CN106520782A - Application of a gene GmRAV1 related to soybean photoperiod regulation - Google Patents

Abstract

The invention discloses an application of a gene GmRAV1 related to soybean photoperiod regulation, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the soybean GmRAV1 gene provided by the present invention is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2. Soybean GmRAV1 gene can be used in regulating plant photoperiod sensitivity and promoting tooth regeneration. Tooth regeneration in plants can be promoted by overexpressing the gene, while silencing the expression of the gene can change the photoperiod sensitivity, plant height, and flowering maturity of the plant, and reduce the photoperiod sensitivity of the plant. This gene can be used to cultivate and improve early-maturing, photoperiod-insensitive new varieties of wide adaptability, solve the problem of flowering in hybrid breeding, the growth period control problem of various crops, vegetables, fruits and flowers, and the problem of photoperiod sensitivity and breeding issues.

Description

Application of a gene GmRAV1 related to soybean photoperiod regulation

technical field

The invention relates to the application of a gene GmRAV1 related to soybean photoperiod regulation, and belongs to the technical field of plant genetic engineering.

Background technique

Generally speaking, the growth and development of short-day plants such as soybeans and rice are very sensitive to photoperiod responses. This characteristic seriously hinders the adaptability of soybean varieties, limits the sowing range of soybeans, and affects the effective production level. Generally, shortening the sunshine time promotes the flowering of plants such as soybeans and rice, and shortens the growth period. On the contrary, increasing the sunshine time inhibits soybean flowering and prolongs the growth period. Therefore, the introduction from north to south (sunshine time from long to short during the growth period) will accelerate maturation, shorten the growth period, reduce yield, and limit the performance of yield traits. On the contrary, the introduction from south to north will prolong the growth period and cannot mature normally. This feature seriously hinders the cross-regional planting of soybean varieties and other plant varieties that are sensitive to photoperiod responses. For example, the adaptability of soybean varieties is narrow, and it is not suitable for large-scale planting in the north and south. Many varieties with different photoperiod responses are required. Adaptation to a variety of different ecological conditions, so changing the photoperiod sensitivity of plants like soybean is very important for the improvement of these plant varieties.

It has been reported that at least 7 gene loci affect the flowering and maturation time of soybean, and these gene loci are called E series. Most dominant E alleles in soybean are sensitive to long-day photoperiod and inhibit flowering. The dominant alleles delayed flowering to varying degrees (Cober et al., 1996a). The soybean E allele appears to function similarly to the photostable phytochrome genes phyB, phyD, and phyE in Arabidopsis thaliana involved in the flowering inhibition pathway under short-day conditions (Devlin et al., 1998). The photoperiod-sensitive traits controlling flowering time and ripening in soybean may be controlled by major QTL and modified by several minor QTL. 10 soybean flowering time genes have been located on the linkage map (Tasma and Shoemaker, 2003), including 4 photoperiod-related genes (PHYA, PHYB, CRY2 and CCA1), 5 flowering time genes (FCA, DET1, COL1, COL2 and LD) and a floral meristem trait gene (AP2).

In the existing conventional breeding, it takes a long time to improve varieties by conventional methods such as selection of early-maturing varieties or late-maturing varieties by hybridization, or mutagenesis by radiation and chemical reagents, and the degree or direction of mutations in the offspring cannot be predicted. Variety improvement using these genes is a particularly effective, simple and reliable method compared to traditional methods.

On the other hand, the genetic transformation research of soybean has been paid more and more attention by countries all over the world. Soybean tissue culture has certain difficulties, and it is recognized as a crop that is difficult to transform. The main reason is that it is difficult to differentiate and regenerate plants from transformed cells or tissues. Researchers have devoted themselves to optimizing the transformation system of soybean, but the frequency of soybean transformation is still very low, limited by genotype, and the reproducibility is poor. Establishing a good receptor system is a prerequisite for gene transformation. Therefore, identifying new genes related to soybean bud regeneration, improving the transformation efficiency of soybean varieties with different genotypes through transgenic methods, and creating high regeneration soybean varieties, thereby improving the genetic transformation efficiency of soybean is another important goal of breeding. At the same time, controlling the plant type of the plant is also a key factor in designing yield in soybean breeding. However, there is a lack of methods that can effectively control the plant type in the prior art.

Contents of the invention

In order to solve the above problems, the present invention provides a kind of application of soybean GmRAV1 gene in controlling plant growth period and growth process, and the technical scheme adopted is as follows:

The gene and protein provided by the present invention, named GmRAV1, are derived from soybean (Glycine max (L.) Merrill.) Dongnong 42. Transfer the gene sequence (GmRAV1) into soybean or other dicotyledonous plants and monocotyledonous plants to be improved, and carry out gene silencing, so that the plants show tall plants, many branches, dark green leaves, increased stem spacing, early Flowers, precocity, photoperiod insensitivity or photoperiod-sensitivity-reduced characteristics; or gene overexpression exhibits the characteristics of enhanced adventitious bud regeneration ability, and this method is used to cultivate and improve the general adaptability or regeneration ability of plants of new varieties.

The purpose of the present invention is to provide the application of soybean GmRAV1 gene in controlling the growth period and growth process of plants.

The nucleotide sequence of the GmRAV1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein in the coding region is shown in SEQ ID NO.2.

Preferably, said use is in particular the use in controlling the photoperiod sensitivity of plants.

Preferably, the application is to adjust and control the photoperiod sensitivity of plants by silencing the expression of GmRAV1 gene after the GmRAV1 gene is transferred into plant cells.

The steps of the application are as follows:

1) connecting the fragment containing the coding region of the GmRAV1 gene or the promoter sequence with the plant RNAi interference expression vector to construct the plant RNAi expression vector;

2) transferring the plant RNAi expression vector obtained in step 1) into plant cells to obtain the cells of the plant RNAi expression vector;

3) Screen the transformed cells with silent expression among the cells of the plant RNAi expression vector obtained in step 2), and cultivate and obtain the plants transfected with the GmRAV1 gene.

Preferably, the nucleotide sequence of the coding region of the GmRAV1 gene in step 1) is 270-1421 in SEQ ID NO.1; the promoter is the CaMSV35 promoter.

Preferably, another application method is the application in the process of enhancing the regeneration ability of adventitious buds of plants.

