Wild soybean water channel protein GsPIP1-4 and its encoding gene and application

Abstract

The present invention relates to the field of biotechnology, and more particularly to a wild soybean water channel protein GsPIP1-4, its encoding gene, and its application. The present invention discloses a wild soybean water channel protein GsPIP1-4, wherein the amino acid sequence of the protein GsPIP1-4 is shown in SEQ ID NO. 1; the nucleotide sequence of the gene encoding the wild soybean water channel protein GsPIP1-4 is shown in SEQ ID NO. 2. The encoding gene is used to improve the drought resistance of plants.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to wild soybean water channel protein GsPIP1-4 and its encoding gene and application. Background Art

[0002] Drought is a significant abiotic environmental stressor affecting plant growth and development. With economic development, population growth, and the intensification of the global greenhouse effect, water shortages are becoming increasingly severe, directly leading to the expansion of drought-prone areas and the worsening of drought conditions. Compared to other crops, soybeans have a high transpiration coefficient and a high water requirement, making them sensitive to water shortages. Drought severely impacts their growth, development, yield, and quality. Therefore, the development of drought-tolerant soybean varieties is crucial. The rapid advancement of molecular biology over the past 20 years, including the cloning of numerous drought-resistant genes, the maturity of Agrobacterium-mediated soybean transformation systems, and the growing acceptance of genetically modified crops, has provided attractive prospects for the breeding of drought-tolerant soybean transgenics.

[0003] Plant aquaporins (AQPs) belong to a family of membrane proteins that transport water and control the rapid movement of water molecules across membranes. Studies have shown that the expression levels of AQP genes are closely correlated with abiotic stress in plants. PIPs are the most common type of AQP found in plants, located on the plasma membrane. PIPs are not only selective channels for water and small neutral molecules but also possess numerous physiological functions, making them multifunctional proteins (Li Bing et al., 2006). Numerous reports have shown that the expression levels of PIP genes are closely correlated with plant growth and development at different stages, various environmental stresses, and responses to hormone signals (Zhang Lili et al., 2017). PIPs can alter root water permeability and participate in stomatal movement and photosynthesis in leaves. Overexpression of HvPIP2;1 in barley not only increases plant permeability to water and carbon dioxide but also promotes water and carbon dioxide exchange within the plant, ultimately enhancing photosynthesis (Hanba et al., 2004). PIPs can also transport small molecules and gases. For example, Gao et al. (2010) found that the TaNIP gene promotes the flow of Na+ from the cytoplasm to the extracellular matrix and increases K+ and Ca2+ levels in tissues, thereby effectively improving plant stress tolerance. Real-time fluorescence quantitative PCR and immunohistochemistry showed that the expression levels of maize ZmPIPs, except for ZmPIP2;7, were much higher in the growing zone of leaves than in raw and mature leaves, suggesting that these proteins may be involved in radial water transport within leaves, particularly in vascular bundles and mesophyll tissues (Hachez et al., 2008). Zhuang et al. (2015) found that transgenic Arabidopsis plants overexpressing FaPIP2;1 maintained higher leaf relative water content, chlorophyll content, net photosynthetic rate, and lower leaf membrane permeability under drought conditions, improving drought tolerance.

[0004] Wild soybean (Glycine soja L.), the ancestor of cultivated soybean (Glycine max L.), is native to East Asia and has a wide geographical distribution, from eastern Russia to southern China, growing in a variety of ecological environments. Compared to cultivated soybean, wild soybean possesses richer genetic diversity. Furthermore, during the process of domestication and utilization, humans have lost many of its beneficial traits, such as drought resistance, salt tolerance, aphid resistance, and cyst nematode resistance. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a wild soybean water channel protein GsPIP1-4 and its encoding gene, and to transfer the gene into cultivated soybean varieties through transgenic technology to improve the drought resistance of soybean plants, thereby increasing soybean yield.

[0006] In order to solve the above technical problems, the present invention provides a wild soybean water channel protein GsPIP1-4, the amino acid sequence of the protein GsPIP1-4 is shown in SEQ ID NO.1.

[0007] The present invention also provides the coding gene of the wild soybean water channel protein GsPIP1-4, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The present invention also provides an expression vector containing the above coding gene.

