CN112321690A - Wild soybean aquaporin GsPIP1-4 and coding gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a wild soybean aquaporin GsPIP1-4 and a coding gene and application thereof. The invention discloses a wild soybean aquaporin GsPIP1-4, wherein the amino acid sequence of the protein GsPIP1-4 is shown in SEQ ID NO. 1; the nucleotide sequence of the coding gene of the wild soybean aquaporin GsPIP1-4 is shown in SEQ ID NO. 2. The coding gene is used for improving the drought resistance of plants.

Description

Wild soybean aquaporin GsPIP1-4 and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a wild soybean aquaporin GsPIP1-4 and a coding gene and application thereof.

Background

Drought is an abiotic environmental stress factor which affects the growth and development of plants, and with the economic development of human beings, population growth and global greenhouse effect are aggravated, and the phenomenon of water resource shortage is more and more serious, which directly leads to the enlargement of drought regions and the aggravation of drought degree. Compared with other crops, the soybean has high transpiration coefficient and large water demand, is a crop which is sensitive to water shortage, and has serious influence on growth, development, yield and quality due to drought. Therefore, the cultivation of drought-resistant soybean varieties is particularly important. The molecular biology develops rapidly in the last 20 years, many genes related to drought resistance are cloned, the soybean agrobacterium-mediated transformation system is mature, and the identity of people to transgenic crops is increased, so that attractive prospects are provided for breeding of drought-resistant transgenic resistance of soybeans.

The Aquaporin (AQP) of the plant belongs to a membrane protein family for transporting water, controls the rapid movement of water molecules across membranes, and researches show that the expression level of aquaporin genes is closely related to abiotic stress of the plant. PIPs are the aquaporins located on plasma membranes which are most found in plants at present, and are not only water and neutral small molecule selective aquaporins, but also have many physiological functions, and are multifunctional proteins (leihousing et al, 2006). There have been many reports to date showing that the expression level of PIPS gene is closely related to the growth and development of plants at different stages, different kinds of environmental stress and hormone signal response process (Charlie et al, 2017). PIPs can cause the change of the permeability of root system water and also participate in the stomatal movement and photosynthesis of plant leaves, namely barley HvPIP 2; overexpression of 1 not only increases the plant's permeability to water and carbon dioxide, but also promotes the exchange of water and carbon dioxide in the plant, thereby ultimately enhancing photosynthesis (Hanba et al, 2004). Meanwhile, the PIPs can also transport small molecular substances and gases, for example, Gao et al (2010) find that the TaNIP gene promotes Na + to flow from cytoplasm to extracellular matrix, and the content of K + and Ca2+ in tissues is increased, so that the stress resistance of the plants is effectively improved. The real-time fluorescent quantitative PCR and immunohistochemical detection show that: except zmpi 2; the expression level of ZmPIPs of maize other than 7 in the growing region of leaves is much greater than that in the immature leaves, indicating that the protein may be involved in the radial transport of water in leaves, especially in vascular bundles and mesophyll tissues (Hachez et al, 2008). The Zhuang et al (2015) study found that arabidopsis overexpresses FaPIP 2; 1 under drought conditions, transgenic plants can keep higher relative water content of leaves, chlorophyll content, net photosynthetic rate and lower plasma membrane permeability of leaves, and improve the drought resistance of arabidopsis thaliana.

The wild soybean (Glycine soja L.) is an ancestor of cultivated soybean (Glycine max L.), is native to east Asia, is widely distributed geographically, is widely distributed from the east of Russia to the south of China, and can grow in various ecological environments. Compared with the wild soybean and the cultivated soybean, the wild soybean has abundant genetic diversity; in addition, in the process of domesticating and utilizing wild soybeans, human beings lose a lot of favorable characters of the wild soybeans, such as drought resistance, salt resistance, aphid resistance, cyst nematode resistance and the like of the wild soybeans.

Disclosure of Invention

The invention aims to solve the problem of providing a wild soybean aquaporin GsPIP1-4 and a coding gene thereof, and the gene is transferred into a cultivated soybean variety through a transgenic technology to improve the drought resistance of a soybean plant, thereby improving the yield of the soybean.

In order to solve the technical problem, the invention provides a wild soybean aquaporin GsPIP1-4, wherein the amino acid sequence of the protein GsPIP1-4 is shown in SEQ ID NO. 1.

