CN106167801A - In Semen sojae atricolor WILLIAMS-DARLING Ton 82, NAC film combines transcription factor gene GmNTL1 and application thereof - Google Patents

Abstract

The invention discloses a NAC membrane binding transcription factor gene GmNTL1 in soybean Williams 82 and a plant expression vector containing the gene GmNTL1. The invention also discloses the application of the gene GmNTL1 to improve salt stress tolerance in Arabidopsis and soybean plants. Experiments have proved that the salt tolerance of the transgenic plants of the present invention has been greatly improved compared with non-transgenic plants, which provides a theoretical basis and a practical basis for improving the salt tolerance of crops, and indicates that the genes of the present invention can be widely used. for breeding salt-tolerant plant varieties.

Description

NAC Membrane-Bound Transcription Factor Gene GmNTL1 in Soybean Williams 82 and Its Application

technical field

The invention belongs to the technical field of biogenetic engineering, and in particular relates to a NAC membrane-bound transcription factor gene in soybean (Glycine max (L.) Merr) Williams 82 (Williams 82)—GmNTL1 and its application.

Background technique

Plants are exposed to the natural environment and are often affected by various environmental stresses, such as drought, extreme temperatures, nutrient deficiencies, and pests and diseases. These environmental stresses not only restrict the growth and development of plants, but also lead to the reduction of food crops. The growth and development of higher plants and their response to environmental changes are achieved by regulating the expression of target genes. The interaction between transcription factors and gene cis-acting elements can be used as a molecular switch for gene expression regulation. It is the interaction between transcription factors and cis-acting elements in the promoter region of stress-resistant functional genes that activates the expression of stress-related genes and improves the comprehensive stress resistance of plants. There is a special class of transcription factors, which are called membrane-bound transcription factors (MTFs) because they contain a transmembrane region. Membrane-bound transcription factors are directly integrated on the membrane structure in the cell (such as cytoplasmic membrane, endoplasmic reticulum membrane, nuclear membrane, etc.), and are in a dormant state. When stimulated by changes in the external environment, membrane-bound transcription factors are released from the membrane. It is transformed into an activated state and transported to the nucleus to perform its function.

MTFs have been found in yeast, prokaryotes, animals and plants, and it has been confirmed that they can perform multiple functions by activating the expression of target genes. Notably, most plant membrane-bound transcription factors play important roles in regulating plant responses to environmental stress. The NAC transcription factor family is a plant-specific transcription factor family. Since Kim et al first discovered the MTFs type of the NAC transcription factor family in Arabidopsis in 2006, and named it NTM1 (NAC WITHTRANSMEMBRANE MOTIF 1), it is currently in plants. , the functions of nine NAC membrane-bound transcription factors have been discovered and reported. As in Table 1:

Table 1 Functions of plant membrane NAC binding transcription factors

Since the first discovery of plant membrane-bound transcription factors, people have made great progress in the research on membrane-bound transcription factors, involving many aspects such as plant growth and development, hormone signal response and so on. It is particularly noteworthy that membrane-bound transcription factors play a very important regulatory role in the adaptation of plants to adversity such as disease resistance, cold resistance, drought resistance and salt tolerance. However, there is still a lack of breakthrough research results in the research on its signal transduction and regulation mechanism, and most of them are concentrated in the model plant Arabidopsis thaliana. After searching, there is no report on the soybean NAC membrane-bound transcription factor and its related functions.

Contents of the invention

In view of the current technical situation, the purpose of the present invention is to provide a soybean (Glycine max (L.) Merr) Williams 82 (Williams 82) NAC membrane-bound transcription factor gene - GmNTL1 and its application.

The NAC membrane-bound transcription factor gene in soybean Williams 82 of the present invention is characterized in that: the gene is named as soybean Williams 82NAC membrane-bound transcription factor gene GmNTL1, and the nucleotide sequence of the gene cDNA is as SEQID No. .1 shown.

The present invention also provides a plant expression vector containing the above-mentioned NAC membrane-bound transcription factor gene in soybean Williams 82, characterized in that: the plant expression vector is named expression vector pK2GW7::GmNTL1, and the expression vector clones the nuclear The nucleotide sequence is shown in SEQ ID No.5.

Alternatively, the present invention also provides a plant expression vector containing the above-mentioned NAC membrane-bound transcription factor gene in soybean Williams 82, characterized in that: the plant expression vector is named as expression vector pB2GW7::GmNTL1, and the expression vector clones The nucleotide sequence of the region is shown in SEQ ID No.6.

The application of the NAC membrane binding transcription factor gene in soybean Williams 82 in improving the salt stress tolerance of the plant.

