WO2015042732A1

WO2015042732A1 - Kandelia candel sodium-hydrogen transport protein nha2, coding gene of same, and application thereof

Info

Publication number: WO2015042732A1
Application number: PCT/CN2013/001151
Authority: WO
Inventors: 崔洪志; 田大翠; 游婵平; 梁远金
Original assignee: 创世纪转基因技术有限公司
Priority date: 2013-09-25
Filing date: 2013-09-25
Publication date: 2015-04-02
Also published as: CN104995205A

Abstract

Provided are a Kandelia candel-sourced sodium-hydrogen transport protein NHA2, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased salt tolerance.

Description

Kandelia sodium hydrogen transporter NHA2 and its coding gene and application

FIELD OF THE INVENTION The present invention relates to plant proteins and their coding genes and applications, and more particularly to a sodium hydrogen transporter NHA2 derived from Kandelia and its coding gene, and its use in the cultivation of transgenic plants having 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 improvement of plant resistance to salt and alkali and the selection of plant species or strains suitable for growth on saline-alkali land with high economic and ecological value are economical and effective measures to utilize saline-alkali land. For most crops, most plants are poorly tolerant to saline and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na ^{+ in the} soil will normalize the growth and metabolism of plants. Produces a toxic effect. 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. Activation of the transcription factor acts on the functional gene and initiates the expression of the stress response gene, thereby increasing the tolerance of the plant. Although researchers have conducted a large number of studies from different sides, due to the complexity of its mechanism, many important issues in plant salt resistance remain to be explored. For example, key factors in plant salt tolerance have not been found; the molecular mechanism of plant salt tolerance is not well understood. SUMMARY OF THE INVENTION The present inventors cloned a Kupffer sodium hydrogen transporter (herein referred to as HA2) encoding gene and determined its DNA by using SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends). sequence. Moreover, it was found that the transgenic plants were significantly improved in salt tolerance by transgenic technology and introduced into the plants, and these traits were stably inherited.

The first aspect of the present invention provides a gene encoding a Kappa sodium hydrogen transporter HA2 (designated herein as KcNHA2) 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-KcNHA2-2300 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.

A 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

Figure 1 shows the construction process of the plant expression vector (35S-KCNHA2-2300) of the ^3⁄42 gene (Fig. la-lb). Figure 2 is a plasmid map of the plant expression vector (35S-KcNHA2-2300) of the ^3⁄42 gene.

Fig. 3 shows the results of salt tolerance test of T1 Arabidopsis plants transgenic with KcNIU2 gene, Tln3 showed significant salt tolerance, and the results of Τ1η7, Τ1η16 were similar thereto, and are not shown here.

Figure 4 shows the results of molecular level detection of the transcription level of the KcNfU2 gene in T1 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 plants ( belong to Tln3, Τ1η7, Τ1η16, respectively), and 9-12 are non-transgenic control Arabidopsis plants. 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 endonucleases mentioned in the examples below were purchased from New England Biolabs. In the present invention, the proportions and percentages are calculated on a weight basis if not stated and not ambiguous in the context. Example 1. Construction of SSH library of Kandelia 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 Kandelia root was used as a sample (Tester) during the experiment, and the mRNA extracted from the untreated K. candelensis was used as a control. Specific steps are as follows:

(1) Test materials:

Kandelia is collected from Futian National Nature Reserve, Shenzhen, Guangdong Province (Ν22° 53 ', E114° 01 '). Collecting a pest-free, well-developed, matured canola (andelia candel hypocotyls, selecting hypocotyls of similar size, length, and weight for experimentation. In plastic buckets (bottle diameter 18 cm, height 15 cm), sand culture, per The bottom of the barrel is padded with plastic trays, the fine sand is river sand, the average particle size is about 1 mm, the tap water is washed, and the hypocotyls are planted in each keg. At 28 ° C, the natural light is 12 hours per day. During the cultivation of the seedlings, water is poured into the tap water every day, and the Hoagland nutrient solution is poured once a week (DR Hoagland and DI Arnon. The water-culture) Method of growing plants without soil. Calif. Agr. Expt. Sta. Circ. 347. 1950).

