation medium for adventitious bud induction. The culture temperature is about 26 ℃, the illumination is carried out for 24 hours, the subculture is carried out once every 14 days, and the fresh culture medium is replaced. Excess shoots are cut off during subculture and new wounds are made at the contact surface of the explant with the medium to promote adventitious shoot induction. Transferring the adventitious bud after inducing for 28 days into an adventitious bud elongation culture medium, cutting off cotyledons, subculturing every 14 days, and replacing fresh culture medium until the adventitious bud elongates to 3-5 cm. Cutting off adventitious bud when it extends to 3-5cm, dipping in proper amount of 1mg/L IBA, transferring into rooting culture medium, rooting for about 20d, transferring into 26 deg.C artificial climate chamber for acclimatization, transplanting, and harvesting T₀Generation seeds and further analysis of the generations.

Example 5: screening and identification of GmMYB68 transgenic soybean

And (3) test strip detection: about 0.2g of tender leaves of the regenerated seedlings are taken and put into a clean centrifuge tube, and 100ul of sterile water is added for grinding. Will be provided with

One end of the test strip with the arrow mark is immersed in the grinding fluid for several minutes, and the result of positive detection is obtained when the color development end presents two red lines, and the result of negative detection is obtained when the color development end presents one red line.

Screening herbicides: when the second pair of compound leaves of the T0 generation transgenic seedling in the greenhouse is completely unfolded, selecting the lobules in the middle of the three compound leaves, smearing glyphosate (1 per thousand m/v) solution, marking, and observing and photographing for recording after smearing for 3 d. The arrow marks the position where the herbicide is applied, the wild plants wither and turn yellow after the herbicide is applied, and the transgenic plants have no obvious change. The transgenic plant carries the gene of the selection marker gene Bar on the plant expression vector and contains herbicide resistance.

Southern blot hybridization: selecting the progeny of positive plants, namely 4-5 mature leaves of T2 transgenic plants, detected by a test strip and screened by herbicide, extracting total DNA, carrying out DNA digestion by using restriction enzyme Hind III, taking the recombinant plasmid pTF101.1-GmMYB68 as a positive control, and taking the DNA single enzyme digestion product of wild plants as a negative control. The transgenic plant contains 1-2 copy strips of the target gene, which indicates that the target gene is integrated into the soybean genome in a single copy mode.

Example 6: saline-alkali tolerance analysis of T3 GmMYB68 transgenic soybean

With 200mM NaCl solution and 100mM Na₂CO₃The solutions are respectively used as a salt stress treatment solution and an alkali stress treatment solution, 9 pots of transgenic soybean seedlings with uniform growth are divided into three groups, when the soybean just enters a four-leaf stage, the soybean is subjected to salt and alkali treatment, only water is poured into a control group, and meanwhile, wild soybeans in the same growth period are subjected to the same treatment to be used as a wild control group. The total treatment time was 20 days.

After the treatment of saline-alkali stress, leaves of wild plants and transgenic plants generate yellow spots and curling phenomena. Wild plants show obvious phenomenon of hypoevolutism, the symptoms of the transgenic plants are light, and the transgenic plants have Na pair₂CO₃The tolerance of the composite is better than that of NaCl.

In addition, NaCl solution and Na in the above concentrations₂CO₃And (4) irrigating the T3 generation transgenic seeds and wild soybean seeds with the solution, treating 100 seeds in each group for 4 days, and counting the germination rate of the seeds.

The germination rates of the transgenic soybean and the wild soybean seeds have no obvious difference under the condition of irrigation water treatment, but the germination rate of the transgenic plant is superior to that of the wild soybean after saline-alkali stress treatment, and the transgenic plant has stronger tolerance to NaCl in a germination rate test.

