Soybean salt-tolerant related gene GmNPF7.5, and coding protein and application thereof

Abstract

The application discloses a soybean salt-tolerant related gene GmNPF7.5, a coding protein and application thereof. The gene GmNPF7.5 is an NDA molecule as described in 1), 2) or 3) below: 1) a DNA molecule with a genomic sequence as shown in SEQ ID NO. 1; 2) a DNA molecule with a CDS sequence as shown in SEQ ID NO. 2; 3) a DNA sequence hybridized with the DNA sequence defined in 1) or 2) under stringent conditions and encoding the DNA molecule. The application provides a genetic engineering application of the gene GmNPF7.5 in regulating soybean salt tolerance, specifically, inhibiting the aforementioned gene GmNPF7.5 to improve the salt tolerance of soybean, and overexpressing the aforementioned gene GmNPF7.5 to improve the salt sensitivity of soybean.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of genetic engineering, and particularly relates to a salt-tolerant related gene GmNPF7.5 of soybean, and a coding protein and application thereof. BACKGROUND

[0002] Soybean is rich in oil and protein, and is one of the main sources of vegetable oil and protein in human food and animal feed, and is widely planted in China. However, in recent years, the problem of soil salinization is becoming increasingly serious, and the rapid increase in population and improper agricultural cultivation measures have caused a large amount of secondary salinization of good farmland, and the area of potential saline-alkali land is increasing day by day, which seriously affects the growth and development of plants and reduces the yield of soybean and other crops. Therefore, using modern biological means to breed soybean varieties suitable for saline-alkali land and to screen out plants with excellent salt tolerance traits will be the primary task and inevitable trend of the development of soybean in China.

[0003] The accumulation of salt in the soil will, on the one hand, cause the water potential around the root system to decrease, affecting the water absorption of plants and causing osmotic stress, and in severe cases, can lead to inhibition of plant growth and development or even death; on the other hand, the plant roots absorb too much Na + Afterwards, Na + is transported to the stem by transpiration and finally excessively accumulates in the cotyledon, thereby causing ion toxicity. Ion toxicity can damage the photosynthetic system of the plant, thereby causing the premature senescence or even death of functional leaves of the plant. These harmful effects of salt stress on plants will also cause some secondary stresses such as oxidative stress, metabolic disorder, nutritional imbalance and membrane tissue damage, which will seriously affect the normal growth and development of plants and the yield of crops.

[0004] The research on salt stress of soybean is mainly used for breeding salt-tolerant soybean varieties, and at present, breeding salt-tolerant varieties is mainly carried out by using breeding and conventional breeding methods. However, genetic engineering methods will become another effective way to improve the stress tolerance of soybean in the future. Finding, analyzing and cloning stress-tolerant genes of soybean to breed excellent stress-tolerant soybean varieties will become the primary task of the research on salt stress of soybean and the direction of future stress-tolerant research. SUMMARY

[0005] To solve the above technical problems in the prior art, the purpose of the present application is to disclose a salt-tolerant related gene GmNPF7.5 of soybean, and a coding protein and application thereof.

[0006] The first purpose of the present application is to provide a salt-tolerant related gene GmNPF7.5, which is a DNA molecule as described in 1) or 2) or 3) below:

[0007] 1) a DNA molecule with a gene sequence as shown in SEQ ID NO. 1;

[0008] 2) a DNA molecule having a CDS sequence as shown in SEQ ID NO. 2;

[0009] 3) a DNA molecule hybridizing under stringent conditions to the DNA sequence defined in 1) or 2) and encoding said protein.

[0010] A second object of the present application is to provide a protein encoded by the aforementioned gene GmNPF7.5.

[0011] In particular, the protein provided by the present application is selected from any one of the following (a) or (b):

[0012] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3;

[0013] (b) a protein derived from the target sequence selected from SEQ ID NO. 2.

[0014] SEQ ID NO. 3 in the sequence listing consists of 597 amino acids.