Preferably, the above application is to transfer the GmRAV1 gene into plant cells, and enhance the growth ability of adventitious buds of plants by overexpressing the GmRAV1 gene.

The steps of the application are as follows:

1) connecting the coding region sequence containing the GmRAV1 gene with the expression vector to construct a plant expression vector;

2) transferring the plant expression vector obtained in step 1) into a plant cell to obtain a plant cell containing the plant expression vector;

3) Screening the plant cells obtained in step 2), cultivating and obtaining plants overexpressing the GmRAV1 gene.

Preferably, the overexpression is to promote the overexpression of GmRAV1 gene under the condition of adding cytokinin and long daylight.

Preferably, another application method is application in the process of controlling plant shape.

Preferably, the steps of the above application method are as follows:

1) Obtained by transferring the GmRAV1 gene into the plant, and transforming the GmRAV1 gene plant;

2) Regulate the plant type by adjusting the expression level of GmRAV1 gene and the usage level of epiBL.

More preferably, the amount of the epibrassinolide epiBL added to the MS solid medium is 10 mmol/L.

In the method of the present invention, the GmRAV1 gene can be utilized as the target gene to construct a plant expression vector, wherein any promoter such as cauliflower mosaic virus (CaMV) 35S promoter, Ubiquitin promoter or other promoters can be used, and in the expression vector Enhancers, whether transcriptional or translational, may be included as desired. As the expression vector used, Ti plasmid, Ri plasmid, plant virus vector and the like can be used. Transformation methods may include Agrobacterium-mediated methods, particle gun methods, pollen tube passage methods, or other methods.

In the method of the present invention, the GmRAV1 gene can be used as the target gene to construct the plant RNAi vector, and the expression vector used can use Ti plasmid, Ri plasmid, plant virus vector and the like. Transformation methods may include Agrobacterium-mediated methods, particle gun methods, pollen tube passage methods, or other methods.

Recipient plants of the GmRAV1 gene of the present invention include monocotyledonous and dicotyledonous plants, such as rice, wheat, corn, soybean, cotton, vegetables, etc., which are used to suppress expression to improve crop yield and accelerate crop maturity, or for RNAi gene silencing to reduce Plant photoperiod sensitivity.

The present invention provides a method for identifying the GmRAV1 gene associated with photoperiod-controlled flowering time and maturation traits in soybean and simultaneously determining what role the gene plays in the growth and development of soybean roots, stems and leaves.

The beneficial effects that the present invention obtains are as follows:

The GmRAV1 gene provided by the present invention (the accession number of the plant genome database phytozome (URL is: https://phytozome.jgi.doe.gov/pz/portal.html) is Glyma01g22260.1) and protein can not only regulate plant photoperiod At the same time, the inventors also found for the first time that by overexpressing the gene, the bud regeneration ability of plants can be enhanced, and the division regeneration ability of plants can be improved. Under LD and SD, GmRAV-i (GmRAV gene silencing) soybean plants were significantly taller than the control, with thin and weak stems, large internode spacing, little difference in the number of nodes, short roots, and small leaves, indicating that the gene has an effect on stem development. Inhibitory effect, Arabidopsis rav mutant plants were significantly less sensitive to photoperiod than the control under LD and SD, and the flowering time was similar under SD and LD. And flowering early under SD.

The present invention provides that compared with SD, LD induces the expression of this gene in soybean leaves, so that the up-regulation of the abundance of this gene in these vegetative organs of soybean inhibits the growth of its leaves and stems, so that the soybean presents a thick leaf. Green and large, the characteristics of long roots, because the expression of GmRAV1 in leaves responds to day length, the abundance of GmRAV1 mRNA is higher under LD than under SD, LD promotes the expression of GmRAV1 and inhibits soybean flowering, and SD inhibits the expression of GmRAV1 and promotes soybean flowering. Soybean blossoms.

The invention provides that the gene is a promoting factor in the pathway of cytokinin (6-BA, 2-ip, etc.) to promote cell division and inhibit the elongation of roots and hypocotyls, and the cytokinin promotes the expression of the gene, adding the cytokinin , the hypocotyl and root shortening range of GmRAV1 gene transgenic plants is larger than that of the control, the growth is slower than that of the control, the leaves are dark green, and the branches are more, indicating that the expression of GmRAV1 enhances the effect of cytokinin, and exogenous application of cytokinin makes GmRAV -i transgenic soybeans were not sensitive to cytokinin, and the height increase of transgenic soybeans was significantly lower than that of control soybeans.

Description of drawings

Fig. 1 is the amino acid sequence comparison result of the GmRAV1 gene and homologous genes in plants by multiple sequence alignment using ClustalX (1.83) software.

Figure 2 Real-time RT-PCR analysis of GmRAV1 gene tissue-specific expression changes;

(Wherein, the black column represents the short day, and the white column represents the long day).

Figure 3 Real-time RT-PCR analysis of the circadian rhythm expression of GmRAV1 gene in DN42 soybean under long day (16h/8h light/dark) (LD) and short day (8h/16h light/dark) (SD);

(In the figure, a) is the expression analysis of soybean GmRAV1 gene under LD and SD, b) LD48h of GmRAV1 gene is transferred to LL (continuous light) rhythmic expression, c) GmRAV1 gene is transferred to DD (continuous dark) after LD48h ) rhythmic expression, d) GmRAV1 gene shifted to LL (continuous light) rhythmic expression after SD48h, e) GmRAV1 gene shifted to DD (continuous dark) rhythmic expression after SD48h).

Fig. 4 Plasmid map of plant expression vector pCAMBIA3301-GmRAV1.

Fig. 5 Plasmid map of plant expression vector pJawoh18-GmRAV1.

Fig. 6 DN50, GmRAV1-ox, GmRAV-i soybean seedlings planted in the soil for 45 days under long daylight (16h/8h light/dark) (LD).

The growth situation of DN42, GmRAV-i and X DN42 soybean seedlings under different sunshine conditions of Fig. 7;

(a) DN50 and GmRAV-i soybean seedlings planted in soil for 1 month under long day (16h/8h light/dark) (LD) and short day (8h/16h light/dark) (SD); (b) DN42 and GmRAV-i, X DN42 soybean seedlings planted in soil for 1 month under long day (16h/8h light/dark) (LD) and short day (8h/16h light/dark) (SD).

