A Baldcypress TdADH3 gene, its expressed protein and application

Abstract

The invention discloses a baldcypress TdADH3 gene and its expression protein and application, belonging to the field of plant genetic engineering technology. The present invention uses baldcypress tissue culture seedling roots as materials, and clones the baldcypress TdADH3 full-length gene by RACE technology. At the same time, the overexpression vector pBWA (V) KS-TdADH3 is constructed by GATEWAY technology, and the gene is located after the promoter P35S. Under the drive of the promoter P35S, TdADH3 is efficiently expressed in poplar. After the transgenic plant grows in a solution containing 100moL / LNaCl for 30 days, the growth is good, and the growth is not significantly affected, indicating that the TdADH3 gene is an important gene for plant response to salt stress. It can be seen that the gene will have a wide range of uses in baldcypress and other plant production and breeding, and can also provide a theoretical basis for subsequent baldcypress stress research, so the present invention has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Baldcypress TdADH3 gene and its expression protein and application. Background Art

[0002] Baldcypress (Taxodium distichum) is a deciduous tree of the Cupressaceae family, also known as larch, and is an ancient "relict plant". It has the advantages of being resistant to flooding, wind, pollution, and pests, and its leaves show different colors in different seasons, making it highly ornamental. In addition, the wood of baldcypress is heavy, coarse, straight, moderately hard, and highly resistant to decay, and can be used for construction, electric poles, furniture, and shipbuilding. In 1917, my country began to introduce and cultivate baldcypress, which has shown strong adaptability and has made positive contributions to the construction of passages, wetland protection, and afforestation in industrial areas in the low-lying and humid areas of Jiangnan, my country.

[0003] Alcohol dehydrogenase (ADH, EC1.1.1.1) is a member of the dehydrogenase / reductase (MDR) protein gene superfamily. ADH gene family members play a very important role in plant resistance to waterlogging, drought, cold damage and other stress environments. In recent years, researchers have found that the ADH3 gene is closely related to the salt tolerance of plants. Overexpression of the ADH3 gene in rice can significantly enhance the adaptability of salt-sensitive rice to salt stress. However, there are few studies on the salt tolerance of ADH3 genes in woody plants, and there are no related reports in the genus Baldcypress. Researchers from the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province mined the whole genome data of Baldcypress, obtained the full length of the TdADH3 gene of Baldcypress, and studied its salt tolerance function, hoping to provide a theoretical basis for in-depth research on the stress resistance mechanism of Baldcypress trees. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Baldcypress TdADH3 gene. Another technical problem to be solved by the present invention is to provide a specific application of the Baldcypress TdADH3 gene.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A Baldcypress TdADH3 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the expressed protein of the Baldcypress TdADH3 gene is shown in SEQ ID NO.2.

[0008] Furthermore, an expression cassette, vector, host bacteria or cell line containing the Baldcypress TdADH3 gene.

[0009] Furthermore, the vector is a plant expression vector.

[0010] Furthermore, the plant expression vector is pBWA(V)KS::TdADH3.

[0011] Furthermore, the Baldcypress TdADH3 gene is used in plant breeding or improving plant salt tolerance.

[0012] Furthermore, the application of the Baldcypress TdADH3 gene in improving plant salt tolerance comprises the following steps:

[0013] 1) Construction of the vector of the Baldcypress TdADH3 gene;

[0014] 2) transforming the constructed vector of the Baldcypress TdADH3 gene into plants;

[0015] 3) Cultivate and screen transgenic plants with higher salt tolerance.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses the root of Baldcypress tissue culture seedling as material, and clones the full-length gene of Baldcypress TdADH3 by RACE technology. At the same time, the overexpression vector pBWA(V)KS-TdADH3 is constructed by GATEWAY technology, and is transferred into the woody model plant poplar. The TdADH3 gene is located after the promoter P35S. Under the drive of the promoter P35S, TdADH3 is efficiently expressed in the poplar. After the transgenic poplar and the non-transgenic poplar are grown in a solution containing 100 mol / L NaCl for 30 days, the growth of the non-transgenic poplar is inhibited, the leaves turn yellow, and death occurs. The transgenic plants grow well, and the growth is not significantly affected. It shows that the TdADH3 gene is an important gene for plant response to salt stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the expression changes of the TdADH3 gene in the roots, stems and leaves of Baldcypress tissue culture seedlings under salt stress;

[0019] Figure 2 Schematic diagram of the plant overexpression vector pBWA(V)KS;

[0020] Figure 3The figure shows the phenotypic differences between pBWA(V)KS::TdADH3 transgenic plants and non-transgenic plants under salt stress. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments. In the following embodiments, operations not described in detail are all routine biological experimental operations, which can be performed with reference to molecular biology experimental manuals and existing published journals and literature.