[0015] A third object of the present application is to provide a recombinant expression vector, an expression cassette or a recombinant bacterium comprising the aforementioned gene GmNPF7.5.

[0016] Further, the recombinant expression vector or expression cassette is obtained by inserting the gene GmNPF7.5 into the recombination site of the vector pBA002 digested with XbaI; and the recombinant expression vector or expression cassette is transformed into an engineering bacterium to obtain the recombinant bacterium.

[0017] The recombinant expression vector containing any one of the aforementioned genes also falls within the protection scope of the present application.

[0018] The recombinant expression vector containing the gene can be constructed by using the existing plant expression vector.

[0019] The plant expression vector includes Agrobacterium binary vector and vector that can be used for plant microprojectile bombardment, etc. The plant expression vector can further comprise the 3' untranslated region of the exogenous gene, i.e. comprising the polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end transcription of the Agrobacterium crown gall inducing (Ti) plasmid gene (such as the Nos gene of the nopaline synthase), the 3' end transcription of the untranslated region of the plant gene (such as the soybean storage protein gene) has similar functions.

[0020] When the gene construct is used to construct a recombinant plant expression vector, any one of the enhanced promoters or constitutive promoters, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize, can be added before the transcription initiation nucleotide, which can be used alone or in combination with other plant promoters; in addition, when the gene construct of the present application is used to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be ATG start codon or adjacent regions start codon, but must be in the reading frame of the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0021] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical reagent marker genes (such as herbicide-resistant genes), etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0022] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene GmNPF7.5 into the recombination site of the restriction enzyme XbaI single enzyme digestion vector pBA002. The pBA002 containing GmNPF7.5 is named pBA002-GmNPF7.5.

[0023] The expression cassette containing any one of the above-mentioned genes GmNPF7.5, the transgenic cell line and the recombinant bacteria all belong to the protection scope of the present application.

[0024] The fourth object of the present application is to provide primers for amplifying the aforementioned gene GmNPF7.5, and the primer pair for amplifying the full length or any fragment of the gene GmNPF7.5 also belongs to the protection scope of the present application.

[0025] The fifth object of the present application is to provide an interfering RNA, an expression vector or a recombinant bacteria for silencing the aforementioned gene GmNPF7.5.

[0026] The aforementioned gene GmNPF7.5 of the present application can be silenced according to the conventional method in the art, for example, designing an interfering RNA or knocking out the gene, etc. The present application provides an interfering RNA of the aforementioned gene GmNPF7.5, the sequence of which is shown in SEQ ID NO. 4.

[0027] The sixth object of the present application is to provide a genetic engineering application of the aforementioned gene GmNPF7.5, the aforementioned protein, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria, or the aforementioned primer, or the aforementioned interfering RNA, expression vector or recombinant bacteria in regulating salt tolerance of soybean.

[0028] Further, inhibiting the expression of the aforementioned gene GmNPF7.5, such as interfering silencing or knock-out, can improve salt tolerance of soybean. Overexpressing the aforementioned gene GmNPF7.5 can improve salt sensitivity of soybean.

[0029] Preferably, the aforementioned interfering RNA, expression vector or recombinant bacteria is introduced into soybean to inhibit the aforementioned gene GmNPF7.5.

[0030] Preferably, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria is introduced into soybean to overexpress the aforementioned gene GmNPF7.5.

[0031] The sixth object of the present application is to provide a method for regulating salt tolerance of soybean, which comprises inhibiting the aforementioned gene GmNPF7.5 in a soybean plant to improve salt tolerance of soybean; or overexpressing the aforementioned gene GmNPF7.5 in a soybean plant to improve salt sensitivity of soybean.

[0032] Inhibiting the aforementioned gene GmNPF7.5 in a soybean plant can be achieved by using common methods in the art, such as silencing or knock-out, etc. In the present patent, the gene in soybean is inhibited by using RNAi interference technology to close the expression of the gene.