(2) Material handling:

The K. candel seedlings with the same growth and development appearance (the seedling height is consistent and each seedling grows to 6 leaves) were divided into two groups. One group was poured with 2 L of 500 mM NaCl, and one group was poured with 2 L of distilled water for 6 hours. Collect the roots of the treatment and control groups. After rapid freezing with liquid nitrogen, it was stored in a -70 ° C refrigerator.

(3) Total RNA extraction:

3.0 g of K. candelensis in the control and salt-treated groups were taken, and total RNA was extracted using a plant RNA extraction kit (purchased from Invitrogen). The absorbance of the total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD ₂₆₀ / OD ₂₈₀ ratio was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose gel was used. Gel electrophoresis detected the integrity of total RNA. The 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. 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 provided as primers) to obtain double-stranded cDNA, and 2 μg of Tester cDNA and 2 μg of Driver cDNA were used as starting materials for subtractive hybridization. . The Tester cDNA and 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 excess Driver cDNA 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 linked to the pGEM-T Easy vector as follows: The following components are sequentially added to the 200 l PCR tube: Purified combined positive subtractive hybridization cDNA fragment second inhibitory PCR product 3 μ 1 , 2 X T4 DNA ligase buffer 5 μl, pGEM-T Easy vector 1 μl, Τ4 DNA ligase 1 μl, ligated overnight at 4 °C. Then, 10 μΐ of the reaction product was ligated, and added to 100 μM E. coli JM109 competent cells (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, and ice bath for 2 minutes. Then add 250 μM LB liquid medium (containing 1% tryptone (Tryptone, purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Sinopharm)) Incubate at 37 ° C for 30 minutes with shaking at 225 rpm, then inoculate 200 μl of the bacterial solution from 50 μg/ml ampicillin, 40 g/mL X-gal (5-bromo-4-chloro-3) -吲哚-β-D-galactoside), 24 g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) LB (same as above) solid (1.5% agar, the same below) Plates (X-gal and IPTG were 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 300 white colonies (number: Kc-SR-001 to Kc-SR-300). The white colonies picked were inoculated into LB liquid medium (same as above) containing 50 μg/ml ampicillin in 96-well cell culture plate (CORNING), and cultured at 37 ° C overnight to add glycerol to the final concentration of glycerol. 20% (by volume), then stored at -80 °C for later use. The nested PCR primers Primer 1 and Primer 2R (PCR-select ^TM cDNA Subtraction Kit from Clontech) were used to perform PCR amplification verification on the cultured cells, and 232 positive clones were obtained, and then all positive clones were sent. Yingjie 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 214 Expressed Sequence Tags (EST) were obtained. Example 2 Cloning of Kappa sodium hydrogen transporter encoding gene KcNiU2

After removing the redundant DNA from the clone designated Kc-SR-132 in the identified Kandelia SSH library, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the sodium hydrogen transporter. Here, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 is named ΜΜ2, and the corresponding protein is named ΝΗΑ2.

SEQ ID No: 3:

