CN107287212A - Salt sward resistant gene of salt HgS3 and its application - Google Patents

Abstract

The present invention relates to the resistant gene of salt HgS3 separated from halophytes salt sward and its application.Its object is to provide new a resistant gene of salt HgS3 and its encoding proteins and its application in plant salt endurance and cultivation New salt-tolerant cultivar (being) is improved.Resistant gene of salt HgS3 includes the nucleotide sequence such as SEQ ID NO.1cDNA, molecular weight is 468bp, its cDNA coded sequence is the nucleotide sequence described in the 208th to the 336th in SEQ ID NO.1, molecular weight is 129bp, its amino acid sequence such as SEQ ID NO.3, it is made up of 42 amino acid, the gene can significantly improve the salt-tolerant trait of transgenic arabidopsis seedling.Resistant gene of salt of the present invention will be helpful to the cultivation of salt-tolerant plant (crop) new lines (kind).

Description

Salt sward resistant gene of salt HgS3 and its application

Technical field

The invention belongs to plant biological engineering and field of transgenic technology, and in particular to halophytes salt sward resistant gene of salt And its application in plant salt endurance is improved.

Background technology

The soil salinization is to influence one of main abiotic stress of plant growth and yield.With global climate by Gradual change is warmed up, and even more exacerbates soil salinization process, for the vast drought-hit area of NORTHWEST CHINA and semiarid zone, former This extremely limited rainfall is further reduced, and evaporation is further to be strengthened, and causes soil salinization situation more acute, and by In traditional agriculture heavy irrigation, only fill and do not arrange, even more allow large area it is valuable can farming land resource swallowed by salination, most Have to be abandoned by agricultural production eventually, be gradually evolved into desertification and desertification land.Therefore, plant (crop) salt tolerant is improved Property so that the salt marsh aerial that plant (crop) can fall into disuse at these, accomplish to make full use of and effectively improve these salt marsh Soil realizes that agricultural sustainable development is significant to ensureing China's grain security and improving the ecological environment.

Widely distributed a variety of salt-tolerant plants in NORTHWEST CHINA area, these plants form to local harsh natural environment Good adaptability, considers from the angle of salt-tolerant plant (crop) cultivation, and these plants are that valuable resistant gene of salt excavation is excellent Different material.And, especially salt resistance pionner salt less on the research that arid region of Northwest China native country halophytes resistant gene of salt is excavated Sward (Halogeton glomeratus), therefrom excavate excellent resistant gene of salt has weight to cultivating salt-tolerant plant (crop) material The applying value wanted.

The content of the invention

A kind of salt sward resistant gene of salt HgS3 is provided it is an object of the invention to avoid the deficiencies in the prior art part, with The current demand to excellent resistant gene of salt of reply.

A further object of the present invention is the provision of a kind of salt sward resistant gene of salt HgS3 preparation method, so as to realize height Imitate, quickly obtain the gene.

The a further object of the present invention is the provision of a kind of described salt sward resistant gene of salt HgS3 and is improving plant salt tolerant The application of property.

The present invention is according to early stage salt sward salt stress response transcription group sequencing result (Transcriptomic profiling of the salt-stress response in the halophyte Halogeton glomeratus [J].BMC genomics,2015,16(1):169), Screening and Identification is to salt stress expression profile HgS3, further in salt HgS3 genes are isolated in sward blade, and the gene is connected on overexpression vector pCAMBIA3300, then pass through agriculture bar Bacterium dip method arabidopsis thaliana transformation, continuous antibiotic Kan screenings, obtains T2 and is sheerly for transgenosis, confirmed through seedling Salt Tolerance Analysis, HgS3 genes can significantly improve the salt tolerance of Arabidopsis plant.

To achieve the above object, the technical scheme taken of the present invention is：A kind of salt sward resistant gene of salt HgS3, it is main special Select and be that the nucleotide sequence SEQ ID NO.1 of isolated HgS3 genes from salt sward blade are as follows, molecular weight 468bp:

Described salt sward resistant gene of salt HgS3, it is characterised in that described gene cDNA encoding sequence is separated to Nucleotide sequence SEQ ID NO.2 in the nucleotide sequence of HgS3 genes described in the 208th to the 336th are as follows, and molecular weight is 219bp：

Described salt sward resistant gene of salt HgS3, the gene coded protein amino acid sequence of described gene cDNA encoding sequence SEQ ID NO.3 are arranged, are made up of 42 amino acid：

Described salt sward resistant gene of salt HgS3, its special primer is：

HgS3-F1 is 5 '-AAAAGACACTCCATAATCTTGTGTT-3 ',

HgS3-R1 is 5 '-TTTTATTGATGCAAATAAGCTACTA-3 '.

By Shanghai, Sheng Gong bio-engineering corporations synthesize.