[0033] Overexpressing the aforementioned gene GmNPF7.5 in a soybean plant can be achieved by introducing the aforementioned recombinant expression vector, expression cassette or recombinant bacteria into soybean to overexpress the aforementioned gene GmNPF7.5 in a soybean plant.

[0034] Any carrier for RNAi interference in plants can be used to inhibit the gene encoding the protein to obtain a transgenic cell line and a transgenic plant. The expression vector carrying the gene can be transformed into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. and the transformed plant tissue is cultivated into a plant.

[0035] Advantages:

[0036] The application discloses a new plant salt tolerance related protein gene GmNPF7.5. The plant salt tolerance related protein of the application influences the salt tolerance of plants. Inhibition of the expression of the gene of the protein can improve the salt tolerance of plants, so that a transgenic plant with salt tolerance can be bred. Introduction of the coding gene of the protein into plants can cause the salt tolerance of the plants to decrease, so that a transgenic plant with salt sensitivity can be bred. The protein and the coding gene thereof can be applied to genetic improvement of plants. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Expression amount of GmNPF7.5 gene in GmNPF7.5-RNAi and GmNPF7.5-OE soybean combination hairy root plants.

[0038] Figure 2 Phenotype (a) and plant fresh weight (b) and plant root length comparison (c) of GmNPF7.5-RNAi and GmNPF7.5-OE soybean combination hairy root plants under salt stress. DETAILED DESCRIPTION

[0039] The following examples facilitate better understanding of the application, but do not limit the application. In the following examples, the experimental methods are conventional methods, and no special instructions are given. In the following examples, the test materials are obtained from conventional biochemical reagent stores, and no special instructions are given.

[0040] Example 1, cloning of soybean gene GmNPF7.5

[0041] The following primers are designed:

[0042] Primer 1: 5'-ATGGGTTGTTTGTATTTTCTTAGA-3';

[0043] Primer 2: 5'-CACTACTTCAGGGTCTTCTTTGTT-3'.

[0044] PCR amplification is performed with primer 1 and primer 2 as primers and the root cDNA of Williams 82 seedlings as a template to obtain the target gene GmNPF7.5.

[0045] PCR amplification reaction was carried out in Bio-rad T100 PCR instrument, and the reaction system (50 μL) was as follows: 25 μL of 2x Phanta Max Buffer, 1 μL of dNTP Mix (10 mM), 1.5 μL of primer 1 (10 μM), 1.5 μL of primer 2 (10 μM), 2 μL of template cDNA (50 ng / uL), 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 18 μL of ddH2O; the program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 2 min, 35 cycles; 72 °C extension for 5 min; 15 °C storage.

[0046] After the PCR product was recovered and purified, it was ligated to pEASY-Blunt (Beijing Zoman Biotech Co., Ltd.), and E. coli DH5α competent cells (CB101, Beijing Tiangen Co.) were transformed. After positive clones were selected, sequencing was performed.

[0047] The sequencing results showed that the GmNPF7.5 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO. 2, encoded a protein (SEQ ID NO. 3) composed of 597 amino acid residues, and the whole genome sequence was shown in SEQ ID NO. 1.

[0048] Example 2, Obtaining and Identifying of Soybean GmNPF7.5 Gene RNAi Hairy Root Combination Plants

[0049] I. Construction of GmNPF7.5 Gene RNAi Vector

[0050] The target sequence of GmNPF7.5 gene (SEQ ID NO. 2) was designed, and the sequence was shown in SEQ ID NO. 4. The genomic cDNA of Williams82 root was used as a template, and primer 3 and primer 4 were used for PCR amplification to obtain the target sequence of GmNPF7.5 gene.

[0051] Primer 3: 5'-ATGGGTTGTTTGTATTTTCT-3';

[0052] Primer 4: 5'-TAAGGAAGGCTCCAAGAAGA-3'.

[0053] The amplification product was ligated to pFGC5941 vector through SwaI, NcoI and BamHI, XbaI twice, and transformed into E. coli DH5α, and positive plasmid was extracted and sequenced. The sequencing result showed that the recombinant expression vector containing the sequence shown in SEQ ID NO. 6 was obtained, and named as pFGC5941-GmNPF7.5.

