CN111117992A - Flood-resistant and drought-resistant related gene ThPDC1 of taxus chinensis and application thereof - Google Patents

Abstract

The invention discloses a flood-resistant and drought-resistant related gene ThPDC1 of taxus chinensis and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the gene ThPDC1 is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. The invention takes the sequoia sempervirens 406 tissue culture seedling leaves as materials, clones the full-length gene of the sequoia sempervirens ThPDC1 by RACE technology, constructs the over-expression vector pH35GS-ThPDC1 by GATEWAY technology, transfers the ThPDC1 gene into the poplar of woody plant, the expression quantity of the ThPDC1 in the transgenic plant is 300 times higher than that of the control poplar by 100 times, the growth of the non-transgenic plant is inhibited under the water flooding stress, and the growth vigor of the transgenic plant is good. Under drought treatment, the growth of non-transgenic plants is inhibited, and leaves are yellow and fall off; the transgenic plants grew well.

Description

Flood-resistant and drought-resistant related gene ThPDC1 of taxus chinensis and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a flooding-resistant and drought-resistant related gene ThPDC1 of taxus chinensis and application thereof.

Background

The Zhongshan fir is a good clone bred from the hybrid combination of the larval fir and the china academy of sciences of Jiangsu province. The work is in the leading position in China and even internationally, a plurality of national-level and provincial-level forest fine varieties are obtained at present, and the second-level prize of science and technology of Jiangsu province is obtained in 2013. Years of indoor comparison tests, intermediate demonstration tests and regional tests show that the hybrid vigor of the taxus chinensis is obvious, and the taxus chinensis has the characteristics of fast growth, water-moisture resistance, drought resistance, salt and alkali resistance and the like.

In a low oxygen environment, an ethanol fermentation pathway is particularly important, which can produce glucose and glycogen which can maintain the activity of plants and is accompanied with ATP production, Pyruvate Decarboxylase (PDC), which is a carboxyl lyase taking thiamine pyrophosphate as a coenzyme, is one of main enzymes of the ethanol fermentation pathway, and thus pyruvate decarboxylase and a coding gene thereof have been widely paid attention at home and abroad. In recent years, researchers have been dedicated to studying the expression and function of members of the PDC gene family under normal and various stress treatment conditions, the PDC gene family is a relatively small number of gene families in plants, but the members of the PDC gene family play an important role in the stress environments such as waterlogging, drought, cold damage resistance and the like of the plants. The rice contains 4 PDC genes, wherein OsPDC1, OsPDC2 and OsPDC4 are all multi-exon genes and are induced by hypoxia and drought stress, and the Arabidopsis contains two PDC genes, and after the deletion mutants AtPDC1 and AtPDC2 are subjected to flooding treatment for 8 days, the survival rate of the deletion mutants is obviously lower than that of the wild type. At present, researches on PDC genes are mainly concentrated in herbaceous model plant Arabidopsis thaliana and main crops of rice and wheat, few researches are carried out in woody plants, and no relevant reports are found in the plants of the genus Hupezia.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a flood-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis. The invention also aims to solve the technical problem of providing the application of the flood-resistant and drought-resistant related gene ThPDC1 of the sequoia intermedia.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a flood-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis is shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the flood-resistant and drought-resistant related gene ThPDC1 of the sequoia intermedia is shown as SEQ ID NO. 2.

The carrier contains the flooding-resistant and drought-resistant related gene ThPDC1 of the sequoia intermedia.

A host bacterium containing the flooding-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis.

Further, the carrier is pH35GS:: ThPDC 1.

The gene ThPDC1 is applied to plant breeding.

The gene ThPDC1 is applied to plant flooding resistance and drought stress resistance.

The invention takes the leaves of the tissue culture seedling of the sequoia zhongshanensis 406 as the material, and clones the full-length gene of the sequoia zhongshanensis ThPDC1 through the RACE technology. Meanwhile, an overexpression vector pH35GS of the gene is constructed by adopting the GATEWAY technology, namely ThPDC, the gene is positioned behind a promoter P35S, and ThPDC1 is efficiently expressed in poplar under the drive of a promoter P35S.

Has the advantages that: compared with the prior art, the invention transfers the ThPDC1 gene into the woody plant poplar, the expression quantity of the transgenic poplar with the over-expression ThPDC1 is increased by 300 times of 100-fold, and the growth of the non-transgenic poplar is inhibited after the plant is subjected to flooding stress for 7 days, so that the plant is short and small and the leaves are yellow; the transgenic plants grew well. After 10 days of drought treatment, the growth of the non-transgenic plants is inhibited, and leaves are yellow and fall off; the transgenic plants grew well and the growth was not affected at all. The ThPDC1 gene is shown to be an important gene of plants responding to stress.

