CN102964437A - Soybean nuclear factor protein and encoding genes of protein and applications of protein and encoding genes - Google Patents

Abstract

The invention discloses a soybean nuclear factor protein, its encoding gene and application. The protein provided by the present invention, named GmNFYB1, is derived from soybean (Glycine max (L.) Merrill.) Dongnong 50, and is the protein of the following 1) or 2): 1) the amino acid residue shown in sequence 2 in the sequence listing 2) A protein derived from 1) in which the amino acid sequence of Sequence 2 is substituted and/or deleted and/or added by one or several amino acid residues and is related to plant stress tolerance. Experiments of the present invention prove that: under drought stress, the transgenic plants transformed into soybean GmNFYB1 (soybean nuclear factor binding protein) have longer roots, larger proline content and higher yield.

Description

A kind of soybean nuclear factor protein and its coding gene and application

technical field

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

Background technique

Abiotic stresses such as drought and salinity seriously affect the yield and quality of crops. It is an effective way to improve the stress resistance of crops by means of genetic engineering. The elucidation of the molecular mechanism of resistance to abiotic stress is the prerequisite for the application of genetic engineering improvement. Transcription factors play a very important role in the signal transduction of abiotic stress. Soybean, as an important food crop, is widely planted all over the world. However, compared with other plants such as Arabidopsis, rice, maize and wheat, there is little information about NFYB transcription factors in soybean. Therefore, it has become an effective breeding method to use bioengineering technology to change the stress resistance response of plants, and then to breed new varieties with excellent traits.

Contents of the invention

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

The protein provided by the present invention, named GmNFYB1, is derived from soybean (Glycine max (L.) Merrill.) Dongnong 50, which is the protein of 1) or 2) as follows:

1) A protein composed of the amino acid residues shown in Sequence 2 in the Sequence Listing;

2) A protein derived from 1) in which the amino acid sequence of Sequence 2 is substituted and/or deleted and/or added by one or several amino acid residues and is related to plant stress tolerance.

The above sequence 2 consists of 174 amino acid residues, and the substitution and/or deletion and/or addition of one or several amino acid residues is no more than 10 amino acid residues.

The coding genes of the above proteins are also within the protection scope of the present invention.

The coding gene is any one of the following 1)-5) DNA molecules:

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

2) The DNA molecule shown in the 22nd-665th nucleotides from the 5' end of sequence 1 in the sequence listing;

3) The DNA molecule shown in the 93rd-617th nucleotides from the 5' end of Sequence 1 in the sequence listing;

4) A DNA molecule that hybridizes with the DNA molecule defined in 1) or 2) or 3) under stringent conditions and is associated with a plant stress tolerance-related protein coding gene;

5) A DNA molecule that has at least 90% homology with the DNA sequence defined in 1) or 2) or 3) and is related to a gene encoding a protein related to plant stress tolerance.

The above sequence 1 consists of 710 nucleotides, and the coding region is the 93rd-617th nucleotides from the 5' end of the sequence 1.

The stringent conditions can also be hybridized at 65°C in a solution of 6×SSC, 0.5% SDS, and then the membrane is washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Recombinant vectors, transgenic cell lines, recombinant bacteria or expression cassettes containing the above coding genes are also within the protection scope of the present invention.

The above-mentioned recombinant vector is a recombinant vector obtained by inserting the above-mentioned coding gene into an expression vector, specifically inserting the DNA molecule shown in the 22nd-665th nucleotide from the 5' end of the sequence 1 in the above sequence listing into the XbaI and XbaI of the pCAMBIA-pBI121 vector. The vector obtained between the Sac I recognition sites.

The pCAMBIA-pBI121 vector was prepared according to the following method: the vectors pCAMBIA3301 and pBI121 were double-digested with restriction endonucleases HindIII and EcoRI respectively, and the pCAMBIA3301 vector large fragment of 11257 bp and the pBI121 vector small fragment of 3032 bp after digestion were recovered. The fragments were connected to construct the intermediate expression vector pCAMBIA-pBI121.

The recombinant vector contains a promoter and a gene encoding GmNFYB1 protein connected downstream of the promoter. The above-mentioned promoter can be any of the following promoters: cauliflower mosaic virus 35S promoter and Ubiquitin promoter, preferably cauliflower mosaic virus 35S promoter.

The above-mentioned recombinant bacteria are recombinant bacteria obtained by introducing the above-mentioned recombinant vectors into host bacteria.

The primer pair for amplifying the full length or any fragment of the above-mentioned coding gene is also within the protection scope of the present invention, and the primer pair is specifically as follows: one primer sequence is shown in sequence 3 in the sequence listing, and the other primer sequence is shown in the sequence listing Sequence 4 is shown.

The application of the above-mentioned proteins, above-mentioned coding genes, above-mentioned recombinant vectors, transgenic cell lines, recombinant bacteria or expression cassettes in regulating plant stress tolerance and/or plant breeding is also within the protection scope of the present invention.

In the above application, the regulation of plant stress tolerance is to improve plant stress tolerance, and the stress tolerance is specifically drought tolerance; the above-mentioned improvement of plant drought tolerance is carried out under drought stress, specifically by increasing root length and increasing germination efficiency and/or increase the proline content; the plant is specifically a dicotyledon or a monocotyledon, and the dicotyledon is further specifically Arabidopsis or soybean;

The application of the above-mentioned protein, the above-mentioned coding gene, the above-mentioned recombinant vector, transgenic cell line, recombinant bacteria or expression cassette in regulating plant yield is also within the protection scope of the present invention, wherein regulating plant yield is to increase plant yield, and the plant is specifically Gemini Leafy plants or monocotyledonous plants, the dicotyledonous plants are further specifically soybeans; the above-mentioned increase in plant yield is carried out in arid areas, specifically in Xinjiang, and other areas with less rain and dry climate conditions are also possible.

Another object of the present invention is to provide a method for breeding transgenic plants.

The method provided by the present invention includes the following steps: in order to introduce the above coding gene into the target plant to obtain a transgenic plant, the stress tolerance of the transgenic plant is higher than that of the target plant.

In the above method, the stress tolerance is drought tolerance; the target plant is specifically a dicotyledonous plant or a monocotyledonous plant, and the specific cotyledonous plant is Arabidopsis thaliana or soybean.

The third object of the present invention is to provide a method for cultivating transgenic plants.

The method provided by the present invention comprises the following steps: to obtain a transgenic plant obtained by introducing the above-mentioned coding gene into a target plant, the yield of the transgenic plant is higher than that of the target plant;

The yield is reflected by increasing plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant and/or 100-grain weight; the target plant is specifically a dicotyledonous plant or a monocotyledonous plant The plant, said dicot is further particularly soybean.

Experiments of the present invention prove that: the present invention has discovered a new gene GmNFYB1, and it is transferred into Arabidopsis or soybean to obtain transgenic GmNFYB1 Arabidopsis or transgenic GNFYB1 soybean; the root length of transgenic GmNFYB1 Arabidopsis after drought resistance treatment increases, The content of proline increased, and the germination rate of seeds increased significantly; the transgenic GmNFYB1 soybean grew as wild-type soybean after drought treatment, and the yield in arid area was higher than that of wild-type soybean; it can be seen that the gene is related to plant drought resistance and can be applied for breeding and improving new varieties of drought-resistant plants.

