CN111500595B - Ephedra sinica gene CeDREB1 and application thereof - Google Patents
Ephedra sinica gene CeDREB1 and application thereof Download PDFInfo
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Abstract
The invention provides an casuarina equisetifolia drought-resistant gene CeDREB1 and application thereof, wherein a cDNA sequence of the casuarina equisetifolia drought-resistant gene CeDREB1 is shown as SEQ ID No.1, or a sequence which is completely complementary and matched with the SEQ ID No.1, or a sequence of which an encoding amino acid sequence is shown as SEQ ID No. 2. Functional research carried out by gene cloning discovers that the CeDREB1 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
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an casuarina equisetifolia gene CeDREB1 and application thereof.
Background
Plants grow in fixed positions for a long time and are relatively vulnerable to adversity stress. Abiotic stress is a harmful factor affecting the growth and productivity of plants and crops, and mainly includes drought stress, water stress, salt stress, heavy metal stress, and the like. Drought stress and salt stress are common stress types, and water loss can cause osmotic pressure change of plant cells and damage of plasma membrane peroxide cell structures, so that physiological activities of plants are influenced, and the growth of the plants is inhibited. Extreme weather is frequent due to global climate change, crops face potential threats of environmental stress, and increasing crop yield is particularly important for guaranteeing grain safety. The research and development of crop varieties with strong stress resistance and high yield can be realized by means of transgenic breeding. The extreme environment has rich plant resources, and the suitable plant species obtain stronger stress resistance through long-term adaptation and evolution to the stress. The method researches the stress-resistant functional genes of the suitable plants in the drought and high-salt environment, further develops the application potential of the genes, and has important significance for cultivating new varieties of drought-resistant crops.
Casuarina equisetifolia is a pioneer species of tropical and subtropical zones, has strong adaptability, and can grow in strong light, drought, sand blown by wind and saline-alkali environments. The casuarina equisetifolia is a tool species for recovering the vegetation of tropical coral islands, and can be used for wind prevention and sand fixation and green land construction. Ephedra sinica is also a medicinal plant, and the bark, branches and leaves of the Ephedra sinica can be used as a medicinal material, so that the Ephedra sinica has a certain medicinal value. At present, important anti-adversity genes of casuarina equisetifolia are still to be excavated and developed.
DREB (dehydration Responsive Element binding) is an important transcription factor, mainly participates in low temperature and drought signal transduction pathways, and can be combined with a promoter of a downstream gene to regulate the gene expression of the downstream gene. Multiple studies indicate that expression of DREB gene is induced by low temperature, drought and high salt stress.
Disclosure of Invention
Based on the above, the invention provides the casuarina equisetifolia gene CeDREB1 and the application thereof, and the functional research carried out by gene cloning finds that the CeDREB1 gene can improve the drought resistance of organisms, can be used for improving the drought resistance of plants, cultivating drought-resistant plant varieties and improving the drought resistance of yeast strains.
The specific technical scheme is as follows:
a casuarina equisetifolia drought-resistant gene CeDREB1 is characterized in that the cDNA sequence of the casuarina equisetifolia drought-resistant gene CeDREB1 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.
The invention also provides an expression protein of the casuarina equisetifolia drought-resistant gene CeDREB1, and the amino acid sequence of the expression protein of the casuarina equisetifolia drought-resistant gene CeDREB1 is shown in SEQ ID No. 2.
The invention also provides application of the casuarina equisetifolia drought-resistant gene CeDREB1 and the casuarina equisetifolia drought-resistant gene CeDREB1 in improving the drought-resistant performance of plants.
The invention also provides application of the casuarina equisetifolia drought-resistant gene CeDREB1 and the casuarina equisetifolia drought-resistant gene CeDREB1 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 CeDREB1 recombinant expression vector.
The specific technical scheme is as follows:
a casuarina equisetifolia drought-resistant gene CeDREB1 recombinant expression vector is inserted with the casuarina equisetifolia drought-resistant gene CeDREB 1.
The casuarina equisetifolia drought-resistant gene CeDREB1 recombinant expression vector is applied to improving the drought-resistant performance 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-CeDREB 1.
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-CeDREB 1.
In some embodiments, in the pCAMBIA 1300-cedram 1 recombinant expression vector, the promoter controlling the expression of the cedram 1 gene is a UBQ promoter.