Drawings

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

FIG. 2 is a graph showing the difference in expression of the gene of interest in a ThPDC1 transgenic plant compared to an untransformed plant, pH35 GS;

FIG. 3 is a graph of the phenotypic differences of ThPDC1 transgenic plants under water-flooding stress from non-transgenic plants at pH35 GS;

FIG. 4 is a graph showing the expression changes of the target gene of a ThPDC1 transgenic plant under water-logging stress at pH35 GS;

FIG. 5 is a graph of the phenotypic differences of ThPDC1 transgenic plants under drought stress from non-transgenic plants at pH35 GS;

FIG. 6 is a graph of the change of gene expression of a ThPDC1 transgenic plant under drought stress at pH35 GS.

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 ThPDC1 Gene by RACE technique

1. Extraction of Total RNA and cDNA Synthesis

1) Sucking 1mL of RNA Plant Reagent lysate in each 2mL of centrifuge tube, adding about 3g of the leaf sample of the Taxus chinensis 406 which is ground by liquid nitrogen, and strongly swirling to fully and uniformly mix the leaf sample;

2) horizontally placing the centrifugal tube for 10min at room temperature to allow the lysate to fully crack the nucleoprotein;

3) centrifuging at 4 deg.C for 5min at 1200rpm with a high-speed refrigerated centrifuge, and transferring the supernatant to a new centrifuge tube;

4) adding 200 μ L of 5M/L sodium chloride solution, mixing gently, adding 300 μ L of chloroform, and mixing by turning upside down;

5) centrifuging at 4 ℃ and 12 × 1000rpm for 10min, gently sucking the supernatant, and transferring to a new 2mL centrifuge tube;

6) repeating the steps 4) and 5);

7) adding isopropanol with the same volume as the obtained supernatant, mixing well, and standing at room temperature for 10 min;

8) centrifuging at 4 deg.C and 12 × 1000rpm for 10min, removing supernatant, and collecting bottom precipitate;

9) followed by extraction with RNeasy plant Mini Kit (QIAGEN, USA);

10) add 0.5mL of freshly prepared RLT buffer (1mL) RLT and 10. mu.L β -mercaptoethanol) and vortex thoroughly;

11) adding 0.5mL of absolute ethyl alcohol, blowing, beating and uniformly mixing;

12) transferring the mixed solution into a collecting column RNeasy mini column (ping), centrifuging at 12000rpm for 15s, discarding the filtrate, and reusing the centrifuge tube;

13) adding 350 mu L of RWI Buffer to RNeasy mini column, centrifuging at 12000rpm for 15s, and discarding the filtrate and centrifuge tube;

14) putting the RNeasy mini column into a new centrifuge tube, adding 500 mu L of buffer RPE.12000rpm, centrifuging for 15s, and removing the filtrate;

15) adding 500 μ L Buffer RPE 12000rpm again, centrifuging for 2min, and removing filtrate and centrifuge tube;

16) putting the RNeasy mini column into a new centrifugal tube for 1min at 13000 rpm;

17) putting the collecting column RNeasy mini gum into a new RNA collecting tube, adding a proper amount of RNase freewater to the center of the membrane, standing for 1min and centrifuging at 12000rpm for 1 min;

18) adding the filtrate into collecting column RNeasy mini column, centrifuging at 12000rpm for 1min, and recovering filtrate to obtain total RNA;

19) and (4) detecting and determining the concentration of the RNA by using an ultraviolet spectrophotometer, and detecting the integrity of the RNA by using agarose gel electrophoresis.

20) The total RNA sample thus obtained was ImProm-II from Promega^TMReverse transcription was performed by Reverse Transcriptase. The reaction system is as follows: total RNA (1 ug); 4.0. mu.L of ImProm-II5 × reaction Buffer; 2.5 μ L Mgcl₂(25mM)；1.0μL dNTP(10mM)；1.0uL Oligo(dT)Adaptor Primer(50ng/μL)；0.5μL Recombinant Rnasin Ribonuclease Inhibitor；1.0μL ImProm-II^TMReverseTranscriptase, plus Nuclear-Free water to make up 20.0. mu.L. The reaction procedure is as follows: 5min at 25 ℃; 42 ℃ for min; 70 ℃; 15 min; and keeping at 4 ℃.

RACE nested PCR amplification

(1) 3' RACE cDNA reverse transcription reaction

Taking total RNA as a template, the reaction system is as follows: 1 μ L of 3' RACE Adaptor (5 μ M); 2 μ L of 5 XM-MLVBuffer; 1 μ L dNTP mix (10mM each); 0.25. mu.L of Reverse Transcriptase M-MLV (RNaseH) (200U/. mu.L); 0.25. mu.L RNase Inhibitor (40U/. mu.L); addition of RNase Free dH₂O make up to 10. mu.L. The reaction procedure is as follows: 60min at 42 ℃; 15min at 70 ℃; and keeping at 4 ℃.

(2)3’RACE Outer PCR

The 3' RACE Outer PCR is a PCR reaction using a known cDNA fragment to design an upstream Outer specific Primer ThPDC 13 ' Outer Primer and a 3' RACE Outer Primer. The reaction system is as follows: 2 mu L of reverse transcription reaction liquid; 8ul 1 × cDNA Dilution buffer II; 2 μ L of 3' RACE Outer Primer (10 μ M); 2 μ L of ThPDC1 outprimer (10 μ M); 4 μ L of 10 × LATaq Buffer II (Mg)²⁺Free)；3μL MgCl₂(25 mM); 0.25 μ L of LATaqpolymerase (5U/. mu.L); water was added to make up 50. mu.L. The reaction procedure is as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 2 min; 10min at 72 ℃; keeping at 4 ℃.