[0022] Example 1: Cloning of TdADH3 gene by RACE technique

[0023] The whole genome data of Baldcypress were screened, and 15 ADH family gene fragments were screened. The 15 gene fragments were compared at the National Center for Biotechnology Information (NCBI https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and finally the target gene was screened out, which had the highest similarity with the Swiss ryegrass LrADH3 gene (Sequence ID: XM_047227036.1).

[0024] 1. Total RNA extraction and cDNA synthesis

[0025] 1) Pipette 1 mL of RNA Plant Reagent lysis solution into each 2 mL centrifuge tube, add about 3 g of Baldcypress leaf sample finely ground with liquid nitrogen, and vortex vigorously to mix thoroughly;

[0026] 2) Place the centrifuge tube horizontally at room temperature for 10 minutes to allow the lysis buffer to fully lyse the nuclear protein;

[0027] 3) Centrifuge at 1200 rpm, 4°C for 5 min in a high-speed refrigerated centrifuge and transfer the supernatant to a new centrifuge tube;

[0028] 4) Add 200 μL of 5 M / L sodium chloride solution, mix gently, then add 300 μL of chloroform, and mix by inverting;

[0029] 5) Centrifuge at 4°C, 12×1000 rpm for 10 min, and gently pipette the supernatant into a new 2 mL centrifuge tube;

[0030] 6) Repeat steps 4) and 5);

[0031] 7) Add an equal volume of isopropanol to the obtained supernatant, mix thoroughly, and let stand at room temperature for 10 min;

[0032] 8) Centrifuge at 4°C, 12×1000 rpm for 10 min, discard the supernatant and keep the precipitate at the bottom;

[0033] 9) Then extract using RNeasy plant Mini Kit (QIAGEN, USA);

[0034] 10) Add 0.5 mL of freshly prepared RLT buffer (1 mL) RLT and 10 μL of β-mercaptoethanol and vortex thoroughly;

[0035] 11) Add 0.5 mL of anhydrous ethanol and mix thoroughly by pipetting;

[0036] 12) Transfer the mixed solution to the collection column RNeasy mini column (pink), centrifuge at 12000 rpm for 15 seconds and discard the filtrate. The centrifuge tube can be reused;

[0037] 13) Add 350 μL RWI Buffer to the collection column RNeasy mini column and centrifuge at 12000 rpm for 15 seconds, then discard the filtrate and centrifuge tube;

[0038] 14) Place the collection column RNeasy mini column into a new centrifuge tube, add 500 μL Buffer RPE, centrifuge at 12000 rpm for 15 seconds, and discard the filtrate;

[0039] 15) Add 500 μL Buffer RPE again and centrifuge at 12000 rpm for 2 min. Discard the filtrate and centrifuge tube.

[0040] 16) Place the collection column RNeasy mini column into a new centrifuge tube and centrifuge at 13000 rpm for 1 min;

[0041] 17) Place the collection column RNeasy mini column into a new RNA collection tube, add an appropriate amount of RNase free water to the center of the membrane, let it stand for 1 minute, and centrifuge at 12000 rpm for 1 minute;

[0042] 18) Add the filtrate back into the collection column RNeasy mini column, centrifuge at 12000 rpm for 1 min, recover the filtrate and obtain total RNA;

[0043] 19) The RNA concentration was determined by UV spectrophotometer and the integrity of RNA was checked by agarose gel electrophoresis.