[0054] II. Obtaining of recombinant Agrobacterium

[0055] The pFGC5941-GmNPF7.5 was transformed into Agrobacterium K599 strain by heat shock method, and the recombinant strain was obtained, and the plasmid was extracted for PCR and enzyme digestion identification, and the recombinant strain with correct identification was named as K5-pFGC5941-GmNPF7.5.

[0056] III. Obtaining of transgenic plants

[0057] The K5-pFGC5941-GmNPF7.5 strain was transformed into soybean variety Williams82, and the specific method was as follows:

[0058] (1) Select mature seeds with large size, fullness and no disease spots, and wash them with deionized water. Then take clean filter paper, spray it with water, and place it in a clean culture dish. Spread the selected soybean seeds evenly on the filter paper, and germinate them in the dark at 26°C.

[0059] (2) After the soybean seeds germinate, select the seeds with consistent growth and sow them in a turnover box containing vermiculite. Cover the seeds with another layer of vermiculite, and place them in a 26°C culture room for 3-4 days of germination. When the soybean cotyledons are about to open but have not yet opened (the color is light green, and lateral roots are growing), the hairy roots can be infected.

[0060] (3) When the seeds germinate for 2-3 days, cultivate the K5-pFGC5941-GmNPF7.5 strain at 28°C and 200r overnight for about 12h; inoculate the bacterial liquid into new LB medium at 1:100, and cultivate it at 28°C and 200r until the OD 600 = 0.6-0.8, and collect the bacterial cells;

[0061] (4) Resuspend the centrifuged bacterial cells with the prepared and sterilized equal volume of co-culture medium (CCM).

[0062] (5) Select the germinated seeds without contamination and with intact cotyledons, cut off the base with a scalpel, and reserve about 2cm of hypocotyls. Soak the soybean hypocotyls in the above resuspended bacterial liquid for 1h. Then transplant the infected explants into moist vermiculite, and cultivate them in a 26°C culture room.

[0063] (6) After culturing for one week, when white callus grows at the hypocotyl wound of the soybean, the callus is cultured in 1 / 2 Hoagland nutrient solution, and after continuing to culture for one week, when soybean root hairs grow at the callus, the root hair is obtained as a transgenic soybean plant.

[0064] IV. Identification of the transgenic plant

[0065] 1. PCR molecular identification

[0066] DNA of the root of the soybean root hair combination plant is extracted as a template for PCR amplification, and a nucleotide fragment of the edited target point and both sides of SEQ ID NO. 6 is amplified by PCR. The PCR primers are as follows:

[0067] Primer5: 5'-ATGGGTTGTTTGTATTTTCT-3';

[0068] Primer6: 5'-TTGGATTTGTAATAATAAAACGCAA-3'.

[0069] The primer5 above is located in the target sequence of the GmNPF7.5 gene shown in SEQ ID NO. 6, and the primer6 is located in the pFGC5941 vector sequence.

[0070] The PCR product is detected by 1% agarose gel electrophoresis, and the target band can be detected in the positive plant, but not in the negative plant.

[0071] 2. Detection of the expression amount of the GmNPF7.5 gene

[0072] The root system sample of the GmNPF7.5 soybean root hair combination plant is frozen by liquid nitrogen, and RNA is extracted by the TRIzol method. An appropriate amount of RNA is used to obtain cDNA by using a reverse transcription kit as a template for fluorescence quantitative RT-PCR detection. An appropriate amount of template cDNA is used to detect the expression amount of the GmNPF7.5 gene by using the Hieff qPCR TM qPCR Green Master Mix (No RoxPlux) kit in a Bio-Rad fluorescence quantitative PCR instrument CFX96. The sequences of the GmELF gene quantitative detection primers (primer7 and primer8) and the GmNPF7.5 gene quantitative detection primers (primer9 and primer10) are as follows:

[0073] Primer7: 5'-GTTGAAAAGCCAGGGGACA-3';

[0074] Primer8: 5'-TCTTACCCCTTGAGCGTGG-3'.