[0009] As an improvement of the expression vector of the present invention: the Agrobacterium recombinant expression vector pPIP1-4, which is obtained by cloning the GsPIP1-4 gene shown in SEQ ID NO.2 into the expression vector pLM-B001 and transferring it into Agrobacterium tumefaciens EHA101.

[0010] The present invention also provides the use of the above encoding gene for improving the drought resistance of plants (soybeans).

[0011] As an improvement of the use of the encoding gene of the present invention: cultivating drought-tolerant soybeans.

[0012] GsPIP1-4 is a water channel protein gene cloned from wild soybean. Studies have shown that when the relevant water channel protein gene is transferred into soybean, the drought resistance is significantly enhanced.

[0013] The coding gene of the present invention is cloned from wild soybean by the following ideas.

[0014] (1) Wild soybean (Glycine soja L.) is the ancestor of cultivated soybean (Glycine max L.). Cultivated soybean evolved from wild soybean through artificial natural selection. Compared with cultivated soybean (Tianlong No. 1), wild soybean (Tonglu wild soybean) shows obvious drought resistance;

[0015] (2) In wild soybean, the expression of GsPIP1-4 genes increased with the extension of drought treatment time;

[0016] (3) In wild soybean, the expression of GsPIP1-4 genes increased with the extension of ABA treatment time;

[0017] (4) Using wild soybean cDNA as a template, primers were designed based on the full-length sequence of GsPIP1-4 gene CDs for PCR amplification, and finally the base sequence of GsPIP1-4 gene SEQ ID NO.2 was obtained.

[0018] (5) Bioinformatics analysis of the nucleotide sequence showed that the GsPIP1-4 genes, as shown in SEQ ID NO. 2, have a high degree of similarity with the PIP genes of cultivated soybean, Arabidopsis, rice, and tea.

[0019] The present invention also provides the use of the cloned wild soybean nucleotide sequence GsPIP1-4 gene shown in SEQ ID NO. 2 in cultivating drought-resistant plants.

[0020] Preferably, the plant is a new drought-tolerant soybean germplasm.

[0021] Specifically include:

[0022] (1) constructing a plant expression vector containing the encoding gene;

[0023] (2) The plant expression vector is transferred into soybean recipient material through the cotyledonary node method mediated by Agrobacterium tumefaciens, using glufosinate as a selection marker, and transgenic T0 generation soybean plants are obtained through co-cultivation, bud induction, bud elongation and bud rooting cultivation;

[0024] (3) T0 generation transgenic soybean plants were identified and self-pollinated to obtain T1 generation seeds;

[0025] (4) After cultivation and screening of T1 generation seeds, a new transgenic drought-tolerant soybean germplasm of GsPIP1-4 was obtained.

[0026] (5) T1 transgenic drought-tolerant soybeans transformed with GsPIP1-4 were exposed to 20% PEG-simulated drought conditions, confirming the drought resistance function of the GsPIP1-4 gene.

[0027] In step (1), when constructing the recombinant expression vector, the original vector used is pLM-B001, and the nucleotide sequence of the encoding gene GsPIP1-4 is shown in SEQ ID NO.2.

[0028] In step (2), the Agrobacterium is Agrobacterium tumefaciens EHA105, and the soybean recipient material is the cotyledonary node of the soybean variety Tianlong No. 1.

[0029] In steps (3) and (4), the identification and screening methods used are three of the following: glufosinate smear method, Bar test strip detection method and PCR identification method.

[0030] The present invention has the following technical advantages:

[0031] The present invention cloned aquaporin genes from wild soybeans and introduced the GsPIP1-4 genes into soybeans using Agrobacterium-mediated transfection. Experiments showed that overexpression of GsPIP1-4 enhanced water absorption by soybean roots, reduced water loss from soybean leaves, and increased net photosynthetic rate, transpiration rate, and stomatal conductance, potentially increasing yield. Furthermore, transgenic soybeans can improve drought resistance by increasing antioxidant enzyme activity and proline content. Therefore, the GsPIP1-4 gene can be used to cultivate new drought-tolerant plant germplasm.

