CN107287212B - Halogeton sativus salt-tolerant gene HgS3 and application thereof - Google Patents
Halogeton sativus salt-tolerant gene HgS3 and application thereof Download PDFInfo
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Abstract
The invention relates to a salt-tolerant gene HgS3 separated from halophyte and application thereof. The purpose is to provide a new salt-tolerant gene HgS3 and its coding protein and application in improving plant salt tolerance and breeding new salt-tolerant species (line). The salt-tolerant gene HgS3 comprises a nucleotide sequence of SEQ ID No.1cDNA, the molecular weight is 468bp, a cDNA coding sequence of the salt-tolerant gene is the nucleotide sequence from 208 th to 336 th in SEQ ID No.1, the molecular weight is 129bp, an amino acid sequence of the salt-tolerant gene is SEQ ID No.3 and consists of 42 amino acids, and the salt-tolerant gene can obviously improve the salt tolerance of transgenic arabidopsis seedlings. The salt-tolerant gene of the invention is beneficial to the cultivation of new strains of salt-tolerant plants (crops).
Description
Technical Field
The invention belongs to the technical field of plant bioengineering and transgenosis, and particularly relates to a halophyte salt-tolerant gene and application thereof in improving the salt tolerance of plants.
Background
Soil salination is one of the most major abiotic stresses affecting crop growth and yield. With the gradual warming of global climate, the soil salinization process is aggravated, especially for vast arid regions and semiarid regions in northwest of China, originally extremely limited rainfall is further reduced, evaporation is further enhanced, so that the soil salinization situation is more severe, and due to the fact that traditional agriculture is over-irrigated, irrigation is not carried out, large-area valuable tileable land resources are phagocytosed by salinization, and finally the land has to be abandoned by agricultural production, and gradually becomes desertification and desertification land. Therefore, the salt tolerance of the plants (crops) is improved, the plants (crops) can grow on the waster saline soil, the full utilization and the effective improvement of the saline soil are realized, and the method has important significance for guaranteeing the food safety of China, improving the ecological environment and realizing the sustainable development of agriculture.
Various salt-tolerant plants are widely distributed in northwest regions of China, the plants form good adaptability to local harsh natural environments, and the plants are valuable excellent materials for salt-tolerant gene discovery from the perspective of salt-tolerant plant (crop) cultivation. And the research on the discovery of salt-tolerant genes of indigenous halophytes in the northwest arid region is less, particularly the discovery of excellent salt-tolerant genes of salt-tolerant pioneer halophytes (haloeton glomeratus) has important application value for cultivating salt-tolerant plant (crop) materials.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provide a halogeton sativus salt-tolerant gene HgS3 to meet the current requirement on excellent salt-tolerant genes.
The invention also aims to provide a preparation method of the halogeton sativus salt-tolerant gene HgS3, so that the gene can be efficiently and quickly obtained.
The invention also aims to provide application of the halogeton sativus salt-tolerant gene HgS3 in improving the salt tolerance of plants.
According to the sequencing result of early-stage halophytic salt stress response transcriptomics (transfection-induced profiling of the salt-stress response in the halophytic halogen on genome [ J ]. BMC genetics, 2015,16(1):169), the salt stress induced expression gene HgS3 is screened and identified, the HgS3 gene is further separated from the halophytic leaves and connected to a super expression vector pCAMBIA3300, then the Arabidopsis is transformed by an agrobacterium-infected method, continuous antibiotic Kan screening is carried out, a T2-generation transgenic pure line is obtained, and seedling salt tolerance analysis proves that the HgS3 gene can obviously improve the salt tolerance of Arabidopsis plants.
In order to achieve the purpose, the invention adopts the technical scheme that: the halogeton sativus salt-tolerant gene HgS3 is mainly characterized in that the nucleotide sequence SEQ ID NO.1 of HgS3 gene separated from halogeton sativus leaves is as follows, and the molecular weight is 468bp:
the halogeton sativus salt-tolerant gene HgS3 is characterized in that the cDNA coding sequence of the gene is the nucleotide sequence SEQ ID NO.2 from 208 th to 336 th in the nucleotide sequence of the isolated HgS3 gene, and the molecular weight is 219 bp:
the halogeton sativus salt-tolerant gene HgS3, the gene coding protein amino acid sequence SEQ ID NO.3 of the gene cDNA coding sequence, consists of 42 amino acids:
the specific primers of the halogeton sativus salt-tolerant gene HgS3 are as follows:
HgS3-F1 is 5'-AAAAGACACTCCATAATCTTGTGTT-3',
HgS3-R1 is 5'-TTTTATTGATGCAAATAAGCTACTA-3'.
