WO2014205599A1 - Tonoplast sodium-hydrogen antiporter in nhx3 of thellungiella halophila and coding gene and use thereof - Google Patents

Abstract

Disclosed in the present invention are a tonoplast sodium-hydrogen antiporter in NHX3 derived from Thellungiella halophila and the coding gene thereof, and the use thereof in the cultivation of transgenic plants having an improved salt tolerance.

Description

FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a vacuolar membrane sodium hydrogen reversion derived from small salt mustard The transporter NHX3 and its coding gene, and its use in the cultivation of transgenic plants with improved salt tolerance. BACKGROUND OF THE INVENTION Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Salted soil is usually dominated by sodium salt, calcium salt or magnesium salt, and is a major factor affecting plant growth and causing food and economic crop yield reduction. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland. Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance. The selection of plant resistance to salt and alkali, drought-tolerant ability and plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land. For most crops, most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of 0.3% or less. Excess Na ^{+ in the} soil will be normal to the plant. Growth metabolism produces toxic effects. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.

The salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules. Scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, which has made a breakthrough in the research in this field (Zhu Ank. 2002. Arab. singapore. H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cell, 23: 396-411). Higher plant cells can sense changes in physicochemical parameters in the external environment through various pathways, thereby transmitting extracellular signals to intracellular signals, and finally transmitting stress signals to the nucleus to activate transcription factors through a series of signal transduction. The activated transcription factor acts on the functional gene to initiate the expression of the stress response gene to increase the tolerance of the plant. Although researchers have conducted a large number of studies from different sides, due to the complexity of the mechanism, many important issues in plant salt resistance remain to be explored. For example, the key factors in plant salt resistance have not yet been found in IJ; the molecular mechanism of plant salt tolerance is not well understood. Summary of the invention

 The present inventors cloned a tonoplast sodium hydrogen anti-transporter (herein named NHX3) encoding gene of small salt mustard using SSH (suppression subtractive hybridization) and RACE (rapid amplification of cDNA ends), and The DNA sequence was determined. Furthermore, it was found that the transgenic plants were introduced into the plants and expressed, and the salt tolerance of the transgenic plants was significantly improved, and these traits were stably inherited.

 The first aspect of the present invention provides a gene encoding a tonoplast sodium hydrogen back transporter NHX3 of small salt mustard (herein named 73⁄4NH 5 ) having the sequence of SEQ ID NO: 2.

A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, and the nucleotide sequence of the gene The expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300 _; preferably, the recombinant expression vector is the 35S-ThNHX3-2300 vector shown in Figure 2.

 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.

 A fourth aspect of the invention provides a method for improving salt tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene Preferably, the plant is Arabidopsis thaliana.

 A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Preferably, the plant is Arabidopsis thaliana.

 A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving salt tolerance of a plant and for use in plant breeding Use; Preferably, the plant is Arabidopsis thaliana.

 The seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is set forth in SEQ ID NO: 1. DRAWINGS

Fig. 1 is a plant expression vector (35S-ThNHX3-2300) construction flow of the Γ5 gene (Fig. la-lb) Fig. 2 is a plasmid map of the plant expression vector (35S-ThNHX3-2300) of the 73⁄4Λ3⁄4Χ5 gene. Fig. 3 shows the results of salt tolerance test of T1 Arabidopsis thaliana plants transgenic with ThNHJG gene, and Τΐβ showed significant salt tolerance, and the results of Tlf7 and Tlf10 were similar thereto, and are not shown here.

 Figure 4 shows the results of molecular level detection of the transcription level of the ΊΉΝΗΧ3 gene in 1\ generation transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. M is DNA Ladder Marker ( DL2000 ) , 1-8 is salt-tolerant T1 transgenic Arabidopsis thaliana plants (three strains of Τ1β, Tlf7, TlflO, respectively), 9 is 35S-ThNHX3-2300 plasmid PCR positive control, 10- 13 is a non-transgenic control Arabidopsis plant. BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

 The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1. Salt salt mustard SSH library construction under salt stress:

 The specific method is:

A method according to PCR-select ^TM cDNA Clontech kit Subtraction Kit's instructions shown by suppression subtractive hybridization libraries constructed SSH (suppression subtractive library). The mRNA extracted from the salt-treated small salt mustard tissue was used as a sample (Tester) during the experiment, and the mRNA extracted from the untreated small salt mustard tissue was used as a control. Specific steps are as follows:

 (1) Test materials:

Small salt mustard (TheUungieUa halophila, purchased from the Yanlan Plant Breeding Center of Ulan Buh and Desert Green Botanical Garden, Bayannao, Inner Mongolia, China) Seeded onto sterilized vermiculite, at 22 ° C, photoperiod 12 hours light / 12 hours dark (Light intensity 3000-4000 Lx) culture, 1/2MS liquid medium per week (containing 9.39 mM KN0 ₃ , 0.625 mM KH ₂ P0 ₄ , 10.3 mM NH4NO3, 0.75 mM MgS0 ₄ , 1.5 mM CaCl ₂ , 50 μΜ KI, 100 μΜ H ₃ B0 ₃ , 100 μΜ MnS0 ₄ , 30 μΜ ZnS0 ₄ , 1 μΜ Na ₂ Mo0 ₄ , 0.1 μΜ CoCl ₂ , 100 μΜ Na ₂ EDTA, 100 μΜ FeS0 ₄ ) once. It was used for experiments when the plant diameter reached 5-6 cm.

 (2) Material handling:

The test plants were divided into 2 groups, 4 pots per group and 3 plants per pot. The first group was the control group, which was normally watered with 1/2 MS liquid medium; the second group was the salt treatment group, and 1/2 MS liquid medium containing 300 mM NaCl was poured. The two groups of plants were cultured for 10 days at 22 ° C, photoperiod of 12 hours light / 12 hours dark (light intensity 3000-4000 Lx), and then two groups of plants were collected in time (washing the roots with steamed water) After liquid nitrogen is rapidly frozen, Store in a -70 ° C refrigerator.

 (3) Total RNA extraction:

3.0 g of each of the small salt mustards of the control and salt treatment groups were taken, and total RNA was extracted using a plant RNA extraction kit (purchased from Invitrogen). The absorbance of total RNA at 260 nm and 280 nm, OD ₂₆ , was determined using a HITACHI UV spectrophotometer U-2001. /OD ₂₈ . The ratio of 1.8-2.0 indicates that the total RNA purity is high. The integrity of total RNA is detected by 1.0% agarose gel electrophoresis. The brightness of the 28S band is about twice that of the 18S band, indicating that the RNA integrity is good. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

The method according to Clontech's PCR-select ^TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization. Driver mRNA and Tester mRNA were reverse transcribed separately (reverse transcription primers were used as primers in the kit) to obtain double-stranded cDNA, and subtraction hybridization was carried out using 2 Tester cDNA and 2 g Driver cDNA as starting materials. The Tester cDNA and P Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with an excess of Driver for the first forward subtractive hybridization. The products of the two first forward subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed genes were amplified by two inhibitory PCR amplifications (PCR). Before, the second forward subtractive hybridization product is end-filled).

 (5) Construction and preliminary screening, cloning and identification of subtractive libraries

The second inhibitory PCR amplification product of the second forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) according to the instructions of the pGEM-T Easy kit (purchased from Promega) The specific steps are as follows: Connect the following components in the 200 μΐ PCR tube: Purify the second PCR product 3 μ1, 2χΤ4 ligase buffer 5 μΚρΟΕΜ-Τ Easy Vector 1 μ1, T4 DNA ligation The enzyme was 1 μl and ligated overnight at 4 °C. Then 10 μΐ of the ligation reaction product was added to 100 μM E. coli JM109 competent cells (purchased from TAKARA), ice bath for 30 minutes, heat shock at 42 ° C for 60 seconds, ice bath for 2 minutes, and then 250 μL LB liquid culture. Base (containing 1% tryptone (purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Chinese medicine), placed in a 37 ° C shaker, Incubate at 225 rpm for 30 minutes, then inoculate 200 μM from the inoculum containing 50 μ _§ /ιη1 ampicillin, 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-half Lactate), 24 μgmL IPTG (isopropyl-β-D-thiogalactopyranoside) on LB (ibid.) solid plate (X-gal and IPTG) All purchased from TAKARA) and incubated at 37 ° C for 18 hours. Count the clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 450 white colonies (number: Th-S001 to Th-S450). The white colonies picked were inoculated into LB liquid medium containing 50 g/ml ampicillin in a 96-well cell culture plate (CORNING), and cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20% glycerol. Ratio), then stored at -80 ° C for later use. The nested PCR primers Primer 1 and Primer 2R (PCR-selectTM cDNA Subtraction Kit from Clontech) were used to verify the PCR amplification of the cultured bacteria, and 342 positive clones were obtained, and then all positive clones were sent to the UK.潍Jieji (Shanghai) Trading Co., Ltd. was sequenced.