Figure 8 Soybean seedlings planted in the transgenic base of Northeast Agricultural University in Harbin.

Fig. 9 applies the regeneration situation of different plant cells after different concentrations of auxins;

(a) Regeneration of three soybean species after exogenously applying different concentrations of cytokinin 6-BA to B ₅ medium; (b) exogenously applying different concentrations of cytokinin 2-ip to MS medium Regeneration in Arabidopsis.

Fig. 10 adds the growth situation of different cytoseparatins plant roots;

(a) The growth of three kinds of soybean roots and hypocotyls after exogenously applying different concentrations of cytokinin 6-BA to B5 medium; (b) exogenously applying different concentrations of cytokinin 6-BA to B5 medium Length diagrams of three soybean roots and hypocotyls after BA; (c) root lengths of four vertically cultured Arabidopsis after exogenously applying different concentrations of cytokinin 6-BA to MS medium; (d ) Statistical graphs of root lengths of four vertically cultured Arabidopsis thaliana after exogenously applying different concentrations of cytokinin 6-BA to MS medium.

Figure 11 Real-time RT-PCR analysis of the expression of GmRAV1 gene in DN42 soybeans exogenously applied with 6-BA (100 μmol 6-BA) and without 6-BA (0 μmol 6-BA).

Fig. 12 Effects of different concentrations of epiBL on plant hypocotyl length;

(a) Actual growth of Col-0, rav, and GmRAV1-ox Arabidopsis after applying different concentrations of epiBL to MS medium under long-day light (16h/8h light/dark) (LD); (b) long-day Histogram of hypocotyl length of Col-0, rav, GmRAV1-ox Arabidopsis after applying different concentrations of epiBL to MS medium under daylight (16h/8h light/dark) (LD).

detailed description

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples.

The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels.

Example 1: Cloning and sequence analysis of the GmRAV1 gene

cDNA was obtained by reverse transcription using soybean leaf transcribed RNA as a template, and the cDNA was used as a PCR reaction template to obtain the gene DNA sequence of the full-length ORF region. The complete DNA sequence of the GmRAV1 transcription factor deduced from the soybean is 1757bp, the ORF is 1155bp, encoding 384 amino acids, containing the AP2/B3 domain (see the 270th-1421st position in the sequence table SEQ ID NO.1), and the sequence is in the plant genome database The accession number of phytozome (URL: https://phytozome.jgi.doe.gov/pz/portal.html) is Glyma01g22260.1. Homologous sequence comparison found (Fig. 1), this sequence and phaseolus vulgaris (XM_007143739), Medicago truncatula (Medicago truncatula) (XM_003591774) RAV1 homology are respectively 50.20% and 42.34%, and corn (Zea mays) (NM_001148270 ) homology is 47.07%.

Example 2: Real-time RT-PCR analysis of the effects of day length and circadian rhythm on GmRAV1 gene expression

Soybean DN42 was cultured in a light incubator at 25°C, 250mol m ^-2 sec ^-1 white light, and long-day light (16h/8h light/dark) (LD). On the 20th day after emergence (determined as 0d), part of it was transferred to short-term Grow under the photoperiod of sunshine (SD) (8h/16h light/dark), and 0, 3, 6, 9, 12, 15, 18, 21, 24 days after the transfer, the LD and SD leaves are taken at the same time, and the liquid nitrogen is quick-frozen , stored at -80°C, extracted RNA, and carried out fluorescent quantitative PCR reaction according to the procedure of SYBR(R)ExScriptTM RT-PCR Kit. The amplified fragment of GmRAV1 is 144bp, and the amplified fragment of the soybean housekeeping gene Actin4 (AF049106) is 214bp. The primers are as follows: GmRAV1 sense primer: GGTGTAGTGGCATAGTGGC, antisense primer:

TAAGAAGGGGAGAAGCTAGA; Actin4 sense primer: GTGTCAGCCATACTGTCCCCATTT, antisense primer: GTTTCAAGCTCTTGCTCGTAATCA. The results are shown in Figure 2 and Figure 3.

Example 3: Construction of plant expression vector pCAMBIA3301-GmRAV1, transformation of soybean cotyledon nodes by Agrobacterium-mediated transformation and transformation of Arabidopsis thaliana by tidbit infection method