Synthesized by Shanghai Bioengineering Co.
The preparation method of the halogeton sativus salt-tolerant gene HgS3 is mainly characterized by comprising the following steps:
using 200-500mM NaCl to treat 3-7d halogeton seedling leaf tissue as a material, extracting total RNA by a Trizol method, carrying out reverse transcription by adopting a cDNA synthesis kit to synthesize a cDNA first strand, and amplifying the gene fragment, wherein a PCR reaction system comprises 5 multiplied PrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-AAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-TTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL, cDNA 1 muL, PrimeSTAR HS polymerase 0.25 muL, ultrapure water 14.75 muL and a total volume of 25 muL; the amplification procedure comprises pre-denaturation at 94 deg.C for 4min, pre-denaturation at 94 deg.C for 50s, pre-denaturation at 60-60.4 deg.C for 15-20s, and pre-amplification at 72 deg.C for 1min for 32-35 cycles, and extension at 72 deg.C for 7-8min, with the amplification results shown in FIG. 1; PCR products were recovered using a gel recovery kit (Dalibao bioengineering, Ltd.), according to the instructions: firstly, detecting the size of a target fragment by using agarose gel, cutting a gel block containing a target gene by using a sterilization blade, and filling the gel block into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume of the rubber blocks according to the proportion that 1mg is 1 mu L, adding 3 times of Buffer GM for melting the rubber blocks, and oscillating or sucking to melt the rubber blocks fully in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu L of Buffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR identification, and sending the bacterial liquid containing a target strip through electrophoresis detection to a Shanghai biological engineering company for sequencing.
The construction method of the expression vector of the halogeton sativus salt-tolerant gene HgS3 comprises the following steps:
BamHI and SacI enzyme cutting sites are respectively added to an upstream primer and a downstream primer of the halogeton salt-tolerant gene HgS3, wherein the upstream primer F1 is 5'-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3', and the downstream primer R1 is 5'-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3':
using 200-500mM NaCl to treat 3-7d halogeton seedling leaf tissue as a material, extracting total RNA by a Trizol method, carrying out reverse transcription by adopting a cDNA synthesis kit to synthesize a cDNA first strand, and amplifying the gene fragment, wherein a PCR reaction system comprises 5 multiplied PrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL, cDNA 1 muL, PrimeSTAR HS polymerase 0.25 muL, ultrapure water 14.75 muL and a total volume of 25 muL; the amplification procedure comprises pre-denaturation at 94 deg.C for 4min, pre-denaturation at 94 deg.C for 50s, pre-denaturation at 60-60.4 deg.C for 15-20s, and pre-amplification at 72 deg.C for 1min for 32-35 cycles, and extension at 72 deg.C for 7-8 min; PCR products were recovered using a gel recovery kit (Dalibao bioengineering, Ltd.), according to the instructions: firstly, agarose gel is used for detecting the size of a target fragment, a film containing a target gene is cut by a sterilization blade, and the film is loaded into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume according to the proportion that 1mg is 1 mu L, adding 3 times of Buffer GM for melting the rubber blocks, and properly oscillating or sucking to melt the rubber blocks in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu LBuffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR identification, and sending the bacterial liquid containing a target strip through electrophoresis detection to a Shanghai biological engineering company for sequencing.