Described salt sward resistant gene of salt HgS3 preparation method, it is mainly characterized by step and is：

Material is organized as with the 200-500mM NaCl salt sward seedling leaves for handling 3-7d, extracts total with Trizol methods RNA, the chains of cDNA first are synthesized using the reverse transcription of cDNA synthetic agent box, expand the genetic fragment, and PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F15 '- AAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R15 '-TTTTATTGATGCAAATAAGCTACTA- 3 ' (10 μM) 11 μ L, PrimeSTAR HS archaeal dna polymerases of μ L, cDNA 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Expand Increasing program is 94 DEG C of pre-degeneration 4min, 94 DEG C of 50s, 60-60.4 DEG C of 15-20s, 72 DEG C of 1min, and common 32-35 is circulated, and 72 DEG C are prolonged 7-8min is stretched, amplification is as shown in Figure 1；PCR productions are reclaimed using glue reclaim kit (Dalian treasured bioengineering Co., Ltd) Thing, to specifications step：Ago-Gel testing goal clip size is used first, contains target gene with sterilizing cut Blob of viscose, and be fitted into 1.5mL centrifuge tubes；The blob of viscose cut is weighed, weight converted its body according to 1mg=1 μ L ratio Product, then add the Buffer GM of 3 times of volumes makes it fully melt for melting to vibrate in blob of viscose, melting process or inhale to beat；Will Spin posts are placed in collecting pipe, then go to all blob of viscose melting liquids in Spin posts, room temperature centrifugation 12000rpm, 1min, outwell Filtrate；Plus 700 μ L Buffer WB in Spin posts and stand 5min after, room temperature centrifugation 12000rpm, 1min, outwell filtrate, weight Multiple previous step；Empty Spin posts are placed on collecting pipe, room temperature centrifugation 12000rpm, 1min place 2min；Spin posts are put again Enter on new centrifuge tube, in film center plus 20-30 μ L sterilizing ultra-pure waters, place 1min；Room temperature 12000rpm, centrifuges 1min, is used for Eluted dna, finally drawing 2-3 μ L is used for electrophoresis detection, other -20 DEG C of preservations of product；Then recovery product and pMD19-T are carried Body is connected, and obtains recombinant plasmid pMD19-HgS3, converts bacillus coli DH 5 alpha competent cell, the blue hickie of screening, and carry out bacterium Fall PCR identifications, the bacterium solution that electrophoresis detection contains target stripe is served Hai Sheng works bio-engineering corporation and is sequenced.

The construction method of described salt sward resistant gene of salt HgS3 expression vector, its step is：

The two digestions of BamHI and SacI are added respectively in described salt sward resistant gene of salt HgS3 upstream and downstream primer Site, sense primer F1 be 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ', anti-sense primer R1 be 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3’:

Material is organized as with the 200-500mM NaCl salt sward seedling leaves for handling 3-7d, extracts total with Trizol methods RNA, the chains of cDNA first are synthesized using the reverse transcription of cDNA synthetic agent box, expand the genetic fragment, and PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F1 5 '- CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R1 5 '- 1 μ L, PrimeSTAR the HS archaeal dna polymerases of μ L, cDNA of CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' (10 μM) 1 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre-degenerations 4min, 94 DEG C of 50s, 60-60.4 DEG C of 15- 20s, 72 DEG C of 1min, common 32-35 circulation, 72 DEG C of extension 7-8min；Using glue reclaim kit, (Dalian treasured bioengineering is limited Company) reclaim PCR primer, to specifications step：Ago-Gel testing goal clip size first, with sterilizing cut Film containing target gene, and be fitted into 1.5mL centrifuge tubes；The blob of viscose cut is weighed, will according to 1mg=1 μ L ratio Then plus the Buffer GM of 3 times of volumes are used to melt in blob of viscose, melting process appropriate vibration or inhaled and beat weight converts its volume, It is set fully to melt；Spin posts are placed in collecting pipe, then gone to all blob of viscose melting liquids in Spin posts, room temperature centrifugation 12000rpm, 1min, outwell filtrate；Plus 700 μ L Buffer WB in Spin posts and stand 5min after, room temperature centrifugation 12000rpm, 1min, outwell filtrate, repeat previous step；Empty Spin posts are placed on collecting pipe, room temperature centrifugation 12000rpm, 1min, places 2min；Spin posts are put on new centrifuge tube again, in film center plus 20-30 μ L sterilizing ultra-pure waters, 1min are placed； Room temperature 12000rpm, centrifuges 1min, for eluted dna, and finally drawing 2-3 μ L is used for electrophoresis detection, other -20 DEG C of guarantors of product Deposit；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid pMD19-HgS3, convert bacillus coli DH 5 alpha sense By state cell, the blue hickie of screening, and bacterium colony PCR identifications are carried out, the bacterium solution that electrophoresis detection contains target stripe serves the raw work life in sea Thing engineering company is sequenced.