Wherein: the plant is preferably soybean or Arabidopsis. Further, the Arabidopsis is Col-0 wild type, and the soybean variety is Ludou 11.

In the present invention, the applicant isolated the NAC membrane-bound transcription factor gene—GmNTL1 from soybean Williams 82, transferred the gene to the model plant Arabidopsis thaliana to prove its function, and then transferred the gene to the original plant soybean, the result Transgenic plants with improved salt tolerance were obtained.

Specifically, an example of the application of soybean Williams 82NAC membrane-bound transcription factor gene GmNTL1 expressing GmNTL1 in Arabidopsis or soybean plants.

The applicant first cloned the NAC membrane-bound transcription factor gene GmNTL1 in the Williams 82 plant; used the Gateway system to connect the GmNTL1 gene to the pDONR221 vector through BP reaction (see Figure 1), and then obtained it in Escherichia coli DH5α by transformation A large number of clones, followed by LR reaction to connect this gene GmNTL1 to the expression vector pK2GW7 (see Figure 2) or pB2GW7 (see Figure 3), and expressed in Escherichia coli DH5α; then screen the transgenic Escherichia coli, and extract the transformation plasmid, and finally The transformation plasmid was transformed into Agrobacterium strain GV3101. The transformed Agrobacterium strains were transformed into Arabidopsis and soybean, and the function of the expressed gene GmNTL1 was verified.

The beneficial effects of the present invention are: using the existing plant genetic engineering technology, the present invention clones and obtains the Williams82NAC membrane-bound transcription factor gene for the first time and expresses it in Arabidopsis, so that the transgenic Arabidopsis obtains the non-transgenic Arabidopsis Does not have the ability to tolerate high-salt stress. At the same time, the exogenous gene transformation method assisted by soybean germination embryo vacuum infiltration is used to transfer the gene into soybean. It is proved by comparative analysis that the salt tolerance of transgenic plants is greatly improved compared with non-transgenic plants. It has been fully proved that the soybean Williams 82NAC membrane-bound transcription factor gene GmNTL1 of the present invention plays an important role in improving plant salt stress tolerance, indicating that the gene of the present invention can be widely used to cultivate salt-tolerant plant varieties .

Description of drawings

Figure 1 is a map of the entry vector pDONR221.

Fig. 2 is a map of the plant expression vector pK2GW7.

Fig. 3 is a map of the plant expression vector pB2GW7.

Figure 4 is the electrophoresis diagram of the CDS full-length clone of the Williams 82NAC transcription factor gene GmNTL1,

Among them: M is Marker, and lanes 1, 2, 3, and 4 are CDS full-length clones of genes.

Fig. 5 is the screening of Arabidopsis pure lines of 35S::GmNTL1 transgene.

Fig. 6 is the RT-PCR expression analysis of Williams 82NAC transcription factor gene GmNTL1 in transgenic Arabidopsis.

Figure 7 shows the salt resistance analysis of 35S::GmNTL1 transgenic Arabidopsis plants under the conditions of 0, 50, 100, 150, and 200 mmol/L NaCl;

Among them: A: under the condition of 0mM NaCl; B: under the condition of 50mM NaCl; C: under the condition of 100mM NaCl; D: under the condition of 150mM NaCl; E: under the condition of 200mM NaCl.

Fig. 8 shows the regenerated plants of 35S::GmNTL1 transgenic soybeans, wherein: A: transgenic plants of T0 generation; B: transgenic plants of T1 generation; C: transgenic plants of T2 generation.

Figure 9 is a PCR detection diagram of Williams 82NAC membrane-bound transcription factor gene GmNTL1 transfected with Ludou 11,

Among them: the upper figure is the result of PCR amplification with 35S-F/R primers, and the lower figure is the result of PCR amplification with 35S-F+GmNTL1-R primers;

Lane M is Marker, and the positive control in the lane is the positive plasmid control; the negative control in the lane is the negative plant control; in the figure above: lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 are GmNTL1 transgenic positive plants . In the lower panel: Lanes 1, 2, 3, 4, 5, 6, and 8 are GmNTL1 transgene-positive plants.

Figure 10 is a Southern blot analysis diagram of Williams 82NAC membrane-bound transcription factor gene GmNTL1 transfected with Ludou 11,

Among them: lane M is Marker, lane "+" is positive plasmid control; lane "-" is negative plant control; lane "1, 2, 3, 4" are GmNTL1 transgene positive plants.

Figure 11 shows the salt tolerance analysis of 35S::GmNTL1 transgenic soybean plants, WT is the negative plant control, and 1 and 2 are transgenic positive plants.