Wherein, 3' RACE outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3', respectively; ThPDC 13' outerPrimer: 5'-GCATAGTTGGGGGCCCCAACTCCAAT-3' are provided.

(3)3’RACE Inner PCR

The 3' RACE Inner PCR takes an Outer PCR reaction product as a template, and utilizes an upstream Inner side specific Primer ThPDC 13 ' Inner Primer and a 3' RACE Inner Primer to carry out PCR amplification on the primers, wherein the reaction system is as follows: 1 mu of LOuter PCR reaction product; 8 μ L dNTP mix (10mM each); 5 μ L of MgCl₂(25mM)；5μL 10×LATaqBuffer II(Mg²⁺Free); 2 μ L of ThPDC 13' Inner Primer (10 μ M); 2 μ L of 3' RACE Inner Primer (10 μ M); 0.5. mu.L of LATaq polymerase; water was added to make up 50. mu.L. The reaction procedure is as follows: 943min at the temperature; 30s at 94 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 32 cycles; 10min at 72 ℃; keeping at 4 ℃. And (3) carrying out 1% agarose gel electrophoresis on the PCR product, reading and shooting on a gel imager, cutting and recovering the gel, connecting a T vector, converting, detecting and sequencing.

Wherein, 3' RACE Inner Primer:

5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3'；

ThPDC13’Inner Primer：

5'-CCAGTGTATATCAGCATTAGCTGTAATCTCCCTGGT-3'。

(4) 5' RACE dephosphorylation reaction

1) The reaction system is as follows: 2-5 μ g Total RNA; mu.L of 10 × Alkaline Phosphatase Buffer (MgCl)₂Free); 0.6. mu.L of Alkaline Phosphatase (Calf intestine) (16U/. mu.L); 1 μ L RNaseInhibitor (40U/. mu.L); addition of RNase Free dH₂O is complemented to 50 mu L; reacting for 1h at 50 ℃;

2) to the reaction solution was added 20. mu.L of 3M CH₃COONa (pH5.2), 130. mu.L of RNase Free dH₂Fully and uniformly mixing after O;

3) add 200. mu.L of phenol: chloroform: isoamyl alcohol, evenly mixing, centrifuging at 13000g for 5min at room temperature, and transferring the upper aqueous phase into a new centrifugal tube of 1.5 mL;

4) adding 200 mu L of chloroform, fully and uniformly mixing, centrifuging at 13000g for 5min at room temperature, and transferring the upper aqueous phase into a new centrifugal tube of 1.5 mL;

5) adding 2 μ L of NA Carrier, mixing, adding 200 μ L of isopropanol, mixing, standing at-40 deg.C for more than 2 hr;

6) centrifuging at 4 deg.C and 13000 Xg for 20min, and removing supernatant;

7) adding 500 μ L of 75% precooled ethanol for rinsing, centrifuging at 4 deg.C and 13000g for 5min, removing supernatant, and drying;

8) the precipitate was dissolved with 7. mu.L of DEPC water to obtain CIAP-treated RNA.

Tobacco Acid Pyrophosphatase (TAP) was used to remove the 5' cap structure of mRNA and retain a phosphate group. The reaction system is as follows: 7 μ L of CIAP-treated RNA; 1 μ L of 10 × TAP Reaction Buffer; 1 μ L of monobacaco Acid Pyrophosphatase (0.5U/. mu.L); 1 μ L RNase Inhibitor (40U/. mu.L); after 1h reaction at 37 ℃, 5. mu.L of the mixture was used for 5' RACE Adaptor ligation reaction.

(5) Ligation of 5' Adaptor

1) The reaction system is as follows: 5 μ L of CIAP/TAP-treated RNA; 1 μ L of 5' RACE Adaptor (15 μ M); 4 μ LRNase Free dH₂O; after 5min of reaction at 65 ℃, the mixture was left on ice for 2min, and the following reagents were added: 8 μ L of 5 XRNA ligation buffer; 20 μ L40% PEG 6000; 1 μ L T4 RNA Ligase; 1 μ L RNase Inhibitor (40U/. mu.L); the total volume is 40 mu L; reacting at 16 ℃ for 1h, and standing overnight at 4 ℃;

2) to the reaction solution was added 20. mu.L of 3M CH₃COONa (pH5.2), 140. mu.L of RNase Free dH₂After O, fully and uniformly mixing;

3) add 200. mu.L of phenol: chloroform: isoamyl alcohol, evenly mixing, centrifuging at 13000g for 5min at room temperature, and transferring the upper aqueous phase into a new centrifugal tube of 1.5 mL;

4) adding 200 mu L of chloroform, fully and uniformly mixing, centrifuging at 13000g for 5min at room temperature, and transferring the upper aqueous phase into a new centrifugal tube of 1.5 mL;

5) adding 2 mu L of NA Carrier and fully mixing; adding 200 μ L of isopropanol, and standing at-40 deg.C for 2 hr;

6) adding 500 μ L of 75% precooled ethanol for rinsing, centrifuging at 4 deg.C and 13000g for 5min, discarding the supernatant, centrifuging for 1min, completely sucking the solution, and drying;

7) add 7. mu.L of RNase Free dH₂And dissolving the precipitate by using O to obtain the Ligated RNA.