1 ACATTGATAT CCTGTTGTAT CATATCTGCA GGTGCTACAC TAGCCTTTGA GAAATTGGAT

61 ATTGGTTCTC TGGATGTTGG GGATTATCTT GCAATTGGTG CAATA TCGC TGCCACCGAT 121 TCTGTTTGCA CATTGCAGGT CCTTGATCAG GATGAGACAC CTTTACTCTA TAGTCTGGTT 181 TCGGAGAAG GTGTTGTAAA TGATGCCACA TCGGT GTGC TCTTTAATGC AATCCAGAGC 241 TTTGATCTCA CTCATCTTAG TCCCAGTATT ACTGGGCAGT TTGTTGGCAG ΟΊΊΊΊΊΆΤΑΤ 301 TTATTITTCA CGAGCACTAT GCTGGGAGTG GTTACTGGTC TGGTTAGTGC CTACATCATC 361 AAAAAACTTT ATTTTGGCAG GCACTCAACA GATCGTGAGG TGCTCTTAT GATCCTTATG 421 GCATACCTTT CGTATATGCT GGCTGAACTT TTCTACTTAA GTGGCATTCT CACTGTATTT 481 TTCTGTGGGA TTGTGATGTC ACATTACACC TGGCACAATG TGACAGAGAG TTCAAGGGTA 541 ACTACCAAGC ATGCTITTGC AACCTTATCA TTGTTGCTG AGATTTTCAT CTTCCITTAT 601 GTTGGCATGG ATGCCTTGGA CATTGAAAAG TGGCGTTTTG TGAGTGATAG CCCGGGAACA

661 TCAATTGCAG TGAGCTCCAT ACTGCTAGCT TTTGTCCTGA TTGGGAGAGC AGCTITTGTC

721 TTTCCATTAT CCTTCATATC CAACCTATCT AAGAAATCAA CTAGCGAAAA GATAGGCATC

781 AAGCAGCAAA TTATAGTATG GTGGGCTGGA CTAATGAGAG GCGCTGTGTC GATGGCACTA

841 GCATACAATA AGTTTACAAG CTTGGGTCAT ACCAATGTGC GAGATAATGC AATAATGATC

901 ACAAGT

Cloning of KcNHA2 full-length coding gene

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.

KcNHA2 GSP1 : SEQ ID No: 4:

GATGTTGGGGATTATCTTGCA

KcNHA2 GSP2: SEQ ID No: 5:

TTGCAGGTCCTTGATCAGGATG Experimental procedure 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 by the salt treatment group was used as a template. Specific steps are as follows:

50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primers SEQ ID NO: 4 and AUAP Each 2.0 μ 1 and 35 μ 1 double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes), 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 by using SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:

50 l PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 5 and P AUAP Each of 2.0 μ 1 and 35 μl 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 60 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

A 1200 bp fragment (Gel Extraction Kit from OMEGA) in the second round of PCR product was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method as above) , and the transformed bacterial solution is coated with 50 μl mL of ampicillin, 40 ^ glmL Screening was performed on LB solid medium of X-gaK 24 g/mL IPTG. 10 white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml 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. . Using SEQ ID NO: 5 and the universal primer AUAP for PCR amplification, 9 positive clones were obtained, and 3 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 gene fragments obtained, the following three specific primers were designed as the 3'-end specific primers for 5 ' RACE.

KcNHA2 GSP3: SEQ ID No: 6:

GGCACTAACC AGACCAGTAA CCACT

KcNHA2 GSP4: SEQ ID No: 7:

CAACAAACTG CCCAGTAATA CTGG

KcNHA2 GSP5 : SEQ ID No: 8:

TCTGGATTGC ATTAAAGAGC ACAACC

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 the mRNA extracted from Kandelia candidum (reverse transcription primer SEQ ID NO: 6, dCTP plus tail) was used as a template. One round of PCR amplification, the specific steps are as follows:

50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 7 and P AAP each with 2.0 μl and 35 μl 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 60 ° 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 μ ΐ 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 yl PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 8 and P AUAP Each of 2.0 μ 1 and 35 μl 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 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

Recover a strip of approximately 800 bp in the second round of PCR product (Gel Extraction Kit was purchased from

OMEGA), and ligated it to pGEM-T Easy vector, then transformed into E. coli JM109 In the cells (the same method as above), the transformed bacterial solution was applied to LB solid medium containing 50 μ _§ /ηΛ ampicillin, 40 μ _§ /ηΛ X-gaK 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml 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. . Using SEQ ID NO: 8 and primer AUAP to verify the bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), 8 positive clones were obtained, and 3 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The 5' end of the cDNA of the 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 KcNHA2 full-length cDNA sequence SEQ ID No _: 9.