 (6) cDNA sequencing analysis of differential clones:

 After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 301 Expressed Sequence Tags (EST) (Unigene) were obtained. Example 2 Small salt mustard vacuolar membrane sodium hydrogen antiporter encoding gene ThNHX3 clone

 After removing the redundant DNA from the identified clone of Th-S173 in the identified SSH library of Arabidopsis thaliana, the sequence is SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belongs to the vacuolar membrane sodium hydrogen anti-transporter. . Here, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named ThNHX3, and its corresponding protein was named NHX3.

 SEQ ID No: 3:

ACGCAACAGT CCTGGCCAGT CGATAGGAGT GAGTTCGATA CTCCTTGGGC TTATTCTTCT

 241 TGGAAACGCT ATCATGATCA CCAGCACCAT CAGTGTTGTT CTTTTCAGT

Cloning of the full-length coding gene of ThNHX3

 Based on the sequence of SEQ ID No: 3 that has been obtained, the following two specific primers were designed as the 5'-end specific primer for 3' RACE.

 ThNHX3 GSP1: SEQ ID No: 4:

 TGGTCTGATG CGTGGGGCAGT G

ThNHX3 GSP2: SEQ ID No _: 5:

 CTTCAGGACA CACGAAGCTT CTT

The experimental procedure was performed according to the kit instructions (3'RACE System for Rapid Amplification of cDNA The Ends kit was purchased from Invitrogen).

 The first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal primer AUAP (provided with the kit), and the cDNA obtained by reverse transcription of the mRNA extracted from the salt-treated group was used as a template. Specific steps are as follows:

 50 μl ΡΟ Reaction system: 5 μΐ 10x ExBuffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 lExTaq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and AUAP each 2.0 μΐ and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50 times with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:

 50 μl ΡΟ Reaction system: 5 μΐ 10xExBuffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 lExTaq, 10 μΜ primer SEQ ID NO: 5 and AUAP each 2.0 μΐ and 35 μΐ double distilled water . PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

A band of about 200 bp in the second round of PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method is the same as above) The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin and 40 μg mL X-gaK 24 g/mL IPTG for screening. Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 μ _§ /ιη1 ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20% (volume ratio), and stored at -80 ° C until use. Using SEQ ID NO: 5 and the universal primer AUAP for bacterial liquid PCR amplification, 7 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and 3 of the cDNA of the gene was obtained. 'end.

 Based on the obtained 73⁄4^3⁄4^5 gene fragment, the following three specific primers were designed as the 3'-end specific primer of 5'RACE.

ThNHX3 GSP3: SEQ ID No _: 6:

 CCAAATGGTT ACCTGTTTCT TC

ThNHX3 GSP4: SEQ ID No _: 7:

 CGACAAGAAC GAAAGAGGGA AC

ThNHX3 GSP5: SEQ ID No _: 8:

 GCCCAAGGAG TATCGAACTC AC

Experimental procedure according to the kit instructions (5'RACE System for Rapid Amplification of cDNA The Ends kit was purchased from Invitrogen).