Extracting T-vector and plant expression vector pCAMBIA3300 plasmid DNA of HgS3 gene containing BamHI and SacI enzyme cutting sites, and performing double enzyme cutting on the extracted plasmid by using BamHI and SacI endonucleases, wherein the enzyme cutting system is 10 XQuickcut Buffer 3 muL, plasmid DNA 4 muL, Quickcut BamH 11 muL, Quickcut Sac 11 muL, ddH2O21 μ L, total volume 30 μ L. And (3) carrying out enzyme digestion for 6-8h at 37 ℃, carrying out electrophoresis detection on the enzyme digestion product, and purifying and recovering. Then, the vector purified and recovered in the previous step and the target fragment of the HgS3 gene were ligated by using T4DNA ligase, i.e., 1. mu.L of Buffer, 7. mu.L of target fragment, 1. mu.L of vector DNA, and 9. mu.L of total volume. Firstly, preserving heat in a water bath at 65 ℃ for 3min, rapidly carrying out ice bath for 1-2min, then adding 1 mu L of T4DNA ligase to connect at 16 ℃ for 12-18h to obtain a recombinant plasmid transformed escherichia coli competent cell, screening blue-white spots and sequencing to confirm that a recombinant plasmid pCAMBIA3300-HgS3 containing a target gene HgS3 is obtained; meanwhile, the recombinant plasmid pCAMBIA3300-HgS3 is cut for 6-8h at 37 ℃, and the double-enzyme cutting system is 10 XQuickcut Buffer 3 muL, plasmid DNA 6 muL, Quickcut BamHI 11.2 muL, Quickcut Sac 11.2 muL, ddH2O18.6. mu.L, total volume 30. mu.L.
The application of the halogeton sativus salt-tolerant gene HgS3 in improving the salt tolerance of plants.
The recombinant plasmid pCAMBIA3300-HgS3 provided by the invention is a nucleotide sequence or cDNA coding sequence of HgS3 gene cDNA inserted as shown in SEQ ID NO. 1. It should be noted that any expression vector capable of introducing foreign genes into plants is suitable for the present invention, including direct gene transfer techniques such as particle gun method, protoplast method, liposome method, pollen tube channel method, electro-stimulation transformation method, PEG-mediated transformation method, etc.; the biological mediated transformation method mainly comprises two transformation methods of agrobacterium-mediated transformation and virus-mediated transformation.
The application of the salt-tolerant gene HgS3 in improving the salt tolerance of plants can be widely applied to breeding salt-tolerant crops and new plant varieties (lines).
The invention has the beneficial effects that a new salt-tolerant gene is cloned from halophyte of northwest indigenous halophyte, and the salt tolerance is identified by transforming the Arabidopsis plant, and the result shows that the Arabidopsis plant strain transforming the HgS3 gene shows excellent salt-tolerant property. Meanwhile, the molecular weight of the HgS3 is only 468bp, so that the method is very suitable for genetic transformation operation, and the acquisition of the salt-tolerant gene HgS3 provides a candidate gene for cultivating new strains of salt-tolerant plants (crops) by a biotechnology means.
Description of the drawings:
FIG. 1 shows the amplification result of the nucleotide sequence encoding cDNA of HgS3 gene. M: DL1000 Marker; 1-3 are HgS3 gene PCR products;
FIG. 2pCAMBIA3300-HgS3 expression vector construction double enzyme digestion verification results. M: DL15000 Marker; 1-2 of pCAMBIA3300-HgS3 plasmid; 3-4: performing enzyme digestion electrophoresis;
FIG. 3 shows the molecular test results of T2 generation positive plants of HgS3 gene-transformed Arabidopsis thaliana. M: DL2000 Marker; 1-12PCR determination sequence is shown in SEQ ID NO.1: resistant seedling PCR products; "-": blank control; "WT": wild type Arabidopsis thaliana; "+": a plasmid;
FIG. 4 growth status of HgS3 gene-transformed Arabidopsis thaliana T2 strain under 400mM NaCl stress.
Detailed Description
The following examples facilitate a better understanding of the invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods and reagents of the following examples are conventional methods and reagents.