[0044] 20) The total RNA sample obtained was reverse transcribed using Promega's ImProm-IITM Reverse Transcriptase. The reaction system was: Total RNA (1ug); 4.0μL ImProm-II5×Reacation Buffer; 2.5μL MgCl2 (25mM); 1.0μL dNTP (10mM); 1.0uL Oligo (dT) Adaptor Primer (50ng / μL); 0.5μL Recombinant RnasinRibonuclease Inhibitor; 1.0μL ImProm-IITM ReverseTranscriptase, and Nuclease-Free water was added to make up to 20.0μL. The reaction procedure was: 25℃, 5min; 42℃, min; 70℃; 15min; 4℃, hold.

[0045] 2. RACE nested PCR amplification

[0046] (1) 3'RACE cDNA reverse transcription reaction

[0047] With total RNA as template, the reaction system is as follows: 1μL 3'RACE Adaptor (5μM); 2μL 5×M-MLVBuffer; 1μL dNTP Mixture (10mMeach); 0.25μL Reverse Transcriptase M-MLV (RNase H) (200U / μL); 0.25μL RNase Inhibitor (40U / μL); add RNase Free dH2O to make up to 10μL. The reaction program is: 42℃, 60min; 70℃, 15min; 4℃, hold.

[0048] (2) 3' RACE Outer PCR

[0049] 3'RACE Outer PCR is a PCR reaction using the known cDNA fragment to design the upstream outer specific primers TdADH3 3'outer Primer and 3'RACE Outer Primer. The reaction system is as follows: 2μL reverse transcription reaction solution; 8μL 1×cDNA Dilution buffer II; 2μL 3'RACE Outer Primer (10μM); 2μL TdADH3 outer Primer (10μM); 4μL 10×LATaq Buffer II (Mg 2+Free); 3 μL MgCl2 (25 mM); 0.25 μL LATaq polymerase (5 U / μL); add water to make up to 50 μL. The reaction procedure is: 94℃ 3min; 94℃ 30s, 60℃ 30s, 72℃ 2min, after 30 cycles; 72℃ 10min; 4℃ hold. Among them:

[0050] 3'RACE outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3';

[0051] TdADH33'outer Primer: 5'-CACCTCTGATTCTTGCCCCTTCCGCAAC-3'.

[0052] (3) 3'RACE Inner PCR

[0053] 3'RACE Inner PCR uses the Outer PCR reaction product as a template and uses the upstream inner specific primer TdADH3 3'Inner Primer and 3'RACE Inner Primer for PCR amplification. The reaction system is: 1μL Outer PCR reaction product; 8μL dNTP Mixture (10mM each); 5μL MgCl2 (25mM); 5μL 10×LATaqBuffer II (Mg 2+ Free); 2μL Gene Specific Inner Primer (10μM); 2μL 3'RACE Inner Primer (10μM); 0.5μL LATaq polymerase; add water to make up to 50μL. The reaction procedure is: 94℃3min; 94℃30s, 55℃30s, 72℃2min, 32 cycles; 72℃10min; 4℃. The PCR product was subjected to 1% agarose gel electrophoresis, and the slide was read and photographed on a gel imager, then the gel was cut and recovered, connected to the T vector, transformed, detected, and sequenced. Among them,

[0054] 3'RACE Inner Primer: 5'-CGCGGATCCTCCACTAGTGATTTCACTA-3';

[0055] TdADH33'Inner Primer: 5'-TTTCCAGCTTCACCGAATACACTGTTGTAGAA-3'.

[0056] (4) 5'RACE dephosphorylation reaction

[0057] 1) The reaction system is as follows: 2-5 μg Total RNA; 5 μL 10×Alkaline Phosphatase Buffer (MgCl2 Free); 0.6 μL Alkaline Phosphatase (Calf intestine) (16 U / μL); 1 μL RNase Inhibitor (40 U / μL); add RNase Free dH2O to make up to 50 μL; react at 50℃ for 1 hour;

[0058] 2) Add 20 μL of 3M CH3COONa (pH 5.2) and 130 μL of RNase Free dH2O to the above reaction solution and mix thoroughly;

[0059] 3) Add 200 μL of phenol:chloroform:isoamyl alcohol, mix thoroughly, centrifuge at 13000g for 5 min at room temperature, and transfer the upper aqueous phase to a new 1.5 mL centrifuge tube;