cDNA was obtained by reverse transcription using soybean leaf transcribed RNA as a template, and the cDNA was used as a PCR reaction template to obtain the gene DNA sequence of the full-length ORF region. The complete DNA sequence of GmRAV1 transcription factor inferred from the soybean is 1757bp, and the ORF is 1155bp. Using Bgl II and BstE II as restriction sites, design cloning primers, and perform PCR reaction at 94°C for 5min; 38 cycles: 94°C for 30s , 58°C for 30s, 72°C for 1min; 72°C for 10min; PCR products were subjected to 1% agarose gel electrophoresis, DNA fragments were recovered according to the instructions of the OMEGA GelExtraction Kit, and TaKaRa’s pMD18-T vector was used for ligation. The molar ratio of the gene and the connection vector is 3:1, calculate the amount of the inserted gene fragment and the connection vector, and incubate at 16°C for 16h. Transform 5ul of the ligation product into 50ul E. coli competent TOP10 of a certain company, culture it upside down at 37°C for 12-16h, pick a single clone, shake the bacteria with LB liquid medium (50mg/L Kan), and preserve the strain, and the single clone The cultured bacteria were sent to BGI for sequencing to identify positive clones. Shake the Escherichia coli strains containing the positive clones, use OMEGA Plasmid MiniKit to extract the plasmid, use the restriction endonuclease Bgl II and BstE II of Fermentas Company according to the ratio of 1:1, and enzyme in the environment of Buffer O of Fermentas Company Cut the plasmid, and use the restriction endonuclease BglII and BstE II of Fermentas Company to digest the pCAMBIA3301 plasmid in the environment of Buffer O of Fermentas Company at the same time according to the ratio of 1:1, and perform 1% agarose gel electrophoresis on the digested fragment According to the instructions of the OMEGA Gel Extraction Kit, DNA fragments were recovered, and TaKaRa’s T4 ligase and corresponding T4 ligated 10X Buffer were used for connection, and the molar ratio of the target fragment of the inserted DNA to the DNA fragment of the vector was 3:1 or 1:1 The ratio of the ligation system was established, incubated at 16°C for 16 hours, and the ligation product was transformed into Escherichia coli. Transform 5ul of the ligation product into 50ul E. coli competent TOP10 of a certain company, culture it upside down at 37°C for 12-16h, pick a single clone, shake the bacteria with LB liquid medium (50mg/L Kan), and save the strain, use Fermentas company The restriction endonucleases Bgl II and BstE II were used in the ratio of 1:1, and positive clones were identified by double enzyme digestion and PCR in the environment of Buffer O of Fermentas Company. The cloning primers are as follows: GmRAV1 sense primer: AGATCTATGGATGCAATTAGTTGCC; antisense primer: GGTCACCCTACAAAAGCACCAATAAT. Prepare Agrobacterium EHA105 competent cells: pick fresh EHA105 single colony from YEP plate (25mg/L Rif, 50mg/L Str) and inoculate in the YEP liquid medium containing 50mg/L Str and 25mg/L Rif, 28 Cultivate overnight at 220 rpm for 24-36 hours with shaking, take 2 mL of overnight activated bacterial solution in the logarithmic growth phase, inoculate it in 50 mL of liquid YEP, and continue to cultivate the OD600 to about 0.4-0.6. Bath 30min, 4℃, centrifuge at 4000rpm for 10min, discard the supernatant, pre-cool 0.05MCaCl ₂ with 10mL of ice to suspend the cells, ice-bath for 30min, centrifuge at 4℃, 4000rpm for 10min, discard the supernatant, and pre-cool 0.05M with 2mL of ice Resuspend the bacteria in M CaCl ₂ , place the prepared competent cells on ice, and use them within 24 to 48 hours for the highest transformation efficiency, or dispense 100 μL per tube into sterile tubes, and add glycerol to make the final concentration 15 % and stored at -70°C. Freeze-thaw transformation of Agrobacterium: Take 2 μL of purified plasmid DNA, add it to a centrifuge tube containing 100 μL of Agrobacterium tumefaciens competent cells, mix gently, ice bath for 30 minutes, freeze in liquid nitrogen for 2 minutes, and quickly place at 37°C Heat shock in water bath for 5 minutes, then quickly ice bath for 2 minutes, add 500 μL of YEP antibiotic-free liquid culture based on 28°C, shake gently at 100 rpm for 3 to 5 hours to recover, take 50-200 μL of bacterial liquid from the culture and evenly spread it on On the surface of the YEP solid medium (50mg/L Str, 25mg/L Rif and 50mg/L Kan) of antibiotics, culture it upside down at 28°C for 1-2 days, and pick white single colonies on the YEP liquid medium (50mg/LStr , cultured in 25mg/L Rif and 50mg/L Kan), positive clones were identified by PCR. The obtained Agrobacterium pCAMBIA3301-GmRAV1 ( FIG. 4 ) vector transformant can be used for plant transformation.

Agrobacterium liquid preparation: Pick a single colony of Agrobacterium containing pCAMBIA3301-GmRAV1 recombinant plasmid and inoculate it in 5mL YEB liquid medium containing rifampicin 50mg/L, streptomycin 25mg/L, kanamycin 50mg/L , 28°C, 180rpm for 48h. Take 1ml of bacterial liquid and inoculate it into 50ml of fresh above-mentioned YEB liquid medium, shake and culture until the OD600 is 0.6-0.8, centrifuge the bacterial liquid in a centrifuge at 5000rpm for 15min, enrich the bacterial cells and reweight it with an equal amount of liquid CCM medium hanging.

Soybean Seed Sterilization: Chlorine Sterilization. Select the seeds that are plump and free of bacteria spots, put them into the desiccator of the fume hood, pour 96ml of sodium hypochlorite into the small beaker of the desiccator, quickly add 4ml of concentrated hydrochloric acid, and then quickly cover it tightly. Soybeans are sterilized in the chlorine gas generated by the reaction for 16 hours, then placed in the ultra-clean bench for 30 minutes of ventilation, and sealed for later use.

Soybean seed germination: plant the sterilized seeds hilum down in the germination medium (GM), plant 8-10 seeds per bottle, and germinate for 4-5 days at 23°C, 16h light/8h dark conditions.

Preparation of soybean cotyledon nodes: take out sterile seedlings, select fully germinated plants, remove the seed coat, cut off the hypocotyl at the lower end of the cotyledon at 5 mm, cut the two midlines along the midline of the hypocotyl, remove the true leaves, and obtain Cotyledonary node explants. , use a scalpel to make 3-5 cuts in the range of about 3 mm in diameter at the junction of cotyledons and hypocotyls. After this operation, two cotyledon node explants for transformation can be obtained from each sterile seedling.

Soybean infection and co-cultivation: Put the prepared explants into the infection solution at 100rpm, shake and culture for 30 minutes, take out the excess infection solution on the surface of the explants with sterile paper, and place them upside down on the co-cultivation medium , and cultured in the dark for 3 days.

Induction and screening of clustered soybean buds: Wash the dark-cultured cotyledon node explants with water and liquid bud induction medium respectively for 3 times, and dry the liquid on the surface of the cotyledon node with sterile paper to make it 45 from the surface of the medium. Bud induction was carried out on recovery medium by oblique insertion at an angle. The clustered buds induced for 7 days were transferred to the selection medium containing 8mg/LPPT, subcultured every 7 days, and selected for 20 days.

Preparation of soybean rootstock: Plant 50 species of Dongnong in vermiculite 5 days in advance, wait for the cotyledons to slightly expand the true leaves, select seedlings whose true leaves do not exceed 2/3 of the cotyledons, remove the true leaves completely with a scalpel, and then remove the true leaves along the stem The axis cuts a 0.8mm-10mm wound on the hypocotyl.

The preparation of soybean scion: the cluster buds through screening, remove dead tissue around, cut off vertically to the surface of cluster buds, the cluster buds are cut into the upper part of 3-7mm, the lower length of about 5-8mm has a wedge shape with complete vascular bundles.

Grafting of soybeans: put the scion vertically into the cut of the rootstock, the surface of the clustered buds is flush with the cotyledon node, wrap it with a sealing film, and transplant it into a plastic pot. Put the grafted plants into a deep box, cover with plastic wrap, seal it properly, and keep a low-temperature and high-humidity growth environment. After a week, when the seedlings have fully recovered, the film can be removed to harden the seedlings.