The preparation method of the halogeton salt-tolerant gene HgS3 in the embodiment 1 is mainly characterized by comprising the following steps:
using leaf tissue of 3-7d halogeton as a material treated by 200mM NaCl, extracting total RNA by a Trizol method, performing reverse transcription by adopting a cDNA synthesis kit (Dalianbao bioengineering Co., Ltd.) to synthesize a first cDNA chain, and amplifying the gene segments, wherein a PCR reaction system comprises 5 XPrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-AAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-TTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL (synthesized by Shanghai Biotech engineering Co., Ltd.), cDNA 1 muL, PrimeSTAR HS DNA polymerase 0.25 muL, ultrapure water 14.75 muL and total volume of 25 muL; the amplification procedure is pre-denaturation at 94 ℃ for 4min, at 94 ℃ for 50s, at 60 ℃ for 20s, at 72 ℃ for 1min, 32 cycles in total, extension at 72 ℃ for 8min, and the PCR amplification result is shown in FIG. 1; PCR products were recovered using a gel recovery kit (Dalibao bioengineering, Ltd.), according to the instructions: firstly, detecting the size of a target fragment by using agarose gel, cutting a gel block containing a target gene by using a sterilization blade, and filling the gel block into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume of the rubber blocks according to the proportion that 1mg is 1 mu L, adding 3 times of Buffer GM for melting the rubber blocks, and oscillating or sucking to melt the rubber blocks fully in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu L of Buffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR identification, and sending the bacterial liquid containing a target strip through electrophoresis detection to a Shanghai biological engineering company for sequencing. The sequence determined by PCR is shown in SEQ ID NO.1:
the halogeton sativus salt-tolerant gene HgS3 is characterized in that the cDNA coding sequence of the gene is the nucleotide sequence SEQ ID NO.2 from the 208 th to the 336 th in the nucleotide sequence of the isolated HgS3 gene, and the molecular weight is 129 bp:
the gene coding protein amino acid sequence SEQID NO.3 of the cDNA coding sequence of the halogeton sativa salt-tolerant gene HgS3 consists of 42 amino acids:
example 2: the preparation method of the halogeton sativus salt-tolerant gene HgS3 is characterized by comprising the following steps:
using leaf tissue of 3-7d halogeton as a material treated by 500mM NaCl, extracting total RNA by a Trizol method, performing reverse transcription by adopting a cDNA synthesis kit (Dalianbao bioengineering Co., Ltd.) to synthesize a first cDNA chain, and amplifying the gene segment, wherein a PCR reaction system comprises 5 XPrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-AAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-TTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL (synthesized by Shanghai Biotech engineering Co., Ltd.), cDNA 1 muL, PrimeSTAR HS DNA polymerase 0.25 muL, ultrapure water 14.75 muL and total volume of 25 muL; the amplification procedure is pre-denaturation at 94 ℃ for 4min, pre-denaturation at 94 ℃ for 50s, amplification at 60.4 ℃ for 15s, amplification at 72 ℃ for 1min, 35 cycles in total, extension at 72 ℃ for 8min, and the PCR amplification result is shown in FIG. 1; PCR products were recovered using a gel recovery kit (Dalibao bioprocess, Inc.) according to the instructions: firstly, detecting the size of a target fragment by using agarose gel, cutting a gel block containing a target gene by using a sterilization blade, and filling the gel block into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume of the rubber blocks according to the proportion that 1mg is 1 mu L, adding 3 times of buffer GM for melting the rubber blocks, and properly oscillating or sucking to melt the rubber blocks in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu L of Buffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR (polymerase chain reaction) identification, carrying out electrophoresis detection on a bacterial liquid containing a target strip, and sending the bacterial liquid to Shanghai biological engineering company for sequencing, wherein the PCR determination sequence is shown as SEQ ID NO. 1.