The invention also provides a coding gene of the wild soybean aquaporin GsPIP1-4, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

The invention also provides an expression vector containing the coding gene.

As an improvement of the expression vector of the present invention: is an agrobacterium recombinant expression vector pPIP1-4, wherein the agrobacterium recombinant expression vector pPIP1-4 is obtained by cloning a GsPIP1-4 gene shown in SEQ ID NO.2 onto an expression vector pLM-B001 and transferring the gene into agrobacterium tumefaciens EHA 101.

The invention also provides the application of the coding gene, which is used for improving the drought resistance of plants (soybean).

Improvement as the use of the coding gene of the present invention: and (5) cultivating drought-enduring soybeans.

GsPIP1-4 is a aquaporin gene cloned from wild soybean, and research shows that the drought resistance is obviously enhanced by transferring the relevant aquaporin gene into the soybean.

The coding gene of the present invention is cloned from wild soybean by the following idea.

(1) The wild soybean (Glycine soja L.) is an ancestor of the cultivated soybean (Glycine max L.), the cultivated soybean is artificially and naturally selected and evolved from the wild soybean, and compared with the cultivated soybean (Tianlong I), the wild soybean (Elaeagnus elaeosporiformis L.) shows obvious drought resistance;

(2) in wild soybeans, the GsPIP1-4 gene has enhanced expression along with the prolonging of the drought treatment time;

(3) in wild soybeans, the GsPIP1-4 gene has enhanced expression along with the prolongation of ABA treatment time;

(4) wild soybean cDNA is used as a template, and a primer designed by a full-length sequence CDs of a GsPIP1-4 gene is used for PCR amplification, so that a base sequence SEQ ID NO.2 of the GsPIP1-4 gene is finally obtained.

(5) Nucleotide sequences are analyzed through bioinformatics, and the GsPIP1-4 gene shown in SEQ ID No.2 has high similarity with the PIP genes of cultivated soybeans, arabidopsis thaliana, rice and tea trees.

The invention also provides application of the cloned wild soybean nucleotide sequence GsPIP1-4 gene shown in SEQ ID NO.2 in cultivating drought-tolerant plants.

Preferably, the plant is a new germplasm for cultivating drought-tolerant soybeans.

The method specifically comprises the following steps:

(1) constructing a plant expression vector containing the coding gene;

(2) transferring the plant expression vector into a soybean receptor material by an agrobacterium tumefaciens mediated cotyledonary node method, and obtaining a transgenic T0 generation soybean plant by adopting glufosinate-ammonium as a screening marker through co-culture, bud induction, bud elongation and bud rooting culture;

(3) identifying transgenic soybean plants of T0 generation, and self-pollinating to obtain seeds of T1 generation;

(4) and culturing and screening the T1 generation seeds to obtain a novel GsPIP1-4 transgenic drought-tolerant soybean germplasm.

(5) T1 GsPIP1-4 transgenic drought-tolerant soybeans prove the drought-resistant function of the GsPIP1-4 gene under the condition of 20% PEG simulated drought.

In the step (1), when the recombinant expression vector is constructed, the original vector adopted is pLM-B001, and the nucleotide sequence of the coding gene GsPIP1-4 gene is shown as SEQ ID NO. 2.

In the step (2), the agrobacterium is agrobacterium tumefaciens EHA105, and the soybean receptor material is cotyledonary node of soybean variety Tianlong I.

In the steps (3) and (4), the identification and screening are carried out by three methods, namely a glufosinate smearing method, a Bar test strip detection method and a PCR identification method.

The invention has the following technical advantages:

according to the invention, an experiment that a aquaporin gene is cloned from wild soybeans and a GsPIP1-4 gene is introduced into the soybeans by adopting an agrobacterium-mediated method shows that the over-expression GsPIP1-4 can enhance the moisture absorption of the roots of the soybeans, reduce the moisture loss of the leaves of the soybeans, enhance the net photosynthetic rate, transpiration rate and stomatal conductance of the soybeans and have the potential of increasing the yield; the transgenic soybean can improve the drought resistance of plants by enhancing the activity of antioxidant enzyme and the content of proline. Therefore, the GsPIP1-4 gene can be used for cultivating new germplasm of drought-tolerant plants.

