CN107177608B

CN107177608B - Thadh4 gene for regulating flooding-resistant characteristic of sequoia intermedia and application thereof

Info

Publication number: CN107177608B
Application number: CN201710547619.8A
Authority: CN
Inventors: 宣磊; 华建峰; 范文才; 王芝权; 杨颖�; 於朝广; 殷云龙
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2017-07-06
Filing date: 2017-07-06
Publication date: 2019-12-27
Anticipated expiration: 2037-07-06
Also published as: CN107177608A

Abstract

The invention discloses a Thadh4 gene for regulating flooding resistance of sequoia intermedia and application thereof. The nucleotide sequence of the Thadh4 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein is shown as SEQ ID NO. 2. The invention takes the taxus chinensis 406 leaves as the material, and clones the taxus chinensis Thadh4 by RACE technology。Meanwhile, an excessive expression vector pH35GS-Thadh4 is constructed by adopting a path cloning technology, the gene is positioned behind a promoter P35S, the Thadh4 is efficiently expressed in poplar under the drive of the promoter P35S, the growth rate is accelerated, the internode is obviously lengthened, the expression level of the gene is increased by hundreds of times under the water-logging stress, and the Thadh4 gene is an important gene of a plant responding to the water-logging stress and has important application value in the field of forest genetic engineering.

Description

Thadh4 gene for regulating flooding-resistant characteristic of sequoia intermedia and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Thadh4 gene for regulating flooding resistance of sequoia intermedia and application thereof.

Background

The Zhongshan fir is a good clone bred from the hybrid combination of the larval hemlock and the china academy of sciences of Jiangsu province and China Mediterranean fir, and has the advantages of fast growth, flooding resistance, salt and alkali resistance, disease and insect resistance and the like. Due to the outstanding flooding resistance, the construction method plays an important role in the construction of wetland ecosystems in areas such as Yunnan pond of Yunnan, Anhui brood lake and three gorges reservoir area in recent years. The test of the hydro-fluctuation belt in the three gorges reservoir area of Chongqing Wanzhou shows that: after the base part is flooded for the longest 149 days, the top is flooded for the longest 122 days, the maximum top depth is 12m, the Zhongshan fir can still obtain 90% afforestation preservation rate, and the annual average breast diameter and tree height growth of the trees respectively reach 1.3cm/a and 0.60m/a, so the Zhongshan fir can be used as an excellent material for researching the woody flooding-resistant property. The early research on the plant flooding-resistant mechanism mainly focuses on herbaceous model plant Arabidopsis thaliana and grain crop rice, and the research on the flooding-resistant characteristic of the taxus chinensis also focuses on the aspects of morphology and physiology, so that the flooding-resistant mechanism cannot be deeply disclosed. Therefore, the excavation of key genes of the sequoia intermedia responding to the flooding stress has important significance for exploring the flooding resistance of the sequoia intermedia and disclosing a flooding resistance mechanism of woody plants.

The plant can induce glycolysis to occur under the stress of water logging so as to adapt to an anoxic environment, and the whole process can provide energy for the plant body and reduce the toxicity of hypoxia to the plant. Thus, genes related to the glycolytic pathway, in which a gene encoding alcohol dehydrogenase is encoded, have been widely isolated and identifiedadhIs of great interest. Although overexpression of adh1 in rice does not have a significant effect on plant water flooding tolerance, the anaerobic tolerance of plants is greatly reduced once adh1 is mutated or deleted. The maize and arabidopsis adh null mutants and the rice rda (adh with low activity) mutant have obviously reduced in-vivo fermentation capacity, the pH value of root cell cytoplasm is rapidly reduced, and the mutant dies obviously earlier than the wild type. The adh1 gene of rice is transferred into tobacco, the flooding resistance of tobacco transgenic plant is obviousThis is highly enhanced and the activity of ADH1 enzyme also increased with increasing flooding time. The transcriptome sequencing result of the water flooding stress of the sequoia zhongshanensis also shows that the higher the water flooding degree is, the higher the RPKM value of the adh gene is. In conclusion: the adh gene plays an important role in responding to hypoxia stress of herbaceous plants, but the research in woody plants is less, and no relevant report is found in the taxus chinensis.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a Thadh4 gene for regulating flooding-resistant property of the sequoia intermedia. The invention also aims to provide application of the Thadh4 gene for regulating flooding resistance of the sequoia intermedia.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a Thadh4 gene for regulating flooding resistance of Sequoia intermedia has a nucleotide sequence shown in SEQ ID NO. 1.