Example 3: the construction method of the expression vector of the halogeton sativus salt-tolerant gene HgS3 comprises the following steps:
BamHI and SacI enzyme cutting sites are respectively added to the upstream primer and the downstream primer of the halogeton salt-tolerant gene HgS3, wherein the upstream primer F1 is 5'-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3', and the downstream primer R1 is 5'-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3' (synthesized by Shanghai Bioengineering Co., Ltd.):
using leaf tissue of 3-7d halogeton as a material treated by 200mM NaCl, extracting total RNA by a Trizol method, performing reverse transcription by adopting a cDNA synthesis kit (Dalianbao bioengineering Co., Ltd.) to synthesize a first cDNA chain, and amplifying the gene segments, wherein a PCR reaction system comprises 5 XPrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL (synthesized by Shanghai Biotech engineering Co., Ltd.), cDNA 1 muL, PrimeSTAR HS DNA polymerase 0.25 muL, ultrapure water 14.75 muL and total volume of 25 muL; the amplification procedure is pre-denaturation at 94 ℃ for 4min, at 94 ℃ for 50s, at 60 ℃ for 20s, at 72 ℃ for 1min, 32 cycles in total, extension at 72 ℃ for 8min, and the PCR amplification result is shown in FIG. 1; PCR products were recovered using a gel recovery kit (Dalibao bioprocess, Inc.) according to the instructions: firstly, detecting the size of a target fragment by using agarose gel, cutting a gel block containing a target gene by using a sterilization blade, and filling the gel block into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume of the rubber blocks according to the proportion that 1mg is 1 mu L, adding 3 times of Buffer GM for melting the rubber blocks, and properly oscillating or sucking to melt the rubber blocks in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu L of Buffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR (polymerase chain reaction) identification, carrying out electrophoresis detection on a bacterial liquid containing a target strip, and sending the bacterial liquid to Shanghai biological engineering company for sequencing, wherein the PCR determination sequence is shown as SEQ ID NO. 1.
Extracting T-vector and plant expression vector pCAMBIA3300 plasmid DNA of HgS3 gene containing BamHI and SacI enzyme cutting sites, and performing double enzyme cutting on the extracted plasmid by using BamHI and SacI endonucleases, wherein the enzyme cutting system is 10 XQuickcut Buffer 3 muL, plasmid DNA 4 muL, Quickcut BamH 11 muL, Quickcut Sac 11 muL, ddH2O21 μ L, total volume 30 μ L (Dalianbao bioengineering Co., Ltd.). The enzyme is cut for 6-8h at 37 ℃, and the cut product is detected by electrophoresis and purified and recovered (figure 2). Then, the vector purified and recovered in the previous step and the target fragment of the HgS3 gene were ligated by using T4DNA ligase, i.e., 1. mu.L of Buffer, 7. mu.L of target fragment, 1. mu.L of vector DNA, and 9. mu.L of total volume. Firstly, preserving heat in a water bath at 65 ℃ for 3min, rapidly carrying out ice bath for 1min, then adding 1 mu L of T4DNA ligase, connecting at 16 ℃ for 18h to obtain a recombinant plasmid transformed escherichia coli competent cell, screening out blue-white spots, sequencing, and confirming to obtain a recombinant plasmid pCAMBIA3300-HgS3 containing a target gene HgS 3; simultaneous counterweightThe plasmid pCAMBIA3300-HgS3 was digested at 37 ℃ for 6h in a double digestion system of 10 XQuickcut Buffer 3. mu.L, plasmid DNA 6. mu.L, Quickcut BamH 11.2. mu.L, Quickcut Sac11.2. mu.L, ddH2O18.6. mu.L, total volume 30. mu.L.
Example 4: the construction method of the expression vector of the halogeton sativus salt-tolerant gene HgS3 comprises the following steps:
BamHI and SacI enzyme cutting sites are respectively added to the upstream primer and the downstream primer of the halogeton salt-tolerant gene HgS3, wherein the upstream primer F1 is 5'-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3', and the downstream primer R1 is 5'-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3' (synthesized by Shanghai Bioengineering Co., Ltd.).
