CN115232822A - Identification and application of wild soybean inositol transporter gene GsINT1 - Google Patents
Identification and application of wild soybean inositol transporter gene GsINT1 Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural biological germplasm resource excavation and innovative utilization, and discloses identification and application of a wild soybean Inositol Transporter gene GsINT1 (Inositol Transporter 1). The Inositol Transporter gene GsINT1 takes wild soybean cDNA as a template, obtains a full-length nucleotide sequence of the GsINT1 gene by a reverse transcription PCR method, and the nucleotide sequence is SEQ ID No.1, finds that the protein is strongly induced by saline-alkali stress and has stronger saline-alkali resistance, and lays a foundation for cultivating new saline-alkali resistance soybean germplasm. The invention proves the function of the GsINT1 gene in the process of saline-alkali stress tolerance of the soybean and lays a foundation for cultivating new germplasm of the saline-alkali tolerant soybean and related breeding work.
Description
Technical Field
The invention belongs to the technical field of agricultural biological germplasm resource excavation and innovative utilization, and particularly relates to identification and application of a wild soybean inositol transporter gene GsINT1.
Background
The salinization of soil is one of the important factors affecting agricultural production, ecological environment and grain safety. At present, about 10 hundred million hectares of saline-alkali soil exist in the world, while the area of the saline-alkali soil in China is 9913 million hectares, which occupies about 10 percent of the total area of the world. Because the plant contains high-concentration sodium ions, the plant generates high ion stress and osmotic stress, the plant is greatly limited from absorbing water and nutrient substances from soil, the phenomena of yellowing, water loss and even necrosis of plant leaves are caused, the yield of crops in China is greatly reduced, and great influence is brought to agricultural production.
The soybean is an important protein and grease source in the world, plays a special important role in agricultural industrial structure and people's life in China, and the annual consumption keeps about 10 percent of the growth rate. However, compared with main crops such as wheat and rice, the soybean belongs to moderate saline-alkali sensitive crops; under the condition of saline-alkali stress, the soybean yield can be reduced by 50 percent seriously, and the soybean yield in China is seriously influenced. Therefore, the saline-alkali tolerance of soybeans in China is improved, functional genes related to the saline-alkali tolerance of the soybeans are explored, and the cultivation of new species of the saline-alkali tolerant soybeans is urgent.
Researches show that the inositol can relieve the reduction of the photosynthetic capacity and chlorophyll content of leaves and reduce growth inhibition; can also participate in abiotic stress such as plant salt, drought, cold damage and the like to improve the antioxidant system of the plant, reduce the oxidative damage caused by ROS accumulation and salt stress, maintain the ion and osmotic balance in the plant body, and improve the plant tolerance. The inositol transporter (INT) is a key transporter of important metabolites such as inositol uptake, fat, minerals, and saccharides in plants. The existing research shows that the apple inositol transporter MdINT1 is positioned in a vacuole membrane, and the expression of the apple inositol transporter MdINT1 is induced by salt stress; after the gene is over-expressed, the ion balance, the activity of an antioxidant system and the accumulation of penetration protective substances are further improved by improving the reutilization of inositol in vacuole, and the salt tolerance of the apple is greatly improved, but related researches are not reported in soybean.
Through the above analysis, the problems and defects of the prior art are as follows: how to effectively excavate the salt and alkali tolerant candidate genes of soybeans, improve the salt and alkali tolerant capability of the soybeans and provide a basis for cultivating high-quality salt and alkali tolerant soybean varieties.
The difficulty in solving the above problems and defects is: the saline-alkali tolerant character of crops is generally controlled by polygene and is closely linked with some undesirable characters, and the soybean variety with saline-alkali tolerant and high quality is difficult to obtain by the conventional breeding method.
The significance of solving the problems and the defects is as follows: neutral salt such as NaCl in coastal region and NaHCO in inland region 3 And the soybean yield can be reduced by 50% seriously under the harm of alkaline salt, which has great influence on the soybean yield in China. Therefore, the method excavates the soybean salt and alkali resistance gene, improves the soybean salt and alkali resistance function, provides high-efficiency candidate genes and a new idea for improving the quality and the efficiency of soybeans in China and cultivating a new salt and alkali resistance soybean variety (line) through molecular breeding, and has important application value.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides identification and application of a wild soybean inositol transporter gene GsINT1.