The vector contains the Thadh4 gene for regulating and controlling the flooding-resistant property of the sequoia intermedia.

The amino acid sequence of the expression protein of the Thadh4 gene for regulating the flooding resistance of the sequoia intermedia is shown as SEQ ID NO. 2.

The Thadh4 gene for regulating and controlling flooding resistance of the sequoia intermedia is applied to plant breeding.

The Thadh4 gene for regulating flooding resistance of the taxus chinensis is applied to waterlogging resistance of plants.

The invention takes the taxus chinensis 406 leaves as the material, and clones the taxus chinensis Thadh4 by RACE technology。Meanwhile, an excessive expression vector pH35GS-Thadh4 is constructed by adopting a channel cloning technology, the gene is positioned behind a promoter P35S, the Thadh4 is efficiently expressed in poplar under the drive of the promoter P35S, the growth rate of a transgenic plant is accelerated along with phenotypic variation, and internodes are obviously lengthened.

Has the advantages that: compared with the prior art, the Thadh4 gene is transferred into the woody plant poplar, the transgenic poplar which overexpresses Thadh4 has the advantages of accelerated growth rate, obviously lengthened internode and hundreds of times increased gene expression amount under the water-logging stress. The Thadh4 gene is an important gene for responding to the water logging stress of plants and has important application value in the field of forest genetic engineering.

Drawings

FIG. 1 is a schematic representation of a plant overexpression vector pH35 GS;

FIG. 2 is a comparison graph of the overall phenotype of transgenic poplar with overexpression Thadh4 and non-transgenic poplar, wherein the left part of the graph is non-transgenic poplar and the right part of the graph is transgenic poplar;

FIG. 3 is a diagram of the gene expression change of transgenic plants overexpressing Thadh4 under water-logging stress.

Detailed Description

The present invention will be further described with reference to the following specific examples.

In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed with reference to molecular biology experimental manuals, published journal literature, and the like.

Example 1 cloning of Thadh4 Gene by RACE technique

1) Extraction of RNA and inversion of cDNA

The process is improved by using the leaf of Taxus chinensis 406 as material, and extracting total RNA from Taxus chinensis with RNeasy Plant Mini Kit (QIAGEN), because the leaf contains more phenolic compounds. Before RNA extraction, all gun heads, centrifuge tubes and mortar are soaked overnight in 0.1% DEPC water, autoclaved for 40min and dried. All solutions should be made up with 0.1% DEPC water. Determination of total RNA concentration and purity was performed with NanoDrop 1000. Total RNA was separated by 1.0% agarose gel electrophoresis, and one RNA with better integrity should observe three clear bands, 5.8SRNA was slightly darker, and 28SRNA band brightness was about twice as bright as 18SRNA, indicating that RNA was not degraded, and then reverse transcribed by using the SMARTer PCR cDNA Synthesis Kit, Advantage 2 PCR Kit from Clontech.