Identification of soybean transformants: plant the seeds harvested from grafted soybeans in the soil, observe whether their growth status is different from that of the control, and use a small amount method to extract the DNA of fresh leaves. According to the DNA sequence of the recombinant plasmid, finally select For the 35S promoter sequence and GmRAV1 gene sequence on the recombinant plasmid, design specific primers, use DNA as a template, and perform PCR amplification according to the instructions of EasyTaq DNA Polymerase. The primer sequences are as follows: sense primer: 5'TATCCTTCGCAAGACCCTTCCTC 3', antisense primer : 5'ACGACGAAGCCATAGTGGTTTGC3', product size 740bp.

Agrobacterium liquid preparation: Pick a single colony of Agrobacterium containing pCAMBIA3301-GmRAV1 recombinant plasmid and inoculate it in 5mL YEB liquid medium containing rifampicin 50mg/L, streptomycin 25mg/L, kanamycin 50mg/L , 28°C, 180rpm for 48h. Take 1ml of the bacterial solution and inoculate it into 50ml of the fresh YEB liquid medium mentioned above, shake and culture until the OD600 is 0.6-0.8, centrifuge the bacterial solution in a centrifuge at 5000rpm for 10min, discard the supernatant, and suspend the precipitate in half the volume of osmotic Medium (0.22% MS, 5% sucrose, 0.02% silwet L-77).

Sterilization of Arabidopsis seeds: 10% NaClO Sterilization method: select full Arabidopsis seeds after vernalization at 4°C for 3 days, put them in a 1.5EP tube, add 10% NaClO solution to it, and vortex for 7-10min , so that the seeds are fully in contact with the solution, fully disinfected, and then rinsed with sterile water 3 to 4 times.

Germination of Arabidopsis thaliana seeds: the sterilized seeds were planted in germination medium (MS) containing 2% sucrose, about 100 seeds were planted per plate, and germinated for 7 days at 22°C, 8h light/16h dark.

Seedling transplantation of Arabidopsis: Mix the soil and vermiculite at a ratio of 1:1, put it in a pot, water it from the bottom to make it fully soaked, and move the seedlings in the medium into it, taking care not to damage the roots. Wrap the Arabidopsis seedlings with plastic wrap, place them at 22°C, 16h light/8h dark conditions to grow for 7 days, and remove the plastic wrap.

Infection and transformation of Arabidopsis thaliana: tidbit infection method: pour the resuspended Agrobacterium solution containing the pCAMBIA3301-GmRAV1 recombinant plasmid into a small porcelain plate. The pot body for cultivating Arabidopsis thaliana was placed horizontally on the side of the porcelain plate, and the freshly white and unopened flowers were soaked in the suspension for 30 seconds. Take out the plants, place them horizontally on a plastic film, cover them with plastic wrap to maintain humidity, and cover the plastic wrap with a layer of opaque paper. Incubate in a constant temperature room for 24 hours in the dark. The next day, uncover the paper and plastic wrap, and culture vertically. After 4-5 days, continue to dip for one or two times according to the growth of Arabidopsis. Cultivate for 3 to 4 weeks. After the seeds mature, collect the seeds and store them in a desiccator.

Identification of Arabidopsis transformants: After vernalization of the infected Arabidopsis seeds at 4°C for 3 days, they were planted in germination medium MS (5 mg/L Kan), and cultured under light at 22°C±2°C for 7d-10d , select Arabidopsis thaliana in good growth state and dark green leaf color, move it to the soil, and cultivate it under long-day light at 22°C±2°C. After it grows up, use a small amount method to extract the DNA of its fresh leaves. Finally, select the 35S promoter sequence and GmRAV1 gene sequence on the recombinant plasmid, design specific primers, use DNA as a template, and perform PCR amplification according to the instructions of EasyTaq DNA Polymerase. The primer sequence is as follows: sense primer: 5'TATCCTTCGCAAGACCCTTCCTC 3', antisense primer: 5'ACGACGAAGCCATAGTGGTTTGC3', the product size is 740bp. Some transgenic soybeans were selected for semi-quantitative RT-PCR analysis. The results showed that the exogenous GmRAV1 gene in the GmRAV1-ox strain had been integrated into the soybean genome, and GmRAV1 was overexpressed in transgenic soybeans compared with non-transgenic soybeans at the transcriptional level. very strong.

Example 4: Construction of plant RNAi expression vector pJawoh l8-GmRAV1 and Agrobacterium-mediated transformation of soybean cotyledon nodes

According to the GmRAV1 gene sequence published on NCBI, select the gene sequence in the ORF to design the target fragment for interference, and use the full-length sequence of the GmRAV1 gene as a template to design primers. According to the Gateway technical specification, a 333bp specific interference fragment was amplified, and the entry clone was constructed by recombination through BP reaction, and then the entry vector was reacted with the RNAi expression vector pJawohl8 through LR to obtain the RNAi vector (destination vector) containing the target gene ). The conditions for PCR amplified fragments were 94°C for 5min; 38 cycles: 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 10min; PCR products were subjected to 1% agarose gel electrophoresis according to the instructions of the OMEGA Gel Extraction Kit To recover DNA fragments, determine the BP reaction system according to the Gateway technical specification, and carry out the BP reaction. Take 2 μL of the BP reaction product into a centrifuge tube, place it on ice, carefully add 100 μL of competent cells to it, and flick the bottom of the tube to mix well. Ice bath for 30min, heat shock in 42°C water bath for 90s (do not shake), immediately place on ice for 2min, add 200μL LB solution to the centrifuge tube, shake and culture at 37°C, 150rpm for 45min, place on ice, and take bacterial solution to coat Put it on the LB plate containing Amp (100mg/L), put it upright at 37°C for 1h until the bacterial solution is completely absorbed by the plate, and culture it upside down overnight for 12h-16h, pick a white single colony, and inoculate it in 5mL of the plate containing Amp (100mg/L). LB liquid medium, 37°C, 220rpm shaking culture for 12-16h, after the medium becomes turbid obviously, take 0.5mL bacterial liquid and add 0.5mL sterile 30% glycerol to a final concentration of 15%, and store the strain at -80°C. Take 2 μL of bacterial liquid for PCR amplification, and sequence the clones with amplified products and the correct fragment size. The positive clone with correct sequencing results was taken to extract the plasmid as the entry vector clone, and the concentration and purity of the plasmid were detected by ultraviolet spectrophotometer and agarose gel electrophoresis. Using GmRAV-attB-Fi and GmRAV-attB-Ri as primers and the transformed bacterial solution after BP reaction as a template, carry out PCR reaction, and take 5 μL of the amplified product for agarose electrophoresis detection. Determine the LR reaction system according to the Gateway technical specification, carry out the LR reaction, transform the reaction product according to the method of transforming Escherichia coli after the BP reaction, spread all the transformed cells evenly on the LB solid medium containing 50mg/mL ampicillin, and keep at 37°C After culturing for 12-16 hours, pick a single colony and inoculate it in LB liquid medium containing 50 mg/mL ampicillin for overnight culture, then take 2 μL of the bacterial liquid for PCR amplification, and sequence the clones with the amplified product and the correct fragment size. And carry out bacterial liquid PCR detection. The plant entry vector pDONR201-GmRAV plasmid was digested with SmaI, the plant expression vector pJawoh18-GmRAV plasmid (Figure 5) was digested with HindIII, and 6-10 μL of the product was detected by electrophoresis. The positive clones were sent to Shanghai Yingjun Company for sequencing. The primer sequence for amplifying the interference fragment is as follows: sense primer: GmRAV-attB-Fi 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTACGCCGTCACCAACTTCA-3', antisense primer: GmRAV-attB-Ri