SEQ ID No: 9:

61 C TCCTCCGG TGTCTGCAAA GCCACACGGC AGCATTAGAT ATGCAAAACT CTGTACCTCC

121 TGAAATGGAC TGTTCAGAGA CATAAAAAAG AAAAAAGGAA ACTGTGAAGA TTTTTACCTC

181 TGTAGAGATA AAGCTGAGTT TTTCATAGAC CCAATTGCCT TTTCATTGGT TTGGAGAGGA

241 AGGGGAGACA GTGAGACATA TACGATGGA TACCTACATA AGCTCGGCCA TGTCGAGATG

301 GCAGATGGTT TTAGCGTCTG ACCACGCCTC TGTGGTATCT ATGAACCTAT TTGTGGCGCT

361 TCTTTGCGCT TGCATTGTGG TTGGTCATCT TTTAGAAGAG AATCGATGGA TGAATGAGTC

421 GATCACCGCC CTCGTCAT G GTGTATGCAC TGGCGTTGTT ATITIGCTGA TCAGTGGAGG

481 AAAAAGCTCG CGTC rr丄 TCTTCAGCGA GGATC rr丄' C TTCATATATC T CTGCCGCC

541 AATTATATTC AATGCTGGGT TTCAGGTGAA GAAGAAGCAG T CI TCGTA ATTTCATTAC

601 CATCATGCTA TTTGGCGCTG TTGGTACATT GATATCCTGT TGTATCATAT CTGCAGGTGC

661 TACACTAGCC TTTGAGAAAT TGGATATTGG TTCTCTGGAT GTTGGGGATT ATCTTGCAAT

721 TGGTGCAATA TCGCTGCCA CCGAT CTGT TTGCACATTG CAGGTCCTTG ATCAGGATGA

781 GACACCTTTA CTCTATAGTC TGGT TTCGG AGAAGGTGTT GTAAATGATG CCACATCGGT

841 TGTGCTCITT AATGCAATCC AGAGCITTGA TCTCACTCAT CTTAGTCCCA GTATTACTGG

901 GCAGTTTGTT GGCAGCrriT TATATTTATT T TCACGAGC ACTATGCTGG GAGTGGTTAC

961 TGGTCTGGTT AGTGCCTACA TCATCAAAAA ΑΟΓΓΤΑΊΊΊΊ' GGCAGGCACT CAACAGATCG

1021 TGAGGTTGCT CTTATGATCC TTATGGCATA CC TTCGTAT ATGCTGGCTG ΑΆΟΊΊΊΊ'ΟΤΑ

1081 CTTAAGTGGC ATTCTCACTG TATTTTTCTG TGGGATTGTG ATGTCACATT ACACCTGGCA

1141 CAATGTGACA GAGAGTTCAA GGGTAACTAC CAAGCATGCT TTTGCAACCT TATCATTTGT

1201 TGCTGAGATT TTCATCTTCC TTTATGTTGG CATGGATGCC TTGGACATTG AAAAGTGGCG

1261 '丄'丄'丄'丄 GTGAGT GATAGCCCGG GAACATCAAT TGCAGTGAGC TCCATACTGC TAGCriTlGT

1321 CCTGATTGGG AGAGCAGCTT TTGTCITTCC ATTATCCTTC ATATCCAACC TATCTAAGAA

1381 ATCAACTAGC GAAAAGATAG GCATCAAGCA GCAAATTATA GTATGGTGGG CTGGACTAAT

1441 GAGAGGCGCT GTGTCGATGG CACTAGCATA CAATAAGTTT ACAAGCTTGG GTCATACCAA

1501 TGTGCGAGAT AATGCAATAA TGATCACAAG TACCATAACT GTTGTTCTCT TCAGCACAGT 1561 GGTGTTTGGT CTGCTGACTA AACCTCTTAT AAGGTGTCTG CTGCCTCATA CAAAACAAAA