 Using SEQ ID NO: 7 and the universal primer AAP (provided with the kit), the cDNA obtained by reverse transcription of mRNA extracted from the salt-treated group of small salt mustard (reverse transcription primer SEQ ID NO: 6, dCTP tail) was used as a template. Perform the first round of PCR amplification, the specific steps are as follows:

 50 μl ΡΟ Reaction system: 5 μΐ 10xEx Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 l Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 7 and AAP each 2.0 μΐ and 35 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and the primer AUAP. The specific steps are as follows:

 50 μl ΡΟ Reaction system: 5 μΐ 10xEx Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primers SEQ ID NO: 8 and AUAP each 2.0 μΐ and 35 μΐ Double steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 Recover a 1100 bp band from the second round of PCR products (Gel Extraction Kit was purchased from

OMEGA), and ligated it into pGEM-T Easy vector, then transformed into E. coli JM109 competent cells (specific method as above), and applied the transformed bacterial solution to 50 g/mL ampicillin, 40 ^ Screening was performed on LB solid medium of glmL X-gaK 24 g/mL IPTG. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 μ _§ /ιη1 ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. spare. Using SEQ ID NO: 8 and primer AUAP for PCR amplification (the reaction system and reaction conditions are the same as above), 7 positive clones were obtained, and 4 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The 5' end of the cDNA of this gene was obtained. After the obtained 5' RACE product clone was sequenced, it was spliced with the above 3' RACE product sequencing result and the SEQ ID No: 3 sequence to obtain the ThNHX3 full-length cDNA sequence SEQ ID No: 9.

SEQ ID No: 9: 361

42 1

 4 81

54 1

601

661

72 1

781

84 1

901

961

102 1

1081

114 1

12 01

1261

132 1

13 81

 144 1 TACTGGAAAA CCAATTCTAC A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:

 SEQ ID No: 10:

 ATGGCTTTGG GATTAAACAC AAT

SEQ ID No: 11:

 CTAGCCGAAA TCTGTATAAA TGAG The ΊΉΝΗΧ3 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.

 The PCR reaction was carried out using TAKARA's PrimeSTAR HS DNA polymerase and using the cDNA of the salt-treated mustard as a template. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primer SEQ ID NO:

10 and SEQ ID NO: 11 each of 2.0 μΐ and 30 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes.

PCR amplification product plus A tail: 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and the resulting precipitate was dissolved in 21 μL of double distilled water. Then add 2.5 μΐ to it ΙΟχΕχ Buffer 0.5 μΐ 5 mM dATP, 1.0 lExTaq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained 1400 bp DNA fragment was recovered (Omega recovery kit), and ligated into the pGEM T-easy vector to obtain a ThNHX3-pGEM plasmid, and then the ligation product was transformed into E. coli JM109 competent cells (method as above) The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin, 40 g/mL X-gal, and 24 g/mL IPTG for screening. Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 μ _§ /ιη1 ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20% (volume ratio), and stored at -80 ° C until use. Using SEQ ID NO: 10 and SEQ ID NO: 11 to confirm the bacterial liquid PCR amplification (reaction system and reaction conditions as above), five positive clones were obtained, and four positive clones were selected and sent to Yingjiejie (Shanghai). The company was sequenced and the resulting sequence was SEQ ID NO: 2, and the amino acid sequence of the encoded protein was SEQ ID NO: 1.