The pCAMBIA1300-CeDREB1 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 CeDREB1 is transferred into the 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 CeDREB1 into a pCAMBIA1300 vector to obtain a pCAMBIA1300-CeDREB1 recombinant expression vector;
(2) transforming agrobacterium with the pCAMBIA1300-CeDREB1 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 cedrreb 1 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 using 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 CeDREB 1.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a casuarina equisetifolia drought-resistant gene CeDREB1 and constructs a recombinant expression vector of the CeDREB1 gene. The inventor discovers that the CeDREB1 gene can improve the drought resistance of organisms through functional research of gene cloning. The recombinant expression vector of the CeDREB1 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 CeDREB1 gene is transferred into a plant to cause the plant to be over-expressed, so that the drought resistance of the plant can be effectively improved. The CeDREB1 gene provided by the invention 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 CeDREB1 gene.
FIG. 2 is a schematic structural diagram of the pYES-DEST52-CeDREB1 recombinant expression vector constructed in example 2.
FIG. 3 is a schematic structural diagram of the pCAMBIA1300-CeDREB1 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 H2O2Susceptible yeast strain yap1 pairsResults of oxidative stress tolerance experiments.
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 CeDREB1 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：
ATGGAGTTCTCTCAGTATTCCTCGGAATCCGAGTGCGGCAGTGCTCGAGCTGTGCACTTGTCGGATGAGGAGATTCTACTCGCCTCTCGGAATCCCAAGAAACGGGCGGGAAGGAAGAAGTTCAAGGAGACCAGGCACCCGGTGTACCGAGGAGTGAGGAGGAGGAACTCCGACAAGTGGGTTTGCGAGGTGCGCGAGCCCAGCAAGCAGGCTAGGATATGGCTTGGGACGTTCCCAACTCCTGAGATGGCTGCCCGAGCTCACGACGTCGCAGCGATTGCACTGCGAGGCCGTTCCGCATGCCTCAACTTCGCAGAATCCGCGTGGCTGCAAGTGCCCACCTCGGGTGACCCCAAGGAGATTCAGCGAATGGCAGCCGAGGCAGCAGAGAAGTTTCGGCCAGAGGAAGCAAAGGTGGAGGAGAAGAGAGAATCGCCGGAAAGCGTGTTCTTCATGGACGAGGAGGCGGTCTTCGGCATGCCAGGGCTCCTTTCAAATATGGCGGAGGGGATGCTCCTGCCTCCTCCTTACTATATTGGAGATGATGGAAGTGGTGGAGATGACAAAGAAGCCAGCGCCGACGTTTCACTGTGGAGTTTTTCCATTTGA
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 CeDREB1 is shown in SEQ ID No. 2.
SEQ ID NO.2：
MEFSQYSSESECGSARAVHLSDEEILLASRNPKKRAGRKKFKETRHPVYRGVRRRNSDKWVCEVREPSKQARIWLGTFPTPEMAARAHDVAAIALRGRSACLNFAESAWLQVPTSGDPKEIQRMAAEAAEKFRPEEAKVEEKRESPESVFFMDEEAVFGMPGLLSNMAEGMLLPPPYYIGDDGSGGDDKEASADVSLWSFSI (. filled.) -end)
Example 1 Ephedra sinica drought-resistant gene CeDREB1 and expression protein thereof
The cDNA sequence of the casuarina equisetifolia drought-resistant gene CeDREB1 is shown in 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 in SEQ ID No. 2.
The amino acid sequence of the expression protein of the casuarina equisetifolia drought-resistant gene CeDREB1 is shown in SEQ ID No. 2.
The cDNA of the casuarina equisetifolia drought-resistant gene CeDREB1 is obtained by the following method:
1. extraction of casuarina equisetifolia 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 in a centrifuge tube and 1mL of cell lysate was added. The homogenate was transferred to a clean 1.5mL centrifuge tube.
(3) Add 300. mu.L of deproteinized solution and 200. mu.L of chloroform to the centrifuge tube, and mix them by shaking for 30 s. Centrifuge at 12000g for 5min at room temperature and transfer the supernatant to a fresh clean 1.5mL centrifuge 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 of column washing solution, centrifuging at 12000g at room temperature for 1min, and removing the liquid. Add 500. mu.L of column wash and repeat one side. Then, the residue was removed by centrifugation at 12000g for 1min at room temperature.