1621 CCCAAAAGGC ATATTGGATT CAACTTCTCC AAAATCAAAT ACAGTGCCAC TCCTTGGAGA

1681 GGGGCAGGAT TCCTTGGATG ACATAGGTGG GCATGAGGTT CTACGCCCAA ACAGTTTGCG

1741 TGCCCTCCTG ACAACCCCAG CACACACCGT TCATTACTAC TGGCGCAAAT T GACGATGC

1801 ATTCATGCGT CCCGTGT TG GCGGTCGGGG '丄'丄'丄'丄 GT CCG T TGT CCCG GCTCACCAAC

1861 AGAACGGAGT GTCCACAATC AGTCTCAATG AAGAGAAAGA CAAAGCAAGA TGTACAAATT

1921 ATGTAAAGTT ATAAGCATCT TGAAGATGGT TAAACAGTAT CACTTATCTT TGGACCTGCT

1981 GCTGAAAATA GTGGTTTTGT 'I'l'l'l'CCTCTC TCACACTCAA CTAATTCCCT GTTAAATTTG

2041 TTACAGTAGA ACATTCTTAT GGCGCCAAAA ΑΑΑΑΑΑΑΑΑΆ 设计 A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:

SEQ ID No: 10:

ATGTCGAGAT GGCAGATGGT TT SEQ ID No: 11:

TCATTGAGAC TGATTGTGGA CA clones the full-length coding gene by SEQ ID NO: 10 and SEQ ID NO: 11.

Using TISTA's PrimeSTAR HS DNA polymerase, the cDNA of the cDNA obtained by reverse transcription of the mRNA extracted by the salt treatment group was used as a template for PCR reaction. 50 yl PCR reaction system: 10 yl 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 10 and P SEQ ID NO: 11 each of 2.0 μ 1 and 30 μ 1 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 then the resulting precipitate was dissolved in 21 μM of double distilled water. Then, 2.5 μl lO X Ex Buffer 0.5 μl 5 mM dATP, 1.0 l Ex Taq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained 1500 bp DNA fragment was recovered (Omega recovery kit), and ligated into pGEM T-easy vector to obtain KcNHA2-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 and 40 g/mL X-gaK 24 g/mL IPTG for screening. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin. After incubation at 37 ° C overnight, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. . The bacterial liquid PCR amplification assay was carried out with SEQ ID NO: 10 and SEQ ID NO: 11 (the reaction system and the reaction conditions were the same as above), and 9 positive clones were obtained, and 3 of them were selected. The positive clones were sent to Ingeki (Shanghai) Trading Co., Ltd. for sequencing, 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 NHA2 protein: SEQ ID NO: 1