The invention is realized in such a way, and discloses the identification and application of one tonoplast inositol transporter GsINT1. The team identified salt and alkali (NaCl + NaHCO 3) and salt (NaCl) tolerance phenotypes of 125 wild soybean linesA tolerant line SWBY032 and a sensitive line SWLJ092 are screened respectively. On the basis, 2 wild soybean line saline-alkali (NaCl + NaHCO) labels are carried out by using LC-MS/MS and TMT (Tandem Mass Tag) 3 ) And proteome quantification under salt (NaCl) stress. Finally, a gene GsINT1 (glysoja _ 027082) for coding wild soybean vacuolar Inositol Transporter1 (inostol Transporter 1) is obtained, and the expression level of the gene GsINT1 (glysoja _ 027082) is highest in roots and leaves under saline-alkali and salt stress. Meanwhile, gsINT1 is respectively subjected to overexpression analysis in yeast and soybean hairy roots, and the result shows that the gene shows a strong saline-alkali resistance function.
The wild soybean inositol transporter gene GsINT1 of the invention takes wild soybean cDNA as a template, and obtains the full-length nucleotide sequence of the GsINT1 gene by a reverse transcription PCR method, wherein the nucleotide sequence is SEQ ID No.1.
The other purpose of the invention is to provide a complementary and reverse sequence containing SEQ ID No.1 applying the wild soybean inositol transporter gene GsINT1.
The other purpose of the invention is to provide an amino acid sequence which is encoded by the gene GsINT1 containing SEQ ID No.1 or reverse and complementary sequences and uses the wild soybean inositol transporter gene GsINT1.
The invention also aims to provide an expression vector, a transgenic cell line and a host bacterium which use the wild soybean inositol transporter gene GsINT1 and contain the inositol transporter gene.
The invention also aims to provide a construction method of the expression vector containing the inositol protein transporter gene, which mainly comprises the following steps:
(1) Cloning of wild soybean GsINT1 Gene
Cloning GsINT1 gene by taking wild soybean cDNA as a template, wherein a PCR amplification system is as follows: template 1. Mu.L, 10xbuffer 2.5. Mu.L, dNTP 2.0. Mu.L, gsINT 1-F1.0. Mu.L, gsINT 1-R1.0. Mu.L, ddH 2 O17.5 μ L; pre-denaturation at 94 ℃ for 10min, annealing at 60 ℃ for 1min, extension for 2min, and the cycle number is 35; detecting the PCR product by agarose gel electrophoresis, and then carrying out the detection on the PCR product and a cloning vector pEASY-T1 is connected to form pEASY-GsINT1, and then sequencing is carried out.
(2) Construction of Yeast vectors
Carrying out double enzyme digestion on pEASY-GsINT1 plasmid and pYES2 plasmid respectively by using Sac I and BamH I, respectively recovering a target gene and a large vector fragment, and connecting under the action of T4 DNA ligase to obtain an expression vector pYES2-GsINT1; empty vectors, namely pYES2 and pYES2-GsINT1, are respectively transformed into Saccharomyces cerevisiae INVSC1 for gene function identification.
(3) Construction of plant expression vectors
Carrying out double enzyme digestion on pEASY-GsINT1 plasmid and pCAMBIA-Flag plasmid by using Nco I and Spe I respectively, recovering a target gene and a large vector fragment, and connecting under the action of T4 DNA ligase to obtain an expression vector pCAMBIA-GsINT1; the empty vectors, namely pCAMBIA-Flag and pCAMBIA-GsINT1 are respectively transformed into agrobacterium rhizogenes K599 for soybean hairy root induction and transformation.