Description of drawings

Figure 1 shows the GUS staining results of transgenic plants and wild-type plant controls

Figure 2 is a comparison of 4-week-old seedlings of transgenic plants expressing GmNFYB1 and wild-type plant controls 4-week-old seedlings under drought stress

Figure 3 is the change of mannitol on the average root length of transgenic plants and wild-type plants

Figure 4 is a picture of the root length changes of transgenic plants and wild-type plant controls under different mannitol concentrations

Fig. 5 is the change of root length of different strains of transgenic plants and wild-type plant control under 300mM Minnitol mannitol concentration treatment

Fig. 6 is the change of germination rate of seeds of transgenic plants and wild-type plant controls under different ABA concentration treatments

Figure 7 is a comparison of the proline content of transgenic plants (TG) and wild-type plants (CK) after being transferred to soil for drought stress

Figure 8 shows the root length change of transgenic (GmNFYB1) Arabidopsis plants under sodium chloride (NaCl) stress treatment

Figure 9 shows the survival rate changes of GmNFYB1 Arabidopsis plants under different sodium chloride (NaCl) concentration stress treatments

Figure 10 shows the homology comparison of GmNFYB1 and AtNFYB1 genes

Figure 11 is the relative expression level of soybean GmNFYB1 gene treated with PEG

Figure 12 is the genetic transformation of soybean

Figure 13 is the transient expression of gus gene

Figure 14 is the PCR detection of the Bar gene of transgenic plants

Figure 15 is the PCR detection of the GUS gene of transgenic plants

Figure 16 is the PCR detection of the GmNFYB1 gene of the transformant

Figure 17 is soybean leaf genome DNA

Figure 18 shows restriction endonuclease digestion of genomic DNA

Figure 19 is the Southern detection of the GmNFYB1 gene of the transformant

Figure 20 is the phenotype result of transgenic GmNFYB1 soybean under drought stress

Figure 21 shows the growth phenotype results of transgenic GmNFYB1 soybeans in arid regions

Figure 22 shows the statistical results of the growth phenotype of GmNFYB1 soybeans in arid regions

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.

Embodiment 1, the acquisition of GmNFYB1

Soybean (Glycine max) extracted with Trizol reagent (Dongnong 50, Wu Tianlong, Yang Qingkai, Ma Zhanfeng, Wu Zongpu, Zhao Shuwen, Gao Fenglan, Li Wenbin, Zhang Guodong, Yang Qi, Meng Qingxi, Wang Jinling. Dongnong Xiaodoudou No. 1 new soybean variety selection Report, [J]. Journal of Northeast Agricultural University, 1994, (25) 1:104; the public can obtain it from Northeast Agricultural University, hereinafter referred to as wild soybean). Total leaf RNA was reverse-transcribed to synthesize the first strand of cDNA as a template, with sense primer: GG TCTAGA CAAAGGTGCATTGGTGGT (the underlined part is the XbaⅠ restriction site, sequence 3), antisense primer: AT GAGCTC CGTACAAGCATTCAAGGGA (the underlined part is SacⅠ Restriction site, sequence 4), perform PCR reaction, PCR condition is 94°C for 5min; 35 cycles: 94°C for 30s, 60°C for 30s, 72°C for 2.5min; 72°C for 5min. The PCR product was detected by electrophoresis on a 0.8% agarose gel, and the result showed that the PCR product was 522bp. After sequencing, the PCR product has the 22nd-665th nucleotide sequence from the 5' end of the sequence 1 in the sequence listing. The coding region of the gene of the PCR product is sequence 1 in the sequence table from the 93rd to 617th nucleotides at the 5' end. The gene is named GmNFYB1, and the protein encoded by the gene is named GmNFYB1. The amino acid sequence of the protein is sequence Sequence 2 in the list.

Example 2, Obtaining and Functional Research of Transgenic GmNFYB1 Arabidopsis

1. Construction of plant expression vector pCAMBIA-GmNFYB1

The vector pCAMBIA3301 vector (sold by CAMBIA Company of Australia) and pBI121 (purchased from CLONETECH Company) were double-digested with restriction endonucleases HindIII and EcoRI respectively, and the large fragment of pCAMBIA-D3 vector of 11257bp after digestion was recovered and digested The 3032bp small fragment of the pBI121 vector was connected to construct the expression vector pCAMBIA-pBI121.

The PCR product obtained in Example 1 was digested with XbaI and SacI, and the obtained fragment was connected with the pCAMBIA-pBI121 vector fragment that had undergone the same digestion, and the ligated product was transformed into Escherichia coli to obtain a transformant, and the plasmid of the transformant was extracted and sent to Sequencing showed that the plasmid was a recombinant vector obtained by inserting the sequence in the sequence table from the 22nd to 665th nucleotides at the 5' end between the XbaI and SacI recognition sites of pCAMBIA-pBI121, and the recombinant vector was named pCAMBIA- GmNFYB1, the promoter of the vector is CaMV35S.

2. Cultivation of transgenic GmNFYB1 Arabidopsis

1. Obtaining transgenic GmNFYB1 Arabidopsis

The plant expression vector pCAMBIA-GmNFYB1 obtained above was transformed into Agrobacterium tumefaciens LBA4404 (Invitrogen Company, product number 18313015) by freeze-thaw method, and the obtained transformant was extracted with a plasmid and sent for sequencing. The result was that the plasmid was pCAMBIA-GmNFYB1, The recombinant bacteria containing this plasmid was named LBA4404/pCAMBIA-GmNFYB1.

Infect wild-type Arabidopsis thaliana with recombinant strain LBA4404/pCAMBIA-GmNFYB1 by Agrobacterium-mediated method

(Columbia ecotype) (hereinafter referred to as wild-type Arabidopsis, Hao Lin, Xu Xin, Cao Jun. A study on the response of an Arabidopsis mutant to high _CO2 concentration, [J]. Journal of Applied Ecology, 2003, 14( 12): 2359-236 (available to the public from Northeast Agricultural University), and 65 T0-transformed Arabidopsis thaliana were obtained.

1) Preparation of Agrobacterium liquid for transformation

Co-transformation of Agrobacterium: inoculate LBA4404/pCAMBIA-GmNFYB1 in a test tube with YEP culture solution at 10ul: 10ml. Shake overnight at 28°C and 220rpm for about 30 hours, then transfer the shaken live bacteria (1:400) and 750ul bacteria liquid to 300ml YEP (RifR, StrR, KanR) medium. The concentration of Rif (rifampicin) in the medium is 25mg/L, the concentration of Str (streptomycin) in the medium is 50mg/L, and the concentration of Kan (kanamycin) in the medium is 50mg/L L. Incubate at 28°C, 220rpm for about 14 hours, measure the OD value, use YEP+Rif+StrR as a blank control, when the bacterial solution reaches OD600 within 1.5-3.0, collect the bacterial cells in a 50ml centrifuge tube (sterilized), 4 Centrifuge at 4000g for 10min. Dilute it with 5% sucrose (containing 0.02% Silwet) until the OD600 is about 0.8-1.0. Select 2ml of the prepared solution above, fully break up the bacteria at the bottom of the tube, then dissolve the mixed bacteria into 50ml of the solution, and then add Silwet (100%) 120ul to a final concentration of 0.02%.