In conclusion, the gene is transferred into a cultivated soybean variety by an agrobacterium-mediated transformation technology, so that the drought resistance function of the gene in the cultivated soybean is determined, the drought resistance of the soybean is enhanced, and the yield of the soybean is increased.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the growth conditions of 2.5M PEG8000 (osmotic potential-0.54 MPa) of cultivated soybean (A drought-sensitive variety Fen bean 93, B drought-resistant variety Tiefeng 31) and Fulu wild soybean (C) after 3 days of simulated drought;

in A, the left picture is FengDo 93 cultured normally for 3 days, and the right picture is FengDo 93 simulated drought after 3 days;

in the B, the left picture is the Feng 31 after normal culture for 3 days, and the right picture is the Feng 31 after simulated drought for 3 days;

the left picture in C is the wild bean of the cottage which is normally cultured for 3 days, and the right picture is the wild bean of the cottage which is simulated to be drought for 3 days.

FIG. 2 shows the expression of the GsPIP1-4 gene in wild soybean leaves under drought stress (A) and ABA (B) treatment;

FIG. 3 is an alignment chart of the amino acid sequence of the protein GsPIP 1-4;

FIG. 4 is a GsPIP gene multi-sequence alignment phylogenetic tree diagram;

FIG. 5 is a GsPIP-4 gene subcellular localization;

a: no-load contrast green fluorescence signal;

b: fusing a red light image of GFP by a plasma membrane positioning Marker;

c: tobacco epidermal cells are imaged under white light;

d: coincidence diagram of A, B and C;

e: in 35s, positioning an image by GsPIP1-4-mGFP under green fluorescence;

f.35s, observing an image under red fluorescence by using GsPIP 1-4-mGFP;

g: tobacco skin image of GsPIP1-4-mGFP under white light for 35s

H: E. g, coincidence diagram.

FIG. 6 shows the recombinant expression vector pPIP1-4 of GsPIP1-4 gene.

FIG. 7 shows the detection results of the T1 GsPIP1-4 gene soybean plant glufosinate smearing method, wherein 1, 2, 5, 6, 12, 13 and 15 are 7 independent transgenic positive plant leaves.

FIG. 8 shows the test results of Bar test strip for soybean plants transformed with GsPIP1-4 gene at the T1 generation, wherein 1, 2, 5, 6, 12, 13 and 15 are 7 independent transgenic positive plants.

FIG. 9 shows the PCR detection result of soybean plants transformed with GsPIP1-4 gene at T0 generation, wherein 1, 2, 5, 6, 12, 13 and 15 are 7 independent transgenic positive plants.

In FIGS. 7-9, plants 8 and 16-18 died during growth and could not be sampled in time for PCR detection, so only the initial Bar test strip test results and partial glufosinate-coating test results were obtained.

FIG. 10 growth vigor of Tianlong No. one and transgenic plants 5 days after 2 days of rehydration with 20% PEG treatment.

FIG. 11 survival status of sand-cultured transgenic and non-transgenic soybeans after natural wilting and rehydration. A is a control variety Tianlong No. I; b is a transgenic strain L12; c is a transgenic strain L15; d is drought-resistant variety Tiefeng 31.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the methods described in the following examples are conventional methods unless otherwise specified.

Wild soybean whole genome sequence:

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

GenBank:KN651006.1

GenBank Graphics

>KN651006.1:c158272-157948,c156900-156605,c156384-156244,c155623-155531Glycine soja cultivar W05 unplaced genomic scaffold scaffold1918,whole genome shotgun sequence。

example 1

1. Cloning, bioinformatics analysis and Gene mapping of the GsPIP1-4 Gene

The following three types of soybean were cultivated: A. drought-sensitive varieties of Fendou 93 and B and drought-resistant varieties of Tiefeng 31 and Turku wild soybeans are subjected to the following simulated drought treatment experiments (conventional technology):

2.5M PEG-8000 (osmotic potential-0.54 MPa) is adopted for simulated drought treatment in the sand culture of the germination box. The size of the germination boxes is 19X 13X 12cm, and each germination box can be loaded with 1.3kg of river sand. Firstly, river sand is cleaned by water for removing impurities, and then high-temperature and high-pressure sterilization is carried out, and the river sand is dried for standby. The surface of soybean seeds is dried and sterilized by adopting chlorine gas and then is directly sown in germination boxes, 20 seeds are sown in each germination box, when two single leaves of the soybean seeds germinate, seedlings are sown to 10 seedlings in each germination box, when the first three-leaf compound leaves grow out, 100ml of 1/2Hogland nutrient solution containing 2.5M PEG8000 (osmotic potential-0.54 MPa) is poured into each germination box to be used as simulated drought treatment, 100ml of 1/2Hogland nutrient solution is normally poured into each germination box to be used as a control, and the plant forms are observed and compared after 3 days.