Example 7: determination of physiological indexes of T3-generation transgenic soybeans after saline-alkali treatment

1) Chlorophyll content determination

Collecting fresh leaves 0.2g, cutting, placing in a clean 50ml centrifuge tube, adding 10ml anhydrous ethanol and 80% acetoneVolume of the mixture. Standing in dark place for 48 hr until chlorophyll is completely extracted, and measuring OD with ultraviolet spectrophotometer₆₆₃And OD₆₄₅In the formula C_T＝20.29OD₆₄₅+8.05OD₆₆₃Calculating the chlorophyll content;

2) determination of soluble sugar content

Weighing about 1.0g of fresh leaves, cutting into pieces, placing in a large test tube, and adding 10ml of H₂Boiling in a boiling water bath for 20min, cooling to room temperature, filtering into a volumetric flask, fixing the volume to 100ml, taking 1ml of the extract to be measured, adding 5ml of anthrone reagent, quickly and uniformly mixing, boiling in the boiling water bath for 10min, taking out, cooling, measuring the OD value under the wavelength of 620nm, and calculating the content of soluble sugar by referring to a standard curve;

3) determination of proline content

Weighing 0.05g of sample, putting the sample into a centrifuge tube, adding 10ml of 3% sulfosalicylic acid, sealing, boiling in a boiling water bath for 30min, cooling to room temperature, centrifuging at 3000rpm for 10min, and taking supernatant to be tested;

putting 1ml of extracting solution into a test tube, adding 1ml of ice vinegar, 1ml of 3% sulfosalicylic acid and 2ml of 2.5% acidic ninhydrin, uniformly mixing, carrying out a boiling water bath color reaction for 60min, cooling, adding 4ml of toluene, oscillating to extract a red substance, standing, taking an upper liquid phase, measuring an OD value under the wavelength of 520nm, finding out the corresponding proline concentration according to a standard curve, and calculating by referring to a formula: proline content (ug g)^-1FW) ═ cxv/2/W/115; wherein C is the proline content (ug/ml) checked by a standard curve, V is the total volume (ml) of the extracting solution, W is the sample mass (g), and 115 is the relative molecular mass of proline;

4) photosynthetic assay

Adopts an LI-6400 type portable photosynthesis measuring system, adopts a fixed red and blue light source, and sets the illumination intensity at 1200 umol.m^-2·s^-1. The determination was made at 10:00 to 12:00 am on day 10 of treatment, the determination being the middle leaflet of the third set of three compound leaves on the plant, 2. The net photosynthetic rate (Pn) of leaves, the conductance (Gs) of stomata, the concentration (Ci) of intercellular carbon dioxide and the transpiration rate (Tr) are directly read by an instrument;

data processing and analysis of variance were done using the statistical program SPSS 21.0. Data in this text are presented as mean ± sem of triplicates. The analysis and comparison of the physiological indexes adopt LSD multiple comparison;

the results show that after the saline-alkali treatment, the chlorophyll content of the wild plants and the transgenic plants is reduced to a certain extent compared with that of the wild plants and the transgenic plants under normal growth conditions, and the reduction range is different in different stress treatments. Passing through NaCl and Na₂CO₃After treatment, the chlorophyll content of the wild plants is respectively reduced by 32.9 percent and 22.5 percent, and the chlorophyll content of the transgenic plants is respectively reduced by 36.1 percent and 0.11 percent;

under the normal growth state, the photosynthetic physiological indexes of the wild type and the transgenic plant have no obvious difference, the net photosynthetic rate (Pn) of leaves shows a descending trend after saline-alkali stress, the net photosynthetic rate of the wild type is respectively reduced by 30.2 percent and 42.1 percent, and the net photosynthetic rate of the transgenic plant is respectively reduced by 27 percent and 21.8 percent. At the level of alkaline stress, there was a significant difference in the net photosynthetic rate of the transgenic plants compared to the wild type. In addition, the stomatal conductance (Gs) and the transpiration rate (Tr) are consistent with the net photosynthetic rate decreasing trend, and the intercellular carbon dioxide concentration (Ci) is opposite to the net photosynthetic rate decreasing trend, so that the intercellular carbon dioxide concentration (Ci) is increased after the stress treatment, the main reason that the photosynthetic rate is decreased under the saline-alkali stress of the wild type and the transgenic plant is not the non-limiting function of stomata, and the carbon-nitrogen metabolic disorder caused by the decrease of the photosynthetic activity of mesophyll cells is presumed. Salt stress and alkali stress have distinct effects on the physiological indicators of plants compared to controls. Relevant documents prove that the photosynthesis capability of plants with stronger stress resistance is less influenced when the plants are stressed by the adversity, and the experiment proves that the transformation of the GmMYB68 gene is beneficial to relieving the influence of the salt and alkali stress on the chlorophyll content and the photosynthesis of the plants.