Among them: the left picture is the phenotype under 0mM NaCl treatment, and the right picture is the phenotype under 150mM NaCl treatment.

detailed description

Cloning of embodiment 1, GmNTL1

1.1 Extraction of Williams 82 total RNA

(1) Put the Williams 82 plant material into a mortar and grind it into powder with liquid nitrogen (directly used in the following experiments or frozen in a -80°C ultra-low temperature refrigerator for future use);

(2) After the liquid nitrogen volatilizes, immediately transfer 100-200mg of plant powder into a 1.5ml centrifuge tube, then quickly add 1ml of Trizol extract, vortex to fully dissolve the sample into the extract, and place it at room temperature for 5 minutes;

(3) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.9ml of the supernatant to a new 1.5ml centrifuge tube, then add 0.2ml of chloroform, vigorously shake and mix for 15sec, and place at room temperature for 2-5min;

(4) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.4ml of supernatant to a new 1.5ml centrifuge tube, add 0.4ml of isopropanol, turn up and down 15 times to mix the solution, and place at room temperature for 15mim;

(5) Centrifuge at 12,000rpm at 4°C for 10min, discard the supernatant, wash the pellet twice with 1ml of 75% ethanol, centrifuge at 8,000rpm at 4°C for 5min;

(6) Discard the supernatant, open the lid and dry the RNA on a clean bench for about 2-5 minutes, add 40 μl RNase-Free water, and fully dissolve the RNA at 60°C for 10 minutes;

(7) Measure the OD value and concentration of the RNA sample with a UV spectrophotometer, A ₂₆₀ /A ₂₈₀ is preferably 1.7-2.0; the quality detected by agarose gel electrophoresis.

1.2 Reverse transcription of RNA

(1) Add the following substances (40μl reaction system) to the centrifuge tube in sequence:

(2) After mixing gently, denature at 65°C for 5 minutes, immediately insert on ice, and ice bath for at least 1 minute;

(3) Add the following substances in turn to the centrifuge tube

(4) After mixing gently, place in a constant temperature water bath at 42°C for 1 hour, denature at 65°C for 10 minutes, and store at -20°C for later use.

1.3GmNTL1 gene cloning

GmNTL1OX-F: 5'-AAAAAGCAGGCTCGATGGGTGCCGTCGTC-3'

GmNTL1OX-R: 5'-AGAAAGCTGGGTTTTAAAGATCTGACATATGCCCA-3'

(1) The reaction system for the first step amplification of the Gateway system with the high-fidelity enzyme Primer Star is as follows (50 μl system):

The amplification conditions are as follows:

After the reaction, the reaction solution was detected by electrophoresis on 0.8% TAE agarose gel.

(2) Purification and recovery of cloned gene fragments (Tiangen kit)

1) Put the cut gel with the target fragment into a 1.5ml centrifuge tube and weigh the gel, add 3 times the volume of sol solution, sol at 60°C for 10 minutes, and keep turning over during the sol;

2) After the gel is completely melted, suck it all into the recovery column and let it stand for a while;

3) Room temperature, 12000rpm, centrifuge for 30Sec, discard the solution;

4) Add 700 μl of rinsing solution to the column, centrifuge at 12000 rpm for 1 min, and discard the rinsing solution;

5) Add 500 μl of rinsing solution to the column, centrifuge at 12000 rpm for 1 min, and discard the rinsing solution;

6) Empty column, 12000rpm, centrifuge for 2min;

7) Open the recovery column and let it dry for 1-2 minutes, put it into a new clean 1.5ml centrifuge tube, add 40μl of sterilized water or EB buffer preheated at 60°C, and let it stand for 2 minutes;

8) Centrifuge at 12000 rpm for 1 min, and the resulting solution is the recovered fragment.

(3) The high-fidelity enzyme Primer Star performs the second step amplification of the Gateway system.

attb primer sequence:

attb-F: 5'-G GGG ACA AGT TTG TAC AAA AAA GCA GGC T-3'

attb-R: 5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3'

The reaction system is as follows (50μl system):

The amplification conditions are as follows:

After the reaction, the reaction solution was detected by 0.8% TAE agarose gel electrophoresis, as shown in FIG. 4 .

1.4 BP reaction of cloned gene fragment

The BP reaction (Gateway system) system is as follows:

React at 25°C for 8 hours-overnight.

1.5 Preparation of competent E. coli (aseptic operation)

(1) Take the DH5α strain and inoculate it in 20ml LB liquid medium, and culture it on a shaker at 37°C overnight;

(2) Inoculate in 50ml LB liquid medium at a ratio of 1:100, culture at 37°C and 200rpm for 1 hour, until the _OD600 value is 0.4-0.6;

(3) Place the bacterial solution on ice for 30 minutes;

(4) 4°C, 4200rpm, centrifuge for 10min, discard the supernatant, add 10ml of pre-cooled 0.1M CaCl ₂ to suspend the bacteria;

(5) Place the bacterial solution on ice for 10 minutes;

(6) 4°C, 4200rpm, centrifuge for 10min, discard the supernatant, add 2ml of pre-cooled 0.1M CaCl ₂ to suspend the bacteria, aliquot into 1.5ml centrifuge tubes, use now or add 30% final volume of glycerol through liquid nitrogen Store at -80°C after quick freezing.