[0075] Primer9: 5'-GCCACAGATGAGATGCCAGG-3';

[0076] Primer10: 5'-TACAAGATCAGCTGCGGTGAG-3'.

[0077] Relative expression level was 2 -△△CT The method is used for quantitative calculation, and the results are as follows: Figure 1 As shown ( Figure 1 (NPF7.5-RNAi-1, NPF7.5-RNAi-2, NPF7.5-RNAi-3).

[0078] Example 3: Obtaining and identifying rooting combinations of soybean GmNPF7.5 gene overexpression

[0079] I. Construction of GmNPF7.5 gene overexpression vector

[0080] Using root genomic cDNA from Williams82 seedlings as a template, PCR amplification was performed using primer1 and primer2 to obtain the full-length CDS sequence fragment of the GmNPF7.5 gene (SEQ ID NO.2).

[0081] Primer1: 5'-ATGGGGTTGTTTGTATTTTCTTAGA-3';

[0082] Primer2: 5'-CACTACTTCAGGGTCTTCTTTGTT-3'.

[0083] The amplified product was ligated into the pBA002 vector by XbaI single enzyme digestion, transformed into E. coli DH5α, and the positive plasmid was extracted and sequenced. The sequencing results showed that a recombinant expression vector containing the sequence shown in SEQ ID NO.2 was obtained and named pBA002-GmNPF7.5.

[0084] II. Obtaining Recombinant Agrobacterium

[0085] pBA002-GmNPF7.5 was transformed into Agrobacterium K599 using the heat shock method to obtain recombinant strains. Plasmids were extracted and identified by PCR and enzyme digestion. The correctly identified recombinant strains were named K5-pBA002-GmNPF7.5.

[0086] III. Obtaining Transgenic Plants

[0087] The K5-pBA002-GmNPF7.5 strain is transformed into soybean variety Williams82, and the specific method is as follows:

[0088] (1) Select mature seeds with large size, fullness, and no disease spots, and wash them with deionized water. Then take clean filter paper, spray it with water, and place it in a clean culture dish. Spread the selected soybean seeds evenly on the filter paper, and germinate them at 26°C in the dark.

[0089] (2) After the soybean seeds germinate, select seeds with consistent growth and sow them in a turnover box containing vermiculite. Cover the seeds with another layer of vermiculite and place them in a 26°C incubator for 3-4 days to germinate. When the soybean cotyledons are about to open but have not yet opened (the color is light green, and lateral roots are growing), the hairy roots can be infected.

[0090] (3) When the seeds germinate for 2-3 days, cultivate the K5-pBA002-GmNPF7.5 strain at 28°C and 200r overnight for about 12 hours. Inoculate the bacterial solution 1:100 into new LB medium and cultivate at 28°C and 200r until the OD 600 = 0.6-0.8, and collect the bacterial cells;

[0091] (4) Resuspend the centrifuged bacterial cells with an equal volume of prepared and sterilized co-culture medium (CCM).

[0092] (5) Select germinated seeds without contamination and intact cotyledons, cut off the base with a scalpel, and reserve about 2cm of hypocotyls. Soak the soybean hypocotyls in the resuspended bacterial solution for 1 hour. Then transplant the infected explants into moist vermiculite and cultivate them in a 26°C incubator.

[0093] (6) After one week of cultivation, white callus tissue grows at the wound site of the soybean hypocotyls. Cultivate them in 1 / 2 Hoagland nutrient solution. After another week of cultivation, soybean root hairs grow at the callus site, and transgenic soybean plants with root hairs are obtained.

[0094] IV. Identification of Transgenic Plants

[0095] 1. PCR molecular identification

[0096] Extract DNA from the roots of the soybean hairy root combination plants, and use it as a template for PCR amplification. The PCR primers are as follows:

[0097] Primer 11: 5'-AAGTGGATTGATGTGATATCTCCA-3';

[0098] Primer 12: 5'-CTTGTAGCAGCCACAAAAACTT-3'.