The invention also aims to provide a method for identifying the salt and alkali tolerance function of the wild soybean inositol transporter gene GsINT1, which comprises the following steps:
step one, phenotypic identification of salt tolerance and salt and alkali resistance of yeast with over-expression GsINT1 gene;
respectively transferring the yeast expression vector pYES2-GsINT1 and the empty vector pYES2 into saccharomyces cerevisiae INVSC1; selecting a monoclonal colony by using an SD-Ura culture medium, and culturing in an SC-Ura + Galactose liquid culture medium; adjusting the OD600 value to 0.5 with 0.9% NaCl, and further diluting the concentration to 10 -1 、10 -2 、10 -3 、10 -4 (ii) a Respectively taking 1 mu L of the strain, dotting the strain on a blank SC-Ura + Galactose culture medium and a stress culture medium, culturing for 3-7 days, taking the SC-Ura + Galactose blank culture medium as a contrast, and observing and recording the growth states of pYES2 yeast cells and GsINT1 gene-transferred yeast cells under different stress conditions; wherein the stress treatment conditions are: 700mM NaCl and 15mM NaHCO 3 +675mM NaCl。
The phenotype identification results of the yeast and soybean hairy roots over-expressing GsINT1 under salt and saline-alkali stress show that the yeast and soybean hairy roots are subjected to NaCl and NaHCO 3 + NaCl stress yeast strain to pYES2-GsINT1The growth vigor of the strain is obviously superior to that of microzyme transferred into pYES2 empty vector, and the strain shows stronger salt and salt tolerance characteristics, thereby indicating that the GsINT1 gene has the functions of salt tolerance and salt tolerance.
And step two, performing phenotypic identification on salt tolerance and salt and alkali resistance of soybean hairy roots of the over-expressed GsINT1 gene.
Respectively transferring the plant expression vector pCAMBIA-GsINT1 and the empty vector pCAMBIA-Flag into agrobacterium rhizogenes K599; with sodium hypochlorite and concentrated hydrochloric acid 1:1, after mixing, carrying out chlorine overnight sterilization on the soybean seeds; cleaning soybean with sterile water, inoculating into vermiculite sterilized at high temperature and high pressure, and moistening with 1/4Hoagland solution; culturing in artificial climate room at 25 deg.C under illumination for 16h for 6 days; culturing agrobacterium rhizogenes K599 in an incubator at 28 ℃ overnight; the bacteria were collected by scraping with a razor blade.
Injecting agrobacterium rhizogenes to the hypocotyl near the cotyledon; after the injection is finished, water is preserved for 5 to 6 days; covering the wound with sterilized vermiculite and retaining water; culturing for 15 days, removing true roots, and transferring to Hoagland culture solution for culturing after 3 days of seedling culture in vermiculite; after 3 days of culture, stress treatment was started under conditions of 120mM NaCl and 70mM NaHCO 3 +50mM NaCl, normal culture as control, the growth status of each plant under different stress conditions was observed and recorded.
The phenotype identification results of salt tolerance and salt and alkali resistance of soybean hairy roots of the over-expressed GsINT1 gene show that the phenotype identification results of the soybean hairy roots of the over-expressed GsINT1 gene in NaCl and NaHCO 3 Under the stress of NaCl, the growth vigor of the agrobacterium rhizogenes induced plant containing the pCAMBIA-GsINT1 plasmid is obviously superior to that of the plant induced by the pCAMBIA-Flag empty vector, the plant induced by the pCAMBIA-Flag empty vector shows stronger salt tolerance and salt and alkali tolerance, and the GsINT1 protein is further proved to have the functions of salt tolerance and salt and alkali tolerance in soybeans.
The invention also aims to provide application of the wild soybean inositol transporter gene GsINT1 in transgenic plants, wherein the plants comprise monocotyledons and dicotyledons.
The invention also aims to provide application of the wild soybean inositol transporter gene GsINT1 in the process of salt and alkali stress tolerance of soybeans.
By combining all the technical schemes, the invention has the advantages and positive effects that: inositol plays an important role as an osmotic adjusting substance in abiotic stress such as salt and alkali tolerance of plants. Furthermore, the Inositol transporters (INT) located on the plasma and vacuolar membranes primarily regulate their distribution in tissues, organs and subcellular organelles in plants. Research shows that the Int1 gene plays a certain role in improving the salt tolerance of arabidopsis thaliana and apples, besides, research on inositol transporters and functions in other plants is rarely reported, and application in soybeans and related agriculture is not reported yet. The group discovers a gene-GsINT 1 related to saline-alkali stress by carrying out proteomics analysis on saline-alkali sensitive and saline-alkali tolerant wild soybeans, discovers the saline-alkali stress tolerance function of the GsINT1 gene in soybeans for the first time, and provides a basis for improving the saline-alkali tolerance of soybeans in China and cultivating high-quality saline-alkali tolerant soybeans. On one hand, the function of the GsINT1 gene in the process of salt and alkali stress tolerance of the soybean is defined; on the other hand, lays a foundation for cultivating new germplasm of saline-alkali tolerant soybeans and related breeding work.