Found on day 3:

the drought-sensitive variety FenDou 93 has wilting plant and dry leaves; the control is normal growth, the leaves are light green;

drooping and wilting 31 leaves of the drought-resistant variety iron Feng; the control is normal growth, the leaves are light green;

the wild cottage soybean has no obvious difference with the contrast after drought treatment, and can normally grow.

Therefore, the above experimental results show that 2.5M PEG8000 (osmotic potential-0.54 MPa) simulates drought of wild soybean for 3 days after drought treatment and shows obvious drought resistance (FIG. 1C).

Wild soybeans growing normally for 11 days are respectively treated by 2.5M PEG-8000 and 100 mu mol/L ABA, different tissues are sampled before PEG or ABA treatment and after 2h, 6h, 12h, 24h and 48h treatment, RNA is extracted for GsPIP1-4 quantitative expression analysis.

The result shows that the PEG treatment shows the trend of increasing firstly and then decreasing, and the expression quantity is increased by 4.8 times when the PEG treatment is carried out for 12 hours than when the PEG treatment is not carried out; the expression of GsPIP1-4 is inhibited in a short time (2h and 6h) after ABA treatment, the expression is reduced by about 50 percent, the expression level is continuously increased after 12h, and the expression level is maximized and increased by 8.3 times when 2d is treated. This indicates that the expression of the GsPIP1-4 gene can generate obvious response after PEG and ABA treatment, and indicates that the GsPIP1-4 gene may play an important role in drought stress.

As shown in fig. 2: the expression of the GsPIP1-4 gene increased first and then decreased with the increase of the drought treatment time (FIG. 2A); expression increased with increasing duration of ABA treatment (fig. 2B), and in view of this, it was concluded that the GsPIP1-4 gene may be involved in drought tolerance.

Wild soybean (cottage wild soybean) cDNA is used as a template, a primer designed by a GsPIP1-4 gene CDs full-length sequence is used for PCR amplification, and finally, the base sequence SEQ ID NO.2 of the GsPIP1-4 gene is obtained.

Full-length sequence of CDs of GsPIP1-4 gene

Analysis of homology multiple sequence alignments by bioinformatics showed that: the sequence homology of the soybean GsPIP1-4 gene and the coding protein of the cultivated soybean, rice and arabidopsis thaliana GsPIP1-4 gene is higher, the protein sequence conservation of the GsPIP1-4 gene is reflected to be higher (figure 3), and the phylogenetic tree (figure 4) can show that the wild soybean GsPIP1-4 gene has closer genetic relationship with the cultivated soybean, arabidopsis thaliana, rice and tea tree PIP genes.

Constructing an expression vector according to a method for positioning a fused GFP reporter gene, positioning a target protein by virtue of the characteristic of green fluorescence of a GFP expression product, and performing subcellular localization on a GsPIP-4 gene, wherein the method specifically comprises the following steps:

(1) tobacco cultivation: and (4) sowing a plurality of tobacco seeds, culturing in a light incubator for 12h, and growing for one month to be used for experiments.

(2) And (3) agrobacterium culture: preparing 35s mGFP and 35s GsPIP1-4-mGFP vectors; the constructed vector plasmid is transferred into agrobacterium (GV3101) by an electric transformation method and cultured for 2d at 30 ℃.

(3) Suspending agrobacterium: the Agrobacterium was scraped from the solid culture dish using an inoculating loop, inoculated into 10ml YEB liquid medium and cultured at 170rpm/min for 1 h.

(4) And (3) collecting thalli: the suspended Agrobacterium was centrifuged at 4000rpm/min for 4min and the supernatant removed.