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGTGCTGCTTCGGTATCACTAA-3'. Prepare Agrobacterium EHA105 Competent Cells: Pick fresh EHA105 single colonies from the YEP plate (50mg/L Str and 25mg/L Rif) and inoculate them in the YEP liquid medium containing 50mg/L Str and 25mg/L Rif, 28 Cultivate overnight at 220 rpm for 24-36 hours with shaking, take 2 mL of overnight activated bacterial solution in the logarithmic growth phase, inoculate it in 50 mL of liquid YEP, and continue to cultivate the OD600 to about 0.4-0.6. Bath 30min, 4℃, centrifuge at 4000rpm for 10min, discard the supernatant, pre-cool 0.05M CaCl2 suspended cells with 10mL ice, ice-bath for 30min, centrifuge at 4℃, 4000rpm for 10min, discard the supernatant, pre-cool 0.05M with 2mL ice Resuspend the bacteria in MCaCl2, place the prepared competent cells on ice, and use them within 24-48 hours for the highest transformation efficiency, or dispense 100 μL per tube into sterile tubes, add glycerol to make the final concentration 15% Store at -70°C. Freeze-thaw transformation of Agrobacterium: Take 2 μL of purified plasmid DNA, add it to a centrifuge tube containing 100 μL of Agrobacterium tumefaciens competent cells, mix gently, ice bath for 30 minutes, freeze in liquid nitrogen for 2 minutes, and quickly place at 37°C Heat shock in water bath for 5 minutes, then quickly ice bath for 2 minutes, add 500μL YEP liquid culture without antibiotics based on 28℃, shake gently at 100rpm and cultivate for 3-5h to recover, take 50-200μL bacterial liquid from the culture and spread evenly on the medium containing appropriate antibiotics On the surface of the YEP solid medium (50mg/L Str, 25mg/L Rif and 100mg/L Amp), culture it upside down at 28°C for 1-2 days, pick white single colonies on the YEP liquid medium (50mg/L Str , 25mg/L Rif and 100mg/L Amp), and positive clones were identified by PCR. The obtained Agrobacterium pJawoh18-GmRAV1 vector transformant can be used for plant transformation.

Agrobacterium liquid preparation: Pick a single colony of Agrobacterium containing the pJawoh18-GmRAV1 recombinant plasmid and inoculate it in 5mL YEB liquid medium containing rifampicin 50mg/L, streptomycin 25mg/L, and ampicillin 100mg/L , 28°C, 180rpm for 48h. Take 1ml of bacterial liquid and inoculate it into 50ml of fresh above-mentioned YEB liquid medium, shake and culture until the OD600 is 0.6-0.8, centrifuge the bacterial liquid in a centrifuge at 5000rpm for 15min, enrich the bacterial cells and reweight it with an equal amount of liquid CCM medium hanging.

The soybean conversion method is specifically shown in Example 3

Identification of soybean transformants: plant the seeds harvested from grafted soybeans in the soil, observe whether their growth status is different from that of the control, and use a small amount method to extract the DNA of fresh leaves. According to the DNA sequence of the recombinant plasmid, design a specific Sexual primers, using DNA as a template, carried out PCR amplification according to the instructions of EasyTaq DNA Polymerase, the primer sequences are as follows: Pat sense primer: 5′-GCACCATCGTCAACCACTAC-3′, antisense primer: 5′-TGAAGTCCAGCTGCCAGAAAC-3′, the product size is 440bp . Some transgenic soybeans were selected for semi-quantitative RT-PCR analysis. The results showed that the exogenous gene GmRAV1 in the GmRAV-i strain had been integrated into the soybean genome. Compared with non-transgenic soybeans, the expression of GmRAV1 was suppressed at the transcriptional level, and the signal was very weak. .

Example 5: Phenotype observation and functional analysis of transgenic GmRAV1 gene overexpression soybean and transgenic GmRAV-i interference soybean

After mixing vermiculite with soil at a ratio of 1:1, transgenic GmRAV1 gene overexpressed soybeans, transgenic GmRAV-i interfering soybeans, and control soybeans DN50 were planted in it, 16h light/8h dark, and grown at 25°C. Observe the transgenic soybeans and control soybeans Compared, there were differences in plant height, leaf color, number of nodes, number of pods, internode distance and flowering time of the plants (Fig. 6).

Example 6: Functional analysis of Arabidopsis thaliana transgenic GmRAV1 gene, mutant Arabidopsis rav, GmRAV1 gene recovery Arabidopsis GmRAV1-ox rav T4 generation

1GmRAV1 gene enhances the effect of cytokinin (2-ip)

Disinfection and germination of seeds: Columbia-0, rav, GmRAV1-ox, GmRAV1-ox rav 4 kinds of Arabidopsis seeds were vernalized at 4°C for 3 days, sterilized with 10% NaClO for 6-8 minutes by vortex shaking, and then sterile Wash 5 times with water, plant on MS medium containing 2% sucrose, plant about 100 grains per plate, culture vertically at 22°C and 16h light/8h dark photoperiod, and wait for germination.