Extract the T- carriers and plant expression vector of the HgS3 genes containing BamHI and SacI restriction enzyme sites PCAMBIA3300 DNAs simultaneously carry out double digestion with two kinds of restriction endonucleases of BamHI and SacI to institute's upgrading grain, and digestion system is 10 1 μ L, QuickCut Sac1 of the μ L of × Quickcut Buffer 3,4 μ L, QuickCut BamH1 of DNA 1 μ L, ddH₂O 21 μ L, the μ L of cumulative volume 30.In 37 DEG C of digestion 6-8h, and electrophoresis detection is carried out to digestion products and recovery is purified.Then T4DNA is used Ligase, is attached to the carrier and HgS3 gene purpose fragments that recovery is purified in previous step, and linked system is 10 × T4DNA The μ L of ligase Buffer 1, the μ L of purpose fragment 7, the μ L of carrier DNA 1, the μ L of cumulative volume 9.First after 65 DEG C of water-bath insulation 3min, Rapid ice bath 1-2min, then adds the μ L of T4DNA ligases 1 in connecting 12-18h at 16 DEG C, obtains recombinant plasmid transformed large intestine bar Bacterium competence cell, the blue hickie of screening is simultaneously sequenced, and confirms to obtain containing the recombinant plasmid with target gene HgS3 pCAMBIA3300-HgS3；Simultaneously to recombinant plasmid pCAMBIA3300-HgS3 in 37 DEG C of digestion 6-8h, its double digestion system is The μ L of 1.2 μ L, QuickCut Sac1 of the μ L of 10 × Quickcut Buffer 3,6 μ L, QuickCut BamH1 of DNA 1.2, ddH₂The μ L of O 18.6, the μ L of cumulative volume 30.

Described salt sward resistant gene of salt HgS3 is improving the application of plant salt tolerance.

The recombinant plasmid pCAMBIA3300-HgS3 that the present invention is provided, the plasmid is shown in insertion SEQ ID NO.1 The nucleotide sequence or cDNA coded sequences of HgS3 gene cDNAs.It should be noted that any can import foreign gene is planted The expression vector of thing is applied to the present invention, including direct gene transfer technology, such as particle bombardment, protoplasm body, liposome Method, pollen tube passage method, Electroporation conversion, PEG mediated transformation methods etc.；The method for transformation of biology mediation, mainly there is Agrobacterium Mediation and virus-mediated two kinds of method for transformation etc..

The resistant gene of salt HgS3 that the present invention is provided can be widely used in salt tolerant crop, plant in the application for improving plant salt endurance The seed selection of thing new varieties (being).

Beneficial effect of the present invention is to be cloned into a new resistant gene of salt from the halophytes salt sward of northwest native country, led to Cross arabidopsis thaliana transformation plant and carry out Salt-Tolerance Identification, as a result show that the arabidopsis strain for converting HgS3 genes is shown excellent resistance to Salt characteristic.Its molecular weight of HgS3 is only 468bp simultaneously, is particularly well suited for resistant gene of salt HgS3 in genetic transformation operation, the present invention Obtain and provide candidate gene to cultivate salt-tolerant plant (crop) new lines (kind) cultivation by animal nutrition.

Brief description of the drawings：

The amplification of Fig. 1 HgS3 gene cDNA encoding nucleotide sequences.M：DL1000Marker；1-3 is HgS3 genes PCR primer；

Fig. 2 pCAMBIA3300-HgS3 expression vector establishment double digestion the results.M：DL15000Marker；1-2: PCAMBIA3300-HgS3 plasmids；3-4：Restriction enzyme digestion and electrophoresis result；

Fig. 3 converts HgS3 gene arabidopsis T2 for positive plant Molecular Detection result.M：DL2000Marker；1-12PCR Sequence is determined as shown in SEQ ID NO.1：Resistance seedling PCR primer；“-”：Blank control；“WT”：Wildtype Arabidopsis thaliana；“+”：Matter Grain；

Fig. 4 converts growth conditions of the HgS3 gene arabidopsis T2 for strain under 400mM NaCl stress.

Embodiment

Following embodiment facilitates a better understanding of the present invention, not limits the present invention.The experimental method of example below With reagent etc., if being conventional method and reagent without specified otherwise.

