CN111500596A

CN111500596A - Ephedra sinica gene CeSC20 and application thereof

Info

Publication number: CN111500596A
Application number: CN202010365498.7A
Authority: CN
Inventors: 罗鸣; 张世柯; 刘楠; 周雯雯
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-07
Anticipated expiration: 2040-04-30
Also published as: CN111500596B

Abstract

The invention provides an casuarina equisetifolia gene CeSC20 and application thereof, wherein the cDNA sequence of the casuarina equisetifolia gene CeSC20 is shown as SEQ ID No.1, or is a sequence which is completely complementary and matched with the SEQ ID No.1, or is a sequence of which the coding amino acid sequence is shown as SEQ ID No. 2. Functional research carried out by gene cloning discovers that the CeSC20 gene can improve the drought resistance of organisms, can be widely applied to improving the drought resistance of organisms, comprises the construction of drought-resistant saccharomyces cerevisiae engineering strains, the cultivation of drought-resistant plant varieties, the improvement of the drought resistance of plants and the like, and has wide application value in the fields of microorganisms and agriculture.

Description

Ephedra sinica gene CeSC20 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an casuarina equisetifolia gene CeSC20 and application thereof.

Background

Environmental factors of plant growth, such as heavy metal, drought, high salt, high temperature and low temperature, can affect the growth and development of plants. Among the many factors that affect plant growth and development, drought and high salt are the most common types. Drought stress affects the distribution and transport of plant photosynthetic products, causing a decrease in plant productivity. Salt stress breaks the ionic balance of plant cells, disrupting the balance of production and clearance of reactive oxygen species in plant cells. The areas of global drought and high-salt regions are huge, the survival, growth and development of plants and crops face the challenges of drought and salt stress, and the research on the stress resistance genes responding to the drought and salt stress is beneficial to cultivating high-yield and high-adaptability crop varieties. The research on the adverse-resistant memory of the probiotics in the extreme environment can provide a research basis for molecular breeding and has guiding significance.

The casuarina equisetifolia grows quickly, has strong adaptability and has low requirements on the growth environment. The casuarina equisetifolia has developed root system and sunken leaf epidermis pores, and has good wind and sand fixing, alkali and drought resistance. The casuarina equisetifolia is an ideal tree species for constructing the wind-proof sand-fixing green land, is also an important tool species for vegetation recovery, is also a production raw material of wood, medicinal materials and the like, and has ecological value and economic value. At present, important anti-adversity genes of casuarina equisetifolia are still to be excavated and developed.

Disclosure of Invention

Based on the above, the casuarina equisetifolia gene CeSC20 and the application thereof are provided, and functional research is carried out through gene cloning to discover that the CeSC20 gene can improve the drought resistance of organisms, can be used for improving the drought resistance of plants and cultivating drought-resistant plant varieties.

The specific technical scheme is as follows:

a casuarina equisetifolia drought-resistant gene CeSC20 is characterized in that the cDNA sequence of the casuarina equisetifolia drought-resistant gene CeSC20 is shown as SEQ ID NO.1, or is a sequence which is completely complementary and matched with SEQ ID NO.1, or is a sequence of which the coding amino acid sequence is shown as SEQ ID NO. 2.

In some embodiments, the amino acid sequence of the expression protein of the casuarina equisetifolia drought-resistant gene CeSC20 is shown as SEQ ID No. 2.

The invention also provides application of the casuarina equisetifolia drought-resistant gene CeSC20 and expression protein of the casuarina equisetifolia drought-resistant gene CeSC20 in improving the drought-resistant performance of plants.

The invention also provides application of the casuarina equisetifolia drought-resistant gene CeSC20 and the casuarina equisetifolia drought-resistant gene CeSC20 in improving plant drought resistance in plant breeding.

In some of these embodiments, the plant is rice, maize, soybean.

The invention also provides a casuarina equisetifolia drought-resistant gene CeSC20 recombinant expression vector.

The specific technical scheme is as follows:

a casuarina equisetifolia drought-resistant gene CeSC20 recombinant expression vector is inserted with the casuarina equisetifolia drought-resistant gene CeSC 20.

The recombinant expression vector is applied to improving the drought resistance of plants.