The invention provides identification and application of a wild soybean inositol transporter gene GsINT1, relates to identification of a plant tonoplast inositol transporter, finds that the protein is strongly induced by saline-alkali stress and has a strong saline-alkali resistance function, and lays a foundation for culturing a new germplasm of saline-alkali resistant soybeans.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the method for identifying the wild soybean inositol transporter gene GsINT1 provided by the embodiment of the invention.
Fig. 2 is a schematic diagram of phenotype identification of saline-alkali tolerant wild soybean SWBY032 and saline-alkali sensitive wild soybean SWLJ092 provided by the embodiment of the invention.
FIG. 3 is a schematic diagram of PCR amplification of a full-length cDNA fragment of the GsINT1 gene provided by the embodiment of the invention.
Fig. 4 is a schematic construction diagram of a GsINT1 gene yeast expression vector provided by an embodiment of the present invention.
Fig. 5 is a schematic diagram of the construction of a soybean hairy root expression vector of the GsINT1 gene provided by the embodiment of the invention.
Fig. 6A and fig. 6B are schematic diagrams of functional identification of GsINT1 gene in yeast and soybean hairy roots provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Aiming at the problems in the prior art, the invention provides identification and application of a wild soybean inositol transporter gene GsINT1, and the invention is described in detail below with reference to the accompanying drawings.
The wild soybean inositol transporter gene GsINT1 provided by the embodiment of the invention is derived from wild soybean, and the nucleotide sequence is SEQ ID No.1.
The strains comprise agrobacterium rhizogenes K599 and saccharomyces cerevisiae INVSC1, and the strains are purchased from Shanghai Haonyu biotechnology limited; the wild soybean saline-alkali tolerant line SWBY032 and the sensitive line SWLJ09 are from agricultural courtyards of Jilin province. The wild soybean salt and alkali tolerant strain SWBY032 and the sensitive strain SWLJ09 are purchased by molecular breeding and germplasm innovation teams of tropical soybeans of the tropical crop institute of Hainan university.
As shown in FIG. 1, the wild soybean inositol transporter gene GsINT1 provided by the embodiment of the invention has the following functional identification:
s101, phenotypic identification of salt tolerance and salt and alkali resistance of yeast with over-expression GsINT1 gene;
s102, performing phenotypic identification on salt tolerance and salt and alkali resistance of soybean hairy roots of the over-expressed GsINT1 gene.
The invention disclosesThe invention discloses identification and application of a tonoplast inositol transporter GsINT1, wherein a wild soybean salt alkali tolerant line SWBY032 and a sensitive line SWLJ092 are used as research materials, and salt and alkali (NaCl + NaHCO) are used for the research materials 3 ) And (2) carrying out proteomic analysis under the stress of salt (NaCl), identifying a gene GsINT1 for coding tonoplast inositol transporter, and finding that the gene has a strong salt and alkali tolerance function. The full-length nucleotide sequence of the GsINT1 gene is obtained by taking wild soybean cDNA as a template and adopting an RNA extraction and reverse transcription PCR method. On one hand, the invention proves the function of the GsINT1 gene in the process of saline-alkali stress tolerance of soybean; on the other hand, lays a foundation for cultivating new germplasm of saline-alkali tolerant soybeans and related breeding work. The invention discloses a wild soybean vacuole inositol transporter GsINT1, and finds that the protein is strongly induced by saline-alkali stress and has a strong saline-alkali resistance function, thereby laying a foundation for cultivating a new saline-alkali resistant soybean germplasm.
The invention aims to provide a plant salt and salt tolerance stress gene GsINT1.
The tonoplast inositol transporter gene GsINT1 provided by the invention is derived from wild soybean, and the nucleotide sequence of the tonoplast inositol transporter gene GsINT1 is shown in SEQ ID No.1.