2) Watering: Water the seedlings of the wild-type Arabidopsis Columbia ecotype that need to be transformed the day before the transformation.

3) Transformation: Soak the inflorescences of wild-type Arabidopsis plants in the prepared bacterial suspension solution for 30 seconds, and grow them under low light (dark culture) to obtain seedlings of T0-transformed Arabidopsis thaliana.

4) After marking, put 65 seedlings of T0 transgenic Arabidopsis into GmNFYB1 flat in the box, seal the upper cover with parafilm, and cultivate in the dark for 24hrs. After 1 day, stand up the plants for normal cultivation and water them.

A total of 65 T0 transgenic Arabidopsis plants were obtained.

2. Identification of transgenic GmNFYB1 Arabidopsis

1) Molecular identification

Genomic DNA of leaves of 65 T0 transgenic Arabidopsis plants was extracted as a template, and PCR identification was performed with sense primers: GG TCTAGA CAAAGGTGCATTGGTGGT and antisense primers: AT GAGCTC CGTACAAGCATTCAAGGGA. The results showed that the 522bp fragment was positive, and a total of 24 positive T0 plants were transformed into GmNFYB1 Arabidopsis plants, and the transformation rate was 36.92%.

Using the same method, the empty vector pCAMBIA--pBI121 was transformed into wild-type Arabidopsis to obtain the empty vector Arabidopsis. After identification by the above method, there was no amplified fragment in the empty vector Arabidopsis thaliana.

2) GUS staining identification

The positive T0 transgenic GmNFYB1 Arabidopsis plants of three lines (L53, L57, L2) numbered 53, 57 and 2 were selected for GUS staining analysis, and the seeds of transgenic GmNFYB1 Arabidopsis were sterilized and sown on MS medium Grow, treat for 3 days at 4°C, and then transfer to a growth chamber. The 16-day-old shoots were used for the treatment to induce expression. Through aseptic operation, the plants to be tested were carefully taken out, transferred to the GUS staining solution, and the volume ratio of the material to the staining solution was less than 1:5, and incubated overnight at 37°C. After GUS penetrates into the material, turn the material into a transparent solution to remove chlorophyll, and place it at room temperature for 5 hours until the green color is completely removed. Finally, the transparent treated plant material was observed under a dissecting microscope, and the part with GUS expression showed a stable and insoluble blue color in the transparent liquid. The image was saved by taking a photo, and it was properly processed by Adobe Photoshop. Wild-type Arabidopsis and empty vector Arabidopsis were used as controls.

The results are shown in Figure 1, where A: GUS staining results of the germinated Arabidopsis thaliana seeds that were positively transduced by T0 generation; B: seedlings and seeds of Arabidopsis thaliana that were positively transduced by T0 generation; C: positively transduced by T0 generation by GmNFYB1 Arabidopsis Seedling; D: Positive T0 generation transfection of rosette leaves of GmNFYB1 Arabidopsis plants; E: Positive T0 generation transformation of leaves of GmNFYB1 Arabidopsis plants; F: Positive T0 generation transduction of GmNFYB1 Arabidopsis rosette leaves and roots; G: Positive T0 generation transduction of leaves and roots of GmNFYB1 Arabidopsis plants Inflorescences of GmNFYB1 Arabidopsis plants. The leaves of the three lines (L53, L57, L2) numbered 53, 57 and 2 (L53, L57, L2) of the positive T0-transformed GmNFYB1 Arabidopsis plants all appeared blue, but the wild-type Arabidopsis had no blue color. There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

3) PT screening concentration

(1) Sow wild-type Arabidopsis seeds on MS medium containing 0mg/L, 1mg/L, 3mg/L, 5mg/L, 7mg/L, 10mg/LPPT, respectively, in the culture room (temperature 22℃±2℃, light intensity 0.3～0.4mmo·lm-2·s-1) let it germinate under the conditions of growth light/dark cycle of 16h/8h, and determine the effective screening of overexpression positive seedlings according to the germination results Concentration, the result is that the PPT of 5mg/mL is the effective screening concentration.

(2) Screening transformants

The T0 transgenic GmNFYB1 Arabidopsis seeds numbered 53, 57 and 2 were treated at 4°C for 3 days at a low temperature, then 10% sodium hypochlorite was added, shaken for 8-10 minutes, and washed twice with sterile water. Then add 75% ethanol, shake well, 30s. Wash 5 times with sterile water, and then evenly sow in the medium containing 5mg/mLPPT. Six days after moving into the growth chamber, the number of healthy green seedlings grown was observed and counted. The normal green seedlings were taken out from the culture medium and cultured in soil: vermiculite (3:1). After a life cycle of about 50 days, T1 generation GmNFYB1 Arabidopsis plants were screened.

The above experiments showed that the exogenous gene GmNFYB1 had been integrated into the genomes of the three lines (L53, L57, L2) numbered 53, 57 and 2 (L53, L57, L2), and was effectively expressed at the transcriptional level.

Three lines (L53, L57, L2) numbered 53, 57, and 2 were positively T0-transferred to GmNFYB1 Arabidopsis plants, and the plants grown from the seeds were called T1 generation, and the T1 generation was transferred to The seeds produced by the GmNFYB1 Arabidopsis plants and the plants grown from the seeds are called the T2 generation.

3. Functional analysis of the T2 generation of transgenic GmNFYB1 Arabidopsis

A: Drought treatment:

1. Phenotype observation of transgenic Arabidopsis plants

Sterilize the seeds of the T2 generation of the L53 strain No. 53 obtained above and transfer GmNFYB1 Arabidopsis thaliana to sow on MS medium, 16h light/8h dark (long daylight), grow at 25°C for 10 days, and then transfer to In the soil, 30 T2 transgenic GmNFYB1 Arabidopsis seedlings were obtained. Drought resistance treatment: no watering for 15 days. The wild-type Arabidopsis and empty vector Arabidopsis were used as controls, with 30 strains for each line, and the experiment was repeated three times.

The 4-week-old seedlings (GmNFYB1) and wild-type Arabidopsis thaliana 4-week-old seedlings (GmNFYB1) of the three lines (L53, L57, L2) of the T2 generation after drought treatment were taken (Wild type) photos were taken, and the results are shown in Figure 2. It can be seen from Figure 2 that compared with wild type Arabidopsis, the roots of Arabidopsis transgenic to GmNFYB1 in the T2 generation of the L53 line numbered 53 became longer.