In some of these embodiments, the recombinant expression vector is a saccharomyces cerevisiae recombinant expression vector.

In some of these embodiments, it is preferred that the recombinant expression vector is pYES-DEST52-CeSC 20.

The saccharomyces cerevisiae recombinant expression vector is applied to improving the drought resistance of the yeast strain.

In some of these embodiments, the recombinant expression vector is pCAMBIA1300-CeSC 20.

In some embodiments, in the pCAMBIA1300-CeSC20 recombinant expression vector, the promoter controlling the expression of the CeSC20 gene is a UBQ promoter.

The pCAMBIA1300-CeSC20 recombinant expression vector is applied to improving the drought resistance of plants.

The invention also provides a method for improving the drought resistance of the plant.

The specific technical scheme is as follows:

a method for improving drought resistance of plants is characterized in that the casuarina equisetifolia drought resistance gene CeSC20 is transferred into plants and expressed to obtain the drought resistance plants.

In some of these embodiments, the method of improving drought resistance in a plant comprises the steps of:

(1) inserting the cDNA sequence of the casuarina equisetifolia drought-resistant gene CeSC20 into a pCAMBIA1300 plasmid to obtain a pCAMBIA1300-CeSC20 recombinant expression vector;

(2) transforming agrobacterium with the pCAMBIA1300-CeSC20 recombinant expression vector in the step (1), and screening positive agrobacterium;

(3) infecting the positive agrobacterium in the step (2) on the plant to obtain the drought-resistant plant.

In some embodiments, the cDNA of the casuarina equisetifolia drought-resistant gene CeSC20 is obtained by:

(1) taking fresh roots and leaves of casuarina equisetifolia, and extracting to obtain RNA;

(2) carrying out reverse transcription on the RNA obtained in the step (1) to obtain cDNA;

(3) and (3) carrying out PCR amplification by using the cDNA obtained in the step (2) as a template and utilizing an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4 to obtain the cDNA of the casuarina equisetifolia drought-resistant gene CeSC 20.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an casuarina equisetifolia drought-resistant gene CeSC20 and constructs a recombinant expression vector of the CeSC20 gene. Functional research is carried out through gene cloning, and the CeSC20 gene can improve the drought resistance of organisms. The recombinant expression vector of the CeSC20 gene is transformed into saccharomyces cerevisiae, and the overexpression of the gene in the saccharomyces cerevisiae is induced by galactose, so that the tolerance of the saccharomyces cerevisiae to oxidation stress can be improved under the stress treatment of high salt and hydrogen peroxide; the recombinant expression vector of the CeSC20 gene is transferred into a plant, so that the CeSC20 gene is over-expressed in the plant, and the drought resistance of the plant can be effectively improved. The CeSC20 gene can be widely applied to improving the drought resistance of organisms, including constructing drought-resistant saccharomyces cerevisiae engineering strains, cultivating drought-resistant plant varieties, improving the drought resistance of plants and the like, and has wide application value in the fields of microorganisms and agriculture.

Drawings

FIG. 1 is a diagram showing the result of agarose gel electrophoresis detection of the PCR amplification product of the CeSC20 gene.

FIG. 2 is a schematic structural diagram of the recombinant expression vector pYES-DEST52-CeSC20 constructed in example 2.

FIG. 3 is a schematic structural diagram of the pCAMBIA1300-CeSC20 recombinant expression vector constructed in example 3.

FIG. 4 is H₂O₂The result of an experiment on the tolerance of the sensitive yeast strain BY4741 to oxidative stress.

FIG. 5 is H₂O₂Results of experiments on the tolerance of susceptible yeast strain yap1 to oxidative stress.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The cDNA sequence of the casuarina equisetifolia drought-resistant gene CeSC20 is shown as SEQ ID No.1, or is a sequence which is completely complementary and matched with the SEQ ID No.1, or is a sequence of which the coding amino acid sequence is shown as SEQ ID No. 2.