[0032] In summary, the present invention transfers the gene into cultivated soybean varieties through Agrobacterium-mediated transformation technology, clarifies the drought resistance function of the gene in cultivated soybeans, enhances the drought tolerance of soybeans, and thus increases soybean yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 The growth status of cultivated soybean (A: drought-sensitive variety Fendou 93, B: drought-tolerant variety Tiefeng 31) and Tonglu wild soybean (C) after 3 days of simulated drought with 2.5 M PEG 8000 (osmotic potential -0.54 MPa);

[0035] The left picture in A shows Fendou 93 cultured normally for 3 days, and the right picture shows Fendou 93 cultured after 3 days of simulated drought.

[0036] The left picture in B shows Tiefeng 31 cultured normally for 3 days, and the right picture shows Tiefeng 31 cultured after 3 days of simulated drought;

[0037] The left picture in C shows the Tonglu wild soybean cultivated normally for 3 days, and the right picture shows the Tonglu wild soybean cultivated after 3 days of simulated drought.

[0038] Figure 2 The expression of GsPIP1-4 genes in wild soybean leaves under drought stress (A) and ABA (B) treatments;

[0039] Figure 3 is the amino acid sequence alignment of GsPIP1-4 proteins;

[0040] Figure 4 This is the phylogenetic tree of the multiple sequence alignment of the GsPIP gene;

[0041] Figure 5 For the subcellular localization of GsPIP-4 gene;

[0042] A: Green fluorescence signal of empty load control;

[0043] B: Image of the plasma membrane localization marker fused with GFP under red light;

[0044] C: Image of tobacco epidermal cells under white light;

[0045] D: Overlapping graph of A, B, and C;

[0046] E: Localization image of 35s::GsPIP1-4-mGFP under green fluorescence;

[0047] F. 35s::GsPIP1-4-mGFP images observed under red fluorescence;

[0048] G: 35s::GsPIP1-4-mGFP tobacco epidermis image under white light

[0049] H: Overlay of E and G.

[0050] Figure 6 The recombinant expression vector pPIP1-4 is used to transfer the GsPIP1-4 gene.

[0051] Figure 7 These are the results of the glufosinate smear test on T1 generation transgenic soybean plants bearing the GsPIP1-4 gene. Leaves 1, 2, 5, 6, 12, 13, and 15 are seven independent transgenic-positive plant leaves.

[0052] Figure 8 These are the Bar test results of T1 generation transgenic soybean plants with GsPIP1-4 genes, where 1, 2, 5, 6, 12, 13 and 15 are 7 independent transgenic positive plants.

[0053] Figure 9 These are the PCR test results of T0 generation transgenic soybean plants carrying the GsPIP1-4 genes, of which 1, 2, 5, 6, 12, 13 and 15 are 7 independent transgenic positive plants.

[0054] Figures 7 to 9 Plants 8, 16-18 died during their growth and could not be sampled for PCR testing in time, so only the initial Bar test strip test results and some glufosinate smear test results were available.

[0055] Figure 10 Growth of Tianlong No. 1 and transgenic plants after 2-day treatment with 20% PEG and 5 days after rehydration.

[0056] Figure 11 Survival of transgenic and non-transgenic soybeans grown in sand culture after natural wilting and rehydration. A: control variety Tianlong No. 1; B: transgenic line L12; C: transgenic line L15; D: drought-resistant variety Tiefeng 31. DETAILED DESCRIPTION

[0057] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0058] The methods described in the following examples are all conventional methods unless otherwise specified.

[0059] Wild soybean complete genome sequence:

[0060] Glycine soja cultivar W05 unplaced genomic scaffold scaffold1918,whole genome shotgun sequence;

[0061] GenBank:KN651006.1

[0062] GenBank Graphics

[0063] >KN651006.1:c158272-157948,c156900-156605,c156384-156244,c155623-155531Glycine soja cultivar W05 unplaced genomic scaffold scaffold1918,wholegenome shotgun sequence.