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

2. Changes in root length of transgenic GmNFYB1 Arabidopsis plants under mannitol (Minnitol) stress treatment

Sterilize the seeds of the T2 generation of the L53 strain with the number 53 obtained above and transfer them to GmNFYB1 Arabidopsis thaliana, sow them on MS medium, grow them in 16h light/8h dark conditions, and grow at 25°C. After 1 week, 50 The T2 generation of the L53 line with the strain number 53 was transformed into GmNFYB1 Arabidopsis thaliana. Then the Arabidopsis seedlings of the T2 generation of the L53 line numbered 53 were transferred to the MS medium containing 0mM Mannitol (Mannitol), 100mM Mannitol, and 200mM Mannitol respectively, and the root length was measured after 1 week , taking wild-type Arabidopsis seedlings and empty vector Arabidopsis seedlings as controls, each strain was 50, the experiment was repeated three times, and the results were averaged. ImageJ software (http://rsbweb.nih.gov/ij/) was used to calculate the average change of root length.

The result is shown in Figure 3,

In MS medium containing 0mM mannitol:

The root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 is 3.01 cm, and the root length of the wild type Arabidopsis (CK) is 3.05 cm;

In MS medium containing 100mM mannitol:

The root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 is 2.51 cm, and the root length of the wild-type Arabidopsis (CK) is 1.86 cm;

In MS medium containing 200mM mannitol:

The root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 is 2.59cm, and the root length of the wild-type Arabidopsis (CK) is 1.58cm;

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

The root length of the T2 generation transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of the L53 strain numbered 53 was photographed and observed by photographing, and the results were shown in Figure 4. As can be seen from the figure, as the culture The concentration of mannitol in the substrate increased, and the root length of the wild-type plants (0mM Mannitol, 100mM Mannitol, 200mM Mannitol) was significantly shortened, while the root length of the T2 transgenic GmNFYB1 Arabidopsis plants of the L53 line numbered 53 decreased. Concentration change: At 0mM Mannitol, the root length of transgenic Arabidopsis plants was similar to that of wild-type plants, and longer than that of wild-type plants. At the concentrations of 100mM Mannitol and 200mM Mannitol, the roots of transgenic Arabidopsis plants were longer than those of wild-type plants.

Using the above method, the T2 transgenic GmNFYB1 Arabidopsis seedlings (L57, L53, L2) of the three lines (L53, L57, L2) numbered 53, 57 and 2 were transferred to MS culture containing 300 mM mannitol. After 7 days, the plants were photographed and observed. As shown in Figure 5, the leaves of wild-type Arabidopsis plants (WT) began to appear yellowish-white, and the root length became shorter, while the three plants numbered 57, 53, and 2 Arabidopsis seedlings of T2 lines (L53, L57, L2) transfected with GmNFYB1 still showed green leaves.

3. ABA (abscisic acid) stress treatment of Arabidopsis transgenic GmNFYB1 seeds

The seeds of the T2 generation of the L53 strain No. 53 obtained above were sterilized and sowed on MS medium containing different concentrations of ABA (abscisic acid): 0 μM ABA , 0.5μMABA, 1.0μMABA, 1.5μMABA, 2.0μMABA), 16h light/8h dark, grow at 25°C for 6 days, and count the germination rate. The wild-type Arabidopsis thaliana (CK) and the empty vector Arabidopsis thaliana were used as controls, with 50 plants for each line. The experiment was repeated three times, and the results were averaged.

The result is shown in Figure 6:

When the ABA concentration was 0 μM, the germination rates of the T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of the L53 line numbered 53 were 100% and 100%, respectively;

When the ABA concentration was 0.5 μM, the germination rates of T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of L53 line No. 53 were 95.37% and 93.26%, respectively;

When the ABA concentration was 1.0 μM, the germination rates of T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of L53 line No. 53 were 93.75% and 91.35%, respectively;

When the ABA concentration was 1.5 μM, the germination rates of T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of L53 line No. 53 were 89.33% and 75.61%, respectively;

When the ABA concentration was 2.0 μM, the germination rates of the T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis of the L53 line numbered 53 were 88.89% and 68.25%, respectively;

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

It can be seen from the figure that the growth of T2 transgenic GmNFYB1 Arabidopsis and wild-type Arabidopsis was also mostly inhibited in the medium containing ABA. But the transgenic Arabidopsis seeds were less suppressed. It was preliminarily determined that the seed germination of transgenic Arabidopsis plants was related to ABA.

4. Changes of proline content in GmNFYB1 Arabidopsis plants under mannitol (Minnitol) stress treatment

Proline is the most important and effective organic osmotic adjustment substance, and it can cause a large amount of proline accumulation in plants under drought stress. These accumulated prolines prevent water loss from the protoplasm and at the same time enhance protein hydration. The level of its content can be used as an indicator of plant drought resistance.

Sterilize the above-mentioned seeds of the L53 strain with the number 53 and transfer them to GmNFYB1 Arabidopsis thaliana, sow them on MS medium, grow them at 25°C under 16h light/8h dark conditions, and obtain 50 strains after 1 week. The T2 generation of the L53 strain of 53 was transferred to GmNFYB1 Arabidopsis, and then the T2 generation of the L53 strain numbered 53 was transferred to GmNFYB1 Arabidopsis to grow in MS medium containing 0mM, 100mM, and 200mM mannitol respectively for 7 sky.

Detect the content of free proline in Arabidopsis plants, the method is as follows: 1) Preparation of standard curve

(1) Take 7 graduated test tubes with stoppers and add each reagent as shown in Table 1. After mixing, heat in boiling water for 40min.

Formation of standard curve in table 1

(2) Take it out and add 5ml of toluene to each tube after cooling, and shake fully to extract the red complex. After standing still until the layers are separated, absorb the toluene layer, and compare the color at a wavelength of 520nm with tube No. 0 as a control.

(3) With the extinction value as the vertical axis and the proline mass as the horizontal axis, draw a standard curve and find the linear regression equation.

2) Sample determination

(1) Proline extraction. Take 0.5 g of the T2 generation GmNFYB1 Arabidopsis thaliana of the L53 strain numbered 53 treated with different mannitol concentrations, add 5 ml of 3% sulfosalicylic acid solution for grinding, and transfer the homogenate into a centrifuge tube. Extract in a boiling water bath for 10 minutes, centrifuge at 3000 rpm for 10 minutes after cooling, and take the supernatant for testing.

(2) Take 2ml of the supernatant, add 2ml of glacial acetic acid and 3ml of ninhydrin chromogenic solution, heat in a boiling water bath for 40min, take it out and add 5ml of toluene to each tube after cooling, fully shake to extract the red complex. After standing still until the layers are separated, absorb the toluene layer, and compare the color at a wavelength of 520nm with tube No. 0 as a control.

(3) Result calculation. Find the mass of proline in the assay solution from the standard curve, and calculate the mass fraction of proline in the sample according to the following formula

The mass fraction of proline in the sample/(μg g-1) = (C V) (a W)-1

In the formula, C-mass of proline in the extract (μg), obtained from the standard curve;

V - the total volume of the extract (ml);

a - the volume absorbed during the measurement (ml);

W - sample mass (g).

Wild-type Arabidopsis and empty vector Arabidopsis were used as controls. There were 50 strains for each strain, the experiment was repeated 3 times, and the results were averaged.