(5) Resuspending: with 10mM MgCl₂The suspension (containing 120. mu.M AS) was resuspended and OD600 was adjusted to about 0.6.

(6) And (3) injection: selecting tobacco plants with good growth conditions, injecting from the lower epidermis of the tobacco leaf by using a 1mL injector with a pipette tip, and marking.

(7) Culturing: and culturing the tobacco plants subjected to injection in a weak light for 2d, and observing.

(8) And (4) observation: the marked tobacco leaves injected by agrobacterium are taken and made into a slide, observed under a laser confocal microscope and photographed.

The results are shown in FIG. 5. Gene mapping analysis indicated that the gene was localized to the cytoplasmic plasma membrane.

2. Acquisition and identification of GsPIP1-4 transgenic soybean

The experiment takes the soybean variety Tianlong No. I bred by oil crops of Chinese academy of agricultural sciences, which is widely popularized and applied in production, as a transformation receptor material.

The vector of the experiment is an agrobacterium tumefaciens recombinant expression vector pPIP1-4 (figure 6), a GsPIP1-4 gene is directly cloned to an expression vector pLM-B001 containing a promoter and a terminator through a polyclonal enzyme cutting site, and plasmid DNA is transferred into agrobacterium tumefaciens EHA101 after the plasmid DNA is qualified through inspection to obtain the agrobacterium tumefaciens. The transformation can be carried out by a conventional Agrobacterium-mediated transformation method. The expression vector pLM-B001 was derived from pTF101.1(Paz MM et al, 2006), and a promoter and a terminator for expressing a target gene were added to the vector of pTF101.1.

Paz MM,Matinez JC,Kalvig AB,Fonger Tm,Wang K(2006)Improved cotyledonary node method using an alternative explants derived from mature seed for efficient Agrobacterium-mediated soybean transformation.Plant Cell Reports,25:206–213。

The agrobacterium-mediated transgenic method adopted in the experiment is the agrobacterium-mediated soybean cotyledonary node transformation system. Mature soybean seeds are subjected to steps of sterilization, germination, explant separation, agrobacterium infection, co-culture, bud induction, bud elongation, bud rooting, greenhouse acclimation of tissue culture seedlings and the like to obtain T0 generation transgenic soybeans, wherein T0 generation transgenic soybeans are quickly identified by glufosinate smearing (figure 7), Bar test paper (figure 8) and target gene PCR (figure 9);

the identification method specifically comprises the following steps:

the experimental mode of glufosinate coating is as follows: preparing the glufosinate-contained stock solution to a concentration of 135mg/L, coating half leaves with a cotton swab or a writing brush dipped with herbicide diluent, and marking the half leaves without glufosinate-contained leaves; continuously growing the soybean plants in a greenhouse for about one week to check the leaf changes; the results obtained are shown in FIG. 7 as: the leaves of 1, 2, 5, 6, 12, 13 and 15 scribbled glufosinate have not become yellow, which indicates that the marker gene Bar of herbicide-resistant glufosinate has been transferred into the plants, and the plants are positive plants.

The experimental mode of the Bar test strip is as follows: taking a small amount of soybean leaves in a centrifuge tube, adding a steel column and 250 mul of extracting solution, grinding in a grinder, inserting a test strip into the centrifuge tube for about 5 minutes, and checking the test strip result; the results obtained are shown in FIG. 8 as: two red strips are arranged on the test strips 1, 2, 5, 6, 12, 13 and 15, which indicates that the plant has Bar protein and is judged as positive plants, and only one strip appears on other test strips, which indicates that the plants are negative plants.

The gene PCR is to amplify target gene transferred into plant by PCR method, extract plant leaf DNA, use the DNA of leaf as template and carrier plasmid containing target gene as positive control, and apply target gene specific primer (the length of amplified fragment is about 855 bp). Thus, whether the expression vector is transferred into a soybean receptor is identified, and the obtained result is shown in figure 9 and is as follows: the target gene segments can be amplified in 1, 2, 5, 6, 12, 13 and 15 plants, and the results completely accord with the test results of leaf smearing gerbera butyl 21213, which indicates that the GsPIP-4 gene is integrated into a soybean receptor.