(6) 5' RACE cDNA reverse transcription reaction

The reaction system is as follows: 6 μ L of Ligate RNA; 2 μ L of 5 XM-MLV Buffer; 1 μ L dNTP (10 μ M each)0.5 μ L Random 9mers (50 μ M); 0.25. mu.L of Reverse Transcriptase M-MLV (RNase H) (200U/. mu.L); 0.25. mu.L RNase Inhibitor (40U/. mu.L); the total volume is 10 μ L. The reaction procedure is as follows: 30 ℃ for 10 min; 42 ℃ for 1 h; 15min at 70 ℃; and keeping at 4 ℃.

(7)5’RACE Outer PCR

The reaction system is as follows: 2.0. mu.L of the reverse transcription reaction solution diluted by 5 times in the step (6); 2.0 μ L of 5' RACE OuterPrimer (10 μ M); 2.0 μ L ThPDC 15' outer Primer(10μM)；5.0μL 10×ExTaq Buffer II(Mg²⁺Free)；4.0μL MgCl₂(25 mM); 4.0. mu.L dNTP mix (10mM each); 0.3. mu.L of ExTaq polymerase; adding dH₂O make up to 50.0. mu.L. The reaction procedure is as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 2 min; 10min at 72 ℃; keeping at 4 ℃.

Wherein, 5' RACE Outer Primer: 5'-CATGGCTACATGCTGACAGCCTA-3', respectively; ThPDC 15' outerPrimer: 5'-AATGAAACATAAACAATC-3' are provided.

(8)5’RACE Inner PCR

The reaction system is as follows: 2.0. mu.L of 5' RACE Outer PCR reaction product; 2.0 μ L of 5' RACE Inner Primer (10 μ M); 2.0 μ L of ThPDC 15' Inner Primer (10 μ M); 5.0 μ L10 XExTaq Buffer II (Mg)²⁺Free)；4.0μL MgCl₂(25 mM); 4.0. mu.L dNTP mix (10mM each); 0.3. mu.L of ExTaq polymerase; adding dH₂O make up to 50.0. mu.L. The reaction procedure is as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 2 min; 10min at 72 ℃; keeping at 4 ℃.

Wherein, 5' RACE Inner Primer: 5'-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3', respectively; ThPDC 15' Inner Primer: 5'-CACAACCAAAAGTGGGTCCAGTTGATAC-3' are provided.

And (3) performing 1% agarose gel electrophoresis on the PCR product, reading and shooting on a gel imager, then recovering by using a gel cutting recovery kit, connecting a T vector, converting, detecting by using a bacteria liquid PCR, and sending to a Invitrogen company for sequencing.

Splicing 3 'RACE and 5' RACE products obtained by sequencing by using Bioedit software to obtain a target gene containing 1518bp base, determining that the target gene is highly homologous with the rice/arabidopsis thaliana PDC1 gene by Blast, and naming the target gene as ThPDC1 gene, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The amino acid sequence is shown as SEQ ID NO. 2.

Example 2: construction of ThPDC1 Gene plant expression vector

ThPDC1 Gene overexpression vector was constructed by first cloning the target fragment into the vector by BP reaction using Gateway technology from Invitrogen (PCR)^TM8/GW/TOPO^TM) Then the target gene is transferred to the transgenic line by LR reactionThe expression vector was measured at pH35Gs (shown in FIG. 1).

(1) Obtaining the fragment of interest

After Escherichia coli with the correct ORF frame sequencing was amplified and cultured on the basis of the full length of ThPDC1 gene, pMD19-T vector plasmid with the full length gene was extracted with the AXYGEN plasmid miniprep purification kit, the desired fragment was amplified with the ORF frame primer (downstream primer containing stop codon) using the plasmid template, and the fragment was recovered by cutting gel after electrophoresis.

(2) BP reaction

1) The reaction system is as follows: 10-20ng Fresh PCR product (purified); 1 μ L Salt solution; 1 μ L pCRTM8/GW/TOPOTM vector; adding nucleic-free Water to make up to 6 μ L;

2) mixing, reacting at 22 deg.C for 60min, and transferring to ice;

3) adding the reaction product of the previous step of 6 mu L into TOP10 competent cells melted on ice, and gently mixing uniformly without blowing;

4) performing ice bath for 15 min;

5) thermally shocking at 42 deg.C for 60sec, and rapidly placing on ice for 3-5 min;

6) adding 800 μ L SOC culture medium, shaking at 37 deg.C 150rmp for 1 h; centrifuging at 4000rmp for 3min, sucking off about 700 μ L SOC at the upper layer, mixing the rest bacteria liquid uniformly, and coating on an LB screening culture plate;

7) carrying out inverted culture at 37 ℃ overnight, selecting clones to carry out bacteria liquid PCR detection, wherein the used primers are gene specific upstream primers ORF-F: 5'-ATGGATTCAAGCTTTGGAGACGTAA-3' and T7 on vector: 5'-TAATACGACTCACTATAGGG-3' a primer;

8) the detected positive clones are further sequenced and verified.