2) Obtaining of target fragment of Thadh4 gene

Screening Unigene sequences of Thadh4 according to sequencing result of Zhongshan transcriptomeAnd using Oligo6 to design a specific primer to amplify a target fragment of Thadh4 gene, wherein the forward primer of the target fragment of Thadh4 is as follows: 5'-GAAGTGATGAGAAGAGCAGGTT-3', 5'-AGGCCTGGTATAATACGTTGGTAT-3' are provided. And (3) PCR reaction system: TakaraLA Taq (5U/. mu.L) 0.5. mu.L, 10 × LA PCR Buffer (Mg)²⁺Free) 5.0μL，MgCl₂5.0. mu.L (25 mM), 8.0. mu.L dNTP mix (2.5 mM reach), 2.0. mu.L Forward Primer (10. mu.M), 2.0. mu.L Reverse Primer (10. mu.M), 1.0. mu.L cDNA template, 26.5. mu.L Milli-Q Water. The reaction procedure is as follows: 3min at 94 ℃; 94 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 2min, 35 cycles; 10min at 72 ℃; forever at 4 ℃. Sequencing the product, wherein the sequence of the target fragment of the Thadh4 gene is shown as SEQ ID NO. 3.

Respectively designing 3'RACE and 5' RACE amplification according to the target fragment of the Thadh4 gene, carrying out cloning and sequencing after PCR screening, and finally splicing the obtained 3'RACE fragment and 5' RACE fragment.

3）3’RACE

3' RACE reverse transcription: taking total RNA as a template, the reaction system is as follows: total RNA 1. mu.g (X. mu.L), 5 XM-MLV Buffer 2. mu.L, dNTP mix (10 mM each) 1. mu.L, 3' RACE adapter (5'-CTGATCTAGAGGTACCGGATCC-3') (5. mu.M) 1. mu.L, Reverse transcription M-MLV (RNase H) (200U/. mu.L) 0.25. mu.L, RNase Inhibitor (40U/. mu.L) 0.25. mu.L, RNase Free H₂O5.5-X muL, and the total volume is 10 muL. The reaction procedure is as follows: 3min at 94 ℃; 30cycles at 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 2 min; 10min at 72 ℃; forever at 4 ℃. 3' RACE cDNA was obtained and stored at 4 ℃ for the next step.

3' RACE nested PCR: the 3'RACE nested PCR is divided into two rounds of 3' RACE Outer PCR and 3'RACE Inner PCR, and after 10. mu.L of 3' RACE reverse transcription product is obtained, appropriate dilution can be performed, generally to 30. mu.L for subsequent reaction.

The 3' RACE Outer PCR is a reaction system as follows: diluted reverse transcription reaction solution 2. mu.L, 3' RACE Outer Primer (10. mu.M) 2. mu.L, Gene Specific Outer Primer (10. mu.M) 2. mu.L, 10 XExTaq Buffer II (Mg)²⁺Free）5μL，MgCl₂（25mM）4μL，dNTP Mixture（10mM each）4μL，ExTaq polymerase 0.3μL，ddH₂O30.7. mu.L, total volume 50. mu.L. The reaction procedure is as follows: 3min at 94 ℃; 30cycles at 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 2 min; 10min at 72 ℃; forever at 4 ℃. The reaction product was diluted to 5. mu.L with an appropriate amount and used in Inner PCR reaction, and the rest was used for agarose gel electrophoresis detection.

The primer sequences are as follows: thadh 43' RACE Outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3', respectively; gene Specific Outer Primer: 5'-CCTCAAAGATAATAGGAATAGA-3' are provided.

The 3' RACE Inner PCR is a reaction system as follows: diluted 2. mu.L of 3'RACE Inner PCR product, 2. mu.L of 3' RACE Inner Primer (10. mu.M), 2. mu.L of Gene Specific Outer Primer (10. mu.M), 10 XExTaq Buffer II (Mg²⁺Free）5μL，MgCl₂（25mM）4μL，dNTP Mixture（10mM each）4μL，ExTaq polymerase 0.3μL，ddH₂O30.7. mu.L, total volume 50. mu.L. The reaction procedure is as follows: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 56 ℃, 2min at 72 ℃ and 32 cycles; 10min at 72 ℃; forever at 4 ℃.

The primer sequences are as follows: thadh 43' RACE Inner Primer: 5'-CAGCTTCGAATGCATTGGAAATACCA-3', Gene Specific Inner Primer: 5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3' are provided.