The preparation method of salt sward resistant gene of salt HgS3 described in embodiment 1, it is mainly characterized by step and is：

Material is organized as with the 200mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts With cDNA synthetic agent box (Dalian treasured bioengineering Co., Ltd) the reverse transcription synthesis chains of cDNA first, the gene piece is expanded Section, PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F1 5 '-AAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R1 5 '- TTTTATTGATGCAAATAAGCTACTA-3 ' (10 μM) 1 μ L (Sheng Gong bio-engineering corporations synthesize by Shanghai), the μ L of cDNA 1, The μ L of PrimeSTAR HS archaeal dna polymerases 0.25, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre-degenerations 4min, 94 DEG C of 50s, 60 DEG C of 20s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C of extension 8min, PCR amplifications are as shown in Figure 1；Profit PCR primer is reclaimed with glue reclaim kit (Dalian treasured bioengineering Co., Ltd), to specifications step：Agarose is used first Gel detection purpose fragment size, the blob of viscose of target gene is contained with sterilizing cut, and is fitted into 1.5mL centrifuge tubes；It is right The blob of viscose cut is weighed, the Buffer GM of its volume, then 3 times of volumes of addition that weight is converted according to 1mg=1 μ L ratio It is set fully to melt for melting to vibrate in blob of viscose, melting process or inhale to beat；Spin posts are placed in collecting pipe, then by all glue Block melting liquid is gone in Spin posts, room temperature centrifugation 12000rpm, 1min, outwells filtrate；Plus 700 μ L Buffer WB in Spin posts And stand after 5min, room temperature centrifugation 12000rpm, 1min outwell filtrate, repeat previous step；Empty Spin posts are placed on collecting pipe On, room temperature centrifugation 12000rpm, 1min place 2min；Spin posts are put on new centrifuge tube again, in film center plus 20-30 μ L Sterilize ultra-pure water, places 1min；Room temperature 12000rpm, centrifuges 1min, for eluted dna, finally draws 2-3 μ L and is examined for electrophoresis Survey, other -20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid pMD19-HgS3, Bacillus coli DH 5 alpha competent cell, the blue hickie of screening are converted, and carries out bacterium colony PCR identifications, electrophoresis detection contains target stripe Bacterium solution serve Hai Sheng works bio-engineering corporation and be sequenced.PCR determines sequence and sees SEQ ID NO.1:

Described salt sward resistant gene of salt HgS3, described gene cDNA encoding sequence is the core for the HgS3 genes being separated to Nucleotide sequence SEQ ID NO.2 in nucleotide sequence described in the 208th to the 336th are as follows, and molecular weight is 129bp：

The gene coded protein amino acid sequence SEQ of described salt sward resistant gene of salt HgS3 gene cDNA encoding sequences ID NO.3, are made up of 42 amino acid：

Embodiment 2：Described salt sward resistant gene of salt HgS3 preparation method, it is characterised in that step is：

Material is organized as with the 500mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts With cDNA synthetic agent box (Dalian treasured bioengineering Co., Ltd) the reverse transcription synthesis chains of cDNA first, the gene piece is expanded Section, PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F1 5 '-AAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R1 5 '- TTTTATTGATGCAAATAAGCTACTA-3 ' (10 μM) 1 μ L (Sheng Gong bio-engineering corporations synthesize by Shanghai), the μ L of cDNA 1, The μ L of PrimeSTAR HS archaeal dna polymerases 0.25, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre-degenerations 4min, 94 DEG C of 50s, 60.4 DEG C of 15s, 72 DEG C of 1min, totally 35 circulations, 72 DEG C of extension 8min, PCR amplifications are as shown in Figure 1； PCR primer is reclaimed using glue reclaim kit (Dalian treasured bioprocess Co., Ltd), to specifications step：Agar is used first Sugared gel detection purpose fragment size, the blob of viscose of target gene is contained with sterilizing cut, and is fitted into 1.5mL centrifuge tubes； The blob of viscose cut is weighed, the Buffer of its volume, then 3 times of volumes of addition that weight is converted according to 1mg=1 μ L ratio GM, which is used to melt in blob of viscose, melting process suitably vibration or inhales to beat, makes it fully melt；Spin posts are placed in collecting pipe, then will All blob of viscose melting liquids are gone in Spin posts, room temperature centrifugation 12000rpm, 1min, outwell filtrate；Plus 700 μ L Buffer WB in Spin posts and stand after 5min, room temperature centrifugation 12000rpm, 1min outwell filtrate, repeat previous step；Empty Spin posts are placed on On collecting pipe, room temperature centrifugation 12000rpm, 1min place 2min；Spin posts are put on new centrifuge tube again, added in film center 20-30 μ L sterilizing ultra-pure waters, place 1min；Room temperature 12000rpm, centrifuges 1min, for eluted dna, finally draws 2-3 μ L and uses In electrophoresis detection, other -20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid PMD19-HgS3, converts bacillus coli DH 5 alpha competent cell, the blue hickie of screening, and carries out bacterium colony PCR identifications, and electrophoresis detection contains The bacterium solution for having target stripe is served Hai Sheng works bio-engineering corporation and is sequenced, and PCR determines sequence as shown in SEQ ID NO.1.