9) The AXYGEN plasmid miniprep kit was used to extract the correctly sequenced plasmids of the entry vector.

(3) LR reaction

BstXI (TaKaRa) restriction enzyme is selected to carry out enzyme digestion reaction on the vector with the pH value of 35GS, the vector is linearized, the reaction system is 10 XH Buffer (5 mu L), the purified vector with the pH value of 35GS is 4 mu g, Bstx1(2 mu L), the reaction solution is supplemented to 50 mu L by water without nuclease, the reaction is carried out for 1H, and then the inactivation is carried out for 15s at the temperature of 85 ℃; after the completion of the digestion reaction, 4. mu.L of the reaction solution was subjected to electrophoresis detection, and then recovered and purified using AXYGEN PCR Purification Kit. LR reaction system: linearized pH35GS vector (100ng), entry vector (150ng) supplemented to 8. mu.L with 1 XTE (pH8.0), mixed well, added with 2. mu.L LR ClonaseII enzyme mix, PCR reaction at 25 ℃ for 1h, added with 1. mu.L Protease K solution at 37 ℃ for 10min to terminate the reaction; then transformation was performed with Top10 competent cells, positive single clones were picked and the expression of 35 sf: 5'-CGCACAATCCCACTATCCTT-3' has primer and downstream primer ORF-R specific to the target gene: 5'-CTGAGGATTTGGTGGCCTGCTGTT-3' and carrying out PCR detection of bacterial liquid, recovering the target vector after LR reaction after sequencing verification, and obtaining the over-expression vector of the target gene with pH35GS, ThPDC 1.

Example 3: obtaining of ThPDC1 transgenic plant and hardening seedling thereof

(1) Liquid nitrogen freeze thawing and agrobacterium transformation

1) Adding at least 100ng of recovered and purified expression vector pH35GS, ThPDC1 into EHA105 competent cell, mixing gently, and ice-cooling for 30 min;

2) quickly freezing for 1min by using liquid nitrogen;

3) thermally shocking at 37 deg.C for 3min, and rapidly placing on ice for 1-2 min;

4) adding 800 μ L LB culture medium, resuscitating at 28 deg.C and 100rmp for 3 h;

5) centrifuging at 4000rmp for 3min, and sucking off 800. mu.L of LB culture medium;

6) uniformly mixing the residual bacteria liquid, and smearing the bacteria liquid on an LB flat plate (spc + str) added with corresponding antibiotics;

7) inversely culturing for 30-48h at 28 ℃ in an incubator;

8) single clones were picked and cloned with 35 sf: 5'-CGCACAATCCCACTATCCTT-3' has primer and downstream primer ORF-R specific to the target gene: 5'-CTGAGGATTTGGTGGCCTGCTGTT-3' PCR detection of bacterial liquid.

(2) Leaf disc method for transforming Populus deltoides

1) Picking single colony from activated LB culture plate, inoculating positive strain detected by PCR into 2mL LB liquid culture medium containing corresponding antibiotic, culturing at 28 deg.C with shake bacteria (250rmp) for about 12h, and inoculating 0.8mL into 50mL LB liquid culture medium containing corresponding antibioticTo OD₆₀₀A value of about 0.5;

2) the bacterial liquid is subpackaged in a 50mL centrifuge tube, centrifuged at 5000rpm for 10min, and thalli are collected;

3) resuspending the cells to OD with a volume of sucrose-free MS solution₆₀₀A value of 0.5;

4) 2-3 unfolded leaves under the terminal bud of the tissue culture seedling of the populus deltoids with basically consistent growth vigor in 4-6 weeks are taken, and a wound is cut along the edge of the leaves;

5) transferring the cut leaf disc into the prepared dip dyeing solution, and oscillating for 30min at 25 ℃ and 90 rmp;

6) taking out the leaf disc, sucking the residual bacterial liquid by using filter paper, and transferring the leaf disc to an MS1 differential culture plate for dark culture for 48 hours;

7) washing bacteria, transferring to MS1 differentiation culture plate, and culturing for 1 week;

8) then transferring the leaf disc to a differentiation screening culture medium MS1 flat plate for screening culture;

9) when the resistant adventitious bud grows out from the edge of the leaf disc, transferring the resistant adventitious bud to a bud elongation screening culture medium MS2 plate for continuous screening culture;

10) when the resistant adventitious bud grows to about 1cm in the stem elongation culture medium, shearing the resistant adventitious bud and transferring the resistant adventitious bud into a rooting screening culture medium MS₀Carrying out rooting screening on the resistant plants on the flat plate so as to obtain complete plants;

11) rooting screening culture medium MS₀Subculturing resistant seedlings on the culture medium, and carrying out plant phenotype observation.