SEQ ID NO.1：

ATGACGGTGAAGCCTGTTAACGTGTCGCTGGACTGTGTTGCTGAAAGTCGGAAGCCGAAGGATTTCTCCTCGCCGGAGTGCGGTGATCTCGCGGGGAAGGTGTCGAGGGATGATCGGAGGGTGGAGGAGACTGAACGGTCGCCTAAGGCGGTGGGGAAATGGGGGAAGTACGTGCATAGCCAGATCCTGAGGATCAGGGAGGAGGACTCGCACCTCGGGGAGGAATTCAGTCTCGGCATCAAGGAGAACGTTCATCTTTCTCACCACCATCATTATGATCACGTGGATTTGCCGGACCCCGCGGTATTCTCCAAGCCGATCTTGCCGAGCTCCCCGCTCAGCCGCAAGACCAGCGCTGTAGAGGCGTTACTTTGA

It is understood that modifications of the base sequence of the above cDNA without changing the amino acid sequence in consideration of the degeneracy of codons also fall within the scope of the present invention.

The amino acid sequence of the expression protein of the casuarina equisetifolia drought-resistant gene CeSC20 is shown in SEQ ID No. 2.

SEQ ID NO.2：

MTVKPVNVS L DCVAESRKPKDFSSPECGD L AGKVSRDDRRVEETERSPKAVGKWGKYVHSQI L RIREEDSH L GEEFS L GIKENVH L SHHHHYDHVD L PDPAVFSKPI L PSSP L SRKTSAVEA LL (. dotted.) represents termination

Example 1 Ephedra sinica drought-resistant gene CeSC20 and expression protein thereof

The cDNA sequence of the casuarina equisetifolia drought-resistant gene CeSC20 is shown in SEQ ID No.1, or is a sequence which is completely complementary and matched with SEQ ID No.1, or is a sequence of which the coding amino acid sequence is shown in SEQ ID No. 2.

The cDNA of the casuarina equisetifolia drought-resistant gene CeSC20 is obtained by the following method:

(1) extracting fresh root and leaf of Ephedra sinica Stapf to obtain RNA

Ephedra sinica RNA was extracted using the Plant RNA Kit, ultrafast Plant RNA extraction Kit, of Beijing Huayuyo Biotech Co., Ltd.

1) Weighing fresh root and leaf of Ephedra sinica Stapf 100mg, quickly placing into mortar, adding liquid nitrogen, and grinding.

2) The ground plant tissue was placed into a centrifuge tube and 1m L cell lysate was added and the homogenate was transferred to a clean 1.5m L centrifuge tube.

3) Add 300. mu. L deproteinized solution and 200. mu. L chloroform to the tube, mix well with shaking 30s, centrifuge at 12000g for 5min at room temperature, transfer the supernatant to a fresh clean 1.5m L tube.

4) Adding the same volume of rinsing liquid, fully reversing and mixing uniformly. The mixture was added to a centrifugal adsorption column, centrifuged at 12000g at room temperature for 1min, and the permeate was discarded.

5) Adding 500 μ L column washing solution, centrifuging at 12000g for 1min at room temperature, discarding the penetrating solution, adding 500 μ L column washing solution, repeating the steps, and centrifuging at 12000g at room temperature for 1min to remove the residual liquid.

6) Transferring the centrifugal adsorption column into an RNase-free collection tube, adding 50 mu L RNA eluent, and standing at room temperature for 3-5 min.

7)12000g, centrifuging for 1min at room temperature, namely obtaining the casuarina equisetifolia RNA sample in a centrifuge tube, and storing at-80 ℃ for standby.

(2) Carrying out reverse transcription on the RNA obtained in the step (1) to obtain cDNA

The Ephedra sinica RNA obtained in step (1) was subjected to reverse transcription using Super M-Mu L V reverse transcriptase from Diamond.

1) A sterile RNase-free centrifuge tube was filled with 2. mu. L oligo (dT)18Primer, 2. mu. L dNTP mix (10mM), 1.5. mu. L casuarina equisetifolia total RNA, and RNase free ddH₂O to a volume of 10 μ L.

2) Keeping the temperature at 65 ℃ for 5min, and then quickly placing on ice for more than 1 min.

3) The denatured solution of RNA was collected at the bottom of the centrifuge tube by centrifugation for several seconds.

4) The reverse transcription reaction Solution is prepared in the centrifuge tube by mixing RNA denaturation Solution 10 Mu L, 5 × Super MM L0 VBuffer of 4 Mu L, 10 × Solution of 2 Mu L, RNase Inhibitor of 1 Mu L (40U/. Mu. L), Super M-Mu L V of 1 Mu L (200U/. Mu. L), and RNase free ddH₂O to 20 μ L.