 Amino acid sequence of NHX3 protein: SEQ ID NO: 1

 1 ALGLNT LE KSETLLGSDH

 21 ASVVS NLFV ALLCACIVLG

 41 HLLGKTRW N ESITALIIGS

 61 CTGIVILLIS EGKSSRILVF

 81 SEDLFFIYLL PPIIFNAGFQ

 101 VKKKHFFRNF TI LFGAIG

 121 TLISFVIISL GAIHFLEK N

 141 IGDLTISDYL AIGAIFSATD

 161 SVCTLQVLKQ EETPLLYSLV

 181 FGEGVVNDAT SVVLFNAIQR

 201 FDLTHINSAI ALEFAGNFFY

 221 LFILSTALGV AAGLLSAFVI

 241 KKLYFESHAT NREVAL LL

 261 AYLSY LAEL FHLSSILTVF

 281 FCGIV SHYT WHNVTDKSKI

 301 TTRHTFAALS FLAEIFIFLY

 321 VG DALDIEK WDVVRNSPGQ

 341 SIGVSSILLG LILLGRAAFV

 361 FPLSFLSNLT KSSTDERIDW

 381 KKQVTIWWAG L RGAVS AL

 401 AYNQFTTSGH TKLLGNAI I

 421 TSTISVVLFS TVVRLKRFSP

 441 NRVCVLIYTD FG

The nucleotide sequence of the ThNHX3 gene SEQ ID NO: 2 61

121

 181

 241

 301

 361

 421

481

 541

 601

 661

 721

781

 841

 901

 961

 1021

1081

 1141

 1201

 1261

 1321 AACCGGGTTT GCGTCCTCAT TTATACAGAT TTCGGCTAG Example 3 Construction of ΊΉΝΗΧ3 gene plant expression vector

 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The 35S promoter and the Tnos terminator were selected as promoters and terminators of the ThNHJG gene, respectively. The construction flow chart is shown in Figure 1.

Pnos was amplified using the primers SEQ ID NO: 12 and SEQ ID NO: 13 with the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, using TAKARA's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121 plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 12 and SEQ ID NO: 13 each 2.0 μΐ and 31 ΐ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with EcoRI, Bglll, and ligated into pCAMBIA2300 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.

 SEQ ID NO: 12

 GCACGAATTC ggcgggaaac gacaatctga

 SEQ ID NO: 13

 ATCCAGATCTAGATCCGGTGCAGATTATTTG

 Tnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 using the pBI121 plasmid as a template, and TAKARA's PrimeSTAR HS DNA polymerase was used. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121 plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 μΐ and 31 μΐ of double distilled water.

PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by Kpnl and EcoRI (Promega T4 ligase kit) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.

SEQ ID NO: 14:

 AAGGGTACCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 15:

 TCAGAATTCCCAGTGAATTCCCGATCTAGTA The 35S promoter was amplified using the primers SEQ ID NO: 16 and SEQ ID NO: 17 using the pCAMBIA2300 plasmid as a template. TAKARA's PrimeSTAR HS DNA polymerase was used. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μΐ and 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by HindIII and Sail (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3. SEQ ID NO: 16:

 ACTAAGCTTTAGAGCAGCTTGCCAACATGGTG SEQ ID NO: 17:

TGAGTCGACAGAGATAGATTTGTAGAGAGAGACT Amplifies the full-length sequence of the 73⁄4NHX5-encoding gene with primers SEQ ID NO: 18 and SEQ ID NO: 19 The plate was the positive ThNHX3-pGEM plasmid obtained in Example 2, and the PrimeSTAR HS DNA polymerase of TAKARA was used. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ ThNHX3-pGEM plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 18 and SEQ ID NO: 19 each 2.0 Μΐ and 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes. The resulting PCR product was ligated by Sal I, BamH I digestion (ligation method as above) to pCAMBIA2300-3, and the plant expression vector 35S-ThNHX3-2300 was obtained after verification (Fig. 2).

SEQ ID NO: 18

 ACTGTCGAC ATGGCTTTGG GATTAAACAC AAT SEQ ID NO: 19

 ACTGGATTC CTAGCCGAAA TCTGTATAAA TGAG Example 4 35S-ThNHX3-2300 expression vector for transformation of Agrobacterium

Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 Incubate at °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 streptomycin, and cultured overnight (about 12-16 hours) to OD ₆ at 28 °C with shaking. . A value of 0.4 forms a seed broth. 5 ml of culture-activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 streptomycin, and cultured at 28 °C. -2.5 hours to OD ₆ . . =0.8. The ice bath liquid was shaken for 10 minutes every 3 minutes to allow the bacteria to enter a dormant state uniformly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; resuspend the cells by adding 1 ml of ice-cold 10% (by volume) glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; Pre-cooled 10% (volume ratio) glycerin was washed 3-4 times repeatedly; then add 10% (by volume) of glycerol to resuspend the bacterial pellet to prepare LBA4404 competent cells at 40 μΐ/tube. It is packaged and stored at -70 °C for later use.