The phenotype of the obtained transgenic poplar has no obvious difference compared with the phenotype of the non-transgenic poplar, and the research is further carried out by a molecular means.

(3) Quantitative PCR detection of transgenic plants

The real-time quantitative primer is designed by adopting Oligo6.6 software, the Tm value of the primer is at 60 ℃, and the sequence of the primer is as follows: q PDC 1F: 5'-TGGGATGTCTGCCCTG-3' q PDC 1R: 5'-AATCAGATGATCCAG-3' are provided. The reference gene of the real-time quantitative PCR adopts the screened APRT gene APRT-F:

5'-TCCACAGGTTCTTGAATCGCT-3'，APRT-R：

5'-TGACTTGAGCCTCATTCGCTC-3' are provided. Utilizing the Analitik Jena qTOWER2.2 (Germany) systemqRT-PCR was performed. Following the protocol of the SYBR Green kit (Rox), the amplification procedure was: 2min at 55 ℃ and 10min at 95 ℃; 40 cycles of 95 ℃ for 15s and 60 ℃ for 1 min. The melting curve was then generated by setting 60 ℃ to 95 ℃. Each sample was subjected to 3 technical replicates, and 20 μ L of the reaction system contained: mu.L of diluted cDNA (dilution ratio 1: 3, diluted cDNA concentration of about 350 ng. mu.L)^-1) 10 μ L FastStart Universal SYBR Green Master (Rox), 6pmol upstream primer, 6pmol downstream primer and 6.8 μ L ddH₂And O. The relative expression amount of the gene was calculated by the 2- Δ Δ Ct method. The test data were statistically analyzed and plotted by the SPSS 19.0 software. The real-time quantitative result is shown in FIG. 2, and the expression level of the gene in ThPDC1 is 100-fold and 300-fold higher than that of the non-transgenic plant.

Example 4: molecular detection of transgenic plants after water-flooding stress

Carrying out a top-free flooding experiment on tissue culture seedlings of ThPDC1 transgenic poplar and a control poplar, wherein the pH value of the tissue culture seedlings is 35GS, the transgenic poplar and the control plant which have basically consistent growth vigor are taken to have top-free flooding in a tissue culture bottle, the temperature is controlled at 25 ℃, the light intensity is 4000lux, the illumination is carried out for 10 hours every day, and after the tissue culture is carried out in an illumination incubator for 7 days, the control poplar is found to be inhibited from growing, and leaves of the control poplar are yellow and fall off. The transgenic poplar grew well (FIG. 3). Further taking the leaves to carry out real-time quantitative molecular detection, wherein the real-time quantitative result is shown in figure 4: the expression quantity of the waterflooding ThPDC1 gene is 300-500 times higher than that of a control in a transgenic plant, which indicates that the ThPDC1 gene of the taxus chinensis is closely related to the waterflooding resistance.

Example 5: hardening and transplanting of transgenic plants

In the process of hardening off the tissue culture seedlings, firstly, the rubber band at the bottle mouth is loosened and is in a semi-sealed state, the culture is carried out in a room temperature laboratory, in this way, after 2 days of culture, the sealing film is completely uncovered, the culture is continued for 4-5 days, and watering is needed in the process of hardening off the seedlings. Taking out the tissue culture seedling, cleaning the residual culture medium at the root, transferring into a sterilized nutrient soil flowerpot, covering the flowerpot with a beaker, removing the beaker after one week, continuing culturing, regularly observing the growth condition of the plant, and counting the transplanting survival rate.

Example 6: molecular detection of transgenic plants after drought stress

The drought stress experiment is carried out on ThPDC1 transgenic poplar and control poplar with the same growth vigor of hardening seedlings and survival pH35GS, the temperature is controlled at 25 ℃, the light intensity is 4000lux, the control poplar is irradiated by light for 10 hours every day, after water is thoroughly watered once, the control poplar is not watered continuously for 10 days, and the control poplar is found to be obviously inhibited in growth, the leaves are yellow, withered and shed, and a large number of leaves are fallen off. The transgenic poplar grew well and was not affected by any drought, as shown in fig. 5. Further taking the leaves to carry out real-time quantitative molecular detection, wherein the real-time quantitative result is shown in figure 6: the expression quantity of the water flooding hypochondrium ThPDC1 gene is 400-600 times higher than that of a control in a transgenic plant, which indicates that the Taxus chinensis ThPDC1 gene is closely related to drought resistance.