And carrying out agarose gel electrophoresis detection on the PCR product, cutting a target fragment, recovering the fragment by using a gel cutting kit, connecting the fragment to a PMD19-T simple Vector of takara, transforming the competence of Top10 escherichia coli of takara, screening the competent escherichia coli by using an LB screening culture plate containing Amp, carrying out PCR detection on the screened monoclonal colony, and then sending the detected colony to Jinsry for sequencing and identification.

4）5’RACE

5' RACE reverse transcription: the dephosphorylation reaction is the first step of the 5' RACE reaction and is catalyzed by the Alkaline Phosphotase. The reaction system is as follows: total RNA 2-5. mu.g (X. mu.L), 10 × Alkaline Phosphatase Buffer (MgCl)₂ Free）5μL，Alkaline Phosphatase（Calf intestine）（16U/μL）0.6μL，RNase Inhibitor（40 U/μL）1μL，RNase Free dH₂O43.4-X μ L, total volume 50 μ L. Reacting at 50 deg.C for 1h, adding 20 μ L of 3M CH₃COONa (pH5.2), 130. mu.L of RNase Free dH₂After O, mix well and add 200. mu.L of phenol: chloroform: isoamyl alcohol, 13000g is centrifuged at room temperature for 5min after being fully mixed, the upper aqueous phase is transferred to a new centrifuge tube with 1.5mL, 200 mu L of chloroform is added, 13000g is centrifuged at room temperature for 5min after being fully mixed, the upper aqueous phase is transferred to a new centrifuge tube with 1.5mL, 2 mu L of NA Carrier is added and then is uniformly mixed, 200 mu L of isopropanol is added, after being fully mixed, the mixture is placed at minus 40 ℃ for more than 2h, 13000g is centrifuged at 4 ℃ for 20min, the supernatant is discarded, 500 mu L of 75% precooled ethanol is added for rinsing, 13000g is added at 4 ℃, centrifuged for 5min, the supernatant is discarded and then is dried, 7 mu L of DEPC is added to dissolve and precipitate, and the CIAP-treated RNA is obtained.

The second reaction in the 5 'RACE is a decapping reaction, in which the 5' cap structure of mRNA is removed with Tobacco Acid Pyrophosphatase and a phosphate group is retained. Reaction system: 7 μ L of CIAP-treated RNA, 1 μ L of 10 × TAPRACTION Buffer, 1 μ L of Tobacco Acid Pyrophosphatase (0.5U/. mu.L), 1 μ L of RNase Inhibitor (40U/. mu.L), and 10 μ L of Total Volume. Reaction at 37 ℃ for 1h, 5. mu.L of the resulting mixture was used for 5' RACE Adaptor ligation, and the remaining 5. mu.L was stored at-80 ℃. The ligation reaction system of 5' Adaptor is as follows: CIAP/TAP-treated RNA 5. mu.L, 5' RACE Adaptor (15. mu.M) 1. mu.L, RNase Free dH₂O4. mu.L, reacted at 65 ℃ for 5min and then placed on ice for 2min, and the following reagents were added: 5 XRNA Ligation Buffer 8. mu.L, 40% PEG 600020. mu.L, T4 RNA Ligase 1. mu.L, RNaseINHIbitor (40U/. mu.L) 1. mu.L in total volume 40. mu.L, reaction at 16 ℃ for 1h, 4 ℃ overnight. To the reaction solution was added 20. mu.L of 3MCH₃COONa (pH 5.2), 140. mu.L of RNase Free dH₂After O, mix well and add 200. mu.L of phenol: chloroform: isoamyl alcohol, 13000g is centrifuged at room temperature for 5min after being fully mixed, the upper aqueous phase is transferred to a new centrifuge tube with 1.5mL, 200 mu L of chloroform is added, 13000g is centrifuged at room temperature for 5min after being fully mixed, the upper aqueous phase is transferred to a new centrifuge tube with 1.5mL, 2 mu L of NA Carrier is added and is fully mixed; adding 200 μ L isopropanol, standing at-40 deg.C for 2h, adding 500 μ L75% precooled ethanol, rinsing, centrifuging at 4 deg.C for 5min, discarding supernatant, centrifuging for 1min, completely sucking dry solution, drying, adding 7 μ L RNase Free dH2O, dissolving, and precipitatingPrecipitated to obtain Ligated RNA.