atgactggggggggtatcactatgcaatcgacgccgggaagctcaggatacttggatttgtatccagagcggaagatgtcctttttcaaaaatccttacattctcggactggctgctgtcgccggcatcggtggtctcctcttcggctacgacactggtgtgatatcaggggctcttttatacattaaggatgattttgaggaggttagaaacagcaatcttctccaggaaacaatagtcagcatggctattgccggggcaatcgtgggagcagcattgggtggttggattaatgatgcctatggtcgaaagaaggccaccctctttgccgatgttatctttaccgccggagcaataatcatggcttctgctccagatccttatgttctcattttgggacgccttctggttggtttgggtgtgggcatagcttctgtcactgctcccgtttatattgcagaagcatcaccttccgaaataaggggttcattagtcagcacaaatgtcctcatgataaccggtggacagtttctttcctatcttgttaacctcgcttttactggtgttcctggtacttggcgatggatgcttggtgtttctggtgtgcctgctgttgttcagtttgttctcatgctcttcctccctgaatccccaagatggctttttgtcaagaataggaaaaatgaagccgttgatgtgctttctaagatctttgacgttgctcgcttagaagacgaagtcgatttcctcactgctcaatccgagcaagagcgccaaagaaggtcaaatatcaaattctgggatgtttttagatccaaagaaatcagactggcattcctcgtcggtgccggacttctggcttttcagcagttcacgggtattaacacagtcatgtactacagcccgacaattgtccaaatggcagggttccatgctaatgaattggctcttcttctatcccttatagttgccggcatgaatgcagccggaacaattcttggcatttaccttatcgaccatgctggacgaaaaaaattagctctttctagtctgggaggagtaattgtatctttggtcatcttggcctttgcattttataagcaatcatccacgtcaaatgagttgtatggatggcttgcagttgtaggcttagccttgtatattggttttttctcgccaggaatggggcctgtgccatggactctgagttcagagatatatccggaagaatatcgaggtatctgtgggggcatgtcagctactgtgtgttgggtctcaaatttgattgtctcagagacctttctttcaattgcggagggtatagggattggttcaacttttctgattattggtgtcatagctgtggttgcatttgtatttgtgcttgtttatgttccagagaccaaaggattgacctttgatgaagtggaagtaatatggagggagagagcttggggcaagaaccccaacacacaaaaccttcttgagcagggaagtagttcctaa
The full-length nucleotide sequence of the GsINT1 gene is obtained by taking wild soybean cDNA as a template and adopting a reverse transcription PCR method.
A complementary and reverse sequence comprising SEQ ID No.1.
An amino acid sequence encoded by SEQ ID No.1 or its reverse and complementary sequences.
Expression vectors, transgenic cell lines and host bacteria comprising the myo-inositol protein transporter gene of the invention are within the scope of the invention.
The application of the tonoplast inositol transporter gene in transgenic plants, namely monocotyledons and dicotyledons, belongs to the protection scope of the invention.
The technical solution of the present invention is further described below with reference to specific examples.
Example 1: identification and proteomics analysis of 125 wild soybean salt and alkali stress phenotypes
5363 identification of salt and alkali stress tolerance phenotype of wild soybean 1.1125
(1) Dividing 30 seeds to be detected of each variety into a control group and a stress group, setting 3 repeats in each group, and sowing 15 seeds in each group; the seeds are sowed in pots filled with perlite, 5 seeds are sowed in each pot.
(2) After the cotyledon is completely unfolded (VC period), pouring 200ml of 1/4Hoagland nutrient solution every two days; meanwhile, each pot of 3 seedlings with consistent expansion potential are fixed for later stage stress experiment.
(3) Stress culture: after the true leaves are completely unfolded (V1 period), 200ml of stress solution is added into each pot, the stress treatment lasts for 10 days, and each treatment is fixed at the same time.
(4) And (3) control culture: after the true leaves were completely developed (V1 period), 200ml of 1/4 Hoagland's culture medium was added to each pot and cultured, and each treatment was fixed at the same time.
(5) And (4) observing the growth condition of the wild soybeans after the stress treatment, and screening out the saline-alkali tolerant wild soybeans.
1.2 proteomics analysis
A saline-alkali tolerant line SWBY032 and a sensitive line SWLJ092 are screened from 125 wild soybean lines by identifying the saline-alkali tolerant phenotype. On the basis, the proteome under saline-alkali stress of the two wild soybean lines is quantitatively analyzed by LC-MS/MS and TMT.