(6) Transferring the centrifugal adsorption column into RNase-free collection tube, adding 50 μ 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. Reverse transcription is carried out on the RNA obtained in the step 1 to obtain cDNA
The Ephedra sinica Stapf RNA obtained in step 1 was subjected to reverse transcription using Super M-MuLV reverse transcriptase from Diamond corporation.
(1) The following system was configured in a sterile RNase-free centrifuge tube: mu.L Oligo (dT)18 Primer, 2. mu.L dNTP mix (10mM), 1.5. mu.L casuarina total RNA, and RNase free ddH2The volume of O is up to 10 mu 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) Preparing a reverse transcription reaction solution in the centrifuge tube: RNA denaturation Solution 10. mu.L, 4. mu.L of 5 XPROMP LV Buffer, 2. mu.L of 10 XPsolving, 1. mu.L of RNase Inhibitor (40U/. mu.L), 1. mu.L of Super M-MuLV (200U/. mu.L), plus RNase free ddH2O is added to the volume of 20 mu 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. Obtaining of Ephedra sinica DREB1 cDNA
And (3) performing PCR amplification by using the cDNA obtained in the step (2) as a template and using an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.
PCR was performed using the high success rate PCR enzyme KOD FX of TOYOBO.
(1) Designing a primer:
SEQ ID NO.3：5’-GCTTGGTACCGAGCTCGGATGGAGTTCTCTCAGTATTCCTCGGAATC-3’
SEQ ID NO.4：5’-TGCAAAGTGACACCTCAAAAAGGTAAACTCTTAAGACGTCTATAGGT-3’
(2) preparation of PCR reaction solution
The following system was placed in a sterile DNase-free centrifuge tube: 25 μ L of 2 XPCR buffer for KOD FX,10 μ L of dNTPs (2mM), 1.5 μ L of primer 1(10 pmol/. mu.L), 1.5 μ L of primer 2(10 pmol/. mu.L), 0.2 μ L of cDNA, 1 μ L of KOD FX (1.0U/. mu.L), plus distilled water to 50 μ 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) preparing agarose gel: to 50mL of TAE (1X) was added 1g of agarose, the mixture was placed in a microwave oven and dissolved by heating, and 2. mu.L of Ethidium Bromide (EB) was added to the dissolved solution, which was then poured into a plate and cooled 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 CeDREB1 is obtained by successful amplification.
The glue recovery kit of the bio-engineering and biological engineering company Limited is used for glue recovery, and the method comprises the following specific steps:
(1) the gel containing the CeDREB1 fragment was cut from the agarose gel and weighed.
(2) Add 600. mu.L of Buffer B2 per 100mg of gel block and water bath at 50 ℃ until the agarose gel dissolves.
(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) Add 500. mu.L of Wash Solution, centrifuge for 30s at 9000g, and pour off the liquid in the collection tube.
(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 1.5mL centrifuge tube, 25. mu.L of precipitation Buffer was added to the center of the adsorption membrane, and after standing at room temperature for 1min, the tube was centrifuged for 1min and the DNA solution in the tube was stored.
Example 2 recombinant expression vector pYES-DEST52-CeDREB1 of CeDREB1 Gene
The structural schematic diagram of the recombinant expression vector of the CeDREB1 gene is shown in figure 2, and the recombinant expression vector is obtained by inserting the casuarina equisetifolia drought-resistant gene CeDREB1 described in the embodiment 1 into a pYES-DEST52 yeast expression vector, and the specific construction method comprises the following steps:
obtaining pYES-DEST52 enzyme digestion vector
Using an endonuclease and a buffer from Thermo Scientific
(1) Preparing pYES-DEST52 enzyme digestion reaction liquid: mu.L of yeast plasmid, 1. mu.L of BamHI, 1. mu.L of EcoRI, 5. mu.L of K buffer (1X), and distilled water 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 CeDREB1 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 formulated on ice: mu.L of the DNA solution obtained from the gel of example 1, 1. mu.L of pYES-DEST52 vector, 2.5. mu.L of 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 Infusion product obtained in step 2 was introduced into E.coli (DH 5. alpha.).
The Escherichia coli competent strain DH 5. alpha. 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) 600. mu.L of LB liquid medium was added to the clean bench.