[0064] Example 1

[0065] 1. Cloning, bioinformatics analysis, and gene mapping of GsPIP1-4 genes

[0066] The following three cultivated soybeans: A, the drought-sensitive variety Fendou 93, B, the drought-tolerant variety Tiefeng 31, and C, the Tonglu wild soybean were subjected to the following simulated drought treatment experiment (conventional technology):

[0067] Drought simulation was performed in sand culture germination boxes using 2.5M PEG-8000 (osmotic potential -0.54 MPa). The boxes measured 19 × 13 × 12 cm and could hold 1.3 kg of river sand. The sand was first cleaned of impurities and washed with water, then sterilized with high temperature and high pressure and dried for later use. Soybean seeds were surface-sterilized using chlorine gas drying and then directly sown in the germination boxes. Each box contained 20 seeds. After germination and the first two leaves had unfolded, the seedlings were transplanted to 10 plants per box. When the first three leaves emerged, each box was watered with 100 ml of 1 / 2 Hogland nutrient solution containing 2.5M PEG 8000 (osmotic potential -0.54 MPa) to simulate drought. A control was treated with 100 ml of 1 / 2 Hogland nutrient solution. Plant morphology was observed and compared after three days.

[0068] Day 3 findings:

[0069] The drought-sensitive variety Fendou 93 plants wilted and their leaves dried up; the control plants grew normally with green leaves.

[0070] The leaves of the drought-tolerant variety Tiefeng 31 drooped and wilted; the control grew normally with tender green leaves;

[0071] There was no significant difference between Tonglu wild soybeans treated with drought and the control, and both grew normally.

[0072] Therefore, the above experimental results show that wild soybean showed obvious drought resistance after 3-day simulated drought treatment with 2.5M PEG 8000 (osmotic potential -0.54MPa) ( Figure 1 C).

[0073] Wild soybeans grown normally for 11 days were treated with 2.5M PEG-8000 and 100μmol / L ABA, respectively. Samples were collected from different tissues before PEG or ABA treatment and 2h, 6h, 12h, 24h, and 48h after treatment, and RNA was extracted for quantitative expression analysis of GsPIP1-4.

[0074] The results showed that after PEG treatment, expression levels initially increased and then decreased, reaching 4.8 times that of the untreated level after 12 hours of treatment. Within a short period of ABA treatment (2 and 6 hours), GsPIP1-4 expression was inhibited, decreasing by approximately 50%. After 12 hours, expression levels continued to rise, reaching a maximum of 8.3-fold after 2 days of treatment. This indicates that GsPIP1-4 gene expression responds significantly to both PEG and ABA treatments, suggesting that GsPIP1-4 may play an important role in drought stress.

[0075] Specific as Figure 2 The expression of GsPIP1-4 genes first increased and then decreased with the extension of drought treatment time ( Figure 2 A); with the extension of ABA treatment time, the expression of Figure 2 B), based on this, it is inferred that the GsPIP1-4 genes may be related to drought tolerance.

[0076] Using wild soybean (Tonglu wild soybean) cDNA as a template, primers were designed based on the full-length sequence of GsPIP1-4 gene CDs for PCR amplification, and finally the base sequence of GsPIP1-4 gene SEQ ID NO.2 was obtained.

[0077] Full-length sequences of GsPIP1-4 gene CDs

[0078] Bioinformatics analysis of homology multiple sequence alignment showed that the sequence homology between soybean GsPIP1-4 genes and the proteins encoded by cultivated soybean, rice, and Arabidopsis GsPIP1-4 genes was high, reflecting that the protein sequence of GsPIP1-4 genes was highly conserved ( Figure 3 ), by the phylogenetic tree ( Figure 4 ) It can be seen that the wild soybean GsPIP1-4 genes are closely related to the PIP genes of cultivated soybean, Arabidopsis, rice, and tea.

[0079] An expression vector was constructed based on the method of fusion GFP reporter gene localization. The green fluorescence characteristics of the GFP expression product were used to locate the target protein and the subcellular localization of the GsPIP-4 gene was performed as follows:

[0080] (1) Tobacco cultivation: Sow a number of tobacco seeds and culture them in a light incubator for 12 hours. After growing for one month, they can be used for experiments.

[0081] (2) Agrobacterium culture: Prepare 35s::mGFP and 35s::GsPIP1-4-mGFP vectors; transform the constructed vector plasmids into Agrobacterium (GV3101) by electroporation and culture at 30°C for 2 days.