Using leaf tissue of 3-7d halogeton as a material treated by 500mM NaCl, extracting total RNA by a Trizol method, performing reverse transcription by adopting a cDNA synthesis kit (Dalianbao bioengineering Co., Ltd.) to synthesize a first cDNA chain, and amplifying the gene segment, wherein a PCR reaction system comprises 5 XPrimeSTAR Buffer 5 muL, dNTP mix (each 2.5mM)2 muL, an upstream primer F15 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3' (10 muM) 1 muL, a downstream primer R15 '-CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3' (10 muM) 1 muL (synthesized by Shanghai Biotech engineering Co., Ltd.), cDNA 1 muL, PrimeSTAR HS DNA polymerase 0.25 muL, ultrapure water 14.75 muL and total volume of 25 muL; the amplification procedure is pre-denaturation at 94 ℃ for 4min, pre-denaturation at 94 ℃ for 50s, amplification at 60.4 ℃ for 15s, amplification at 72 ℃ for 1min, 35 cycles in total, extension at 72 ℃ for 8min, and the PCR amplification result is shown in FIG. 1; and (3) recovering the PCR product by using the gel recovery kit, and according to the steps of the specification: firstly, detecting the size of a target fragment by using agarose gel, cutting a gel block containing a target gene by using a sterilization blade, and filling the gel block into a 1.5mL centrifuge tube; weighing the cut rubber blocks, converting the weight into the volume of the rubber blocks according to the proportion that 1mg is 1 mu L, adding 3 times of Buffer GM for melting the rubber blocks, and properly oscillating or sucking to melt the rubber blocks in the melting process; placing the Spin column in a collecting pipe, transferring all the gel block melt liquid into the Spin column, centrifuging at the room temperature of 12000rpm for 1min, and pouring off the filtrate; adding 700 mu L of Buffer WB into Spin column, standing for 5min, centrifuging at room temperature at 12000rpm for 1min, pouring off the filtrate, and repeating the previous step; placing the empty Spin column on a collection tube, centrifuging at 12000rpm for 1min at room temperature, and standing for 2 min; putting Spin column on new centrifuge tube, adding 20-30 μ L sterilized ultrapure water in the center of the membrane, and standing for 1 min; centrifuging at 12000rpm for 1min at room temperature for eluting DNA, sucking 2-3 μ L for electrophoresis detection, and storing other products at-20 deg.C; and then connecting the recovered product with a pMD19-T vector to obtain a recombinant plasmid pMD19-HgS3, transforming Escherichia coli DH5 alpha competent cells, screening blue-white spots, carrying out colony PCR (polymerase chain reaction) identification, carrying out electrophoresis detection on a bacterial liquid containing a target strip, and sending the bacterial liquid to Shanghai biological engineering company for sequencing, wherein the PCR determination sequence is shown as SEQ ID NO. 1.
The HgS3 was PCR amplified again and linked to T-vector for sequencing, and the sequence of the obtained HgS3 gene is shown in SEQ ID NO. 1. Extracting T-vector and plant expression vector pCAMBIA3300 plasmid DNA of HgS3 gene containing BamHI and SacI enzyme cutting sites, and performing double enzyme cutting on the extracted plasmid by using BamHI and SacI endonucleases, wherein the enzyme cutting system is 10 XQuickcut Buffer 3 muL, plasmid DNA 4 muL, Quickcut BamH 11 muL, Quickcut Sac 11 muL, ddH2O21 μ L, total volume 30 μ L (Dalianbao bioengineering Co., Ltd.). The enzyme is cut for 6-8h at 37 ℃, and the cut product is detected by electrophoresis and purified and recovered (figure 2). Then, the vector purified and recovered in the previous step and the target fragment of the HgS3 gene were ligated by using T4DNA ligase, i.e., 1. mu.L of Buffer, 7. mu.L of target fragment, 1. mu.L of vector DNA, and 9. mu.L of total volume. Firstly, preserving heat in a water bath at 65 ℃ for 3min, rapidly carrying out ice bath for 2min, then adding 1 mu L of T4DNA ligase, connecting at 16 ℃ for 12h to obtain a recombinant plasmid transformed escherichia coli competent cell, screening out blue-white spots, sequencing, and confirming to obtain a recombinant plasmid pCAMBIA3300-HgS3 containing a target gene HgS 3; meanwhile, the recombinant plasmid pCAMBIA3300-HgS3 is digested for 8h at 37 ℃, and the double digestion system is 10 XQuickcut Buffer 3 muL, plasmid DNA 6 muL, Quickcut BamHI 11.2 muL, Quickcut Sac11.2 muL, ddH2O18.6. mu.L, total volume 30. mu.L.
Example 5 Agrobacterium Arabidopsis transformation and culture
Preparing LBA4404 agrobacterium, activating, transferring the recombinant plasmid pCAMBIA3300-HgS3 into competent agrobacterium, preparing agrobacterium staining solution, infecting arabidopsis inflorescence by using a flower dipping method, covering infected plants with a plastic film, culturing for 24h in a dark place, transferring to a greenhouse for conventional culture, and obtaining seeds by separating plants after the arabidopsis plants are mature. The harvested arabidopsis seeds are subjected to low-temperature treatment, sterilized and then sown in 1/2MS solid culture medium containing antibiotic Kan (50ug/mL) to screen resistant seedlings, the seedlings are transplanted and cultured, after the seedlings grow up, plant leaves are cut to extract DNA, and the HgS3 segment in the expression vector pCAMBIA3300-HgS3 is amplified by using the upstream and downstream primers of the HgS3 gene in the embodiment 1. Continuous screening and target gene PCR detection (figure 3) are carried out until T2 transgenic HgS3 gene Arabidopsis pure line is obtained.