1.6 Plasmid transformation of Escherichia coli (sterile operation)

(1) Add 1-5 μl of plasmid DNA or ligation product to 50 μl of competent cells, flick the centrifuge tube to mix well, and place in ice bath for 30 minutes;

(2) At 42°C, heat shock in a warm water bath for 90 sec, then immediately ice bath for 2-3 min;

(3) Add 1ml of LB medium and incubate at 37°C for 40-50min;

(4) room temperature, 4000rpm, centrifuge 3min, collect bacterial cell;

(5) Spread the bacteria on a culture plate containing the corresponding antibiotics, and culture them upside down at 37°C overnight.

1.7 Escherichia coli PCR verification

The reaction system is as follows (20μl system):

The amplification conditions are as follows:

After the reaction, the reaction solution was detected by electrophoresis on 0.8% TAE agarose gel.

1.8 DNA sequencing

Pick a positive single colony containing the recombinant plasmid and shake it overnight with liquid LB containing Kan (25mg/L), then send it to Shanghai Boya Biotechnology Co., Ltd. for sequencing, and obtain the sequencing result: the gene cDNA sequence is as shown in the sequence table SEQ ID No.1 Show.

After sequence analysis, the above-mentioned cDNA sequence is 100% homologous to the nucleotide homology of soybean Willms82. Three experiments have proved that what is obtained is the Willms82NAC membrane-bound transcription factor gene, which is named GmNTL1. The nucleotide sequence of the cDNA of the gene GmNTL1 is shown as SEQ ID Shown in No.1.

1.9 Extraction of Escherichia coli plasmid DNA

(1) Pick a single clone and inoculate it in 10ml of LB liquid medium containing corresponding antibiotics, and culture at 37°C for 8h;

(2) room temperature, 12,000rpm, centrifuge for 1min, collect the bacteria;

(3) Discard the supernatant, add 100 μl of low-temperature pre-cooled solution I, and oscillate to suspend the bacteria;

(4) Add 200 μl of freshly prepared solution II, quickly turn over and mix well, and ice-bath for 5 minutes;

(5) After the solution is clarified, add 150 μl of low-temperature pre-cooled solution III, mix gently and then ice-bath for 5 minutes;

(6) 4°C, 12000rpm, centrifuge for 10min, pipette the supernatant into a new 1.5ml centrifuge tube;

(7) Add an equal volume of phenol/chloroform/isoamyl alcohol (25/24/1), shake and mix;

(8) Centrifuge at 12,000 rpm for 10 minutes at room temperature, transfer the upper aqueous phase to another new 1.5ml centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), extract once more, and shake to mix;

(9) Centrifuge at 12,000 rpm for 10 minutes at room temperature, transfer the upper aqueous phase to another new 1.5ml centrifuge tube, add an equal volume of isopropanol, mix well and place at -20°C for 30 minutes;

(10) Centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant and keep the precipitate.

(11) The precipitate was washed twice with 75% ethanol, dried in vacuum for 5 minutes, dissolved in 40 μl of sterilized water, and stored at -20°C for later use. 1.10 LR reaction of cloned gene fragment

The LR reaction (Gateway system) system is as follows:

React at 25°C for 8 hours.

1.11 Competent Escherichia coli preparation and plasmid transformation (same as 1.5, 1.6)

1.12 Escherichia coli PCR verification (same as 1.7)

1.13 Extraction of Escherichia coli plasmid DNA (same as 1.9)

The nucleotide sequence of the cloning region of the positive pK2GW7::GmNTL1 plasmid transformed into the pK2GW7 vector is shown in SEQID No.5.

The nucleotide sequence of the cloning region of the positive pB2GW7::GmNTL1 plasmid transformed into the pB2GW7 vector is shown in SEQID No.6.

1.14 Preparation of competent Agrobacterium (aseptic operation)

(1) Get the Agrobacterium GV3101 strain and inoculate it in 10ml YEP liquid medium, and culture it on a shaking table at 28°C overnight;

(2) Inoculate in 50ml YEP liquid medium at a ratio of 1:50, culture with shaking at 28°C for 3-4 hours, until the _OD600 value is 0.4-0.6;

(3) 4°C, 4,200rpm, centrifuge for 10min, collect the bacteria;

(4) Discard the supernatant, add 10ml of pre-cooled 0.15mol/L NaCl suspension bacteria;

(5) Repeat step 3;

(6) Discard the supernatant, add 2ml of pre-cooled 20mmol/L CaCl ₂ to suspend the bacteria, divide into 1.5ml centrifuge tubes, use now or add 7% DMSO to the final volume, quick-freeze with liquid nitrogen, and store at -80°C for later use .