1 MSRWQMVLAS DHASWSMNL

21 FVALLCACIV VGHLLEENRW

41 MNES ITALVI GVCTGWILL

61 ISGGKSSRLL VFSEDLFFIY

81 LLPPI I FNAG FQVKKKQFFR

101 NFITIMLFGA VGTLISCCI I

121 SAGATLAFEK LDIGSLDVGD

141 YLAIGAI FAA TDSVCTLQVL

161 DQDETPLLYS LVFGEGWND

181 ATSWLFNAI QSFDLTHLSP

201 S ITGQFVGSF LYLFFTSTML

221 GWTGLVSAY I IKKLYFGRH

241 STDREVALMI LMAYLSYMLA

261 ELFYLSGILT VFFCGIVMSH

281 YTWHNVTESS RVTTKHAFAT

301 LSFVAEI FI F LYVGMDALDI

321 EKWRFVSDSP GTS IAVSS IL

341 LAFVLIGRAA FVFPLSFISN

361 LSKKSTSEKI GIKQQI IVWW

381 AGLMRGAVSM ALAYNKFTSL

401 GHTNVRDNAI MITSTITWL

421 FSTWFGLLT KPLIRCLLPH

441 TKQNPKGILD STSPKSNTVP

461 LLGEGQDSLD DIGGHEVLRP

481 NSLRALLTTP AHTVHYYWRK

501 FDDAFMRPVF GGRGFVPFVP

521 GSPTERSVHN QSQ*

Nucleotide sequence of KcNHA2 gene SEQ ID NO: 2

1 ATGTCGAGAT GGCAGATGGT TTTAGCGTCT GACCACGCCT CTGTGGTATC TATGAACCTA

61 TTTGTGGCGC TCTTTGCGC TTGCATTGTG GTTGGTCATC TTTTAGAAGA GAATCGATGG

121 ATGAATGAGT CGATCACCGC CCTCGTCA T GGTGTATGCA CTGGCGTTGT TATTTTGCTG

181 ATCAGTGGAG GAAAAAGCTC GCGTCTITTG GTCTTCAGCG AGGATCTITT CTTCATATAT 241 C TCTGCCGC CAATTATATT CAATGCTGGG TTTCAGGTGA AGAAGAAGCA GT CI TCGT