Culture of callus: Add 2.2 μmol/L 2,4-D, 0.2 μmol/L KT, 2% sucrose, 0.59 g/L MES, 0.8% agar to the medium containing B5, pH 5.5. The roots of the four kinds of Arabidopsis thaliana with a seedling age of 8 days were removed from the head and tail of about 5 mm, and the remaining parts were cut into about 5 mm segments, and inoculated in callus culture medium, and each plate was inserted into more than 40 segments for explantation. Each Arabidopsis thaliana was replicated three times and cultured for 3 days at 22°C under a photoperiod of 16 h light/8 h dark.

Induction of adventitious buds: 3 days later, the callus was transferred to the adventitious bud induction medium containing B5, which was added with different concentrations of 2-ip (0, 1, 5, 10 μmol/L), 0.75 μmol/L IAA, 2 % sucrose, 0.59g/L MES, 0.8% agar, pH5.6. 10 explants of each type of Arabidopsis were inoculated in each plate, and the explants of 4 kinds of Arabidopsis were inoculated in each plate at the same time, with 5 replicates for each treatment, at 22°C and 16h light/8h Cultured under a dark photoperiod, waited for the germination of the explants, observed their differentiation, determined the optimum concentration of 2-ip and counted the regeneration efficiency of the four species of Arabidopsis at the optimum concentration. When the shoot induction medium does not contain 2-ip, the callus will not differentiate into shoot tissue. When the concentration of 2-ip is 1 μmol/L, adventitious shoots will appear. When the concentration of 2-ip is less than 5 μmol/L, the proposed The number of adventitious buds of A. thaliana WT increased with the increase of 2-ip concentration, and the color gradually turned green. The induction of adventitious buds was the best when the 2-ip concentration was 5 μmol/L. When the 2-ip concentration was 10 μmol/L The growth of Arabidopsis WT adventitious buds showed a downward trend, and the 2-ip concentration of 5μmol/L was determined to be the optimum concentration for inducing adventitious buds. Four different concentrations of 2-ip were selected for the regeneration experiment of adventitious buds in root explants of Arabidopsis thaliana, the regeneration of adventitious buds was observed in Figure 9a, and the regeneration rate was calculated (Table 1).

Table 1 Regeneration rate of three kinds of soybean after exogenous application of different concentrations of cytokinin ₆ -BA to B5 medium

2. GmRAV1 gene enhances the effect of cytokinin (6-BA)

Columbia-0, rav, GmRAV1-ox, GmRAV1-ox rav 4 kinds of Arabidopsis seeds were vernalized at 4°C for 3 days, sterilized with 10% NaClO for 6-8 minutes by vortex shaking, washed 5 times with sterile water, and planted in On the MS medium containing 2% sucrose, about 100 grains were planted on each plate, cultured vertically at 22°C under a photoperiod of 16h light/8h dark, and waited for germination. After germination, Arabidopsis thaliana was transferred to MS solid medium containing 0, 0.5, and 1 mg/L 6-BA, cultured vertically at 22°C and under a photoperiod of 16h light/8h dark, and the root length was counted after 6 days, and photographed ( Figure 10a and b). Under 6-BA treatment of 0.5mg/L and 1.0mg/L, the rav root inhibition was not significant compared with the control, the RAV1ox root inhibition was significant compared with the control, and the GmRAV1-ox rav root inhibition was not significant compared with the control; This indicated that the mutant rav was not sensitive to 6-BA inhibition of root elongation, and further indicated that the GmRAV1 gene was a positive regulator of 6-BA inhibition of root elongation.

3. GmRAV1-ox gene is insensitive to the effect of epiBL

Columbia-0, rav, and GmRAV1-ox Arabidopsis seeds were vernalized at 4°C for 3 days, sterilized by vortex shaking with 10% NaClO for 6-8 minutes, washed 5 times with sterile water, and planted in a container containing 2% sucrose. About 100 grains were planted on each plate on MS medium, cultured at 22°C and a photoperiod of 16h light/8h dark, and waited for germination. After germination, Arabidopsis thaliana was transferred to MS solid medium containing 0, 5, 10, 50mmol/L epiBL, and half of them were cultured vertically under the photoperiod of 22°C and 16h light/8h dark, and the other half were grown in the dark After 10 days, the hypocotyls were counted and photographed (Fig. 12(a)). With the appropriate increase of epiBL concentration, the hypocotyl elongation of WT Arabidopsis was longer than that without BR, and the optimal concentration was reached at 10mmol/L. When the concentration was increased to 50mmol/L, the The elongation effect becomes worse (Fig. 12(b)). Under 10mmol/L epiBL treatment, the rav hypocotyl promotion was significant compared with the control, but the GmRAV1-ox hypocotyl promotion was not significant compared with the control; it indicated that the mutant rav was sensitive to 6-BA inhibition of root elongation and was overexpressed GmRAV1-ox is ultra-insensitive. That is, by expressing GmRAV1, the plant type of the plant can be made to grow in a short direction, and by inhibiting or interfering with the expression of the GmRAV1 gene, at the same time, 10 mmol/L of epi-brassinolide epiBL can be used to promote the plant's plant type to grow in a tall direction.

Example 7: Functional Analysis of GmRAV1 Gene-transferred Soybeans, Trans-GmRAV-i Gene Interference Soybeans, and DN50 Control Soybeans

1GmRAV1 gene enhances the regeneration-promoting effect of cytokinin (6-BA)

Soybean seeds are sterilized, and the sterilized seeds are planted in germination medium (GM) with the hilum down, 8-10 seeds are planted in each bottle, and germinated at 23°C, 16h light/8h dark conditions for 4-5 days.

Preparation of soybean cotyledon nodes: take out sterile seedlings, select fully germinated plants, remove the seed coat, cut off the hypocotyl at the lower end of the cotyledon at 5 mm, cut the two midlines along the midline of the hypocotyl, remove the true leaves, and obtain Cotyledonary node explants. , use a scalpel to make 3-5 cuts in the range of about 3 mm in diameter at the junction of cotyledons and hypocotyls. After this operation, two cotyledon node explants for transformation can be obtained from each sterile seedling.