The results show that under the saline-alkali stress, 332 and 208 differentially expressed proteins exist in the tolerant line SWBY032, wherein the up-regulation expression is 198 and 128 respectively; under the saline-alkali stress, 320 and 119 differentially expressed proteins of the sensitive line SWLJ092 respectively exist, wherein the up-regulated expression is 182 and 84 respectively. Therefore, the amount of the differentially expressed protein or the up-regulated protein of the tolerant line is higher than that of the sensitive line under the saline-alkali stress. Therefore, the specific 35 differential expression proteins of the tolerant line SWBY032 can be the coding products of the wild soybean salt and alkali tolerant genes of the line, except for the 37 common differential expression proteins of the sensitive line under the condition of salt and alkali stress. For this reason, we first analyzed the expression level of mRNA encoding 18 genes of up-regulated expression proteins, and as a result, found that GsINT1 (glysoja _ 027082) encoding wild soybean tonoplast Inositol Transporter1 (Inositol Transporter 1) is the highest in expression level in both roots and leaves under saline-alkali stress. Therefore, it is preliminarily presumed that GsINT1 can be used as a candidate gene affecting the saline-alkali tolerance of wild soybean.
Example 2: extraction of total RNA from wild soybean
(1) Fresh wild soybean leaf tissue (50-100 mg) is taken and placed under liquid nitrogen for full grinding until the powder has no obvious granular sensation.
(2) An appropriate volume of RNAasso tissue fluid was added to homogenize the mixture, and the mixture was allowed to stand at room temperature for 5 minutes, and then centrifuged at 12,000g and 4 ℃ for 5 minutes.
(3) The supernatant was transferred to a new 1.5ml centrifuge tube, mixed with 200. Mu.l of chloroform and allowed to stand at room temperature for 5 minutes, and centrifuged at 12,000g and 4 ℃ for 15 minutes.
(4) The supernatant was transferred to a new centrifuge tube, isopropanol equivalent to the supernatant solution was added, and the mixture was allowed to stand at room temperature for 10 minutes, centrifuged at 12,000g and centrifuged at 4 ℃ for 10 minutes.
(5) Discarding the supernatant, slowly adding 1ml of 75% ethanol along the wall of the centrifuge tube, gently and slowly inverting the centrifuge tube, mixing, centrifuging at 12,000g at 4 ℃ for 5 minutes, and then pouring off the ethanol on the upper layer.
(6) Drying the precipitate for 2-5 min, adding 50ul RNase-free water to dissolve the precipitate, and storing at-80 ℃ for later use after the RNA precipitate is completely dissolved.
Example 3: cloning of wild soybean GsINT1 Gene
The RNA is reversely transcribed into cDNA, the cDNA is used as a template to clone a GsINT1 gene, a PCR amplification system is shown in Table 3, mainly pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, extension for 2min and cycle number of 35, a PCR product is subjected to agarose gel electrophoresis detection and then is connected with a cloning vector pEASY-T1 to form pEASY-GsINT1, and then sequencing is carried out, and the sequencing result is shown in SEQ ID No.1.
TABLE 3GsINT1 Gene PCR amplification System
Example 4: construction of inositol transporter GsINT1 expression vector
4.1 construction of Yeast vectors
Carrying out double enzyme digestion on pEASY-GsINT1 plasmid and pYES2 plasmid respectively by using Sac I and BamH I, respectively recovering a target gene and a large vector fragment, and connecting under the action of T4 DNA ligase to obtain an expression vector pYES2-GsINT1. Empty vectors, namely pYES2 and pYES2-GsINT1, are respectively transformed into Saccharomyces cerevisiae INVsc1 for gene function identification.
4.2 construction of plant expression vectors
The pEASY-GsINT1 plasmid and the pCAMBIA-Flag plasmid are subjected to double enzyme digestion by Nco I and Spe I respectively, a target gene and a large vector fragment are recovered, and the target gene and the large vector fragment are connected under the action of T4 DNA ligase to obtain the expression vector pCAMBIA-GsINT1. The empty vectors, namely pCAMBIA-Flag and pCAMBIA-GsINT1 are respectively transformed into agrobacterium rhizogenes K599 for soybean hairy root induction and transformation.