[0082] (3) Suspending Agrobacterium: Use an inoculation loop to scrape the Agrobacterium from the solid culture dish and inoculate it into 10 ml of YEB liquid culture medium. Incubate at 170 rpm / min for 1 hour.

[0083] (4) Collecting bacteria: Centrifuge the suspended Agrobacterium at 4000 rpm / min for 4 min and remove the supernatant.

[0084] (5) Resuspension: Resuspend the cells in 10 mM MgCl2 (containing 120 μM AS) suspension and adjust the OD600 to about 0.6.

[0085] (6) Injection: Select tobacco plants with good growth conditions and use a 1 mL syringe with a pipette tip to inject into the lower epidermis of the tobacco leaves and mark them.

[0086] (7) Cultivation: Cultivate the injected tobacco plants under weak light for 2 days and then observe them.

[0087] (8) Observation: Take the labeled tobacco leaves injected with Agrobacterium, make them into slides, observe them under a laser confocal microscope, and take pictures.

[0088] The results are as follows Figure 5 Gene localization analysis showed that the gene was located on the cytoplasmic membrane.

[0089] 2. Acquisition and identification of transgenic soybeans with GsPIP1-4 genes

[0090] In this experiment, the soybean variety Tianlong No. 1, which was developed by the Oil Crops Institute of the Chinese Academy of Agricultural Sciences and has been widely promoted and applied in production, was used as the transformation receptor material.

[0091] The vector of this experiment is the Agrobacterium recombinant expression vector pPIP1-4 ( Figure 6 ) The GsPIP1-4 genes were directly cloned into the expression vector pLM-B001, which contains a promoter and terminator, using multiple cloning restriction sites. After passing the test, the plasmid DNA was transformed into Agrobacterium tumefaciens EHA101 to obtain the plasmid. Conventional Agrobacterium-mediated transformation methods can be used. The expression vector pLM-B001 is derived from pTF101.1 (Paz MM et al., 2006), with the promoter and terminator for expressing the target gene added to pTF101.1.

[0092] Paz MM, Matinez JC, Kalvig AB, Fonger Tm, Wang K (2006) Improved cotyledonary node method using an alternative explants derived from matureseed for efficient Agrobacterium-mediated soybean transformation. Plant CellReports, 25:206–213.

[0093] The Agrobacterium-mediated transgenic method used in this experiment was the aforementioned Agrobacterium-mediated soybean cotyledonary node transformation system. Mature soybean seeds were sterilized, germinated, isolated from explants, infected with Agrobacterium, co-cultivated, bud induction, bud elongation, rooting, and greenhouse acclimation of tissue culture seedlings to obtain T0 generation transgenic soybeans. The T0 generation was treated with glufosinate ( Figure 7 )、Bar test strips( Figure 8 ) and target gene PCR ( Figure 9 ) Rapid identification;

[0094] The identification methods are as follows:

[0095] The experimental method of glufosinate application is as follows: prepare the glufosinate stock solution to a concentration of 135mg / L, apply the diluted herbicide solution to half a leaf with a cotton swab or brush, and then mark the half leaf without glufosinate. The soybean plants continue to grow in the greenhouse for about a week to observe the changes in the leaves. The results are as follows: Figure 7 , as follows: The leaves of plants 1, 2, 5, 6, 12, 13 and 15 coated with glufosinate did not turn yellow, indicating that the marker gene Bar for herbicide resistance to glufosinate has been transferred into these plants, and they are positive plants.

[0096] The experimental method of the Bar test strip is as follows: take a small amount of soybean leaves in a centrifuge tube, add a steel column and 250μl of extract, grind it in a grinder, insert the test strip into the centrifuge tube for about 5 minutes, and check the test strip results; the results are as follows Figure 8 , as follows: there are two red strips on test strips 1, 2, 5, 6, 12, 13 and 15, indicating that the plant has Bar protein and is judged to be a positive plant. Only one strip appears on the other test strips, indicating that these plants are negative plants.