Table 3 effect of GmMYB68 on photosynthetic physiological indices under saline and alkaline stress:

note: marked as significant difference P < 0.05, marked as significant difference P < 0.01

A large number of related documents prove that the accumulation of soluble sugar and proline is beneficial to maintaining the cell structure and the viability of plants under the stress condition and improving the stress tolerance of the plants. After saline-alkali treatment, a large amount of soluble sugar and proline in wild type and GmMYB68 plants are accumulated. Under the normal growth condition, the content of the wild type and the transgenic plant has no obvious difference, after the alkali stress treatment, the content of soluble sugar and the content of proline in the transgenic plant are respectively increased by 76.54 percent and 50.91 percent, and the increase amplitude is obviously higher than that of the wild type plant under the same stress treatment (respectively 62.38 percent and 43 percent). In addition, the increase of proline of the GmMYB68 transgenic plant after NaCl treatment is obviously higher than that of the wild type plant under the same treatment. Taken together, the results indicate that the overexpression of GmMYB68 in soybean favors the synthesis and accumulation of soluble sugars and proline under saline-alkaline conditions.

Table 4 effect of salt, alkali stress on leaf osmoregulation substances of GmMYB68 transgenic plants:

example 8: survey of agronomic traits of GmMYB68 transgenic plants

Passing through NaCl and Na₂CO₃After treatment, the development of the plants is significantly affected, which is specifically characterized by reduced lateral roots, limited root elongation and accelerated lignification. The agronomic character investigation is carried out on the transgenic plant, the main indexes comprise five aspects of plant height, branch number, single plant pod number, single plant grain number and hundred grain weight, and the data result is subjected to statistical analysis by adopting an LSD method. The results are shown in the table, although the indexes are reduced after salt and alkali stress treatment, the transgenic plant and the wild plant have no significant difference, and the result shows that the agronomic characters of the soybean variety are not influenced while the saline-alkali tolerance of the soybean variety is enhanced by the insertion of the GmMYB 68.

Table 5 agronomic trait survey in GmMYB68 transgenic and wild type plants:

example 9: expression of anti-stress related gene in GmMYB68 transgenic plant

The expression levels of the anti-inversion related genes in the transgenic plants and the wild plants are detected by fluorescent real-time quantitative PCR, and the relative expression quantity is obtained by comparing the transgenic plants and the wild plants under the same treatment condition. Wherein: GmABI5 is a key regulatory factor of an ABA signal pathway; DREB2 is a molecular response regulation gene for high salt, drought and low temperature stress of plants; GmNHX1 is Na⁺a/H transporter gene; PAL is a key enzyme phenylalanine ammonia lyase of a phenylalkane metabolic pathway; GmNPR1-1 and GmNPR1-2 are related to the disease course response of plants, and the above 5 genes have been proved to have important relation with the stress response of plants under adversity stress in literature.

Table 6 primers used in the assay:

all the stress-resistance related genes respond to salt stress and alkali stress to different degrees, and the stress-resistance related genes accumulate higher expression levels in GmMYB68 transgenic plants, so that the transcription levels of the genes are influenced by the overexpression of GmMYB68 and participate in a salt and alkali stress response mechanism regulated by GmMYB 68. Among them, GmABI5 and DREB2 have obvious expansion in transgenic plants compared with wild type. In addition, two transcription factors which play a role in broad-spectrum disease resistance are found, namely GmNPR1-1 and GmNPR1-2 have obvious high transcription level (P is less than 0.01) in transgenic plants, and the GmNMB 68 plays a certain role in the aspect of biological stress.