The cultivation of adventitious buds: Cut the cotyledon nodes of soybean 7 days after germination, transfer them directly to the bud induction medium, carry out bud cultivation, and observe their differentiation. The bud induction medium used B5 as the basic medium, added 5 different concentrations of 6-BA (0, 0.0835, 0.167, 1.67, 3.34mg·L ^-1 ), 3% sucrose, 0.59g·L-1MES, 8% Agar, pH 5.6. Each treatment was replicated 3 times, and 10 pieces of callus were inoculated for each replicate. The soybean callus after 7-10 days of regeneration was taken, the regeneration of adventitious buds was observed, and the regeneration efficiency was counted (Fig. 9(b) and Table 2).

Table 2 Regeneration rates of four species of Arabidopsis after exogenously applying different concentrations of cytokinin 2-ip to MS medium

2GmRAV1 gene enhances the effect of cytokinin (6-BA) on root and hypocotyl inhibition

Disinfection and sowing of seeds: Select plump and spot-free seeds, put them into the desiccator of the fume hood, pour 96ml of sodium hypochlorite into the small beaker of the desiccator, quickly add 4ml of concentrated hydrochloric acid, and then quickly cover it tightly. Soybeans are sterilized in the chlorine gas generated by the reaction for 16 hours, then placed in the ultra-clean bench for 30 minutes of ventilation, and sealed for later use. Sow the sterilized seeds hilum down in the germination medium (GM), plant 8-10 seeds per bottle, and germinate for 1-2 days at 23°C, 16h light/8h dark conditions.

Transfer germinated soybeans to media containing different concentrations of 6-BA: take out the sterile seedlings, select plants that have germinated sufficiently, transfer them to media containing different concentrations of 6-BA, and observe their growth. The medium used B5 as the basic medium, added three different concentrations of 6-BA (0, 0.5, 1.0mg·L-1), 3% sucrose, 0.59g·L-1MES, 8% agar, pH 5.6. Each treatment was replicated 3 times, and each replicate was inoculated with 20 soybeans.

Observation of the growth of roots and hypocotyls: observe the growth of soybean roots and hypocotyls after 7 days of growth, and count the lengths of hypocotyls and roots (Fig. 10(c), (d) and (e).

Example 8: Real-time RT-PCR Analysis of GmRAV1 Gene Expression in DN42 Soybeans Exogenously Applied with 6-BA (100 μmol 6-BA) and Without 6-BA (0 μmol 6-BA)

Soybean DN42 was cultivated in a light incubator at 25°C, 250 μmol m-2sec-1 white light, long-day light (16h/8h light/dark) (LD) and grown in vermiculite until the first three compound leaves were fully developed To quickly remove residual vermiculite from soybean roots, put some soybean roots in 1/4MS aqueous solution containing 100μmol 6-BA, and place the other part of soybean roots in 1/4MS aqueous solution without 6-BA At 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after transfer, the leaves were taken at the same time, quick-frozen in liquid nitrogen, stored at -80°C, and RNA was extracted, according to SYBR(R)ExScriptTM The program of RT-PCR Kit carries out the fluorescent quantitative PCR reaction. GmRAV1 amplified fragment length is 144bp, soybean housekeeping gene Actin4 (AF049106) amplified fragment length is 214bp, the primers are as follows: GmRAV1 sense primer: GGTGTAGTGGCATAGTGGC, antisense primer: TAAGAAGGGGAGAAGCTAGA; Actin4 sense primer: GTGTCAGCCATACTGTCCCCATTT, antisense primer: GTTTCAAGCTCTTGCTCGTAATCA .

Real-time RT-PCR analysis of the expression of GmRAV1 gene in DN42 soybeans exogenously applied with 6-BA (100 μmol 6-BA) and without 6-BA (0 μmol 6-BA) is shown in Figure 11 .

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore Therefore, the protection scope of the present invention should be defined by the claims.

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Claims (10)

1. application of the Semen sojae atricolor GmRAV1 genes in control plant growth period and growth course；The GmRAV1 genes, nucleotide Sequence is as shown in SEQ ID NO.1.

2. apply according to claim 1, it is characterised in that the application during control plant photoperiod sensitivity.

3. apply according to claim 2, it is characterised in that be by GmRAV1 gene transferred plant cells after, by silence The photoperiod sensitivity expressed to adjust control plant of GmRAV1 genes.

4. apply according to claim 3, it is characterised in that step is as follows：

1) expression vector is interfered to be connected with plant RNA i the fragment of the coding region containing GmRAV1 genes or promoter sequence Connect, build plant RNA i expression vectors；

2) by step 1) obtained by plant RNA i expression vectors be transferred in plant cell, obtain plant RNA i expression vectors it is thin Born of the same parents；

3) screen step 2) in gained plant RNA i expression vectors cell in expression silencing transformed cells, cultivate and obtain Turn the plant of GmRAV1 genes.

5. apply according to claim 4, it is characterised in that the coding region of the GmRAV1 genes, nucleotides sequence sequence is 270-1421 positions in SEQ ID NO.1；The promoter, is CaMSV35 promoteres.

6. apply according to claim 1, it is characterised in that the application during plant adventitious shoot regeneration ability is strengthened.

7. apply according to claim 6, it is characterised in that by GmRAV1 gene transferred plant cells, by overexpression GmRAV1 genes are strengthening the adventitious bud energy for growth of plant.

8. apply according to claim 7, it is characterised in that step is as follows：

1) coding region sequence containing GmRAV1 genes and expression vector are attached, build plant expression vector；

2) by step 1) obtained by plant expression vector be transferred in plant cell, obtain the plant containing plant expression vector it is thin Born of the same parents；

3) screen step 2) obtained by plant cell, cultivate and obtain the plant of overexpression GmRAV1 genes.

9. apply according to claim 1, it is characterised in that the application during control plant plant type.

10. apply according to claim 9, it is characterised in that step is as follows：

1) GmRAV1 genes are transferred to obtain in plant and turn GmRAV1 gene plants；

2) usage amount by adjusting the expression and Epibrassinolide epiBL of GmRAV1 genes adjusts the plant type of plant.