(6) Shaking and culturing at 37 deg.C for 45 min.
(7) Taking out the cultured DH5 alpha, centrifuging by 10000g for 1 min.
(8) The supernatant was discarded in the clean bench (pipette, 20-50. mu.L remaining).
(9) Resuspend, pipette to bottom without precipitation.
(10) All were plated out in carbenicillin (cab) resistant (1. mu.L/mL) 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-CeDREB1 plasmid with CeDREB 1.
Single colonies were picked from the resistant medium, 600. mu.L of LB broth and 6. mu.L of cab antibiotic were added to a 1.5mL sterile centrifuge tube, and single colonies were added to the centrifuge tube and incubated overnight in a shaker 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 of Buffer P1 to thoroughly suspend the cells.
(3) Add 250. mu.L of Buffer P2, mix by immediately and gently inverting 5-10 times, and let stand at room temperature for 2-4 min.
(4) Add 350. mu.L of 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) mu.L of Buffer DW1 was added and centrifuged at 9000g for 30s and the collection tube was decanted.
(7) Add 500. mu.L of Wash Solution, centrifuge for 30s at 9000g, and pour off the liquid in the collection tube.
(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 1.5mL centrifuge tube, 50. mu.L of precipitation Buffer was added to the center of the adsorption membrane, allowed 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. Sequencing was performed by Ongchow, Inc.
The plasmid with correct sequencing result is the pYES-DEST52-CeDREB1 recombinant expression vector.
Example 3 recombinant expression vector pCAMBIA1300-CeDREB1 of CeDREB1 Gene
The structural schematic diagram of the recombinant expression vector of the cedrenb 1 gene is shown in fig. 3, and the recombinant expression vector is obtained by inserting the casuarina equisetifolia drought-resistant gene cedrenb 1 described in example 1 into a pCAMBIA1300 plasmid, 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-CeDREB1 of the CeDREB1 gene.
Example 4 overexpression of the CeDREB1 Gene in Yeast improves the tolerance of Yeast to oxidative stress
The pYES-DEST52-CeDREB1 recombinant plasmid with correct sequencing result was 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, respectively, with 20mL of liquid YPD medium and overnight at 28 ℃ as mother liquors.
(2) Taking part of the mother liquor to 20mL of liquid YPD medium, and adjusting 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 is subpackaged into 15mL centrifuge tubes, 6000g is centrifuged for 5min, and the supernatant is discarded and then the yeast is resuspended by Li salt.
(4) mu.L of carp sperm DNA, 4. mu.L of empty pYES-DEST52 vector plasmid (control) or pYES-DEST52-CeDREB1 recombinant plasmid (experimental), 100. mu.L of competent cells and 600. mu.L of PEG were added to the resuspended tubes. Mixing, and shake culturing at 28 deg.C 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.L of 1 XTE, and plating onto solid YNB medium to which the corresponding amino acid had been added (830. mu.L each of histidine, leucine and methionine per 100ml of 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 1ml of YNB broth.
(7) Placing the centrifuge tube with bacteria in 28 deg.C oven, shaking overnight, and adjusting to the same OD600And (6) finally. The bacterial solution was diluted to 10 ×, 100 × and 1000 ×, respectively, and spotted to H2O2YNB solid medium (830. mu.L each of histidine, leucine and methionine per 100ml of 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 plasmid containing pYES-DEST52-CeDREB1 (shown BY CeDREB1 in figure 4) and the growth of the strain liquid containing BY4741 with no-load pYES-DEST52 plasmid (shown BY pYES2 in figure 4) on the YNB medium added were almost the same; 0.75mM H2O2Under the condition, the growth conditions of the two are similar; but at 1mM and 1.5mM H2O2BY4741, into which pYES-DEST52-CeDREB1 recombinant plasmid was introduced, grew better than BY4741, into which empty vector pYES-DEST52 plasmid was introduced, on YNB medium at higher concentration.
As shown in FIG. 5, pYES-DEST52-CeDREB1 recombinant plasmid (shown as CeDREB1 in FIG. 5) was introduced 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-CeDREB1 recombinant plasmid2O2The growth of the sensitive yeast yap1 low concentration bacterial liquid is better than that of BY4741 containing the empty vector pYES-DEST52 plasmid. At 1mM and 1.5mM H2O2Of concentrationOn YNB medium, the diluted yap1 bacterial liquid containing pYES-DEST52-CeDREB1 recombinant plasmid grew better than BY4741 containing empty vector pYES-DEST52 plasmid.