Embodiment 3：The construction method of described salt sward resistant gene of salt HgS3 expression vector, its step is：

The two digestions of BamHI and SacI are added respectively in described salt sward resistant gene of salt HgS3 upstream and downstream primer Site, sense primer F1 be 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ', anti-sense primer R1 be 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' (by Shanghai, Sheng Gong bio-engineering corporations synthesize):

Material is organized as with the 200mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts With cDNA synthetic agent box (Dalian treasured bioengineering Co., Ltd) the reverse transcription synthesis chains of cDNA first, the gene piece is expanded Section, PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F1 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R1 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' (10 μM) 1 μ L (Sheng Gong bio-engineering corporations synthesize by Shanghai), cDNA 1 μ L, PrimeSTAR HS archaeal dna polymerases 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre- changes Property 4min, 94 DEG C of 50s, 60 DEG C of 20s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C of extension 8min, PCR amplifications are as shown in Figure 1； PCR primer is reclaimed using glue reclaim kit (Dalian treasured bioprocess Co., Ltd), to specifications step：Agar is used first Sugared gel detection purpose fragment size, the blob of viscose of target gene is contained with sterilizing cut, and is fitted into 1.5mL centrifuge tubes； The blob of viscose cut is weighed, the Buffer of its volume, then 3 times of volumes of addition that weight is converted according to 1mg=1 μ L ratio GM, which is used to melt in blob of viscose, melting process suitably vibration or inhales to beat, makes it fully melt；Spin posts are placed in collecting pipe, then will All blob of viscose melting liquids are gone in Spin posts, room temperature centrifugation 12000rpm, 1min, outwell filtrate；Plus 700 μ L Buffer WB in Spin posts and stand after 5min, room temperature centrifugation 12000rpm, 1min outwell filtrate, repeat previous step；Empty Spin posts are placed on On collecting pipe, room temperature centrifugation 12000rpm, 1min place 2min；Spin posts are put on new centrifuge tube again, added in film center 20-30 μ L sterilizing ultra-pure waters, place 1min；Room temperature 12000rpm, centrifuges 1min, for eluted dna, finally draws 2-3 μ L and uses In electrophoresis detection, other -20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid PMD19-HgS3, converts bacillus coli DH 5 alpha competent cell, the blue hickie of screening, and carries out bacterium colony PCR identifications, and electrophoresis detection contains The bacterium solution for having target stripe is served Hai Sheng works bio-engineering corporation and is sequenced, and PCR determines sequence as shown in SEQ ID NO.1.

Extract the T- carriers and plant expression vector of the HgS3 genes containing BamHI and SacI restriction enzyme sites PCAMBIA3300 DNAs simultaneously carry out double digestion with two kinds of restriction endonucleases of BamHI and SacI to institute's upgrading grain, and digestion system is 10 1 μ L, QuickCut Sac1 of the μ L of × Quickcut Buffer 3,4 μ L, QuickCut BamH1 of DNA 1 μ L, ddH₂O 21 μ L, the μ L of cumulative volume 30 (Dalian treasured bioengineering Co., Ltd reagent).Electricity is carried out in 37 DEG C of digestion 6-8h, and to digestion products Swimming detects and purifies recovery (Fig. 2).Then T4DNA ligases are used, carrier and HgS3 gene mesh to purifying recovery in previous step Fragment be attached, linked system be the μ L of 10 × T4DNA ligases Buffer 1, the μ L of purpose fragment 7, the μ L of carrier DNA 1, always The μ L of volume 9.First after 65 DEG C of water-bath insulation 3min, then rapid ice bath 1min adds the μ L of T4DNA ligases 1 to connect at 16 DEG C 18h is met, recombinant plasmid transformed competent escherichia coli cell is obtained, the blue hickie of screening is simultaneously sequenced, and confirmation is obtained containing with mesh Gene HgS3 recombinant plasmid pCAMBIA3300-HgS3；Simultaneously to recombinant plasmid pCAMBIA3300-HgS3 in 37 DEG C of digestions 6h, its double digestion system be the μ L of 10 × Quickcut Buffer 3, the μ L of 6 μ L, QuickCut BamH1 of DNA 1.2, QuickCut Sac1 1.2 μ L, ddH₂The μ L of O 18.6, the μ L of cumulative volume 30.

Embodiment 4：The construction method of described salt sward resistant gene of salt HgS3 expression vector, its step is：

The two digestions of BamHI and SacI are added respectively in described salt sward resistant gene of salt HgS3 upstream and downstream primer Site, sense primer F1 be 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ', anti-sense primer R1 be 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' (by Shanghai, Sheng Gong bio-engineering corporations synthesize).