Transformation of Agrobacterium: The LBA4404 competent cells were thawed on ice, and 1 μM of the plasmid 35S-ThNHX3-2300 obtained in Example 3 was added to 40 μΐ of competent cells, and the mixture was mixed and ice-cooled for about 10 minutes. Transfer the mixture of competent cells after ice bath and 35S-ThNHX3-2300 plasmid to a pre-cooled 0.1 cm size electric shock cup (purchased from Bio-Rad) using a micropipette, tapping to bring the suspension to an electric shock The bottom of the cup (be careful not to have bubbles). Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber. Set the program of MicroPulser (purchased from Bio-Rad) to "Agr" and apply an electric shock once. Take out the electric shock immediately Cup, add 200 μΙ Β 预 medium pre-warmed at 28 °C. Quickly and gently mix the competent cells with a micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 1 hour at 225 rpm. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 g/ml rifampicin, 50 μ _§ /ιη1 streptomycin, 50 μ _§ /ιη1 card Natamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Receptor Material Arabidopsis Culture

 Choose the rock with good water absorption and soft soil and the nutrient soil (1: 1) as the soil for Arabidopsis planting. Using a 9 cm diameter pot, 20-30 Arabidopsis seeds were seeded per pot (Columbia type, from the Arabidopsis Bioresource Center, Ohio State University). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22 ° C, light intensity 3500-4000 k, photoperiod of 12 hours dark, 12 hours light culture, 1/2 MS liquid medium per 7 days. After 30 days of culture, 4-5 plants were kept per pot, and the photoperiod was adjusted to 8 hours of darkness and 16 hours of light culture. After most of the plants were bolted, the entire main moss was cut off at the base of the inflorescence, and the top edge was approximated. After 1 week, 4-6 new side mosses grow in the axillary bud, and when the flower buds form part of the flower buds and partially flower or form 1-2 pods, they can be used for transformation. Example 6 Arabidopsis flower leaching transformation

The LBA4404 Agrobacterium liquid of the transformed 35S-ThNHX3-2300 expression vector obtained in Example 4 was inoculated to an LB liquid medium containing 50 μ _§ /ιη1 kanamycin (kan), and the next morning, 1 was pressed. :50 Inoculate into a new LB medium (1 L) containing 50 μ _§ /ιη1 kanamycin, and culture for about 8 hours to Agrobacterium OD ₆ . . Between 1.0 and 1.2. Centrifuge at 5000 rpm for 5 minutes at room temperature, discard the supernatant, and suspend the Agrobacterium pellet in the impregnation medium (1/2MS liquid medium and contain 5% sucrose; adjust to pH 5.7 with KOH; 0.02% Silwet L-77) In the middle, the OD _{6QQ is} around 0.8. The upper part of the Arabidopsis thaliana prepared for transformation prepared in Example 5 was slowly and spirally immersed in the impregnation medium containing the Agrobacterium, and gently shaken clockwise for about 2 minutes, and covered with a transparent plastic cover to maintain Humidity, put in the greenhouse overnight. After 24 hours, remove the plastic transparent cover and pour through the water. After 2-3 weeks, ensure that the plants are hydrated. When the plant stops flowering and the first fruit pod matures and turns yellow, it is covered with a paper bag. When all the pods in the paper bag turn yellow, the watering is stopped. After 1-2 weeks of drying, the seeds are collected and the transformants are screened. Untransformed Arabidopsis fruit pods were taken as controls. Example 7 Screening of transgenic positive transformants from Arabidopsis thaliana Seed disinfection: Soak for 10 minutes with 70% ethanol, and occasionally suspend the seeds; then wash with sterile water four times, and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on the surface of 1/2MS solid screening medium containing 50 μ _§ /ιη1 kanamycin (a maximum of 1500 seeds were seeded in a 150 mm diameter plate), and vernalized at 4 °C. After 2 days, it was cultured for 7-10 days at a constant temperature of 22 ° C, an illumination intensity of 3500-4000 k, and a photoperiod of 12 hours of darkness/12 hours of light. After germination of the transgenic seeds on the screening medium for 2 weeks, the plants capable of germination and normal growth were transferred to soil for further cultivation. 1-2 leaves of each plant capable of normal growth on the screening medium were extracted, DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and Conditions are the same as above), PCR-negative plants are removed, and the seeds of PCR-positive plants are collected and numbered (T0fl-T0£20) and stored. Example 8 Planting of transgenic Arabidopsis thaliana T1 plants overexpressing ΊΉΝΗΧ3