The results are shown in Figure 7A, and some of the results are as follows:

In the MS medium containing 0mM mannitol, the proline contents of T2 transgenic GmNFYB1 Arabidopsis (TG) and wild type Arabidopsis (CK) numbered 53 were 7.5373 μg/g and 9.6567 μg/g, respectively;

In the MS medium containing 100mM mannitol, the proline content of T2 transgenic GmNFYB1 Arabidopsis (TG) and wild type Arabidopsis (CK) of L53 line numbered 53 were 50.5787g/g and 35.0949 μg/g;

In the MS medium containing 200mM mannitol, the proline content of T2 transgenic GmNFYB1 Arabidopsis (TG) and wild type Arabidopsis (CK) of L53 line numbered 53 were 65.5529μg/g and 47.2121 μg/g;

Fig. 7B is the proline extract of Arabidopsis plants in 200mM mannitol MS medium, showing that with the increase of mannitol concentration, the proline content of transgenic Arabidopsis plants increases.

As the concentration of mannitol in the medium increased (0mM Mannitol, 100mM Mannitol, 200mM Mannitol), proline accumulated in the plant in large quantities. At 200mM Mannitol, the T2 generation of the L53 line numbered 53 was transformed into GmNFYB1 Arabidopsis The proline content of wild-type Arabidopsis was 1.47 times, and the proline content increased significantly, indicating that the drought resistance was enhanced.

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

B: Salt treatment:

5. Changes in root length of transgenic GmNFYB1 Arabidopsis plants under sodium chloride (NaCl) stress

Sterilize the seeds of the T2 transgenic GmNFYB1 Arabidopsis obtained from the above-mentioned L53 strain No. 53 and sow them on MS medium, grow them under 16h light/8h dark conditions at 25°C, and obtain them after 1 week The T2 generation of 50 L53 strains numbered 53 were transformed into GmNFYB1 Arabidopsis thaliana. Then the T2 generation of the L53 strain that is numbered 53 is transferred to GmNFYB1 Arabidopsis seedlings to contain 0mM sodium chloride, 50mM sodium chloride, growth in the MS medium of 75mM sodium chloride, treat to measure root length after 1 week, The wild-type Arabidopsis seedlings and the empty vector Arabidopsis seedlings were used as controls, and each strain was 50. The experiment was repeated three times, and the results were averaged. The average change of root length was counted by ImageJ software.

The result is as follows:

In MS medium containing 0mM NaCl:

The average root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 is 3.11cm, and the average root length of the wild-type Arabidopsis (CK) is 3.13cm;

In MS medium containing 50mM NaCl:

The average root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 was 1.85 cm, and the average root length of the wild-type Arabidopsis (CK) was 1.96 cm;

In MS medium containing 75mM NaCl:

The average root length of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 is 1.45 cm, and the average root length of the wild-type Arabidopsis (CK) is 1.52 cm;

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis.

The root length after the treatment of the T2 generation of the L53 strain numbered 53 to GmNFYB1 Arabidopsis and wild-type Arabidopsis was photographed, and the results were shown in Figure 8. As can be seen from the figure, as the culture The concentration of sodium chloride in the base increased, (0mM NaCl, 50mM NaCl, 75mM NaCl) the root length of transgenic Arabidopsis plants was close to that of wild-type plants, and there was no great change. It shows that the transgenic Arabidopsis plants are not resistant to salt at the seedling stage under salt stress.

6. Germination rate of transgenic GmNFYB1 Arabidopsis plants under sodium chloride (NaCl) stress

After sterilizing the seeds of the T2 generation of the L53 and L57 strains obtained from the above-mentioned numbers 53 and 57, the seeds of the GmNFYB1 Arabidopsis were sown in different concentrations of 0mM sodium chloride, 50mM sodium chloride, and MS culture of 75mM sodium chloride. Basically, 16h light/8h dark, grow under the condition of 25°C, count the germination rate of 50 T2 generation Arabidopsis seedlings transfected with GmNFYB1 of L53 and L57 lines numbered 53 and 57 on the 5th day. The wild-type Arabidopsis seedlings and the empty vector Arabidopsis seedlings were used as controls, and each strain was 50. The experiment was repeated three times, and the results were averaged.

In MS medium containing 0mM NaCl:

The germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line numbered 53 was 100.00%, and the germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L57 line No. 57 was 100.00%. The germination rate of wild-type Arabidopsis (CK) was 100.00%; (Fig. 9A)

In MS medium containing 50mM NaCl:

The germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L57 line numbered 57 was 38.31%, and the germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line No. 53 was 39.31%. The germination rate of wild-type Arabidopsis (CK) was 41.67%; (Fig. 9B)

In MS medium containing 75mM NaCl:

The germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L57 line numbered 57 was 15.38%, and the germination rate of the T2 transgenic GmNFYB1 Arabidopsis (TG) of the L53 line No. 53 was 16.38%. The germination rate of wild-type Arabidopsis (CK) was 20.83%; (Fig. 9C)

There was no significant difference in the results between wild-type Arabidopsis and empty vector Arabidopsis. The results showed that: under salt stress, the germination rate of transgenic Arabidopsis plants was very close to that of wild-type plants. It further demonstrated that Arabidopsis seeds are salt-intolerant or salt-resistant at the seedling stage.

The article on nuclear factor-Y (AtNFYB1) published by Donald E. et al. and the article on AtNFYB5 published by Wen-XueLi. et al. have shown that this family gene is related to drought resistance and is a drought stress-related transcription factor. Homologous phylogenetic tree analysis of soybean (GmNFYB1) protein and Arabidopsis (AtNFYB1) protein. The MEGA4.0 software was used for analysis, and the results showed that the homology between GmNFYB1 gene and Arabidopsis thaliana (AtNFYB1) was very high (as shown in Figure 10). In order to verify whether this gene is related to salt stress, another salt tolerance or salt resistance experiment was done. Transgenic Arabidopsis seedlings did not show good resistance to salt stress under sodium chloride (NaCl) treatment.

In summary, Arabidopsis transgenic for GmNFYB1 did not show great advantages in salt tolerance or salt resistance compared with wild-type Arabidopsis, and showed stronger performance in drought resistance, indicating that this gene (GmNFYB1) is a Nuclear factors involved in drought resistance.

4. Expression of GmNFYB1 gene in transgenic GmNFYB1 Arabidopsis

In order to further determine whether the GmNFYB1 gene is involved in the drought stress response of soybean, the expression of the GmNFYB1 gene was analyzed according to the method of fluorescent quantitative PCR.

After sterilizing the Dongnong 50 small bean seeds, they were planted in MS medium, and the hydroponic treatment was started when the plants began to grow the second and third compound leaves. Add 15% PEG to the culture medium. Soybean leaves treated at different times were collected for RNA extraction and reverse transcription into cDNA. Fluorescence quantitative PCR specific primers were designed according to the cDNA sequence. Real-time PCR GmNFYB1 gene amplification sense primer: GCCTCCCAATGGCAAGAT, antisense primer: CATTCGCCTCGCTGGTAAT for PCR identification. Real-time PCR for actin gene amplification sense primer: GTGTCAGCCATACTGTCCCCATTT antisense primer: GTTTCAAGCTCTTGCTCGTAATCA for PCR identification. Designed using primer premier 5.0 software. For the method of extracting total RNA, refer to the instructions of Trizol reagent, and the integrity of RNA was detected by 0.8% agarose gel electrophoresis. The soybean housekeeping gene actin was used as an internal control.