Sequence listing

<110> Jilin university

<120> soybean salt tolerance related MYB transcription factor, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 780

<212> DNA

<213> Artificial Synthesis (Artificial sequence)

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cagacttacg gccctaggaa ctggtccgtt ataagcaaat ccattccggg tcggtctggg 120

aaatcgtgcc gcttgcggtg gtgcaaccag ctgtctccgg aggtggagcg ccggcctttc 180

acggcggagg aagacgaggc gatcctgaag gctcacgcca ggttcgggaa caagtgggcc 240

accatcgcgc gcttcctcaa tggccgcacc gacaacgcca tcaagaacca ttggaattcc 300

accctcaaga ggaagtgctc cgagcctctc tccgagcctc ggcctctgaa gagatccgcc 360

accgtttcgg gtagtcaatc cggatccgat ttgagcgatt cgggtttacc cattattctt 420

gcaagaagcg tgagcgtgac ggtagctccc tctaaccacc ttgcggaaac cgcgtcttct 480

tcagtaactg atcctgccac gttattgagt ttgtctctac cgggattcga ttcgtgcgat 540

ggggctaata atgggcctgg gccgaatcag gggcccagtt gcggcccgtt ccaggagata 600

ccgatgcttg gttcccaaaa gcagttgttc agccaagagt ttatgaaggt gatgcaagag 660

atgatacgag tggaagtgag aaattacatg tctgtactgg aacgtaatgg tgtgtgtatg 720

caaaccgatg ccattaggaa ctcggtgttg gagaggatgg gaattggtag ggttgaataa 780

<210> 2

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<212> PRT

<213> Artificial Synthesis (Artificial sequence)

<400> 2

Met Asp Arg Ile Lys Gly Pro Trp Ser Pro Glu Glu Asp Glu Ala Leu

1 5 10 15

Arg Arg Leu Val Gln Thr Tyr Gly Pro Arg Asn Trp Ser Val Ile Ser

20 25 30

Lys Ser Ile Pro Gly Arg Ser Gly Lys Ser Cys Arg Leu Arg Trp Cys

35 40 45

Asn Gln Leu Ser Pro Glu Val Glu Arg Arg Pro Phe Thr Ala Glu Glu

50 55 60

Asp Glu Ala Ile Leu Lys Ala His Ala Arg Phe Gly Asn Lys Trp Ala

65 70 75 80

Thr Ile Ala Arg Phe Leu Asn Gly Arg Thr Asp Asn Ala Ile Lys Asn

85 90 95

His Trp Asn Ser Thr Leu Lys Arg Lys Cys Ser Glu Pro Leu Ser Glu

100 105 110

Pro Arg Pro Leu Lys Arg Ser Ala Thr Val Ser Gly Ser Gln Ser Gly

115 120 125

Ser Asp Leu Ser Asp Ser Gly Leu Pro Ile Ile Leu Ala Arg Ser Val

130 135 140

Ser Val Thr Val Ala Pro Ser Asn His Leu Ala Glu Thr Ala Ser Ser

145 150 155 160

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

165 170 175

Asp Ser Cys Asp Gly Ala Asn Asn Gly Pro Gly Pro Asn Gln Gly Pro

180 185 190

Ser Cys Gly Pro Phe Gln Glu Ile Pro Met Leu Gly Ser Gln Lys Gln

195 200 205

Leu Phe Ser Gln Glu Phe Met Lys Val Met Gln Glu Met Ile Arg Val

210 215 220

Glu Val Arg Asn Tyr Met Ser Val Leu Glu Arg Asn Gly Val Cys Met

225 230 235 240

Gln Thr Asp Ala Ile Arg Asn Ser Val Leu Glu Arg Met Gly Ile Gly

245 250 255

Arg Val Glu

Claims (1)

1. The application of a soybean salt tolerance related MYB transcription factor in the cultivation of salt tolerance soybean varieties has an amino acid sequence of SEQ ID NO. 2 and a nucleotide sequence of SEQ ID NO. 1.

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