1.15 Plasmid transformation of Agrobacterium (sterile operation)

(1) Add 10 μl of plasmid DNA to 50 μl of competent cells, flick the centrifuge tube to mix well, and ice-bath for 30 minutes;

(2) Liquid nitrogen quick freezing for 1 min; then 37 ° C water bath for 5 min, immediately ice bath for 2-3 min;

(3) Add 1ml of YEP medium and incubate at 28°C for 2-4h;

(4) room temperature, 4,000rpm, centrifuge for 3min, collect the bacteria;

(5) Spread the bacteria on the YEP culture plate containing the corresponding antibiotics, and incubate them upside down at 28°C for 48 hours.

1.16 PCR verification of transformed Agrobacterium

The reaction system is as follows (20μl system):

The amplification conditions are as follows:

After the reaction, the reaction solution was detected by electrophoresis on 0.8% TAE agarose gel.

Example 2. Functional verification of expressing Williams 82NAC membrane-bound transcription factor gene GmNTL1 in Arabidopsis

2.1 Transformation of Arabidopsis thaliana by flower infection method

(1) When Arabidopsis thaliana (Col-0 wild type) grows to 1 cm of bolting, the top is subtracted to induce the formation of lateral inflorescences;

(2) One day before transformation, take 1ml of activated Agrobacterium GV3101 containing the expression vector plasmid and add it to 40ml of YEP medium containing corresponding antibiotics and 50μg/ml rifampicin, and culture with shaking at 28°C until the _OD600 is about 1.0 -1.2;

(3) Centrifuge at 4,200 rpm for 10 minutes at room temperature, collect the cells, and resuspend the cells with the liquid (5% sucrose, 0.05% Silwet L-77) to make the OD ₆₀₀ about 0.8;

(4) Use a pipette to drop the Agrobacterium on the inflorescences for dipping, and after all the inflorescences are infected, put the Arabidopsis thaliana into a vacuum desiccator to evacuate for 1 min;

(5) Cover the inflorescence with a fresh-keeping bag, and cultivate in the dark at 20-22°C for one day, cut off the top to expose the inflorescence, remove the fresh-keeping bag after another day of cultivation, and cultivate until the seeds mature.

2.2 Surface disinfection of Arabidopsis seeds

Put an appropriate amount of Arabidopsis thaliana neutrons to be sterilized into a 1.5ml centrifuge tube, add 1ml of 75% ethanol (containing 0.03% volume ratio of TritonX-100) to sterilize by shaking for 1min, and then use 70% ethanol to sterilize by shaking for 1min ( Twice), and finally use a suction tip to suck the seeds onto sterile filter paper to dry, and then use a sterile toothpick to spot them into the culture medium.

2.3 Screening of transgenic plants

The harvested T0 generation seeds were surface sterilized, and then evenly spread on 1/2 MS plates (containing the corresponding antibiotic Baste). After 3 days of vernalization, they were moved into an artificial climate chamber for growth. About 10 days after germination, the plants with dark green cotyledons are transgenic plants, while the plants with light green or even yellowed cotyledons are non-transgenic plants. Transfer the transgenic plants into the soil and grow until the T1 generation transgenic seeds are harvested. The T1 generation plants receive seeds individually, and the seeds harvested from each plant continue to be screened, and the positive plants with a segregation ratio of offspring of 3:1 (positive:negative) are transplanted. After growing to harvest T2 generation transgenic seeds, after single plant harvesting, the seeds harvested by each plant can be screened to obtain pure line T2 generation transgenic seeds, as shown in Figure 5.

2.4 Extraction of plant RNA

(1) Put the plant material into a mortar, and use liquid nitrogen to grind it into powder (directly used in the following experiments or frozen in a -80°C ultra-low temperature refrigerator for later use);

(2) After the liquid nitrogen volatilizes, immediately transfer 100-200mg of plant powder into a 1.5ml centrifuge tube, then quickly add 1ml of Trizol extract, vortex to fully dissolve the sample into the extract, and place it at room temperature for 5 minutes;

(3) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.9ml of the supernatant to a new 1.5ml centrifuge tube, then add 0.2ml of chloroform, vigorously shake and mix for 15sec, and place at room temperature for 2-5min;