Ligated RNA reverse transcription reaction, reaction system: 6 μ L of Ligate RNA, 5 XM-MLV Buffer2 μ L, 1 μ L of dNTP (10 μ M each), 0.5 μ L of Random 9 mers (50 μ M), 0.25 μ L of Reverse Transcriptase M-MLV (RNase H) (200U/μ), 0.25 μ L of RNase Inhibitor (40U/μ), and 10 μ L of total volume. Reaction procedure: 10min at 30 ℃; 1h at 42 ℃; 15min at 70 ℃; forever at 4 ℃. 5' RACE cDNA was obtained for storage at 4 ℃ and the next step was performed.

The 5 'RACE nested PCR is also divided into two rounds of 5' RACE Outer PCR and 5 'RACE Inner PCR, and the reaction conditions of the reaction system are consistent with those of the 3' RACE nested PCR.

The primer sequences are as follows: thadh 45' RACE Outer Primer: 5'-ACACAAGCATAATCCACGACTGTATA-3', Gene Specific Outer Primer: 5'-CGCGGATCCATGGCTACATGCTGACAGCCTA-3' are provided. Thadh 45' RACE Inner Primer: 5'-TGGTACACCGGCTTCCCACCCAAAGA-3', Gene Specific Inner Primer: 5'-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3' are provided.

Splicing 3'RACE and 5' RACE products obtained by sequencing by using Bioedit software to obtain a target gene containing 1506bp bases, determining high homology of the target gene and a rice/Arabidopsis adh4 gene by Blast, and naming the target gene as a Thadh4 gene, wherein the nucleotide sequence of the target gene is shown as SEQ ID NO.1 and comprises a complete coding reading frame of 1212bp, and the sequence of a protein compiled and expressed by the target gene is shown as SEQ ID NO. 2.

Example 2Thadh4 Gene plant expression vector construction

Transferring the target gene segment to a target expression vector by using Gateway directional cloning technology, wherein the target expression vector comprises a BP reaction part and an LR reaction part. The carrier used in this example is shown in FIG. 1.

1) The purpose of the BP reaction was to transfer the gene fragment to an entry vector, reaction: 10-20ng of vector Thadh4 ORF fragment, 1 muL of Salt solution, 1 muL of pCRTM8/GW/TOPOTM vector (entry vector), adding nucleic-free Water to the total volume of 6 muL, mixing evenly, reacting at 22 ℃ for 60min, and transferring to ice; and (3) transforming the reaction product of 6 mu L in the previous step into TOP10 competent cells, coating the competent cells on an LB screening culture plate, selecting a single clone to perform bacterial liquid PCR detection, wherein the primers are a gene specific upstream primer and a T7 primer on a carrier, and the detected positive clone is further subjected to sequencing verification.

2) The LR reaction aims at re-cloning a target gene recombined into a portal vector into the target vector, and the reaction system comprises: 100ng of plasmid after purification of an entry vector, 1.5 mu L of a target vector (100 ng/. mu.L), 2 mu L of LR clone II enzyme mix, 1 XTE (pH8.0) to the total volume of 8 mu L, vortex, then carrying out short-time centrifugation, carrying out warm bath at 25 ℃ for 1h, adding 1 mu L of LProteinase K solution, mixing uniformly, and carrying out warm bath at 37 ℃ for 10min to stop the reaction; and transforming Top10 competent cells with 2. mu.L of LR reaction product, coating the competent cells on an LB screening culture plate, selecting a single clone to perform PCR detection on bacterial liquid, wherein the primers are a Thadh4 gene specific upstream primer and a 35s primer on a carrier, and further sequencing and verifying the detected positive clone. After verification, the recombinant vector after LR reaction is recovered and purified, and the expression vector of the target gene is obtained and stored at-20 ℃ for later use.