5) Flick the centrifuge tube, and keep the temperature at 37 ℃ for 60 min.

6) Keeping the temperature at 80 ℃ for 15min, and cooling on ice to obtain cDNA.

(3) Taking the cDNA obtained in the step (2) as a template, and carrying out PCR amplification by using an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO.4

PCR amplification was performed using the high success rate PCR enzyme KOD FX of TOYOBO.

1) An amplification primer:

SEQ ID NO.3：5，-GCTTGGTACCGAGCTCGGATGACGGTGAAGCCTGTTAA-3，

SEQ ID NO.4：5，-GACATCTCCGCAATGAAACTCTTAAGACGTCTATAGGT-3，

2) preparation of PCR reaction solution

A sterile DNase-free centrifuge tube was filled with 25. mu. L of 2 × PCR buffer for KODFX, 10. mu. × 0 of dNTPs (2mM), 1.5. mu. L of primer 1(10 pmol/. mu. L), 1.5. mu. L of primer 2(10 pmol/. mu. L), 0.2. mu. L of cDNA, 1. mu. L of KOD FX (1.0U/. mu. L), and distilled water to 50. mu. L.

3) Setting PCR program

Step 1: preheating at 94 deg.C for 2 min;

step 2: denaturation, 98 ℃, 10 sec;

and step 3: annealing at 55 ℃ for 30 sec;

and 4, step 4: extension, at 68 ℃ for 2 min;

and 5: repeating the steps 2-4 for 40 cycles.

The agarose gel electrophoresis detection of the PCR reaction product comprises the following steps:

(1) agarose gel was prepared by adding 1g of agarose to 50m L of TAE (1 ×), heating and dissolving in a microwave oven, adding 2. mu. L of Ethidium Bromide (EB) to the dissolved solution, and cooling on a plate for 30 min.

(2) And putting the cooled agarose gel into an electrophoresis tank, and spotting the PCR product and the DNA marker into the tank for electrophoresis.

(3) Observing the electrophoresis result, and recovering the gel after cutting the strip.

The agarose gel electrophoresis detection result is shown in figure 1, and the casuarina equisetifolia drought-resistant gene CeSC20 is obtained by successful amplification.

The method for recovering the agarose gel electrophoresis detection strip by using the gel recovery kit of the biological engineering company Limited comprises the following steps:

(1) the gel containing the CeSC20 gene fragment was excised from the agarose gel and weighed.

(2) 600. mu. L of Buffer B2 was added to each 100mg of the gel block and the gel was washed with water at 50 ℃ until the agarose gel was dissolved.

(3) The sol solution was transferred to an adsorption column, centrifuged at 8000g for 30s and the liquid in the collection tube was decanted.

(4) 500 μ L Wash Solution was added and centrifuged at 9000g for 30s and the tube was decanted.

(5) Repeat step 4 once.

(6) The empty adsorption column was centrifuged at 9000g for 1 min.

(7) The adsorption column was placed in a clean 1.5m L centrifuge tube, 25. mu. L ElutionBuffer was added to the center of the adsorption membrane, and after standing at room temperature for 1min, the DNA solution in the tube was centrifuged for 1min and stored.

Example 2 recombinant expression vector pYES-DEST52-CeSC20 of CeSC20 Gene

The structural schematic diagram of the recombinant expression vector of the CeSC20 gene is shown in FIG. 2, and the recombinant expression vector is obtained by inserting the casuarina equisetifolia drought-resistant gene CeSC20 described in example 1 into a pYES-DEST52 yeast expression vector, and the specific construction method comprises the following steps:

1. obtaining of pYES-DEST52 enzyme digestion vector

Using an endonuclease and a buffer from Thermo Scientific

(1) pYES-DEST52 was prepared as a restriction enzyme, 5.8. mu. L of yeast plasmid, 1. mu. L of BamHI, 1. mu. L of EcoRI, 5. mu. L of K buffer (1 ×), and distilled water was added to 50. mu. L.