(4) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.4ml of supernatant to a new 1.5ml centrifuge tube, add 0.4ml of isopropanol, turn up and down 15 times to mix the solution, and place at room temperature for 15mim;

(5) Centrifuge at 12,000rpm at 4°C for 10min, discard the supernatant, wash the pellet twice with 1ml of 75% ethanol, centrifuge at 8,000rpm at 4°C for 5min;

(6) Discard the supernatant, open the lid and dry the RNA on a clean bench for about 2-5 minutes, add 40 μl RNase-Free water, and fully dissolve the RNA at 60°C for 10 minutes;

(7) Measure the OD value and concentration of the RNA sample with a UV spectrophotometer, A ₂₆₀ /A ₂₈₀ is preferably 1.7-2.0; the quality detected by agarose gel electrophoresis.

2.5 Reverse transcription of RNA

(1) Add the following substances (40μl reaction system) to the centrifuge tube in sequence:

(2) After mixing gently, denature at 65°C for 5 minutes, immediately insert on ice, and ice bath for at least 1 minute;

(3) Add the following substances in turn to the centrifuge tube

(4) After mixing gently, place in a constant temperature water bath at 42°C for 1 hour, denature at 65°C for 10 minutes, and store at -20°C for later use.

2.6 RealTime-PCR detection of transgenic plants

Screening of transgenic positive plants by PCR method: the cDNA of the resistant plants was detected by PCR with RT-PCR primers of GmNTL1 gene.

GmNTL1 gene RT-PCR primer sequence:

GmNTL1RT-F: 5'-GCCGTTAGGGTTCCGTTTC-3'

GmNTL1RT-R: 5'-AAGGCTCCCATTTGCAGACA-3'

The reaction of common Taq enzyme for RealTime-PCR amplification is as follows (15μl system):

The amplification conditions are as follows:

Note: GmTUB was selected as the internal standard gene.

The results are shown in Figure 6.

2.7 Mannitol and NaCl treatment of Arabidopsis

(1) Surface disinfection of Arabidopsis seeds (same as 2.2).

(2) Mannitol and NaCl treatment

Spread the sterilized Col-0, GmNTL1-1, and GmNTL1-2 seeds evenly on 1/2 MS plate (containing the corresponding antibiotic Baste). After 3 days of vernalization, they were moved into an artificial climate chamber for growth. After germination for about 4 days and the root length reaching about 1 cm, the Arabidopsis seedlings were moved to 1/3 of the concentration of mannitol (250, 300, 350, 400 mmol/L) or NaCl (50, 100, 150, 200 mmol/L). 2MS medium plate. When the seedlings grow to 11 days, observe the growth of the seedlings.

The results are shown in Figure 7.

Example 3, Functional Verification of Expressing Williams 82NAC Transcription Factor Gene GmNTL1 in Soybean

3.1 Transformation of Soybean by Vacuum Infiltration-Assisted Transformation of Germinated Soybean Embryos

(1) Seed disinfection and pre-cultivation treatment

Soybean seeds were sterilized with 70% ethanol for 5 minutes, and then sterilized with 0.1% HgCl ₂ for 10 minutes after removing the ethanol, rinsed with sterile water 5-6 times, and soaked in sterile water at 25°C-28°C for 12 hours.

Soybeans whose embryos swelled and germinated after soaking were put into the pre-cultivation medium, and cultured in light at 28°C for 1 day, wherein the formula of the pre-cultivation medium was: MS+3.0mg/L 6-BA(6-benzylaminoadenine)+ 20g/L sucrose+7g/L agar, pH5.8.

(2) After the seed embryo swells and germinates, it is cut to expose or damage the germinated embryo part

When the whole pre-cultivated soybean embryo is enlarged and the radicle grows to 0.5±0.1mm without breaking through the seed coat, peel off the seed coat, cut off the radicle, keep the embryo, hypocotyl and 1/2 of the cotyledon, and use a dissecting needle Puncture the needle at the tip of the germ so that the eye of the needle covers the tip of the germ.

(3) Vacuum infiltration assisted method to infect and germinate embryos with Agrobacterium containing the target gene

Streak the Agrobacterium with plant expression vector stored at 4°C on the YEP solid medium supplemented with 50mg/L rifampicin, pick a single colony after 3 days of dark culture at 28°C, and inoculate it with 50mg/L rifampicin Flat YEP liquid medium, cultured in the dark at 28°C, 200r/min shaking for 24 hours, then retransplanted, and cultured under the same conditions for 16 hours. Place the bacterial solution in a sterilized centrifuge tube, centrifuge at 5,000 rpm for 5 minutes to collect the bacterial cells, resuspend the bacterial cells with the infection solution, adjust the OD ₆₀₀ value to 0.6, and add 30 mg/L acetosyringone for later use. The formula of the infection solution is: 0.1MS in large amount + 0.1MS in trace amount + B5 vitamin + 0.5MES + 1% glucose + 2% sucrose, pH 5.4.