[0097] Gene PCR is a method of amplifying the target gene transferred into the plant by PCR. The DNA of the plant leaves is extracted, and the DNA of the leaves is used as a template and the vector plasmid containing the target gene is used as a positive control. The target gene specific primers (the amplified fragment length is about 855bp) are used to identify whether the expression vector has been transferred into the soybean recipient. The results are as follows: Figure 9 , as follows: the target gene fragment can be amplified in plants 1, 2, 5, 6, 12, 13 and 15. The results are completely consistent with the test results of applying glufosinate on the leaves, indicating that the GsPIP-4 gene has been integrated into the soybean receptor.

[0098] Therefore, it can be concluded that 1, 2, 5, 6, 12, 13 and 15 are 7 independent GsPIP-4 gene-positive plants, while the remaining 11 plants are negative plants, indicating that the exogenous gene and Bar gene have not been transferred into the soybean recipient.

[0099] In the present invention, T1 generation seeds are obtained by self-pollination, and the T1 generation seeds are then verified to be GsPIP-4 gene-transformed plants for the identification of drought resistance.

[0100] 3. Identification of drought tolerance of transgenic GsPIP1-4 plants

[0101] Chlorine-dried T1 transgenic seeds (obtained from self-pollination of the T0 generation) and non-transgenic soybean seeds (i.e., Tianlong No. 1 seeds) were sown in sterilized sand and germinated in an artificial climate chamber. The roots of the soybean plants 4-5 days after germination were washed and transplanted into a hydroponic nutrient solution (1 / 2 Hoagland) for cultivation, and ventilation was maintained at all times.

[0102] Transgenic soybeans and wild-type soybeans (Tianlong No. 1) that had been hydroponically cultured for 11 days were treated with 20% PEG8000 for 2 days and then grown normally (i.e., cultured in an incubator at 25±1°C and a light intensity of 14h / 10h (day / night)) for 5 days. It was found that drought caused the fresh weight of the stems and leaves of the three transgenic lines L5, L12 and L15 (randomly selected) to decrease by 18.7%, 18.1% and 24.2%, respectively, while the wild-type soybean decreased by 47.5%, reaching a significant level; 20% PEG8000 simulated drought, the fresh weight of the roots of L5, L12 and L15 decreased by 15.8%, 16.9% and 13.0%, respectively, while the wild-type soybean decreased by 37.6% (Table 1).

[0103] Table 1. Reduction in plant height, root length, stem and leaf fresh weight, and root fresh weight of Tianlong No. 1 and transgenic strains after PEG-simulated drought treatment and rehydration

[0104]

[0105] To further clarify the drought resistance function of the GsPIP1-4 gene, the present invention conducted a dehydration and rehydration experiment using the non-transgenic soybean Tianlong No. 1, the drought-tolerant soybean variety "Tiefeng 31" (Wang Guifeng, 2019), and the transgenic GsPIP1-4 gene lines L12 and L15. These four lines were planted in sand culture and allowed to grow normally to the three-leaf stage. Watering was then stopped until the leaves wilted, and then rewatered. Two days later, the number of plants that revived and grew normally was counted. Statistics showed that the survival rates after rehydration for Tianlong No. 1, "Tiefeng 31," and transgenic lines L12 and L15 were 42.1%, 47.5%, 54.4%, and 53.7%, respectively. Therefore, it can be demonstrated that "Tiefeng 31" has better drought resistance than "Tianlong No. 1," and that transgenic GsPIP1-4 soybean plants have a better ability to recover growth after drought rehydration than "Tiefeng 31," showing a certain advantage in drought resistance. Details are shown in Table 2.