(2) Placing the enzyme digestion reaction solution in a water bath kettle at 37 ℃ and carrying out water bath for 3 h.

2. The casuarina equisetifolia CeSC20 gene is connected to the digested pYES-DEST52 vector.

The ligation was performed using Ready-to-Use Seamless Cloning Kit (Biotechnology engineering Co., Ltd.).

(1) The Infusion system was prepared on ice, 1. mu. L of the DNA solution recovered from the gel of example 1, 1. mu. L of the digested pYES-DEST52 vector, and 2.5. mu. L of the Seamless Cloning Master Mix (2X).

(2) And (3) putting the prepared Infusion system into a water bath kettle at 50 ℃ and carrying out water bath for 20 min.

(3) After completion of the water bath, ice bath was carried out for 2 min.

3. The product obtained in step 2 was introduced into E.coli (DH5 α).

The Escherichia coli competent strain DH5 α was used.

(1) The Infusion product was added to DH5 α.

(2) Ice-cooling for 30 min.

(3) Water bath at 42 deg.c for 1 min.

(4) Ice bath for 2 min.

(5) L B liquid culture medium 600 mu L was added in the clean bench.

(6) Shaking and culturing at 37 deg.C for 45 min.

(7) Taking out cultured DH5 α, centrifuging by 10000g for 1 min.

(8) The supernatant was discarded in the clean bench (pipette aspiration, 20-50. mu. L remaining).

(9) Resuspend, pipette to bottom without precipitation.

(10) All were plated out in carbenicillin (cab) resistant (1. mu. L/m L) medium.

(11) After the medium was dried, the cells were cultured overnight in an incubator at 37 ℃.

4. Obtaining a yeast expression vector pYES-DEST52-CeSC20 plasmid with CeSC20 gene.

Single colonies were picked from the resistant medium, added to a 1.5m L sterile centrifuge tube with 600. mu. L L B broth and 6. mu. L cab antibiotic, and added to the centrifuge tube for overnight shake cultivation at 37 ℃.

Plasmids were extracted using a crude SanPrep column plasmid DNA miniprep kit.

(1) The overnight cultured broth was centrifuged at 8000g for 2min, and the cells were collected and the medium was discarded.

(2) To the pellet was added 250. mu. L Buffer P1 to thoroughly suspend the cells.

(3) Adding 250 μ L Buffer P2, immediately and gently inverting for 5-10 times, mixing, and standing at room temperature for 2-4 min.

(4) Add 350. mu. L Buffer P3 and mix by immediately inverting gently 5-10 times.

(5) Centrifuging at 12000g for 5-10min, transferring supernatant into adsorption column, centrifuging at 8000g for 30s, and pouring out liquid in collection tube.

(6) 500 μ L Buffer DW1 was added and 9000g centrifuged for 30s and the collection tubes were decanted.

(7) 500 μ L Wash Solution was added and centrifuged at 9000g for 30s and the tube was decanted.

(8) Repeat step 7 once.

(9) The empty adsorption column was centrifuged at 9000g for 1 min.

(10) The adsorption column was placed in a clean 1.5m L centrifuge tube, 50. mu. L ElutionBuffer was added to the center of the adsorption membrane, and the tube was left to stand at room temperature for 1min, centrifuged at 9000g for 1min, and the DNA solution in the tube was stored.

(11) The obtained plasmid was sent to sequencing company for sequencing, which was done by Ongzhou Ongji Biopsis.

The plasmid with the correct sequencing result is the pYES-DEST52-CeSC20 recombinant expression vector.

Example 3 recombinant expression vector pCAMBIA1300-CeSC20 of CeSC20 Gene

The recombinant expression vector of the CeSC20 gene of the embodiment is shown in fig. 3, and is obtained by inserting the casuarina equisetifolia drought-resistant gene CeSC20 of embodiment 1 into a pCAMBIA1300 vector, and specifically comprises the following steps: the DNA solution obtained by recovering the glue in the embodiment 1 reacts with the pCAMBIA1300 vector through an Infusion system, then an Infusion product is introduced into a competent cell, a positive single colony is selected, amplification culture is carried out, plasmids are extracted, the obtained plasmids are sent to a sequencing company for sequencing, the sequencing is completed by Guangzhou Pongke biology company, and the plasmid with the correct sequencing result is the recombinant expression vector pCAMBIA1300-CeSC20 of the CeSC20 gene.