[0099] The primers described above, primer 11, are located in the pBA002 vector sequence, and primer 12 is located in the CDS sequence of the GmNPF7.5 gene shown in SEQ ID NO.2.

[0100] The PCR products were detected by 1% agarose gel electrophoresis. The target band could be detected in positive plants, but not in negative plants.

[0101] 2. Detection of GmNPF7.5 gene expression level

[0102] Root samples were collected from soybean plants of the GmNPF7.5 rooting combination. After homogenization by liquid nitrogen freezing, RNA was extracted using the TRIzol method. An appropriate amount of RNA was used to obtain cDNA using a transcription kit, which was then used as a template for quantitative RT-PCR detection. An appropriate amount of template cDNA was taken, and soybean GmELF was used as an internal control gene for analysis using Hieff. TM qPCR The Green Master Mix (No RoxPlux) kit was used to detect the expression level of the GmNPF7.5 gene in a Bio-Rad CFX96 real-time PCR instrument. The sequences of the primers used for quantitative detection of the GmELF gene (primer 7 and primer 8) and the primers used for quantitative detection of the GmNPF7.5 gene (primer 9 and primer 10) are as follows:

[0103] Primer7:5'-GTTGAAAAGCCAGGGGACA-3';

[0104] Primer8: 5'-TCTTACCCCTTGAGCGTGG-3'.

[0105] Primer9: 5'-GCCACAGATGAGATGCCAGG-3';

[0106] Primer10: 5'-TACAAGATCAGCTGCGGTGAG-3'.

[0107] Relative expression level was 2 -△△CT The method is used for quantitative calculation, and the results are as follows: Figure 1 As shown ( Figure 1 (NPF7.5-OE1, NPF7.5-OE2, NPF7.5-OE3).

[0108] Example 4: Salt tolerance identification of GmNPF7.5 gene RNAi and overexpression rooting combination plants.

[0109] The identified GmNPF7.5-OE and NPF7.5-RNAi hairy root combination plants are further cultured with 1 / 2 Hoagland nutrient solution to the 2-3rd leaf expansion, wherein the soybean hairy root combination plants transformed with the empty strain K599 are used as a control, and then the soybean hairy root combination plants with more consistent growth are selected and moved to 1 / 2 Hoagland nutrient solution and 1 / 2 Hoagland nutrient solution containing 100 mM NaCl for further culture. After 7-10 days of further growth, the phenotypes of the soybean hairy root combination plants are observed by taking photos, and the related physiological indexes are determined.

[0110] Figure 2 The experimental results show that, compared with the soybean hairy root combination plants transformed with the empty strain, the GmNPF7.5-RNAi soybean hairy root plants exhibit lighter leaf chlorosis and wilting symptoms under salt stress, and the fresh weight and root length of the plants are significantly higher than those of the control group.

[0111] Compared with the soybean hairy root combination plants transformed with the empty strain, the GmNPF7.5-OE soybean hairy root combination plants exhibit heavier leaf chlorosis and wilting symptoms under salt stress, and the fresh weight and root length of the plants are significantly lower than those of the control group.

[0112] Therefore, according to these results, it can be determined that the GmNPF7.5 gene has an important negative regulatory effect on the salt tolerance of soybean, and inhibiting the expression of the protein coding gene can improve the salt tolerance of plants, and overexpression of the protein coding gene can increase the salt sensitivity of soybean.

Claims

1. The application of interfering RNA, expression vector or recombinant bacteria of silencing gene GmNPF7.5 in improving salt tolerance of soybean, characterized in that, The gene GmNPF7.5 is a DNA molecule as shown in SEQ ID NO.

2.

2. A method for improving salt tolerance in soybean, characterized by, The method is to inhibit the gene GmNPF7.5 as shown in SEQ ID NO. 2 in the soybean plant, thereby improving the salt tolerance of the soybean.

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