[0106] Table 2. Survival statistics of Tianlong No. 1, Tiefeng 31, and transgenic lines L12 and L15 after drought treatment and rewatering

[0107]

[0108] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention. Sequence Listing <110> Zhejiang University <120> Wild soybean water channel protein GsPIP1-4 and its encoding gene and application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 284 <212> PRT <213> Glycine L. (soybean) <400> 1 Met Glu Gly Lys Glu Glu Asp Val Arg Val Gly Ala Asn Arg Tyr Gly 1 5 10 15 Glu Arg Gln Pro Ile Gly Thr Ala Ala Gln Ala Lys Asp Tyr Arg Glu 20 25 30 Pro Pro Ser Ala Pro Leu Phe Glu Pro Gly Glu Leu Ser Ser Trp Ser 35 40 45 Phe Tyr Arg Ala Gly Ile Ala Glu Phe Val Ala Thr Phe Leu Phe Leu 50 55 60 Tyr Ile Thr Val Leu Thr Val Met Gly Val Phe Lys Ser Lys Ser Lys 65 70 75 80 Cys Ser Thr Val Gly Ile Gln Gly Ile Ala Trp Ala Phe Gly Gly Met 85 90 95 Ile Phe Ala Leu Val Tyr Ser Thr Ala Gly Ile Ser Gly Gly His Ile 100 105 110 Asn Pro Ala Val Thr Phe Gly Leu Phe Leu Ala Arg Lys Leu Ser Leu 115 120 125 Thr Arg Ala Ile Phe Tyr Ile Ile Met Gln Cys Leu Gly Ala Ile Cys 130 135 140 Gly Ala Gly Val Val Lys Gly Phe Glu Pro His Leu Tyr Glu Arg Leu 145 150 155 160 Gly Gly Gly Ala Asn Thr Ile Ala Lys Gly Tyr Thr Asn Ser Ala Gly 165 170 175 Leu Gly Ala Glu Ile Val Gly Thr Phe Val Leu Val Tyr Thr Val Phe 180 185 190 Ser Ala Thr Asp Ala Lys Arg Asn Ala Arg Asp Ser His Val Pro Ile 195 200 205 Leu Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Leu Val His Leu Ala 210 215 220 Thr Ile Pro Val Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Leu Gly 225 230 235 240 Ala Ala Ile Ile Phe Asn Lys Asp Gln Ala Trp Asp Asp His Trp Ile 245 250 255 Phe Trp Val Gly Pro Phe Ile Gly Ala Ala Leu Ala Ala Leu Tyr His 260 265 270 Gln Ile Val Ile Arg Ala Ile Pro Phe Ser Ser Lys 275 280 <210> 2 <211> 855 <212> DNA <213> Soybean (Glycine L.) <400> 2 atggagggaa aagaagagga cgtaagagtt ggagccaaca ggtacggaga gaggcagcca 60 atagggaccg ctgctcaggc taaagactac agagagccac cgtcggcgcc tctcttcgaa 120 ccgggagagt tgtcatcgtg gtctttctat agggctggca tagcagagtt tgtggccact 180 ttcttgttcc tctacatcac agtgctgact gtgatgggtg tgttcaaatc taagagcaag 240 tgttccactg tgggtatcca aggcattgct tgggcttttg ggggaatgat ctttgctctt 300 gtttattcca ctgctggaat ctcagggggt catattaacc cagcagtgac gtttgggctg 360 ttcttggcac gcaagctctc tctgacaagg gcaatttttt acataatcat gcagtgcttg 420 ggagctatat gtggtgctgg tgtagttaag gggttcgagc cacacctcta tgagaggctt 4​​​​​​ gtgcacttgg ctacaattcc tgttacaggg actggtatca accctgctag aagtctaggt 720 gcagccatta tcttcaacaa ggaccaagct tgggatgacc attggatatt ttgggttggg 780 cctttcattg gggcagcact tgcagctttg taccatcaga tagtgatcag ggccatcccc 840 ttctcgtcga agtga 855

Claims

1. The use of the gene encoding wild soybean water channel protein GsPIP1-4, characterized in that: Used to improve the drought resistance of plants; the plants are soybeans; it can reduce the loss of fresh weight of soybean stems and leaves caused by drought, reduce the loss of fresh weight of soybean roots caused by drought, reduce the shortening of soybean root length caused by drought, and improve the survival rate after rehydration; It can enhance water absorption by soybean roots, reduce water loss from soybean leaves, and increase soybean net photosynthetic rate, transpiration rate, and stomatal conductance, potentially increasing yield. Genetically modified soybeans can improve plant drought resistance by increasing antioxidant enzyme activity and proline content. The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The use of the coding gene according to claim 1, characterized in that: Cultivating drought-tolerant soybeans.

Images