Example 4 overexpression of the CeSC20 Gene in Yeast improves the tolerance of Yeast to oxidative stress

The pYES-DEST52-CeSC20 recombinant expression plasmid with correct sequencing result is introduced into H₂O₂Sensitive strains BY4741 and yap1, empty pYES-DEST52 vector plasmid was introduced into the wild type BY4741 yeast strain as a control.

(1) Single colonies of BY4741 and yap1 were shaken in small conical flasks each with 20m L liquid YPD medium and shaken overnight at 28 ℃ as mother liquors.

(2) Taking part of the mother liquor to a 20m L liquid YPD medium, and adjusting its initial OD₆₀₀The value is 02-0.3. Shaking at 28 deg.C for 1.5-2h to OD₆₀₀Is 0.4-0.6.

(3) The yeast in the conical flask was dispensed into 15m L centrifuge tubes, 6000g centrifuged for 5min, the supernatant was discarded and resuspended with L i salt.

(4) Add 10 u L Cyprinus Carpio sperm DNA, 4 u L empty pYES-DEST52 vector plasmid (control group) or pYES-DEST52-CeSC20 recombinant expression plasmid (experimental group), 100 u L competent cells and 600 u L PEG into the tube after resuspension, mix well and shake culture at 28 ℃ for 30 min.

(5) Heat shock at 42 ℃ for 15min, cooling at 4 ℃ for 2min, centrifugation at 6000g for 30s at room temperature, discarding the supernatant, blotting the PEG, resuspending with 70. mu. L1 × TE, and applying to solid YNB medium supplemented with the corresponding amino acids (830. mu. L each for histidine, leucine and methionine per 100m L YNB).

(6) The medium was placed in an oven at 28 ℃ for 3 days and single colonies were picked up in a centrifuge tube containing 1m L YNB broth.

(7) Placing the centrifuge tube with bacteria in 28 deg.C oven, shaking overnight, and adjusting to the same OD₆₀₀Then, the bacterial liquid is respectively diluted to 10 ×, 100 × and 1000 ×, and the bacterial liquid is added to H₂O₂YNB solid medium (830. mu. L each of histidine, leucine and methionine per 100m L YNB) at concentrations of 0.75mM, 1mM and 1.5mM was oven-cultured at 37 ℃ for one week.

As shown in FIG. 4, in the absence of H₂O₂The growth of the recombinant expression plasmid containing pYES-DEST52-CeSC20 (represented BY CeSC20 in FIG. 4) and the growth of the strain of BY4741 containing the unloaded pYES-DEST52 plasmid (represented BY pYES2 in FIG. 4) were substantially the same in the YNB medium added. At 0.75mM H₂O₂Under the condition, the growth conditions of the two are similar; but at 1mM and 1.5mMH₂O₂The BY4741 growth of the recombinant expression plasmid pYES-DEST52-CeSC20 introduced on YNB medium was significantly better than the BY4741 introduced on the unloaded pYES-DEST52 plasmid.

As shown in FIG. 5, pYES-DEST52-CeSC20 recombinant expression plasmid (shown in FIG. 5)Represented by CeSC 20) into H₂O₂In the sensitive yeast yap1, the empty vector pYES-DEST52 plasmid (indicated BY pYES2 in FIG. 5) was introduced into BY 4741. In the absence of added H₂O₂The growth vigor of the YNB medium is basically consistent. At 0.75mM H₂O₂Under the conditions, H containing pYES-DEST52-CeSC20 recombinant expression plasmid₂O₂The growth condition of the sensitive yeast yap1 low-concentration bacterial liquid is better than that of BY4741 containing an empty vector pYES-DEST52 plasmid; at 1mM and 1.5mM H₂O₂The growth of the diluted yap1 yeast containing the recombinant expression plasmid pYES-DEST52-CeSC20 was better than that of the BY4741 yeast containing the empty vector pYES-DEST52 in YNB medium at higher concentrations.