Example 5: phenotypic identification of yeast and soybean hairy root with overexpression GsINT1 under salt and saline-alkali stress
5.1 phenotypic identification of salt tolerance and salt and alkali resistance of yeast overexpressing GsINT1 gene
The yeast expression vector pYES2-GsINT1 and the empty vector pYES2 were transferred into Saccharomyces cerevisiae INVsc1, respectively. Monoclonal colonies were picked using SD-Ura medium and cultured in SC-Ura + Galactose liquid medium. Adjusting the OD600 value to 0.5 with 0.9% NaCl, and further diluting the concentration to 10 -1 、10 -2 、10 -3 、10 -4 . Respectively spotting 1 μ L of the cells on a blank SC-Ura + Galactose medium and a stress medium, culturing for 3-7 days, observing and recording the growth states of pYES2 yeast cells and GsINT1 gene-transferred yeast cells under different stress conditions (stress treatment conditions: 700mM NaCl and 15mM NaHCO) 3 +675mM NaCl)。
The results show that in NaCl and NaHCO 3 The growth vigor of the microzyme transformed into pYES2-GsINT1 is obviously better than that of the microzyme transformed into pYES2 empty vector under the stress of NaCl, and the microzyme shows stronger salt tolerance and salt and alkali tolerance characteristics, thereby indicating that the GsINT1 gene has the functions of salt tolerance and salt and alkali tolerance.
5.2 phenotypic identification of salt tolerance and salt and alkali resistance of soybean hairy root with overexpression GsINT1 gene
On the basis of the predecessors, the invention slightly changes the process of hairy root induction to meet the requirements of the subsequent experiments on salt tolerance and salt and alkali resistance of the soybean hairy root. Firstly, the plant expression vector pCAMBIA-GsINT1 and the empty vector pCAMBIA-Flag are respectively transferred into agrobacterium rhizogenes K599.
(1) With sodium hypochlorite and concentrated hydrochloric acid 1:1, after mixing, carrying out chlorine overnight sterilization on the soybean seeds;
(2) Cleaning soybean with sterile water, inoculating into vermiculite sterilized at high temperature and high pressure, and moistening with 1/4Hoagland solution;
(3) Culturing at 25 deg.C for 16h in artificial climate chamber for about 6 days;
(4) Culturing Agrobacterium rhizogenes (K599) in an incubator at 28 ℃ overnight;
(5) Scraping bacteria with a blade to collect thalli;
(6) Injecting agrobacterium rhizogenes to the hypocotyl near the cotyledon;
(7) After the injection is finished, water is required to be preserved for 5-6 days;
(8) Then covering the wound with sterilized vermiculite and preserving water;
(9) Culturing for about 15 days, removing true roots, and transplanting to Hoagland culture solution after 3 days.
(10) After culturing for about 3 days, stress treatment was started (stress treatment conditions: 120mM NaCl and 70mM NaHCO) 3 +50mM NaCl), with normal culture as control, the growth status of each plant under different stress conditions was observed and recorded. The results show that in NaCl and NaHCO 3 Under the stress of NaCl, the growth vigor of the agrobacterium rhizogenes induced by the pCAMBIA-GsINT1 plasmid is obviously superior to that of the plant induced by the pCAMBIA-Flag empty vector, the plant induced by the pCAMBIA-Flag empty vector shows stronger salt tolerance and salt and alkali tolerance, and the GsINT1 gene is further proved to have the functions of salt tolerance and salt and alkali tolerance in soybeans.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
<110> university of Hainan
<120> identification and application of wild soybean inositol transporter gene GsINT1
<160> 1
<210> 1
<211> 1506
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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Claims (10)
1. An inositol transporter gene GsINT1 is characterized in that the inositol transporter gene GsINT1 is derived from wild soybean, and the nucleotide sequence is SEQ ID No.1.
2. The inositol transporter gene GsINT1 according to claim 1, wherein the inositol transporter gene GsINT1 is the full-length nucleotide sequence of GsINT1 gene obtained by reverse transcription PCR method using wild soybean cDNA as template:
cloning GsINT1 gene by taking wild soybean cDNA as a template, wherein a PCR amplification system is as follows: template 1. Mu.L, 10xbuffer 2.5. Mu.L, dNTP 2.0. Mu.L, gsINT 1-F1.0. Mu.L, gsINT 1-R1.0. Mu.L, ddH 2 O17.5 μ L; pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, extension for 2min, and cycle number of 35; and (3) detecting the PCR product through agarose gel electrophoresis, connecting the PCR product with a cloning vector pEASY-T1 to form pEASY-GsINT1, and then sequencing.
3. Use of the complementary and reverse sequence of the inositol transporter gene GsINT1SEQ ID No.1 according to any of claims 1-2.
4. An amino acid sequence encoded by the inositol transporter gene GsINT1SEQ ID No.1 or the reverse and complementary sequences according to any one of claims 1-2.
5. An expression vector, a transgenic cell line and a host bacterium using the inositol transporter gene GsINT1 as defined in any one of claims 1 to 2.