Material is organized as with the 500mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts With cDNA synthetic agent box (Dalian treasured bioengineering Co., Ltd) the reverse transcription synthesis chains of cDNA first, the gene piece is expanded Section, PCR reaction systems are 5 × PrimeSTAR Buffer 5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, sense primer F1 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ' (10 μM) 1 μ L, anti-sense primer R1 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' (10 μM) 1 μ L (Sheng Gong bio-engineering corporations synthesize by Shanghai), cDNA 1 μ L, PrimeSTAR HS archaeal dna polymerases 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre- changes Property 4min, 94 DEG C of 50s, 60.4 DEG C of 15s, 72 DEG C of 1min, totally 35 circulation, 72 DEG C extension 8min, PCR amplifications such as Fig. 1 institutes Show；PCR primer is reclaimed using glue reclaim kit, to specifications step：It is big with Ago-Gel testing goal fragment first It is small, the blob of viscose of target gene is contained with sterilizing cut, and be fitted into 1.5mL centrifuge tubes；The blob of viscose cut is weighed, according to 1mg=1 μ L ratio converts weight its volume, and then adding the Buffer GM of 3 times of volumes is used to melt blob of viscose, melts Appropriate vibration or suction, which are beaten, in journey makes it fully melt；Spin posts are placed in collecting pipe, then all blob of viscose melting liquids are gone to In Spin posts, room temperature centrifugation 12000rpm, 1min outwell filtrate；Plus 700 μ L Buffer WB in Spin posts and stand 5min Afterwards, room temperature centrifugation 12000rpm, 1min, outwell filtrate, repeat previous step；Empty Spin posts are placed on collecting pipe, room temperature from The heart 12000rpm, 1min, place 2min；Spin posts are put on new centrifuge tube again, it is ultrapure in film center plus 20-30 μ L sterilizings Water, places 1min；Room temperature 12000rpm, centrifuges 1min, for eluted dna, and finally drawing 2-3 μ L is used for electrophoresis detection, other - 20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid pMD19-HgS3, convert large intestine Bacillus DH5 α competent cells, the blue hickie of screening, and bacterium colony PCR identifications are carried out, the bacterium solution that electrophoresis detection contains target stripe is sent Shanghai Sheng Gong bio-engineering corporations are sequenced, and PCR determines sequence as shown in SEQ ID NO.1.

Enter performing PCR amplification again to HgS3, be linked to the sequencing of T- carriers, the HgS3 gene orders such as SEQ ID of acquisition Shown in NO.1.Extract the T- carriers and plant expression vector of the HgS3 genes containing BamHI and SacI restriction enzyme sites PCAMBIA3300 DNAs simultaneously carry out double digestion with two kinds of restriction endonucleases of BamHI and SacI to institute's upgrading grain, and digestion system is 10 1 μ L, QuickCut Sac1 of the μ L of × Quickcut Buffer 3,4 μ L, QuickCut BamH1 of DNA 1 μ L, ddH₂O 21 μ L, the μ L of cumulative volume 30 (Dalian treasured bioengineering Co., Ltd reagent).Electricity is carried out in 37 DEG C of digestion 6-8h, and to digestion products Swimming detects and purifies recovery (Fig. 2).Then T4DNA ligases are used, carrier and HgS3 gene mesh to purifying recovery in previous step Fragment be attached, linked system be the μ L of 10 × T4DNA ligases Buffer 1, the μ L of purpose fragment 7, the μ L of carrier DNA 1, always The μ L of volume 9.First after 65 DEG C of water-bath insulation 3min, then rapid ice bath 2min adds the μ L of T4DNA ligases 1 to connect at 16 DEG C 12h is met, recombinant plasmid transformed competent escherichia coli cell is obtained, the blue hickie of screening is simultaneously sequenced, and confirmation is obtained containing with mesh Gene HgS3 recombinant plasmid pCAMBIA3300-HgS3；Simultaneously to recombinant plasmid pCAMBIA3300-HgS3 in 37 DEG C of digestions 8h, its double digestion system be the μ L of 10 × Quickcut Buffer 3, the μ L of 6 μ L, QuickCut BamH1 of DNA 1.2, QuickCut Sac1 1.2 μ L, ddH₂The μ L of O 18.6, the μ L of cumulative volume 30.

The Agrobacterium transformation of Arabidopsis thaliana of embodiment 5 and culture

Prepare LBA4404 Agrobacteriums and activate, recombinant plasmid pCAMBIA3300-HgS3 is transferred in competence Agrobacterium, During Agrobacterium liquid is prepared, arabidopsis floral is infected using colored method is dipped in, then the plant plastic covering film lucifuge infected is trained Cellar culture in greenhouse is moved to after supporting 24h, point individual plant harvest seed after Arabidopsis plant is ripe.The arabidopsis seed of harvest is low Screening resistance seedling in Kan containing antibiotic (50ug/mL) 1/2MS solid mediums is sown in after temperature processing, sterilizing, and transplants training Support, after seedling grows up, clip plant leaf extracts DNA, and expression vector is expanded with HgS3 genes upstream and downstream primer in embodiment 1 HgS3 fragments in pCAMBIA3300-HgS3.Step sizing, and target gene PCR detections (Fig. 3) are carried out, turn until obtaining T2 HgS3 genes arabidopsis is sheerly.