301 AATTTCATTA CCATCATGCT ATTTGGCGCT GTTGGTACAT TGATATCCTG TTGTATCATA

361 TCTGCAGGTG CTACACTAGC CTTTGAGAAA TTGGATATTG GTTCTCTGGA TGTTGGGGAT

421 TATCTTGCAA TTGGTGCAAT A TCGCTGCC ACCGAT CTG TTTGCACATT GCAGGTCCTT

481 GATCAGGATG AGACACCTTT ACTCTATAGT CTGGT TTCG GAGAAGGTGT TGTAAATGAT

541 GCCACATCGG TTGTGCTCTT TAATGCAATC CAGAGCITTG ATCTCACTCA TCTTAGTCCC

601 AGTATTACTG GGCAGTTTGT TGGCAGCITT TTATATTTAT T TTCACGAG CACTATGCTG

661 GGAGTGGTTA CTGGTCTGGT TAGTGCCTAC ATCATCAAAA AACTTTATTT TGGCAGGCAC

721 TCAACAGATC GTGAGGTTGC TCTTATGATC CTTATGGCAT ACC TTCGTA TATGCTGGCT

781 GAAC'l'l'l'l'CT ACTTAAGTGG CATTCTCACT GTATTTTTCT GTGGGATTGT GATGTCACAT

841 TACACCTGGC ACAATGTGAC AGAGAGTTCA AGGGTAACTA CCAAGCATGC TTTTGCAACC

901 TTATCA.TTTG TTGCTGAGAT TTTCATCTTC CTTTATGTTG GCATGGATGC CTTGGACATT

961 GAAAAGTGGC GTTTTGTGAG TGATAGCCCG GGAACATCAA TTGCAGTGAG CTCCATACTG

1021 CTAGCriTlG TCCTGATTGG GAGAGCAGCT TTTGTCTrTC CATTATCCTT CATATCCAAC

1081 CTATCTAAGA AATCAACTAG CGAAAAGATA GGCATCAAGC AGCAAATTAT AGTATGGTGG

1141 GCTGGACTAA TGAGAGGCGC TGTGTCGATG GCACTAGCAT ACAATAAGTT TACAAGCTTG

1201 GGTCATACCA ATGTGCGAGA TAATGCAATA ATGATCACAA GTACCATAAC TGTTGTTCTC

1261 TTCAGCACAG TGGTGTTTGG TCTGCTGACT AAACCTCTTA TAAGGTGTCT GCTGCCTCAT

1321 ACAAAACAAA ACCCAAAAGG CATATTGGAT TCAACTTCTC CAAAATCAAA TACAGTGCCA

1381 CTCCTTGGAG AGGGGCAGGA TTCCTTGGAT GACATAGGTG GGCATGAGGT TCTACGCCCA

1441 AACAGTTTGC GTGCCCTCCT GACAACCCCA GCACACACCG TTCATTACTA CTGGCGCAAA

1501 T TGACGATG CATTCATGCG TCCCGTGT T GGCGGTCGGG GTTTTGTTCC GTTTGTTCCC

1561 GGCTCACCAA CAGAACGGAG TGTCCACAAT CAGTCTCAAT GA Example 3 Construction of KcNHA2 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 gene, respectively. The construction flow chart is shown in Figure 1.

Pnos was amplified using the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 12 and SEQ ID NO: 13, using TAKARA's PrimeSTAR HS DNA polymerase. 50 l PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 12 and SEQ ID NO : 13 each of 2.0 μ ΐ and 31 μ ΐ of double distilled water. PCR reaction conditions: 94 ° C pre-denaturation 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 with the pBI121 plasmid as a template, using TIAK's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 14 and P SEQ ID NO: 15 each of 2.0 μ 1 and 31 μl 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 obtained 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 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 1.0 μl pCAMBIA2300 plasmid, 1.0 μl PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 16 and P SEQ ID NO: 17 each of 2.0 μ ΐ and 31 μ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds), 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 The full-length sequence of the coding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive dVH^^-pGEM plasmid obtained in Example 2), using TAKARA's PrimeSTAR HS DNA polymerase. 50 l PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 KcNHA2-pGEM plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 18 and SEQ ID NO: 19 each of 2.0 μ 1 and 31 μl 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. The resulting PCR product was ligated by Sall and Kpnl (connection method as above) to pCAMBIA2300-3, and the plant expression vector 35S-KcNHA2-2300 was obtained after verification (Fig. 2).

SEQ ID NO: 18

ACTGTCGAC ATGTCGAGAT GGC AGATGGT TT

SEQ ID NO: 19

ACTGGTACC TCATTGAGAC TGATTGTGGA CA Example 4 35S-KcNHA2-2300 expression vector for transformation of Agrobacterium

Agrobacterium GV3101 (purchased from Shanghai Maiqi Biotechnology Co., Ltd.) Preparation of Competent Cells: Agrobacterium GV3101 was drawn on LB solid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 gentamicin Single spot inoculation, culture at 28 °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 gentamicin, 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 gentamicin, and cultured at 28 ° C with shaking. 2-2.5 hours to OD _6QQ = 0.8. The ice bath solution 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 pre-cooled 10% (by volume) glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; 10% (by volume) of the pre-cooled glycerin was washed 3-4 times; then, the appropriate amount of ice pre-cooled 10% (volume ratio) of glycerol was used to resuspend the bacterial pellet to produce GV3101 competent cells at 40 μΐ/tube. It is packaged and stored at -70 °C for later use.

Transformation of Agrobacterium: The GV3101 competent cells were thawed on ice, and 1 μM of the plasmid 35S-KcNHA2-2300 obtained in Example 3 was added to 40 μΐ of the competent cells, and the mixture was mixed and ice bathed for about 10 minutes. Transfer the mixture of competent cells after ice bath and 35S-KcNHA2-2300 plasmid to a ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) with a micropipette, tapping to bring the suspension to 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 room, push the slide to place the electric shock cup on the base of the electric shock room. Extreme. Set the program of MicroPulser (purchased from Bio-Rad) to "Agr" and apply an electric shock once. Immediately remove the electric shock cup and 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 gentamicin, 50 μ _§ /ιη1 Kanamycin), 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