Soybeans in step (2) were immersed in the Agrobacterium bacterial liquid, vacuumed and maintained at a pressure of 0.05 MPa for 5-8 minutes, cultured with shaking at 28°C and 200 r/min in the dark, and infected for 15-20 minutes.

(4) Co-cultivation

The soybeans in step (3) are taken out, the excess bacterial liquid is sucked off with sterile filter paper, the cut side is placed on the co-cultivation medium, and dark culture is carried out at 25°C-28°C for 4 days. Wherein the co-cultivation medium formula is: MS+30mg/L acetosyringone+20g/L sucrose+7g/L agar, pH5.8.

(5) Bulk bud regeneration, screening and seedling rooting, screening

The co-cultivated soybeans were washed 4 times with sterile water, then blotted dry with sterile filter paper, transferred to cluster bud induction medium, and replaced with new soybeans every two weeks at 25°C and under the condition of 14h of daily light. culture medium, continuously cultured for 20±2d, and clustered shoots were differentiated. Wherein the cluster bud induction screening medium formula is: MS+3.0mg/L 6-BA (6-benzylaminoadenine)+0.2mg/L IAA (indole acetic acid)+0.125μL/mL Baste+200mg/L Cefotaxime sodium+20g/L sucrose+7g/L agar, pH5.8.

Select clustered shoots that grow to 1-2 cm after screening, cut off the small shoots individually, transfer them to the rooting screening medium, and culture them continuously for 14 days at 25°C and under the condition of 14±1 hours of light per day. Wherein said rooting screening medium formula is: MS+0.4mg/LIBA (etodobutyric acid)+0.125 μ L/mL Baste+200mg/L cefotaxime sodium+20g/L sucrose+7g/L agar, pH5.8 .

(6) Strong seedlings and transplanting

Select seedlings with well-grown roots and transfer them to strong seedling medium, and cultivate them for 7-10 days at 25°C and 14 hours of light per day. When the seedlings survive through screening and the root system grows well, take them out without hurting the roots, remove the remaining medium and move them into the soil, and cover the flowerpot with a film to increase the humidity. Wherein said strong seedling medium formula is: MS+2.0mg/L KT (kinetin)+0.4mg/L NAA (alpha-naphthalene acetic acid)+20g/L sucrose+7g/L agar, pH5.8, see figure 8.

3.2 CTAB method to extract genomic DNA from transgenic plants

Weigh 0.5g of regenerated plant leaves, cut them into pieces and grind them with liquid nitrogen, quickly transfer the powder to a 1.5ml centrifuge tube, then add 700μl of 2xCTAB extraction buffer preheated to 65°C and 0.1% mercaptoethanol, gently Gently invert the centrifuge tube to mix, then place in a water bath at 65°C for 2 hours, and mix by inverting from time to time. After the mixture was cooled to room temperature, an equal volume of phenol:atmoform:isoamyl alcohol (25:24:1) was added, and centrifuged at 10,000 g for 10 min at 4°C. Transfer the aqueous phase to a clean centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge at 10,000 g for 10 min at 4°C. Transfer the aqueous phase to a clean centrifuge tube, add twice the volume of absolute ethanol, mix gently, and place at -20°C for 30 minutes to precipitate DNA. Centrifuge at 10,000g for 10min at 4°C, discard the liquid, wash twice with 70% ethanol, dry the DNA at room temperature, add 100ul TE solution to dissolve, and bathe in water at 37°C for 30min. Add 200 μl of absolute ethanol, mix gently, and place at -20°C for 30 minutes to precipitate DNA. Centrifuge at 10,000 g for 10 min at 4°C, discard the liquid, and wash twice with 70% ethanol. After drying the DNA at room temperature, add 40 μl sterile water to dissolve the DNA, and store at 4°C until use.

3.3 Detection of transgenic plants by PCR and Southern blot

3.3.1 PCR detection of positive transgenic plants

The total DNA of the resistant plants was detected by PCR using 35S promoter primers, GmNTL1 gene primers and sequence primers connected with GmNTL1 gene fragments and plant expression vector fragments, respectively.