6 turns of HgS3 genes arabidopsis thaliana salt-tolerance identifications of embodiment

Will in embodiment 3 obtain T2 turn HgS3 genes arabidopsis pure lines plant and wildtype Arabidopsis thaliana (Col-1) seed broadcast Plant in culture matrix (turf：Vermiculite：Perlite is 1:3:0.5 volume ratio is mixed) in, cultivated in greenhouse, treat seedling length to 1 After individual month, 400mM NaCl solutions are poured respectively, are observed Seedling Growth Characteristics, are as a result found, with the lasting exacerbation of salt stress, Wildtype Arabidopsis thaliana growth seriously slows down.After NaCl Stress treatments 6d, it is found that wild-type Arabidopsis plants are seriously wilted, plant Partial blade turn yellow it is withered, when after stress to 9d, the withered death of wildtype Arabidopsis thaliana, and turn HgS3 genes Arabidopsis thaliana Seedlings according to Old survival (Fig. 4).It is therefore seen that conversion HgS3 genes can improve the salt-tolerant trait of Arabidopsis plant, the gene can be used Into other plant (crop) salt tolerance breeding work.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Gansu Agriculture University

<120>Salt sward resistant gene of salt HgS3 and its application

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 468

<212> DNA

<213>Salt sward resistant gene of salt HgS3 nucleotide sequence

<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>Salt sward resistant gene of salt HgS3 coded sequence

<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>The protein amino acid sequence of salt sward resistant gene of salt HgS3 coded sequences

<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 (7)

1. a kind of salt sward resistant gene of salt HgS3, it is characterised in that the nucleotides for the HgS3 genes being separated to from salt sward blade Sequence is as follows, molecular weight 468bp:

。

2. salt sward resistant gene of salt HgS3 as claimed in claim 1, it is characterised in that described salt sward resistant gene of salt HgS3 Coded sequence be nucleotide sequence in claim 1 described in the 208th to the 336th, molecular weight is 129bp：

。

3. salt sward resistant gene of salt HgS3 as claimed in claim 2, it is characterised in that described salt sward resistant gene of salt HgS3 The protein amino acid sequence of coded sequence, is made up of 42 amino acid：

。

4. salt sward resistant gene of salt HgS3 as claimed in claim 1, it is characterised in that special primer is：

HgS3-F1 is 5 '-AAAAGACACTCCATAATCTTGTGTT-3 ',

HgS3-R1 is 5 '-TTTTATTGATGCAAATAAGCTACTA-3 '.

5. salt sward resistant gene of salt HgS3 as claimed in claim 1 preparation method, it is characterised in that step is：

Material is organized as with the 200-500mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts The chains of cDNA first are synthesized with the reverse transcription of cDNA synthetic agent box, the genetic fragment is expanded, PCR reaction systems are 5 × 5 μ L, dNTP Mixture 2.5mM of PrimeSTAR Buffer2.5mM 2 μ L, sense primer F15 '- AAAAGACACTCCATAATCTTGTGTT-3 ' 10 μM of 1 μ L, anti-sense primer R15 '-TTTTATTGATGCAAATAAGCTACTA- 3 ' 10 μM of 11 μ L, PrimeSTAR HS archaeal dna polymerases of μ L, cDNA 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Expand Increasing program is 94 DEG C of pre-degeneration 4min, 94 DEG C of 50s, 60-60.4 DEG C of 15-20s, 72 DEG C of 1min, and common 32-35 is circulated, and 72 DEG C are prolonged Stretch 7-8min；PCR primer is reclaimed using glue reclaim kit, to specifications step：Ago-Gel testing goal is used first Clip size, the blob of viscose of target gene is contained with sterilizing cut, and is fitted into 1.5mL centrifuge tubes；The blob of viscose cut is claimed Weight, converts weight its volume according to 1mg=1 μ L ratio, and then adding the Buffer GM of 3 times of volumes is used to melt blob of viscose, Being vibrated in melting process or inhaling to beat makes it fully melt；Spin posts are placed in collecting pipe, then all blob of viscose melting liquids are gone to In Spin posts, room temperature centrifugation 12000rpm, 1min outwell filtrate；Plus 700 μ L Buffer WB in Spin posts and stand 5min Afterwards, room temperature centrifugation 12000rpm, 1min, outwell filtrate, repeat previous step；Empty Spin posts are placed on collecting pipe, room temperature from The heart 12000rpm, 1min, place 2min；Spin posts are put on new centrifuge tube again, it is ultrapure in film center plus 20-30 μ L sterilizings Water, places 1min；Room temperature 12000rpm, centrifuges 1min, for eluted dna, and finally drawing 2-3 μ L is used for electrophoresis detection, other - 20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains recombinant plasmid pMD19-HgS3, convert large intestine Bacillus DH5 α competent cells, the blue hickie of screening, and bacterium colony PCR identifications are carried out, the bacterium solution that electrophoresis detection contains target stripe is entered Row sequencing.