Select the vermiculite with good water absorption and soft soil to match 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 22V, light intensity 3500-4000 lx, 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 dark/16 hours 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 GV3101 Agrobacterium liquid of the transformed 35S-KcNHA2-2300 expression vector obtained in Example 4 was inoculated to contain rifampicin containing 50 g/ml, 50 μ _§ /ιη1 gentamicin, 50 g/ml Cannamycin Incubate overnight in LB liquid medium, and inoculate 1:50 in the morning to contain rifampicin containing 50 g/ml, 50 μ _§ /ιη1 gentamicin, 50 μ _§ /ιη1 kanamycin In the new LB medium (1 L), the culture was carried out for about 8 hours until the Agrobacterium liquid OD _{6 (K) was} 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) , so that 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 Agrobacterium-containing dyeing medium, and gently shaken clockwise for about 2 minutes, and covered with a transparent plastic cover. 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 (T0nl-T0n20) and stored. Example 8 Planting of Transgenic Arabidopsis thaliana T1 Plants Recording KcNHA2

Choose the rock with good water absorption and soft soil and the nutrient soil (1: 1) as the soil for Arabidopsis planting. Each transformant numbered T0nl-T0n20 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). PCR-negative plants were removed, and 7-8 PCR-positive vaccines were retained in each pot. After 10 days of 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 of KcNHA2

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 Tln3, Τ1η7, Τ1η16 showed significant salt tolerance (see Figure 3, with Tln3). For example, the results of Τ1η7, Τ1η16 are similar, not shown here). Example 10 Verification of gene expression at the transcriptional level

Eight T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (three salt-tolerant strains belonging to the above Τ1η3, Tln7, and η1η16, respectively), and the control plants in Example 9 were randomly selected from 4 plants, each of which was cut. The leaves (0.05 mM) were treated with salt (150 mM NaCl) for 14 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). 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. Reverse transcription was performed according to the Invitrogen reverse transcription assay [J Box Superscript III Reverse Transcriptase method, and 1 total RNA was used as a template for reverse transcription. The fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: ATTATCTCGC ACATTGGTAT GACC), and its transcription was examined. AUT2 fragment was amplified using SEQ ID NO: 21 (SEQ ID NO: 21: 5-GCCATCCAAGCTGTTCTCTC-3) and SEQ ID NO: 22 (SEQ ID NO: 22: TTCTCGATGGAAGAGCTGGT) ( Arabidopsis housekeeping gene: Http:〃 www.ncbi.nlm. nih.gov/nuccore/AK317453.1 ) , as a photo. The PCR reaction was carried out using the Ex DNA polymerase of TAKARA and using the cDNA obtained by the above reverse transcription as a template. 50 μl ΡΟ Reaction system: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 0.3 μΐ Ex DNA polymerase, 10 μΜ primer SEQ ID NO: 10 and P SEQ ID NO: 20 each 2.0 μl, and 35.7 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 30 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 Fig. 4: 1-8 are salt-tolerant T1 transgenic Arabidopsis plants ( belong to Tln3, Τ1η7, Τ1η16, respectively), and 9-12 are non-transgenic control Arabidopsis plants. The results showed that KcNIU2 was significantly transcribed in salt-tolerant T1 transgenic Arabidopsis plants, and no transcription of KcNHA2 in non-transgenic control Arabidopsis plants.

Claims

Claim

A protein encoding a gene encoding a Kandelia sodium hydrogen transporter, the sequence of which is SEQ ID NO: 1.

2. A gene encoding the protein of claim 1, the sequence of which is SEQ ID NO: 2.

A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and wherein the nucleotide sequence of the gene and the expression control sequence of the expression vector are operably Preferably, the expression vector is pCAMBIA2300.

The recombinant expression vector of claim 3 which is the 35S-KcNHA2-2300 vector shown in Fig. 2.

A recombinant cell comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.

A method for improving salt tolerance of a plant, 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 is Arabidopsis.

A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.

8. The method of claim 7, wherein the plant is Arabidopsis thaliana.

9. The gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 4 for use in improving plant salt tolerance and for use in plant breeding.

10. The use of claim 9, wherein the plant is Arabidopsis thaliana.