Example 3 genetic transformation of Thadh4 Gene

The Thadh4 gene is transformed into agrobacterium by a liquid nitrogen freeze-thawing method, and then the hybrid populus davidi is transformed by a leaf disc method. The method comprises the following operation steps: adding at least 100ng of recovered and purified expression vector into EHA105 competent cells, gently mixing, ice-bathing for 30min, quickly freezing for 1min with liquid nitrogen, heat shocking for 3min at 37 ℃, rapidly placing on ice for 1-2min, adding 800 μ L of LB culture medium, recovering for 3h at 28 ℃, 100rmp, centrifuging for 3min at 4000rmp, sucking off 800 μ L of LB culture medium, mixing the rest bacteria liquid uniformly, smearing on LB plate containing antibiotics, performing inverted culture for 30-48h at 28 ℃, detecting positive clone by PCR, expanding and propagating the colony of the positive clone, inoculating into 120mL of LB liquid culture medium containing corresponding antibiotics, culturing for about 24h with bacteria shaking (220 rmp) at 28 ℃ until OD is reached₆₀₀The value is about 0.5, and the bacterial liquid is subpackaged in a centrifugal tube of 50mL and 140Centrifuging at 0rcf for 10min, collecting thallus, resuspending thallus with a certain volume of MS (without adding sucrose) solution to OD600 value of 0.5, adding acetosyringone (As) to final concentration of 20 μ M, gently oscillating at 25 deg.C for 45min to obtain bacterial solution, culturing tissue culture seedling of David populus deltoids with growth potential of 4-6 weeks, taking 2-3 unfolded leaves under apical bud, cutting wound along the edge of the leaf, shearing the leaf into unequal sides, transferring into prepared staining solution, gently oscillating at 25 deg.C for 30min (250 mL filter paper, 90 rmp), taking out leaf disc, sucking dry residual bacterial solution, dark culturing on MS1 differentiation culture plate without antibiotic for 48h, washing, transferring into MS1 differentiation culture plate without antibiotic, culturing for 1W, transferring the leaf disc onto MS1 culture plate with antibiotic, culturing, screening and culturing when resistant adventitious buds grow on the edge of the leaf disc, transferring the plant into an MS2 stem elongation culture plate containing antibiotics for culture, when the resistant adventitious bud grows to about 1cm in a stem elongation culture medium, shearing the plant, transferring the cut plant into the MS culture plate containing the antibiotics for rooting and screening of resistant plants, thereby obtaining complete plants, and carrying out phenotypic observation of cuttings on the seedlings of the cutting resistant plants on the MS culture medium. After 4W of growth, as can be seen from FIG. 2, the overexpression of Thadh4 has a certain influence on the phenotype of the plants, and compared with the control plants, the transgenic plants grow faster, the internode variation occurs, and the internode length is obviously longer than that of the control plants.