Study the relative expression level of soybean GmNFYB1 gene under 15% PEG drought stress and non-stress treatment conditions (Figure 11), it shows that at 0h, the expression level of GmNFYB1 in the treatment group and the control group is exactly the same; The expression level was 1.03 times that of the control, and the gene expression level of soybean seedlings treated with PEG was 0.59 times that of the control at 4 h; the expression level of GmNFYB1 gene in soybean seedlings treated with PEG increased rapidly at 6 h, and the expression level reached 3 times that of the control. The expression level of GmNFYB1 in soybean seedlings treated with PEG decreased again, only 0.58 times and 0.29 times that of the control. At 12h, the expression level of GmNFYB1 gene in PEG-treated soybean seedlings increased rapidly again, reaching 3.1 times that of the control, and then the expression level of GmNFYB1 gene in PEG-treated soybean seedlings decreased again. The expression of GmNFYB1 gene increased at 6h and 12h after PEG treatment, while the expression of GmNFYB1 gene decreased at 4h, 8h, and 10h after PEG treatment, indicating that the expression of GmNFYB1 gene in soybean seedlings changed periodically when drought stress was induced, and the change rule was similar to that of The plant circadian clock coincided, indicating that GmNFYB1 was induced by PEG drought stress, which may be involved in the early drought resistance response of soybean plants.

Example 3, Obtaining and Functional Research of Transgenic GmNFYB1 Soybean Plants

1. Genetic transformation of soybean cotyledon nodes mediated by Agrobacterium

1. Preparation and transformation of soybean cotyledon nodes

(1) Bacterial liquid preparation

Pick a single colony of Agrobacterium and inoculate it in 3mLYEP liquid medium containing rifampicin 50mg/L, streptomycin 25mg/L and ampicillin 100mg/L, cultivate overnight at 28°C and 200rpm, and then transfer the bacterial solution to 100ml Shake the above-mentioned fresh YEP liquid medium to A600=0.8, centrifuge at 5000rpm for 10min, and resuspend in the infection solution (liquid CCM medium), adjust the OD600 to 0.6.

(2) Preparation of soybean cotyledon nodes

Take the mature soybean Dongnong 50 seeds with smooth surface and no disease spots, put them in NaClO/HCl (24:1), and sterilize them in a filter with slightly excess HCl for 8 hours, then inoculate them into the germination medium (GM) with the hilum down. Cultivate for 5 days under the conditions of light at 25°C, 16h light/8h dark, take out the sterile seedlings, remove the seed coat, cut off the upper 1/3 of the cotyledon and most of the hypocotyl, leaving only the 3-hypocotyl near the cotyledon axis, two cotyledons were cut from the midline of the hypocotyl, and the terminal bud and axillary bud were removed to obtain the cotyledon node explant. , use a scalpel to make 7 cuts in the range of about 3mm in diameter at the junction of cotyledons and hypocotyls. After this operation, each sterile seedling can produce 2 cotyledon node explants for transformation.

(3) Conversion

Put the prepared cotyledon node explants into the resuspended infection solution at 28° C. and shake at 200 rpm for 30 minutes. Pour off the bacterial liquid, put the explants into a large Petri dish covered with sterile filter paper above and below, and absorb the excess bacterial liquid. The explants were then inoculated adaxially side down on co-cultivation medium (CCM) covered with a layer of sterile filter paper. The co-cultivation medium was the same as the Agrobacterium resuspension medium, solidified with 0.8% agar. Cultures were co-cultivated for 3 days at 24-25°C in the dark. After 3 days of co-cultivation, transfer the explants into a sterile Erlenmeyer flask, add sterile water to wash 2-3 times, and then wash 2-3 times in liquid shoot induction medium (SIM), and then the cotyledons are adaxially facing Above, hypocotyls were inserted into solid shoot induction medium (SIM). Restore the culture for 7 days, transfer to solid selection (SIM+) medium containing 0, 1, 3, 5, 7, 10 mg/LPPT, and after one week of selection, transfer the explants to solid shoot elongation medium (SEM) middle. Subculture once every 10 days, until the seedling grows to 6cm, transfer it to the rooting medium (RM) for cultivation, until the root is strong enough, transfer it to the liquid 1/2MS and open the seedling for 5-7 days, Transplanted into vermiculite watered by MS for cultivation (cover with plastic wrap to keep moisture in the first 3 days of cultivation, and cultivate under low light).

2. Determination of PPT screening concentration

After germination, co-cultivation, and restoration of wild-type soybeans, they were transferred to PPT containing 0mg/L, 1mg/L, 3mg/L, 5mg/L, 7mg/L, and 10mg/L for screening culture, and the cotyledon nodes were observed For the growth situation, according to the PPT screening situation of soybean according to the statistical method of Arabidopsis thaliana, tabular statistics are made to determine that the final screening concentration of soybean is 5 mg/L.

2. Obtaining of transgenic GmNFYB1 soybeans

1. Obtaining of explants

Select healthy and plump Dongnong 50 seeds harvested in the same year with smooth and disease-free surfaces, and sterilize them with chlorine gas disinfection: put the selected bean seeds in a petri dish, weigh 96ml of sodium hypochlorite and put them in a jar, put them Place the petri dish with the seeds in a closed container in the fume hood stably, weigh 4ml of concentrated hydrochloric acid and mix it in a jar filled with sodium hypochlorite, seal the container quickly, and the disinfection time is the best sterilizing time determined in the preparation test. Bacteria time.

The sterilized seeds were inoculated on the germination medium containing 6-BA, and the concentration of 6-BA was the optimal concentration determined in the preparation test. The culture conditions are: temperature 24±1°C; photoperiod 16/8h. After the seeds have germinated for 7 days, remove the seed coat, cut off the hypocotyl at 4 mm below the cotyledon node, cut the cotyledon longitudinally, and remove the terminal bud and axillary bud. Two cotyledonous node explants were obtained per seed. The basic components of the germination medium are: B5 massive salt and trace salt, MS iron salt, B5 vitamin, 2% sucrose, 0.7mg/L 6-BA, 0.8% agar, PH=5.8.

2. Infection and co-cultivation

Lightly scratch the wound near the growth point of the cotyledon node, and mix it with the pre-prepared LBA4404/pCAMBIA-GmNFYB1 bacterial solution. Place it on a shaker and infect for 30 minutes at 120 rpm. The explants were taken out, the bacterial solution was blotted dry, placed upside down on the co-cultivation medium covered with a layer of filter paper, and cultured in the dark for 3 days. The co-cultivation medium is: 1/10 concentration of B5 macro salt and trace salt, 1/10 concentration of MS iron salt, B5 vitamin, 3% sucrose, 3.9g/L Mes, 40mg/L AS, 0.25ug/L GA3 , 1mM/L DTT, 8.8mM/L L-cysteine, 1.70mg/L 6-BA, 0.5% agar, PH=5.4.