[0060] 4) Add 200 μL of chloroform, mix thoroughly, and centrifuge at 13,000 g for 5 min at room temperature. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube;

[0061] 5) Add 2 μL of NA Carrier and mix evenly, then add 200 μL of isopropanol, mix thoroughly, and place at -40°C for more than 2 hours;

[0062] 6) Centrifuge at 13,000 × g for 20 min at 4°C and discard the supernatant;

[0063] 7) Add 500 μL of 75% pre-cooled ethanol for rinsing, centrifuge at 13,000 g for 5 min at 4°C, discard the supernatant and dry;

[0064] 8) Add 7 μL of DEPC water to dissolve the precipitate to obtain CIAP-treated RNA.

[0065] Tobacco Acid Pyrophosphatase (TAP) was used to remove the 5′ cap structure of mRNA and retain a phosphate group. The reaction system was: 7 μL CIAP-treated RNA; 1 μL 10×TAP Reaction Buffer; 1 μL Tobacco Acid Pyrophosphatase (0.5 U / μL); 1 μL RNase Inhibitor (40 U / μL); react at 37°C for 1 hour, and take 5 μL for 5′ RACE Adaptor ligation reaction.

[0066] (5) 5' Adaptor connection

[0067] 1) The reaction system is: 5μL CIAP / TAP-treated RNA; 1μL 5'RACE Adaptor (15μM); 4μL RNase Free dH2O; react at 65℃ for 5min, place on ice for 2min, add the following reagents: 8μL 5×RNA Ligation Buffer; 20μL 40% PEG6000; 1μL T4 RNA Ligase; 1μL RNase Inhibitor (40U / μL); the total system is 40μL; react at 16℃ for 1h, and at 4℃ overnight;

[0068] 2) Add 20 μL of 3M CH3COONa (pH 5.2) and 140 μL of RNase Free dH2O to the above reaction solution and mix thoroughly;

[0069] 3) Add 200 μL of phenol:chloroform:isoamyl alcohol, mix thoroughly and centrifuge at 13000g for 5 min at room temperature, and transfer the upper aqueous phase to a new 1.5 mL centrifuge tube;

[0070] 4) Add 200 μL of chloroform, mix thoroughly, and centrifuge at 13,000 g for 5 min at room temperature. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube;

[0071] 5) Add 2 μL of NA Carrier and mix thoroughly; then add 200 μL of isopropanol and place at -40°C for 2 h;

[0072] 6) Add 500 μL of 75% pre-cooled ethanol for rinsing, centrifuge at 13,000 g for 5 min at 4°C, discard the supernatant and centrifuge again for 1 min, then completely drain the solution and dry;

[0073] 7) Add 7 μL of RNase Free dH2O to dissolve the precipitate and obtain Ligated RNA.

[0074] (6) 5'RACE cDNA reverse transcription reaction

[0075] The reaction system is: 6μL Ligate RNA; 2μL 5×M-MLV Buffer; 1μL dNTP (10μM each) 0.5μL Random 9 mers (50μM); 0.25μL Reverse Transcriptase M-MLV (RNase H) (200U / μL); 0.25μL RNase Inhibitor (40U / μL); the total system is 10μL. The reaction program is: 30℃, 10min; 42℃, 1h; 70℃, 15min; 4℃, hold.

[0076] (7) 5' RACE Outer PCR

[0077] The reaction system was as follows: 2.0 μL of the reverse transcription reaction solution diluted 5 times in step (6); 2.0 μL of 5'RACE Outer Primer (10 μM); 2.0 μL of TdADH3 5'outer Primer (10 μM); 5.0 μL of 10×ExTaq Buffer II (Mg 2+ Free); 4.0μL MgCl2 (25mM); 4.0μL dNTP Mixture (10mM each); 0.3μL ExTaq polymerase; add dH2O to make up to 50.0μL. The reaction program is: 94℃3min; 94℃30s, 60℃30s, 72℃2min, 30 cycles; 72℃10min; 4℃ hold. Among them:

[0078] 5'RACE Outer Primer: 5'-CATGGCTACATGCTGACAGCCTA-3';

[0079] TdADH3 5'outer Primer: 5'-AAGACCATGAAAAGCCTGTTCACCAGGT-3'.