6. A method of constructing an expression vector containing an inositol protein transporter gene according to claim 5, wherein the method comprises:
(1) Construction of Yeast vectors
Carrying out double enzyme digestion on a pEASY-GsINT1 plasmid and a pYES2 plasmid respectively by using Sac I and BamH I, respectively recovering a target gene and a large vector fragment, and connecting under the action of T4 DNA ligase to obtain an expression vector pYES2-GsINT1; respectively transforming empty vectors pYES2 and pYES2-GsINT1 into Saccharomyces cerevisiae INVSC1 for gene function identification;
(2) Construction of plant expression vectors
Carrying out double enzyme digestion on pEASY-GsINT1 plasmid and pCAMBIA-Flag plasmid respectively by using Nco I and Spe I, recovering a target gene and a large vector fragment, and connecting under the action of T4 DNA ligase to obtain expression vector pCAMBIA-GsINT1; the empty vectors, namely pCAMBIA-Flag and pCAMBIA-GsINT1 are respectively transformed into agrobacterium rhizogenes K599 for soybean hairy root induction and transformation.
7. An identification method of the inositol transporter gene GsINT1 according to any one of claims 1-2, wherein the identification method of salt tolerance and salt and alkali resistance of the inositol transporter gene GsINT1 in yeast comprises the following steps:
respectively transferring the yeast expression vector pYES2-GsINT1 and the empty vector pYES2 into saccharomyces cerevisiae INVSC1; selecting a monoclonal colony by using an SD-Ura culture medium, and culturing in an SC-Ura + Galactose liquid culture medium; adjusting OD600 to 0.5 with 0.9% NaCl and further diluting to 10% -1 、10 -2 、10 -3 、10 -4 (ii) a Respectively taking 1 mu L of the strain, dotting the strain on a blank SC-Ura + Galactose culture medium and a stress culture medium, culturing for 3-7 days, taking the SC-Ura + Galactose blank culture medium as a contrast, and observing and recording the growth states of pYES2 yeast cells and GsINT1 gene-transferred yeast cells under different stress conditions; wherein the stress treatment conditions are: 700mM NaCl and 15mM NaHCO 3 +675mM NaCl。
8. An identification method using the inositol transporter gene GsINT1 as claimed in any of claims 1-2, characterized in that the identification of salt tolerance and salt and alkali resistance of the inositol transporter gene GsINT1 in soybean hairy roots. The method comprises the following steps:
respectively transferring the plant expression vector pCAMBIA-GsINT1 and the empty vector pCAMBIA-Flag into agrobacterium rhizogenes K599; with sodium hypochlorite and concentrated hydrochloric acid 1:1, after mixing, carrying out chlorine overnight sterilization on the soybean seeds; cleaning soybean with sterile water, inoculating into vermiculite sterilized at high temperature and high pressure, and moistening with 1/4Hoagland solution; culturing in artificial climate room at 25 deg.C under illumination for 16h for 6 days; culturing agrobacterium rhizogenes K599 in an incubator at 28 ℃ overnight; scraping bacteria with a blade to collect thalli;
injecting agrobacterium rhizogenes to the hypocotyl near the cotyledon; after the injection is finished, water is preserved for 5 to 6 days; covering the wound with sterilized vermiculite and retaining water; culturing for 15 days, removing true roots, and transferring to Hoagland culture solution after 3 days of seedling revival in vermiculite; after 3 days of culture, stress treatment was started under conditions of 120mM NaCl and 70mM NaHCO 3 +50mM NaCl, normal culture as control, the growth status of each plant under different stress conditions was observed and recorded.
9. Use of the inositol transporter gene GsINT1 according to any one of claims 1-2 in soybean salt tolerance and salt and alkali stress tolerance processes.
10. Use of the myo-inositol transporter gene GsINT1 according to any of claims 1-2 in transgenic plants, characterized in that said plants comprise monocotyledonous and dicotyledonous plants.
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| 范龙;孙天杰;杨郡;张洁;王冬梅;: "大豆GmNHX1基因克隆及其在酵母中的耐盐性分析", 河北农业大学学报, no. 06 * |
| 赵甜甜;范会芬;孙天杰;肖付明;张洁;王冬梅;: "GmVP1基因克隆与转GmVP1/GmNHX1双价基因的大豆发状根耐盐性分析", 河北农业大学学报, no. 02 * |
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