Example 4 molecular detection of transgenic plants after Water flooding stress

And (3) carrying out a waterless flooding experiment on the transgenic Thadh4 gene poplar with phenotypic variation on the screening culture medium and a control poplar, wherein leaf leaves of the control poplar show wilting and yellowing after 1W, while the transgenic poplar has no obvious change, and sampling for carrying out real-time quantitative molecular detection. The real-time quantitative primer is designed by adopting Oligo6.6 software, the length of an amplification product is 156bp, the Tm value of the primer is 60 ℃, and the sequence of the primer is as follows: q adh4F: 5'-CATAATAGAGAGCGTGG-3' q adh4R: 5'-TGACTGTTCTCATGATT-3'. The selected 18S gene is used as the reference gene for real-time quantitative PCR. Real-time quantification was performed on ABI 7500 Real time PCR Systems, three replicates per reverse transcription sample, and 7500 System SDS software was used for data extraction and analysis. The reaction system is as follows: sYBR Premix Ex TaqTM (2X) 10. mu.L, PCR Forward Primer (10. mu.M) 0.4. mu.L, PCR Reverseprimer (10. mu.M) 0.4. mu.L, ROX Reference Dye II (50X) 0.4. mu.L, DNA template 2. mu.L, dH₂O6.8. mu.L, total volume 20. mu.L. Reaction procedure: 30s at 94 ℃; 94 ℃ for 5s, 60 ℃ for 34s, 95 ℃ for 15s, 40 cycles; 60 ℃ for 1min, 95 ℃ for 15 s. The real-time quantification results are shown in fig. 3: after the water logging stresses 1W, the leaf of the control plant presents a wilting state, and the transgenic plant grows well. The expression level of Thadh4 gene is 220-506 times higher than that of contrast in transgenic plant, which shows that the Thadh4 gene of the Taxus chinensis is closely related to flooding resistance property, and the Thadh4 gene is predicted to have important application value in forest genetic engineering field.

SEQUENCE LISTING

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<211> 24

<212> DNA

<213> Artificial

<220>

<223> Forward primer

<400> 4

aggcctggta taatacgttg gtat 24

<210> 5

<211> 22

<212> DNA

<213> Artificial

<220>

<223> 3'RACE Adaptor

<400> 5

ctgatctaga ggtaccggat cc 22

<210> 6

<211> 23

<212> DNA

<213> Artificial

<220>

<223> Thadh4 3'RACE Outer Primer

<400> 6

taccgtcgtt ccactagtga ttt 23

<210> 7

<211> 22

<212> DNA

<213> Artificial

<220>

<223> Gene Specific Outer Primer

<400> 7

cctcaaagat aataggaata ga 22

<210> 8

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Thadh4 3'RACE Inner Primer

<400> 8

cagcttcgaa tgcattggaa atacca 26

<210> 9

<211> 32

<212> DNA

<213> Artificial

<220>

<223> Gene Specific Inner Primer

<400> 9

cgcggatcct ccactagtga tttcactata gg 32

<210> 10

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Thadh4 5'RACE Outer Primer

<400> 10

acacaagcat aatccacgac tgtata 26

<210> 11

<211> 31

<212> DNA

<213> Artificial

<220>

<223> Gene Specific Outer Primer

<400> 11

cgcggatcca tggctacatg ctgacagcct a 31

<210> 12

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Thadh4 5'RACE Inner Primer

<400> 12

tggtacaccg gcttcccacc caaaga 26

<210> 13

<211> 34

<212> DNA

<213> Artificial

<220>

<223> Gene Specific Inner Primer

<400> 13

cgcggatcca cagcctactg atgatcagtc gatg 34

<210> 14

<211> 17

<212> DNA

<213> Artificial

<220>

<223> q adh4F

<400> 14

cataatagag agcgtgg 17

<210> 15

<211> 17

<212> DNA

<213> Artificial

<220>

<223> q adh4R

<400> 15

tgactgttct catgatt 17

Claims

1. A Thadh4 gene for regulating flooding resistance of Sequoia intermedia has a nucleotide sequence shown in SEQ ID NO. 1.

2. The expression protein of Thadh4 gene for regulating flooding resistance of sequoia intermedia as claimed in claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.

3. A vector containing Thadh4 gene for regulating flooding resistance of Taxus chinensis as claimed in claim 1.

4. The use of Thadh4 gene for regulating flooding resistance of Taxus chinensis as claimed in claim 1 in plant breeding.

5. The use of the Thadh4 gene for regulating flooding resistance of a plant as claimed in claim 1.

6. A host bacterium containing Thadh4 gene for controlling flooding resistance of Taxus chinensis as claimed in claim 1.