Example 6 identification of salt tolerance of HgS3 transgenic Arabidopsis thaliana
The T2 transgenic HgS3 gene Arabidopsis pure line plant obtained in example 3 and the seeds of wild type Arabidopsis (Col-1) were sown in a culture medium (peat: vermiculite: perlite mixed at a volume ratio of 1:3: 0.5), cultured in a greenhouse, and after the seedlings grew for 1 month, 400mM NaCl solution was separately poured to observe the growth characteristics of the seedlings, and as a result, it was found that the growth of the wild type Arabidopsis was severely slowed down as the salt stress continued to increase. After 6 days of NaCl stress treatment, the wild type Arabidopsis plants were found to be severely wilted, and parts of the leaves of the plants became yellow and dry, and when stressed to 9 days, the wild type Arabidopsis plants died, while the HgS3 gene-transferred Arabidopsis seedlings survived (FIG. 4). Therefore, the transformed HgS3 gene can improve the salt tolerance of Arabidopsis plants, and the gene can be applied to the salt tolerance breeding work of other plants (crops).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of agriculture in Gansu province
<120> halogeton sativus salt-tolerant gene HgS3 and application thereof
<130>
<160>3
<170>PatentIn version 3.3
<210>1
<211>468
<212>DNA
<213> nucleotide sequence of halogeton salt-tolerant gene HgS3
<400>1
aaaagacact ccataatctt gtgtttgcat ggattcttta aatttgcaag gctcattctt 60
attatcttat ttaagcacct aacatttact acataaccac gaataagcag acaatacagt 120
gttcttgtat caagttttgc ttagaagtat tgttattcga cacaagtttc gatttgtcat 180
agaaataacg tagcaactag caatcaaatg gcagtaagag gagctttatc gacaccaacc 240
tcatctcttc aagcagcact tacccaattg tctttcataa agaaaactca ccctaaacag 300
gtcttatttt tactacttag cttgaaattg ttataaatgg ggacagtata tattgttcta 360
gtcattacga taaagagata tatgtcctat taatggatga atcctcgtat tttacctcct 420
cgttgtaaat atgtactcgt ccttgtagta gcttatttgc atcaataa 468
<210>2
<211>129
<212>DNA
<213> coding sequence of halogeton salt-tolerant gene HgS3
<400>2
atggcagtaa gaggagcttt atcgacacca acctcatctc ttcaagcagc acttacccaa 60
ttgtctttca taaagaaaac tcaccctaaa caggtcttat ttttactact tagcttgaaa 120
ttgttataa 129
<210>3
<211>42
<212>PRT
<213> protein amino acid sequence of halogeton salt-tolerant gene HgS3 coding sequence
<400>3
Met Ala Val Arg Gly Ala Leu Ser Thr Pro Thr Ser Ser Leu Gln Ala
1 5 10 15
Ala Leu Thr Gln Leu Ser Phe Ile Lys Lys Thr His Pro Lys Gln Val
20 25 30
Leu Phe Leu Leu Leu Ser Leu Lys Leu Leu
35 40
Claims (1)
1. The application of the halophyte HgS3 in improving the salt tolerance of plants is characterized in that the halophyte HgS3 gene sequence is shown in a sequence table SEQ ID NO.1, and the salt tolerance of plants to salt stress can be improved.
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Non-Patent Citations (3)
| Title |
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| GenBank：APW84885.1;Wang，J.等;《GenBank》;20170128;全文 * |
| GenBank:KX233498.1;Wang,J.等;《GenBank》;20170128;全文全文 * |
| 基于转录组测序盐生草（Halogeton glomeratus）耐盐基因的克隆与验证;李爱博;《中国优秀硕士学位论文全文库》;20170315;全文 * |
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