3. Induction and screening of clustered buds

The cotyledon nodes after co-cultivation were washed twice with sterile water, and then washed twice with liquid cluster bud induction medium added with fungicide, blotted dry with sterile filter paper, and inoculated in the The recovery culture was carried out on the bud induction medium of the agent. Bud induction medium is: B5 massive salt and trace salt, MS iron salt, B5 vitamin, 3% sucrose, 0.59g/L Mes, 1.7mg/L 16-BA, 100mg/L cefotaxime, 0.8% agar, PH=5.6 . After being cultured on the shoot induction medium for 7 days, they were transferred to the shoot induction medium containing PPT at a concentration of 5 mg/L (ie selection medium) for 7 days of selection. The culture conditions are: temperature 24±1°C; photoperiod 16/8h. The screening medium is: B5 massive salt and trace salt, MS iron salt, B5 vitamin, 3% sucrose, 0.59g/L Mes, 1.7mg/L 6-AP, 100mg/L cefotaxime, 5mg/L PPT, 0.8% agar ,PH=5.6.

4. Elongation and rooting of resistant shoots

The explants that passed the screening were transferred to the shoot elongation medium, and the cotyledon and the aging tissue at the base of the hypocotyl were cut off when transferring, and subcultured once every 7 days. Bud elongation medium is: MS large amount of salt and trace salt, MS iron salt, B5 vitamin, 3% sucrose, 0.59g/L Mes, 0.5mg/L GA3, 0.1mg/L IAA, 1mg/L ZR, 50mg/L L L-asparagine, 100mg/L L-pyroglutamic, 100mg/L cefotaxime, 0.8% agar, PH=5.6.

When the buds elongate to 3-4 cm, they are cut off against the roots and transferred to the rooting medium for rooting culture. The culture conditions are: temperature 24±1°C; photoperiod 16/8h. The rooting medium is: 1/2 concentration of B5 major salt and trace salt, MS iron salt, 2% sucrose, 0.59g/L Mes, 1mg/L IAB, 0.8% agar, PH=5.6.

5. Domestication of resistant plants

After the roots are sufficiently developed, wash the medium of the roots, move the seedlings into the liquid culture medium to harden the seedlings for 3-5 days, and open the bottle at the same time, so that the aseptic seedlings gradually adapt to the external environment with bacteria. Then the plants were taken out and transferred to vermiculite for cultivation and acclimatization in the dark, and at the same time, they were bagged to keep moisture, and then the humidity was gradually reduced and the light was increased. After the small plants survive, they are transferred to normal soil for cultivation, and the temperature, humidity and normal light are maintained until the pods mature.

Using Agrobacterium-mediated method, the cotyledon node of Dongnong 50 was used as an explant to transfer the GmNFYB1 gene into soybean. After a series of processes of germination, infection, cluster bud induction, elongation and rooting, 30 resistant transformed plants were obtained to transform GmNFYB1 soybeans in the T0 generation. The transformation process (as shown in Figure 12) is shown. B: The cotyledon nodes were cultured on the co-culture medium; C: The cotyledon nodes were screened in the resistance medium containing PPT; D: The cotyledon nodes were cultured in the elongation medium; E: The soybean sterile seedlings were refined in 1/2MS seedlings; F is the transgenic seedling T ₀ obtained by transplanting aseptic soybean seedlings into vermiculite.

Using the same method, the empty vector pCAMBIA-pBI121 was transformed into wild-type soybean to obtain the empty vector soybean. After identification by the above method, there was no amplified fragment in soybean transformed from empty vector.

3. Detection of transgenic GmNFYB1 soybeans

1. GUS staining of soybeans transfected with GmNFYB1

30 T0 soybeans transfected with GmNFYB1 were subjected to GUS staining, and wild-type soybeans and soybeans transformed with empty vector were used as controls.

The results are shown in Figure 13. It can be seen that the blue ones are GUS positive, and a total of 10 positive T0 soybeans transformed with GmNFYB1 were obtained.

2. PCR amplification of GmNFYB1 soybean

Extract DNA from 10 positive T0 transgenic GmNFYB1 soybean leaves, use pCAMBIA-GmNFYB1 as a positive control, wild-type Dongnong 50 plants as negative controls 1 and 2, and water as negative control 3, and use Bar, gus and GmNFYB1 gene-specific primers for PCR detection.

Bar gene sense primer: GCGGTACCGGCAGGCTGAAG Antisense primer: CCGCAGGAACCGCAGGAGTG

PCR identification was carried out, and the results are shown in Figure 14, 403bp positive transgenic GmNFYB1 soybeans were obtained.

Gus gene sense primer: 5' AGCAACGCGTAAACTCGACCCG 3', antisense primer: 5' CTGACGCGATCAAAGACGCGGT 3' for PCR identification, the results are shown in Figure 15, and a 333bp positive transgenic GmNFYB1 soybean was obtained.

GmNFYB1 gene sense primer: 5'GG TCTAGA CAAAGGTGCATTGGTGGT3', antisense primer: 5'AT GAGCTC CGTACAAGCATTCAAGGGA3' for PCR identification, the results are shown in Figure 16, and a 522bp positive transgenic GmNFYB1 soybean was obtained.

According to statistics, the above three genes are all positive, and a total of 5 PCR identified positive T0 transgenic GmNFYB1 soybeans.

3. Southern hybridization detection of transgenic GmNFYB1 soybean

The seeds numbered DL-18, DL-26, DL-16(2), DL-16(c), DL-16(d) PCR identification positive T0 generation transfected GmNFYB1 soybeans were propagated in the greenhouse, and the T2 generation The genomic DNA of the plants was detected by Southern analysis. Digoxigenin labeling and detection kit (DIG High Prime DNA Labeling and Detection Starter Kit II) was used.

Preparation of hybridization probe: pCAMBIA-GmNFYB1 plasmid was used as a template, Bar gene sense primer: GCGGTACCGGCAGGCTGAAG, antisense primer: CCGCAGGAACCGCAGGAGTG for PCR identification, and a 403bp product was obtained as a probe.

1) Extract the genomic DNA of the positive T0-transformed GmNFYB1 soybeans identified by PCR (Figure 17, 1-8 are all PCR-positive T0-transformed GmNFYB1 soybeans), and the extraction effect is good.

2) HindIII digested the above genomic DNA, and the results are shown in Figure 18. It can be seen that the genomic DNA was evenly distributed in the gel, indicating that the genomic DNA was fully digested by the restriction endonuclease HindIII.

3) The 403bp fragment is used as a probe for Southern hybridization with the digested genomic DNA fragment.