[0080] (8) 5' RACE Inner PCR

[0081] The reaction system was as follows: 2.0 μL 5'RACE Outer PCR reaction product; 2.0 μL 5'RACE Inner Primer (10 μM); 2.0 μL TdADH3 5'Inner Primer (10 μM); 5.0 μL 10×ExTaq Buffer II (Mg 2+ Free); 4.0μL MgCl2 (25mM); 4.0μL dNTP Mixture (10mM each); 0.3μL ExTaq polymerase; add dH2O to make up to 50.0μL. The reaction procedure is: 94℃3min; 94℃30s, 55℃30s, 72℃2min, 30 cycles; 72℃10min; 4℃ hold. Among them:

[0082] 5′RACE Inner Primer: 5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′;

[0083] TdADH3 5'Inner Primer: 5'-GTTTCCAGCTTCACCGAATACACTGTTGTA-3'.

[0084] The PCR products were subjected to 1% agarose gel electrophoresis and read and photographed on a gel imager. They were then recovered using a gel excision recovery kit, connected to a T vector, transformed, tested by PCR in the bacterial solution, and sent to Yingjun Company for sequencing.

[0085] The 3'RACE and 5'RACE products obtained by sequencing were spliced ​​using Bioedit software to obtain a target gene containing 1434bp of bases. Blast confirmed that the target gene was homologous to the rice ADH3 gene and was named TdADH3, as shown in SEQ ID NO.1. It contains a complete coding reading frame of 1182bp, which is 394bp after translation into amino acids. The amino acid sequence is shown in SEQ ID NO.2. It contains a complete coding reading frame of 1182bp (156-1337), which is 394bp after translation into amino acids.

[0086] Example 2: Molecular detection of Baldcypress tissue culture seedlings after salt stress

[0087] Eight Baldcypress tissue culture seedlings with basically the same growth were taken for salt stress treatment, the temperature was controlled at 25°C, the light intensity was 4000 lux, the light was 10 hours per day, 4 were cultured with deionized water, and 4 were cultured with 100 mol / L NaCl solution. After 7 days of culture in a light incubator, the plants were tested by quantitative PCR. The real-time quantitative primers were designed using Oligo6.6 software, the primer Tm value was 60°C, and the primer sequences were as follows:

[0088] q ADH3F: 5′-TGGAAGTGGGGTGGATTA-3′,

[0089] qADH3R: 5′-AATCAGATGATCCAG-3′.

[0090] The internal reference gene for real-time quantitative PCR uses the screened APRT gene:

[0091] APRT-F: 5′-TCCACAGGTTCTTGAATCGCT-3′,

[0092] APRT-R: 5′-AGTCTATGGGAATGTCTGT-3′.

[0093] qRT-PCR was performed using the Analitik Jena qTOWER2.2 (Germany) system. According to the instructions of the SYBR Green kit (Rox), the amplification program was: 55°C for 2 min, 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 40 cycles. Then the temperature was set from 60°C to 95°C to generate a melting curve. Each sample was technically replicated three times, and the 20 μL reaction system contained: 2 μL of diluted cDNA (the dilution ratio was 1:3, and the concentration of diluted cDNA was approximately 350 ng / μL), 10 μL FarstStart Universal SYBR Green Master (Rox), 6 pmoL upstream primer, 6 pmoL downstream primer, and 6.8 μL ddH2O. The relative expression of the gene was calculated according to the 2-ΔΔCt method. The experimental data were statistically analyzed and plotted using SPSS19.0 software. The real-time quantitative results are shown in the figure. Figure 1 As shown, it was found that the expression level of the gene in the salt stress-treated tissue culture seedlings TdADH3 increased by 15-40 times compared with the control plants.

[0094] Example 3: Construction of TdADH3 gene plant expression vector

[0095] The TdADH3 gene overexpression vector was constructed using the Gateway technology of Invitrogen. First, the target fragment was cloned into the entry vector (PCRTM8 / GW / TOPOTM) through BP reaction, and then the target gene was transferred to the overexpression vector pBWA(V)KS through LR reaction (as shown in Figure 2). Figure 2 shown).