Sequence listing

<110> institute of plant of Chinese academy of sciences of Jiangsu province

<120> Chinese torreya flooding-resistant and drought-resistant related gene ThPDC1 and application thereof

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<170>SIPOSequenceListing 1.0

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gaaaagcagt ggtatcaacg cagagtacat ggggattggt tcaagagatc tacattccca 60

tagactgtcc gcctgctgca tcagattctg aaaaggaata tgaacttccc agttgctcca 120

tggattcaag ctttggagac gtaaattcgc acaggtctga gagcaatgat gtgggatgtc 180

tgccctgcga caaatctgta aacagcgtgt cgcatagcgt tgttcagggt gcggcggttg 240

ccacgctggg gcgccacctg ccccggaggc ttgtccaggt ggccgtgcgt gatgtgttct 300

cagtgccaag ggacttcaat ctgacgtggc tggatcatct gattgcagag cctgacctaa 360

ttttggtggg ttgctgcaac gagctcaatg ccggctacgc cgcggatggc tatgcgcgct 420

cctatggcgt gggagcctgc gctgtcacct tcactgttgg tggcctctct gtgatcaacg 480

caattgcagg ggcttacaat gagaatctgc cggtcatatg catagttggg ggccccaact 540

ccaatgacta tggcaccaac cgtattcttc atcataccat tgcccttcct gactttagcc 600

aggagtatcg atgctttcag tctgtaacat gtgctcaggt gcttgtgaac aatttggaag 660

atgccaacga gctcattgat catgctatat ctactgcact tatggagagc aaaccagtgt 720

atatcagcat tagctgtaat ctccctggta ttccccatca cacattcact catgaacctg 780

ttccatacag tcttacaccc aggcaaagca atccacagag cttgcaatca gctgtagaac 840

tcactgcacg aatcctaaat gcagcggtta agccagtctt ggttggaggc ccaaagctca 900

gggctgcaaa ggcaaaatct gcatttgcag aattagcaaa tgcttcaggt tatgccgtgg 960

ctgtaatgcc atctgctaag gggcaggtta tagagaccca cccacatttc atagggactt 1020

actggggtgc agttagtacc gcattctgtg ctgaaatagt tgagtctgca gatgcatatt 1080

tgttcgcagg gcctattttt aatgattata gttcagtggg gtattcattg ctcttgaaga 1140

aggagaaagc tataattgta caccctaata gggtaacagt atcaactgga cccacttttg 1200

gttgtgtgct gatgaaagat ttcttagaag cccttgcaaa gaagttaaag cacaacaaaa 1260

cggcctttga gaattaccag cgcatctttg tacctgaagg tgttcccttg aaaggtgctc 1320

cacatgaggc tctaagagtc aatgtgctat tcaaacacat tcaggccatg ttatcaaatg 1380

atacagcttt aattgctgaa actggtgact cttggttcaa ttgccagaag cttaaactgc 1440

cagaggaatg cgggtatgaa tcccagatgc agtatggatc aattggttgg tctgtaggtg 1500

aggccaccaa atcctcagcc actttgggat atgctcaagc aactccaaag aagcgagcca 1560

ttgcctgtat tggtgttgga agcttccagg tgacagccca ggatatttat accatgattc 1620

gatatgggca gaaaagtata attttcctga ttaataatgg aggatatact atagaggttg 1680

aaattcatga tggaccatac aatgtaataa agaattggaa ttacactgga ttcaagcttg 1740

ggactctagt agatgctatt cataatggtg aaggaaaatg ttggactacc aaggttaaga 1800

cagaagaaga ggctattgag gctattaaaa ctgcccaggg acctaaaaag gattgtttat 1860

gtttcattga agttattgta cacaaagatg acacaagcaa agagttgctt gaatggggat 1920

ctagggtagc ggcagcaaac agcaggccac caaatcctca gtagttttta aaaaacaagc 1980

tcaggtgggt tagggcgatt ttgtagatta gtatctcaga gtaatttcgc agcagcctgt 2040

tttcccgaga tgatcttttg agcttatgtg aggtatattg accaaggtga cagtccttgg 2100

cttacggtgg attgtttatt ttttagtatg taagactgag ttttggttag gatggtagca 2160

tatttcaagg aacggaagag agtcatgagt gagaatgaat acggtattgc cagtcggtga 2220

cttgggtttt ctttgtgctt cagagccctc caagtcattg actgtgcttc cttccaagaa 2280

ggagaaattg tgccgtagcc ttttatctct tgtaccgtgt ggtttagtga ataggtaatt 2340

ctttatactt tgtatccatg gttgaatcaa agaaatatgt gtgtgagcat gaaaagagtt 2400

tcttgctcac tatcaagtaa tacaagatgg atgatatatt tatgaaaaaa aaaaaaaaaa 2460

aaaaa 2465

<210>2

<211>614

<212>PRT

<213>ThPDC1

<400>2

Met Asp Ser Ser Phe Gly Asp Val Asn Ser His Arg Ser Glu Ser Asn

1 5 10 15

Asp Val Gly Cys Leu Pro Cys Asp Lys Ser Val Asn Ser Val Ser His

20 25 30

Ser Val Val Gln Gly Ala Ala Val Ala Thr Leu Gly Arg His Leu Pro

35 40 45