The hybridization result is shown in Figure 19, 0 and 1: plasmid pCAMBIA-GmNFYB1 is not digested; 2: DL-18 plant (HindIII digestion); 3: DL-26 plant (HindIII digestion); 4: DL-16 ( 2) plant (digested with HindIII); 5: DL-16 (c) plant (digested with HindIII); 6: DL-16 (d) plant (digested with HindIII); 7: wild-type soybean (digested with HindIII); It can be seen that the five transgenic plants that were positive in the PCR test were all positive in the Southern hybridization test, and there was one band in each enzyme digestion sample, which indicated that the Bar gene existed in the form of a single copy in the soybean genome.

Transplant the positive T0 generation coded DL-18, DL-26, DL-16(2), DL-16(c), DL-16(d) into GmNFYB1 soybeans for sowing and passage, and obtain the coded DL-18, DL-26 , DL-16(2), DL-16(c), DL-16(d) T3 transgenic GmNFYB1 soybeans.

4. Functional analysis of T3 transgenic GmNFYB1 soybean

1. Drought stress

The seeds of T3 generation transgenic GmNFYB1 soybeans numbered DL-18, DL-26, DL-16(2) were sown in drought-resistant sheds, and when the seedlings grew to the second and third compound leaves, watering was stopped and drought stress treatment was carried out , and the wild-type soybean Dongnong 50 (WT) and the empty vector soybean were used as controls.

The results are shown in Figure 20, after 3 weeks of drought stress, the T3 transgenic GmNFYB1 soybean line still grew well, with occasional slightly wilted leaves. But it can quickly return to normal after resuming watering. In contrast, the wild-type soybean Dongnong 50 had a severe wilting phenomenon, and it took a long time to recover after resuming watering.

There was no significant difference in the results between wild-type soybean Dongnong 50 (WT) and the empty vector soybean.

The T3 generation numbered DL18 with significant drought resistance effect was transferred to GmNFYB1 soybean (NFYB1) for addition. The obtained T5 generation seeds were subjected to field drought resistance experiments in 2012. Xinjiang was selected for planting plots in Xinjiang with less rain and drought. The plot area is 0.5 mu, 6 meters long long, with a plant-to-plant distance of 10 cm, and the wild-type soybean Dongnong 50 (CK) was used as a control. There were 300 strains for each strain, the experiment was repeated three times, and the results were averaged.

It was harvested 105 days after sowing, and the photographic observation results are shown in Figure 21. It can be seen that compared with wild-type soybean Dongnong 50 (CK), T5 transgenic GmNFYB1 soybean (NFYB1) showed a significant yield advantage. High growth, increased grain number, well-developed root system, deep rooting, and increased yield;

Statistical phenotype data, the results are shown in Figure 22,

Under normal irrigation conditions:

The plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant and average 100-grain weight of T5 transgenic GmNFYB1 soybean (GmNFYB1) numbered DL18 were 85 cm, 19 nodes, 4.7 branches, 112 pods, 353 grains, 28.8g, 8.2g.

The plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant, and average 100-grain weight of wild-type soybean Dongnong 50 (CK-DN50) were: 80 cm, 17 nodes, 5 branches, 115 pods, 292 grains, 24.3g, 8.3g;

In dry conditions:

The plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant, and average 100-grain weight of T5 transgenic GmNFYB1 soybean (GmNFYB1) numbered DL18 were 38 cm, 14 nodes, 5 branches, 78 pods, 210 grains, 14.2g, 6.8g.

The plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant, and average 100-grain weight of wild-type soybean Dongnong 50 (CK-DN50) were: 32cm, 13 nodes, 4 branches, 52 pods, 137 grains, 9g, 6.6g;

It can be seen from the figure that under natural drought conditions, the transgenic GmNFYB1 soybeans showed significant yield advantages, with increased plant height, increased grain number, well-developed root system, deep rooting, and increased yield.

In the above experiments, the overexpression vector pCAMBIA-GmNFYB1 of the GmNFYB1 gene was constructed using plant overexpression technology, and Arabidopsis and soybean were transformed through Agrobacterium-mediated transformation, and the long-day plant Arabidopsis and short-day plant soybean were carried out. Gene overexpression analyzes the function of GmNFYB1 gene, clarifies the mechanism of nuclear factor-Y drought resistance, and lays a theoretical foundation for the use of GmNFYB1 gene to cultivate drought-resistant and high-yield soybean varieties.

Claims (10)

1. A protein, which is a protein of the following 1) or 2): 1) A protein composed of the amino acid sequence shown in Sequence 2 in the sequence listing; 2) A protein derived from 1) in which the amino acid sequence of Sequence 2 is substituted and/or deleted and/or added by one or several amino acid residues and is related to plant stress tolerance. 2. the coding gene of protein described in claim 1. 3. The coding gene according to claim 2, characterized in that: the coding gene is the following 1), 2), 3), 4) or 5) gene: 1) The DNA molecule shown in sequence 1 in the sequence listing; 2) The DNA molecule shown in the 22nd-665th nucleotides from the 5' end of sequence 1 in the sequence listing; 3) The DNA molecule shown in the 93rd-617th nucleotides from the 5' end of Sequence 1 in the sequence listing; 4) A DNA molecule that hybridizes with the DNA molecule defined in 1) or 2) or 3) under stringent conditions and is associated with a plant stress tolerance-related protein coding gene; 5) A DNA molecule that has at least 90% homology with the DNA sequence defined in 1) or 2) or 3) and is related to a gene encoding a protein related to plant stress tolerance. 4. A recombinant vector, a transgenic cell line, a recombinant bacterium or an expression cassette containing the coding gene of claim 2 or 3. 5. The pair of primers for amplifying the full-length or any fragment of the coding gene described in claim 2 or 3. 6. Application of the protein according to claim 1, the coding gene according to claim 2 or 3, the recombinant vector, transgenic cell line, recombinant bacterium or expression cassette according to claim 4 in regulating plant stress tolerance and/or plant breeding . 7. The application of the protein according to claim 1, the coding gene according to claim 2 or 3, the recombinant vector, transgenic cell line, recombinant bacterium or expression cassette according to claim 4 in regulating plant yield. 8. The application according to claim 6 or 7, characterized in that: The regulation of plant stress tolerance is to improve plant stress tolerance, and the stress tolerance is specifically drought tolerance; The regulation of plant yield is to increase plant yield, The plant is specifically a dicot or a monocot, and the dicot is further specifically Arabidopsis or soybean. 9. A method for cultivating a transgenic plant, which is a transgenic plant obtained by introducing the coding gene described in claim 2 or 3 into a target plant, and the stress tolerance of the transgenic plant is higher than that of the target plant; the stress tolerance is specifically for drought tolerance; The target plant is specifically a dicot or a monocotyledon, and the dicot is further specifically Arabidopsis or soybean. 10. A method for cultivating a transgenic plant, which is a transgenic plant obtained by introducing the coding gene described in claim 2 or 3 into a target plant, and the yield of the transgenic plant is higher than that of the target plant; The yield is reflected by increasing plant height, number of main stem nodes, effective branches, effective pod number, grain number per plant, grain weight per plant and/or 100-grain weight; The target plant is specifically a dicotyledon or a monocotyledon, and the said dicotyledon is further specifically soybean.

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