Therefore, it can be seen that: 1, 2, 5, 6, 12, 13 and 15 are 7 independent positive plants with the GsPIP-4 transgene. While the remaining 11 plants were negative, indicating that the foreign gene and Bar gene were not transferred into the soybean receptor.

In the invention, self-pollination is carried out to obtain seeds of T1 generation, and the seeds of T1 generation are verified to be plant of GsPIP-4 gene, which is used for identifying drought resistance.

3. Drought tolerance identification of GsPIP1-4 transgenic plant

Dry sterilizing chlorine gas₁Transgenic seeds (obtained by T0 generation selfing) and non-transgenic soybean seeds (namely seeds of Tianlong No. one) are sown in sterilized sand, germinated in an artificial climate chamber, roots of germinated soybean plants for 4-5 days are cleaned and transplanted into a water culture nutrient solution (1/2Hoagland) for culture, and aeration is kept all the time.

Transgenic soybeans hydroponically cultured for 11 days and wild type soybeans (Tianlong No. I) were treated with 20% PEG8000 for 2 days, and then grown normally (i.e., cultured in an incubator at 25 + -1 ℃ C. and 14h/10h (day/night) of light) for 5 days, and it was found that drought caused the fresh weights of stems and leaves of three transgenic lines L5, L12, and L15 (randomly selected) to be reduced by 18.7%, 18.1%, and 24.2%, respectively, while wild type soybeans were reduced by 47.5% to a significant level; the 20% PEG8000 simulated drought, and the fresh weights of L5, L12, and L15 were reduced by 15.8%, 16.9%, and 13.0%, respectively, while the wild-type soybean was reduced by 37.6% (table 1).

Table 1, Tianlong No. one and the reduction of plant height, root length, fresh weight of stem leaf and root system of transgenic plant after PEG simulated drought treatment and rehydration

In order to further determine the drought resistance function of the GsPIP1-4 gene, the method adopts a soybean variety 'Tiefeng 31' (Wangguifeng, 2019) with non-transgenic soybean Tianlong I and strong drought resistance and GsPIP1-4 gene strains L12 and L15 to carry out a water loss rehydration experiment, plants the four strains in sand culture, stops watering until leaves wither after normal growth reaches three leaves, rehydrates, and counts the number of revived and normally grown plants after two days. Statistics shows that the survival rates of the Tianlong I, the Tiefeng 31, the transgenic lines L12 and L15 after rehydration are 42.1%, 47.5%, 54.4% and 53.7% respectively, so that the Tianlong I proves that the Tiefeng 31 has better drought resistance than the Tianlong I, and the GsPIP1-4 transgenic soybean plant material has better growth recovery capability than the Tianlong 31 after drought rehydration and has certain advantages in drought resistance. The details are shown in Table 2.

TABLE 2 plant survival statistics of Tianlong No.1, Tiefeng 31, transgenic lines L12 and L15 after drought treatment and rehydration

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Zhejiang university

<120> wild soybean aquaporin GsPIP1-4, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 284

<212> PRT

<213> Soybean (Glycine L.)

<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

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Tyr Ile Thr Val Leu Thr Val Met Gly Val Phe Lys Ser Lys Ser Lys

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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

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Leu Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Leu Val His Leu Ala

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ttcttgttcc tctacatcac agtgctgact gtgatgggtg tgttcaaatc taagagcaag 240

tgttccactg tgggtatcca aggcattgct tgggcttttg ggggaatgat ctttgctctt 300

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Claims (6)

1. Wild soybean aquaporin GsPIP1-4, characterized in that: the amino acid sequence of the protein GsPIP1-4 is shown in SEQ ID NO. 1.

2. The gene encoding the wild soybean aquaporin GsPIP1-4 of claim 1, wherein: the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

3. An expression vector comprising the gene according to claim 2.

4. The expression vector of claim 3, wherein: is an agrobacterium recombinant expression vector pPIP1-4, wherein the agrobacterium recombinant expression vector pPIP1-4 is obtained by cloning a GsPIP1-4 gene shown in SEQ ID NO.2 onto an expression vector pLM-B001 and transferring the gene into agrobacterium tumefaciens EHA 101.

5. Use of the gene encoding of claim 2, wherein: is used for improving the drought resistance of plants.

6. Use of the coding gene according to claim 5, characterized in that: and (5) cultivating drought-enduring soybeans.

Images