 Select the vermiculite with good water absorption and soft soil to match the nutrient soil (1:1) as the soil for Arabidopsis planting. Each transformant of TOfl-TOGO and non-transgenic control Arabidopsis seeds were sown in 2 pots (20-30 seeds per pot). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22 ° C, light intensity 3500-4000 lx, photoperiod of 12 hours dark, 12 hours light culture, 1/2 MS liquid medium per 7 days. After culturing for 25 days, 1-2 leaves were cut per plant and DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and conditions are the same as above). The PCR-negative plants were removed, and 7-8 PCR-positive vaccines were retained in each pot. After 10 days of continuous culture, 5-7 transgenic Arabidopsis thaliana or non-transgenic control Arabidopsis thaliana seedlings with uniform size were kept in each pot for salt tolerance experiments. Example 9 Salt tolerance test of transgenic Arabidopsis T1 plants overexpressing ThNHX3

 The transgenic Arabidopsis thaliana and the control Arabidopsis thaliana each of the plants in Example 8 were left untreated, and 1/2 MS liquid medium was normally poured, and one pot of each plant was irrigated with 1/2 MS liquid medium containing 150 mM NaCl. The temperature was 22 ° C, the light intensity was 3500-4000 k, the 12-hour light culture/12-hour dark culture cycle, and the experimental results were observed after 14 days. The salt tolerance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the T1 transgenic plants showed significant salt tolerance in the three strains of Τ1β, Tlf7 and TlflO (see Figure 3, with Τΐβ For example, the results of Tlf7 and Tlf10 are similar, not shown here. Example 10 Validation of expression of the 73⁄4Λ3⁄4Χ5 gene at the transcriptional level

8 of the T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (respectively belonging to the above Τ1β, Tlf7, TlflO three salt-tolerant strains, the control plants in Example 9 were randomly selected from 4 plants, each of which was treated with a salt (150 mM NaCl) for 15 days, 0.05 g, and extracted with the plant RNA extraction kit (Invitrogen). RNA. The absorbance values of total RNA obtained at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. The reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay [J Box Superscript III Reverse Transcriptase, and 1 total RNA was used as a template, and the reverse transcription primer was SEQ ID NO: 11. The ThNHX3 fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20, and its transcription was examined.

 PCR was carried out using TAKARA's PrimeSTAR HS DNA polymerase using the cDNA obtained by the above reverse transcription as a template. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 20 each 2.0 μΐ and 30 μΐ Double steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 32 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 The electrophoresis results of the PCR products are shown in Figure 4: M is the DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), and 1-8 is the salt-tolerant T1 transgenic Arabidopsis plants ( belonging to Τ1β, Tlf7, TlflO, respectively). Three strains), 9 is the 35S-ThNHX3-2300 plasmid PCR positive control, and 10-13 is the non-transgenic control Arabidopsis plant. The size of the band shown is the same as the size of the positive control (approximately 750 bp). The results showed that the salt-tolerant T1 transgenic Arabidopsis plants had significant transcription in 73⁄4Λ3⁄4Χ5, and there was no transcription of ThNHJG in non-transgenic control Arabidopsis plants.

SEQ ID NO: 20: CGATTAGTAG CGTGACTTTC GAA

Claims

claims 1. A tonoplast sodium-hydrogen antiporter of Salina mustard, its sequence is SEQ ID NO: 1. 2. The gene encoding the tonoplast sodium-hydrogen antiporter of claim 1, whose sequence is SEQ ID NO: 2. 3. A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and the nucleotide sequence of the gene is operably linked to the expression control sequence of the expression vector For connection, preferably, the expression vector is pCAMBIA2300. 4. The vector according to claim 3, which is the 35S-ThNHX3-2300 vector shown in Figure 2. 5. A recombinant cell containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4; Preferably, the recombinant cell is a recombinant Agrobacterium cell. 6. A method for improving salt tolerance of plants, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, The plant in question is Arabidopsis thaliana. 7. A method for preparing transgenic plants, comprising: cultivating plants or plant tissues containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions that are effective for producing plants. 8. The method of claim 7, wherein the plant is Arabidopsis thaliana. 9. The use of the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for improving plant salt tolerance and for plant breeding. 10. The use of claim 9, wherein the plant is Arabidopsis thaliana.