[0096] (1) Obtaining the target fragment

[0097] After obtaining the full-length TdADH3 gene, the E. coli with the correct ORF frame sequencing was expanded and cultured, and the pMD19-T vector plasmid containing the full-length gene was extracted using the AXYGEN plasmid mini-purification kit. The target fragment was amplified using the plasmid template and the ORF frame primer (the downstream primer contained a stop codon), and then the fragment was recovered by gel cutting after electrophoresis.

[0098] (2) BP reaction

[0099] 1) The reaction system is: 10-20ng Fresh PCR product (purified); 1μL Salt solution; 1μL pCRTM8 / GW / TOPOTM vector; add Nuclease-free Water to make up to 6μL;

[0100] 2) Mix gently, react at 22°C for 60 min, and then transfer to ice;

[0101] 3) Add 6 μL of the reaction product from the previous step to the TOP10 competent cells melted on ice and mix gently without pipetting;

[0102] 4) Ice bath for 15 minutes;

[0103] 5) Heat shock at 42°C for 60 seconds, then quickly place on ice for 3-5 minutes;

[0104] 6) Add 800 μL SOC medium, shake at 37°C 150 rpm for 1 hour; centrifuge at 4000 rpm for 3 minutes, remove about 700 μL SOC from the upper layer, mix the remaining bacterial solution, and apply it to the LB screening culture plate;

[0105] 7) Incubate the cells at 37°C overnight, pick clones for bacterial liquid PCR detection, and use the gene-specific upstream primer ORF-F and M13F on the vector. The sequences are as follows:

[0106] ORF-F: 5'-ATGCTACACAGTCCACAATTACATACAGCCATGGA-3',

[0107] M13F: 5'-TGTAAAACGACGCGCCAGT-3'.

[0108] 8) The positive clones after detection are further sequenced for verification;

[0109] 9) Use the AXYGEN Plasmid Mini-Purification Kit to extract the plasmid of the entry vector that has been sequenced correctly.

[0110] (3)LR reaction

[0111] AseI (TaKaRa) restriction endonuclease was used to perform restriction digestion on the pBWA(V)KS vector to linearize it. The reaction system was 10×H Buffer (5μL), 4μg of purified pBWA(V)KS vector, AseI (2μL), and the reaction solution was supplemented to 50μL with nuclease-free water. The reaction was incubated at 45℃ for 1h and then inactivated at 85℃ for 15s. After the restriction digestion reaction, 4μL of the reaction solution was taken for electrophoresis detection and then recovered and purified using AXYGEN PCR Purification Kit. LR reaction system: linearized pBWA(V)KS vector (100ng), entry vector (150ng) were supplemented with 1×TE (pH8.0) to 8μL, mixed thoroughly, and added with 2μL of LR Clonase II enzyme mix. After PCR reaction at 25℃ for 1h, 1μL of Proteinase K solution was added to react at 37℃ for 10min to terminate the reaction. Then Top 10 competent cells were used for transformation, positive single clones were picked, and bacterial liquid PCR detection was performed using the M13F: 5'-TGTAAAACGACGGCCAGT-3' primer on the vector and the downstream primer ORF-R: 5'-CTGCAATGTCCTGTTCATCCACAT-3' specific for the target gene. After sequencing verification, the target vector after LR reaction was recovered and purified to obtain the target gene overexpression vector pBWA(V)KS::TdADH3.

[0112] Example 4: Obtaining TdADH3 transgenic plants and salt stress treatment

[0113] (1) Liquid nitrogen freeze-thaw transformation of Agrobacterium

[0114] 1) Add at least 100 ng of the purified expression vector pBWA(V)KS::TdADH3 to the EHA105 competent cells, mix gently, and place on ice for 30 min;

[0115] 2) Quick freezing in liquid nitrogen for 1 min;

[0116] 3) Heat shock at 37°C for 3 min, then quickly place on ice for 1-2 min;

[0117] 4) Add 800 μL LB medium and resuscitate at 28°C, 100 rpm for 3 h;

[0118] 5) Centrifuge at 4000 rpm for 3 min and remove 800 μL LB medium;

[0119] 6) Mix the remaining bacterial solution and apply it on an LB plate (spc+str) containing the corresponding antibiotics;

[0120] 7) Incubate the cells in an incubator at 28°C for 30-48 hours;

[0121] 8) Pick a single clone and perform PCR detection on the bacterial solution using the upstream primer M13F: 5'-TGTAAAACGACGGCCAGT-3' on the vector and the downstream primer 5'-CTGCAATGTCCTGTTCATCCACAT-3' specific for the target gene.