Arg Arg Leu Val Gln Val Ala Val Arg Asp Val Phe Ser Val Pro Arg

50 55 60

Asp Phe Asn Leu Thr Trp Leu Asp His Leu Ile Ala Glu Pro Asp Leu

65 70 75 80

Ile Leu Val Gly Cys Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp

85 90 95

Gly Tyr Ala Arg Ser Tyr Gly Val Gly Ala Cys Ala Val Thr Phe Thr

100 105 110

Val Gly Gly Leu Ser Val Ile Asn Ala Ile Ala Gly Ala Tyr Asn Glu

115 120 125

Asn Leu Pro Val Ile Cys Ile Val Gly Gly Pro Asn Ser Asn Asp Tyr

130 135 140

Gly Thr Asn Arg Ile Leu His His Thr Ile Ala Leu Pro Asp Phe Ser

145 150 155 160

Gln Glu Tyr Arg Cys Phe Gln Ser Val Thr Cys Ala Gln Val Leu Val

165 170 175

Asn Asn Leu Glu Asp Ala Asn Glu Leu Ile Asp His Ala Ile Ser Thr

180 185 190

Ala Leu Met Glu Ser Lys Pro Val Tyr Ile Ser Ile Ser Cys Asn Leu

195 200 205

Pro Gly Ile Pro His His Thr Phe Thr His Glu Pro Val Pro Tyr Ser

210 215 220

Leu Thr Pro Arg Gln Ser Asn Pro Gln Ser Leu Gln Ser Ala Val Glu

225 230 235 240

Leu Thr Ala Arg Ile Leu Asn Ala Ala Val Lys Pro Val Leu Val Gly

245 250 255

Gly Pro Lys Leu Arg Ala Ala Lys Ala Lys Ser Ala Phe Ala Glu Leu

260 265 270

Ala Asn Ala Ser Gly Tyr Ala Val Ala Val Met Pro Ser Ala Lys Gly

275 280 285

GlnVal Ile Glu Thr His Pro His Phe Ile Gly Thr Tyr Trp Gly Ala

290 295 300

Val Ser Thr Ala Phe Cys Ala Glu Ile Val Glu Ser Ala Asp Ala Tyr

305 310 315 320

Leu Phe Ala Gly Pro Ile Phe Asn Asp Tyr Ser Ser Val Gly Tyr Ser

325 330 335

Leu Leu Leu Lys Lys Glu Lys Ala Ile Ile Val His Pro Asn Arg Val

340 345 350

Thr Val Ser Thr Gly Pro Thr Phe Gly Cys Val Leu Met Lys Asp Phe

355 360 365

Leu Glu Ala Leu Ala Lys Lys Leu Lys His Asn Lys Thr Ala Phe Glu

370 375 380

Asn Tyr Gln Arg Ile Phe Val Pro Glu Gly Val Pro Leu Lys Gly Ala

385 390 395 400

Pro His Glu Ala Leu Arg Val Asn Val Leu Phe Lys His Ile Gln Ala

405 410 415

Met Leu Ser Asn Asp Thr Ala Leu Ile Ala Glu Thr Gly Asp Ser Trp

420 425 430

Phe Asn Cys Gln Lys Leu Lys Leu Pro Glu Glu Cys Gly Tyr Glu Ser

435 440 445

Gln Met Gln Tyr Gly Ser Ile Gly Trp Ser Val Gly Glu Ala Thr Lys

450 455 460

Ser Ser Ala Thr Leu Gly Tyr Ala Gln Ala Thr Pro Lys Lys Arg Ala

465 470 475 480

Ile Ala Cys Ile Gly Val Gly Ser Phe Gln Val Thr Ala Gln Asp Ile

485 490 495

Tyr Thr Met Ile Arg Tyr Gly Gln Lys Ser Ile Ile Phe Leu Ile Asn

500 505 510

Asn Gly Gly Tyr Thr Ile Glu Val Glu Ile His Asp Gly Pro Tyr Asn

515 520 525

Val Ile Lys Asn Trp Asn Tyr Thr Gly Phe Lys Leu Gly Thr Leu Val

530 535 540

Asp Ala Ile His Asn Gly Glu Gly Lys Cys Trp Thr Thr Lys Val Lys

545 550 555 560

Thr Glu Glu Glu Ala Ile Glu Ala Ile Lys Thr Ala Gln Gly Pro Lys

565 570 575

Lys Asp Cys Leu Cys Phe Ile Glu Val Ile Val His Lys Asp Asp Thr

580 585 590

Ser Lys Glu Leu Leu Glu Trp Gly Ser Arg Val Ala Ala Ala Asn Ser

595 600 605

Arg Pro Pro AsnPro Gln

610

Claims (7)

1. A flood-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis is shown in SEQ ID NO. 1.

2. The expressed protein of the Chinese fir flooding-resistant drought-resistant related gene ThPDC1 in the claim 1, wherein the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2.

3. A vector containing the flooding-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis as claimed in claim 1.

4. A host bacterium containing the flooding-resistant and drought-resistant related gene ThPDC1 of the taxus chinensis as claimed in claim 1.

5. The vector of claim 3, wherein the vector is ThPDC1 at pH35 GS.

6. Use of the gene ThPDC1 of claim 1 in plant breeding.

7. The application of the gene ThPDC1 of claim 1 in plant flooding and drought stress resistance.

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