6. the construction method of salt sward resistant gene of salt HgS3 as claimed in claim 1 expression vector, it is characterised in that step For：

Add the two digestions of BamHI and SacI position respectively in described salt sward resistant gene of salt HgS3 upstream and downstream primer Point, sense primer F1 be 5 '-CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ', anti-sense primer R1 be 5 '- CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3’:

Material is organized as with the 200-500mM NaCl salt sward seedling leaves for handling 3-7d, total serum IgE is extracted with Trizol methods, adopts The chains of cDNA first are synthesized with the reverse transcription of cDNA synthetic agent box, the genetic fragment is expanded, PCR reaction systems are 5 × 5 μ L, dNTP Mixture2.5mM of PrimeSTAR Buffer2.5mM 2 μ L, sense primer F15 '- CGGGATCCAAAAGACACTCCATAATCTTGTGTT-3 ' 10 μM of 1 μ L, anti-sense primer R1 5 '- μ L, PrimeSTAR the HS archaeal dna polymerases of CGAGCTCTTTTATTGATGCAAATAAGCTACTA-3 ' 10 μM of 1 μ L, cDNA 1 0.25 μ L, the μ L of ultra-pure water 14.75, the μ L of cumulative volume 25；Amplification program is 94 DEG C of pre-degenerations 4min, 94 DEG C of 50s, 60-60.4 DEG C of 15- 20s, 72 DEG C of 1min, common 32-35 circulation, 72 DEG C of extension 7-8min；PCR primer is reclaimed using glue reclaim kit, according to saying Bright book step：Ago-Gel testing goal clip size, the film of target gene is contained with sterilizing cut first, is loaded In 1.5mL centrifuge tubes；Then plus 3 times of bodies the blob of viscose cut is weighed, weight converted its volume according to 1mg=1 μ L ratio, Long-pending Buffer GM, which are used to melt in blob of viscose, melting process to vibrate or inhale to beat, makes it fully melt；Spin posts are placed in collection Pipe, then goes to all blob of viscose melting liquids in Spin posts, room temperature centrifugation 12000rpm, 1min, outwells filtrate；Plus 700 μ L Buffer WB are in Spin posts and stand after 5min, room temperature centrifugation 12000rpm, 1min, outwell filtrate, repeat previous step；Will Empty Spin posts are placed on collecting pipe, room temperature centrifugation 12000rpm, 1min, place 2min；Spin posts are put on new centrifuge tube again, In film center plus 20-30 μ L sterilizing ultra-pure waters, 1min is placed；Room temperature 12000rpm, centrifuges 1min, for eluted dna, finally inhales 2-3 μ L are taken to be used for electrophoresis detection, other -20 DEG C of preservations of product；Then recovery product is connected with pMD19-T carriers, obtains weight Group plasmid pMD19-HgS3, converts bacillus coli DH 5 alpha competent cell, the blue hickie of screening, and carry out bacterium colony PCR identifications, electrophoresis Bacterium solution of the detection containing target stripe is sequenced.

Extract the T- carriers and plant expression vector pCAMBIA3300 matter of the HgS3 genes containing BamHI and SacI restriction enzyme sites Grain DNA simultaneously carries out double digestion with two kinds of restriction endonucleases of BamHI and SacI to institute's upgrading grain, and digestion system is 10 × Quickcut 1 μ L, QuickCut Sac1 of the μ L of Buffer 3,4 μ L, QuickCut BamH1 of DNA 1 μ L, ddH₂The μ L of O 21, cumulative volume 30μL；In 37 DEG C of digestion 6-8h, and electrophoresis detection is carried out to digestion products and recovery is purified；Then T4 DNA ligases are used, it is right The carrier and HgS3 gene purpose fragments that recovery is purified in previous step are attached, and linked system is 10 × T4 DNA ligases The μ L of Buffer 1, the μ L of purpose fragment 7, the μ L of carrier DNA 1, the μ L of cumulative volume 9；First after 65 DEG C of water-bath insulation 3min, rapid ice 1-2min is bathed, then adds the μ L of T4 DNA ligases 1 in connecting 12-18h at 16 DEG C, the impression of recombinant plasmid transformed Escherichia coli is obtained State cell, the blue hickie of screening is simultaneously sequenced, and confirms to obtain containing the recombinant plasmid pCAMBIA3300- with target gene HgS3 HgS3；Simultaneously to recombinant plasmid pCAMBIA3300-HgS3 in 37 DEG C of digestion 6-8h, its double digestion system is 10 × Quickcut 1.2 μ L, QuickCut Sac1 of the μ L of Buffer 3,6 μ L, QuickCut BamH1 of DNA 1.2 μ L, ddH₂The μ L of O 18.6, The μ L of cumulative volume 30.

7. salt sward resistant gene of salt HgS3 as claimed in claim 1 or 2 is improving the application of plant salt tolerance.