[0122] (2) Transformation of Populus davidianus by leaf disc method

[0123] 1) Pick a single colony from the activated LB culture plate, inoculate the positive strain detected by PCR into 2 mL of LB liquid culture medium containing the corresponding antibiotics, and culture the bacteria at 28°C (250 rpm) for about 12 hours. Then take 0.8 mL and inoculate it into 50 mL of LB liquid culture medium containing the corresponding antibiotics until the OD 600 The value is about 0.5;

[0124] 2) The bacterial solution was divided into 50 mL centrifuge tubes and centrifuged at 5000 rpm for 10 min to collect the bacteria;

[0125] 3) Resuspend the cells in a certain volume of MS solution without sucrose to an OD of 600 value 0.5;

[0126] 4) The growth potential of the 4-6-week-old Populus shanxin tissue culture seedlings is basically the same. Take 2-3 stretched leaves under the terminal bud and cut the wound along the edge of the leaf;

[0127] 5) Transfer the cut leaf discs into the prepared impregnation solution, temperature 25°C, oscillate at 90 rpm for 30 min;

[0128] 6) Take out the leaf disc, absorb the residual bacterial solution with filter paper, and transfer it to MS1 differentiation culture plate for dark culture for 48 hours;

[0129] 7) After washing, transfer the cells to MS1 differentiation culture plates and culture for 1 week;

[0130] 8) The leaf disc is then transferred to the differentiation screening medium MS1 plate for screening culture;

[0131] 9) When resistant adventitious buds grow on the edge of the leaf disk, transfer them to the bud elongation screening medium MS2 plate for further screening and culture;

[0132] 10) When the resistant adventitious buds grow to about 1 cm in the stem elongation medium, they are cut off and transferred to the rooting screening medium MS0 plate for rooting screening of resistant plants, thereby obtaining complete plants;

[0133] 11) Subculture the resistant seedlings on the rooting screening medium MS0 medium and observe the plant phenotype.

[0134] After the transgenic poplars grew for 30 days, there was no significant difference in phenotype compared with the non-transgenic poplars, so they were further treated with salt stress. The pBWA(V)KS::TdADH3 transgenic poplars and control poplars with the same growth were selected for salt stress treatment experiments. The temperature was controlled at 25°C, the light intensity was 4000 lux, the light was 10 hours a day, and 100 mol / L NaCl solution was used for hydroponics. After 30 days, it was found that the growth of the control poplars was significantly inhibited, the leaves turned yellow, withered, a large number of leaves fell off, and death occurred, while the transgenic poplars grew well and their growth was not significantly affected ( Figure 3 ), indicating that the TdADH3 gene of Baldcypress is closely related to plant salt tolerance and can be used in the breeding of salt-tolerant plants.

Claims

1. A TdADH3 gene of Baldcypress, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the Baldcypress TdADH3 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. An expression cassette, vector, host bacteria or cell line containing the Baldcypress TdADH3 gene according to claim 1.

4. The vector containing the Baldcypress TdADH3 gene according to claim 3, characterized in that: The vector is a plant expression vector.

5. The vector containing the Baldcypress TdADH3 gene according to claim 4, characterized in that: The plant expression vector is pBWA(V)KS::TdADH3.

6. Use of the Baldcypress TdADH3 gene of claim 1 in plant breeding or improving plant salt tolerance; the plant is Baldcypress or poplar.

7. The use of the Baldcypress TdADH3 gene in improving plant salt tolerance according to claim 6, characterized in that: The following steps are involved: 1) Construction of the vector of the TdADH3 gene of Baldcypress; 2) Transforming the constructed vector of the Baldcypress TdADH3 gene into plants; 3) Cultivate and screen transgenic plants with higher salt tolerance.

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