35S fragment promoter primer sequence:

35S-F: 5'-GCAGAGGCATCTTCAACG-3'

35S-R: 5'-GACGATCTACCCGAGCAA-3'

GmNTL1 gene primer sequence:

Gm NTL1-F: 5'-AAAAAGCAGGCTCGATGGGTGCCGTCGTC-3'

Gm NTL1-R: 5'-AGAAAGCTGGGTTTTAAGATCTGACATATGCCCA-3'

35S promoter F+GmNTL1 gene R primer sequence:

35S-F: 5'-GCAGAGGCATCTTCAACG-3'

Gm NTL1-R: 5'-AGAAAGCTGGGTTTTAAGATCTGACATATGCCCA-3'

The reaction system is as follows (20μl system):

The amplification conditions are as follows:

After the reaction, the reaction solution was detected by 0.8% TAE agarose gel electrophoresis, as shown in FIG. 9 .

3.3.2 Southern blot hybridization verification

Three positive plants (GmNTL1OX-1, GmNTL1OX-2, GmNTL1OX-3) were selected for Southern blot hybridization verification. Proceed as follows:

(1) CTAB method to extract DNA from positive plants and wild-type plants

(2) DNA of EcoRI digested positive plants and wild-type plants, electrophoresis

(3) transfer film

① Alkaline denaturation: Immerse the gel in several times the volume of denaturing solution for 30 minutes at room temperature. Denaturing solution: 0.5mol/L NaOH; 1.5mol/L NaCl.

②Neutralization: transfer the gel to the neutralizing solution for 15 minutes. Neutralizing solution: 1mol/L Tris-HCl (pH 7.4); 1.5mol/L NaCl;

③Transfer: Cut the NC membrane or nylon membrane according to the size of the gel and cut off a corner as a mark. After soaking in water, immerse in the transfer solution for 5 minutes. Cut a strip of Whatman 3mm filter paper slightly wider than the membrane as a salt bridge, then cut 3-5 pieces of filter paper according to the size of the gel and a lot of paper towels for use. to transfer. (The transfer process generally takes 8-24 hours, and the wet paper towels should be replaced every few hours. Use 20×SSC for the transfer solution. Note that there should be no air bubbles between the membrane and the glue. During the entire operation, prevent the membrane from being contaminated with other pollutants. Material) transfer solution (20×SSC): 175.3g NaCl; 82.2g trisodium citrate, adjust the pH to 7.0 with NaOH, add ddH2O to 1,000ml.

④ After the transfer, take out the NC membrane, immerse in 6×SSC solution for several minutes, wash off the gel particles contaminated on the membrane, place it between two filter papers, bake at 80°C for 2 hours, and then sandwich the NC membrane between the two layers of filter paper, Store in a dry place.

(4) Label GmNTL1 according to the instructions of Roche DIG High Prime DNA Labeling and Detection Starter Kit II, and perform hybridization and detection.

It was detected that the genomes of the selected GmNTL1 transgenic plants (GmNTL1OX-1, GmNTL1OX-2, GmNTL1OX-3) were all integrated with the target gene, as shown in FIG. 10 .

3.4 Salt tolerance analysis of transgenic soybean plants

Transgenic soybean seeds of the T1 generation and control seeds were soaked and accelerated germination, and then transplanted into plastic pots (1/2 Hogland liquid medium) respectively, and allowed to grow for 2 weeks at a room temperature of 25°C until they reached the stage of two leaves and one heart. Watered with 1/2 Hogland liquid medium containing NaCl50rnmol/L, in order to avoid the salt shock effect, the concentration was increased by 25rnmol/L every day, until the predetermined concentration of treatment was 250rnmol/L, and the phenotypes of transgenic plants and wild-type plants were observed . See Figure 11.

1/2 Hogland liquid medium formula:

Claims (5)

1. in a Semen sojae atricolor WILLIAMS-DARLING Ton 82, NAC film combines transcription factor gene, it is characterised in that: described unnamed gene is Semen sojae atricolor WILLIAMS-DARLING Ton 82NAC film combines transcription factor gene GmNTL1, the nucleotide sequence of this gene cDNA such as SEQ ID No.1 institute Show.

2. the plant expression load combining transcription factor gene containing NAC film in Semen sojae atricolor WILLIAMS-DARLING Ton 82 described in claim 1 Body, it is characterised in that: described plant expression vector named expression vector pK2GW7::GmNTL1, this expression vector cloning region Nucleotide sequence is as shown in SEQ ID No.5.

3. the plant expression load combining transcription factor gene containing NAC film in Semen sojae atricolor WILLIAMS-DARLING Ton 82 described in claim 1 Body, it is characterised in that: described plant expression vector named expression vector pB2GW7::GmNTL1, this expression vector cloning region Nucleotide sequence is as shown in SEQ ID No.6.

4. in Semen sojae atricolor WILLIAMS-DARLING Ton 82 described in claim 1, NAC film combines transcription factor gene in raising plant salt stress tolerance Application in property.

Apply the most as claimed in claim 4, it is characterised in that: described plant is Semen sojae atricolor or arabidopsis.