The experimental results show that the pYES-DEST52-CeSC20 recombinant expression plasmid can obviously improve the anti-oxidative stress capability of the yeast, and the pYES-DEST52-CeSC20 recombinant expression plasmid can obviously improve the drought resistance of the yeast.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> south China plant garden of Chinese academy of sciences

<120> casuarina equisetifolia gene CeSC20 and application thereof

<130>2020-04-08

<160>4

<170>PatentIn version 3.3

<210>1

<211>375

<212>DNA

<213>Artificial Sequence

<400>1

atgacggtga agcctgttaa cgtgtcgctg gactgtgttg ctgaaagtcg gaagccgaag 60

gatttctcct cgccggagtg cggtgatctc gcggggaagg tgtcgaggga tgatcggagg 120

gtggaggaga ctgaacggtc gcctaaggcg gtggggaaat gggggaagta cgtgcatagc 180

cagatcctga ggatcaggga ggaggactcg cacctcgggg aggaattcag tctcggcatc 240

aaggagaacg ttcatctttc tcaccaccat cattatgatc acgtggattt gccggacccc 300

gcggtattct ccaagccgat cttgccgagc tccccgctca gccgcaagac cagcgctgta 360

gaggcgttac tttga 375

<210>2

<211>124

<212>PRT

<213>Artificial Sequence

<400>2

Met Thr Val Lys Pro Val Asn Val Ser Leu Asp Cys Val Ala Glu Ser

1 5 10 15

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

20 25 30

Lys Val Ser Arg Asp Asp Arg Arg Val Glu Glu Thr Glu Arg Ser Pro

35 40 45

Lys Ala Val Gly Lys Trp Gly Lys Tyr Val His Ser Gln Ile Leu Arg

50 55 60

Ile Arg Glu Glu Asp Ser His Leu Gly Glu Glu Phe Ser Leu Gly Ile

65 70 75 80

Lys Glu Asn Val His Leu Ser His His His His Tyr Asp His Val Asp

85 90 95

Leu Pro Asp Pro Ala Val Phe Ser Lys Pro Ile Leu Pro Ser Ser Pro

100 105 110

Leu Ser Arg Lys Thr Ser Ala Val Glu Ala Leu Leu

115 120

<210>3

<211>38

<212>DNA

<213>Artificial Sequence

<400>3

gcttggtacc gagctcggat gacggtgaag cctgttaa 38

<210>4

<211>38

<212>DNA

<213>Artificial Sequence

<400>4

gacatctccg caatgaaact cttaagacgt ctataggt 38

Claims

1. The casuarina equisetifolia drought-resistant gene CeSC20 is characterized in that the casuarina equisetifolia drought-resistant gene CeSC20 has a cDNA sequence shown as SEQ ID No.1, or is a sequence which is completely complementary and matched with the SEQ ID No.1, or is a sequence with an encoded amino acid sequence shown as SEQ ID No. 2.

2. An expression protein of casuarina equisetifolia drought-resistant gene CeSC20 is characterized in that the amino acid sequence of the expression protein of casuarina equisetifolia drought-resistant gene CeSC20 is shown in SEQ ID No. 2.

3. The use of the Ephedra sinica drought-resistant gene CeSC20 according to claim 1 or the expression protein of the Ephedra sinica drought-resistant gene CeSC20 according to claim 2 for improving the drought-resistant performance of plants.

4. Use of the Ephedra sinica Stapf drought-resistant gene CeSC20 according to claim 1 or the expression protein of the Ephedra sinica Stapf drought-resistant gene CeSC20 according to claim 2 in plant breeding for improving plant drought resistance.

5. Use according to claim 3 or 4, wherein the plant is rice, maize, soybean.

6. An casuarina equisetifolia drought-resistant gene CeSC20 recombinant expression vector, wherein the casuarina equisetifolia drought-resistant gene CeSC20 of claim 1 is inserted into the recombinant expression vector.

7. The recombinant expression vector of claim 6, wherein the recombinant expression vector is a Saccharomyces cerevisiae recombinant expression vector.

8. The use of the recombinant expression vector of Saccharomyces cerevisiae according to claim 7 for improving drought resistance of a yeast strain.

9. The recombinant expression vector of claim 6, wherein the recombinant expression vector is pCAMBIA1300-CeSC 20.

10. Use of the recombinant expression vector according to claim 6 or 9 for improving drought resistance in plants.