The experimental results show that the pYES-DEST52-CeDREB1 recombinant expression plasmid can improve the anti-oxidative stress capacity of the yeast, and the pYES-DEST52-CeDREB1 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 CeDREB1 and application thereof
<140> 202010365496.8
<141> 2020-04-30
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 609
<212> DNA
<213> Artificial Sequence
<400> 1
atggagttct ctcagtattc ctcggaatcc gagtgcggca gtgctcgagc tgtgcacttg 60
tcggatgagg agattctact cgcctctcgg aatcccaaga aacgggcggg aaggaagaag 120
ttcaaggaga ccaggcaccc ggtgtaccga ggagtgagga ggaggaactc cgacaagtgg 180
gtttgcgagg tgcgcgagcc cagcaagcag gctaggatat ggcttgggac gttcccaact 240
cctgagatgg ctgcccgagc tcacgacgtc gcagcgattg cactgcgagg ccgttccgca 300
tgcctcaact tcgcagaatc cgcgtggctg caagtgccca cctcgggtga ccccaaggag 360
attcagcgaa tggcagccga ggcagcagag aagtttcggc cagaggaagc aaaggtggag 420
gagaagagag aatcgccgga aagcgtgttc ttcatggacg aggaggcggt cttcggcatg 480
ccagggctcc tttcaaatat ggcggagggg atgctcctgc ctcctcctta ctatattgga 540
gatgatggaa gtggtggaga tgacaaagaa gccagcgccg acgtttcact gtggagtttt 600
tccatttga 609
<210> 2
<211> 202
<212> PRT
<213> Artificial Sequence
<400> 2
Met Glu Phe Ser Gln Tyr Ser Ser Glu Ser Glu Cys Gly Ser Ala Arg
1 5 10 15
Ala Val His Leu Ser Asp Glu Glu Ile Leu Leu Ala Ser Arg Asn Pro
20 25 30
Lys Lys Arg Ala Gly Arg Lys Lys Phe Lys Glu Thr Arg His Pro Val
35 40 45
Tyr Arg Gly Val Arg Arg Arg Asn Ser Asp Lys Trp Val Cys Glu Val
50 55 60
Arg Glu Pro Ser Lys Gln Ala Arg Ile Trp Leu Gly Thr Phe Pro Thr
65 70 75 80
Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg
85 90 95
Gly Arg Ser Ala Cys Leu Asn Phe Ala Glu Ser Ala Trp Leu Gln Val
100 105 110
Pro Thr Ser Gly Asp Pro Lys Glu Ile Gln Arg Met Ala Ala Glu Ala
115 120 125
Ala Glu Lys Phe Arg Pro Glu Glu Ala Lys Val Glu Glu Lys Arg Glu
130 135 140
Ser Pro Glu Ser Val Phe Phe Met Asp Glu Glu Ala Val Phe Gly Met
145 150 155 160
Pro Gly Leu Leu Ser Asn Met Ala Glu Gly Met Leu Leu Pro Pro Pro
165 170 175
Tyr Tyr Ile Gly Asp Asp Gly Ser Gly Gly Asp Asp Lys Glu Ala Ser
180 185 190
Ala Asp Val Ser Leu Trp Ser Phe Ser Ile
195 200
<210> 3
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 3
gcttggtacc gagctcggat ggagttctct cagtattcct cggaatc 47
<210> 4
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 4
tggatatctg cagaattctc aaatggaaaa actccacagt gaaacgt 47
Claims (7)
1. The casuarina equisetifolia gene CeDREB1 is characterized in that the casuarina equisetifolia gene CeDREB1 has a cDNA sequence shown as SEQ ID No.1, or the casuarina equisetifolia gene CeDREB1 has a nucleotide sequence shown as an encoding amino acid sequence SEQ ID No. 2.
2. An expression protein of casuarina equisetifolia gene CeDREB1 is characterized in that the amino acid sequence of the expression protein of casuarina equisetifolia gene CeDREB1 is shown as SEQ ID No. 2.
3. Use of the Ephedra sinica Stapf gene CeDREB1 as claimed in claim 1 or the expression protein of the Ephedra sinica Stapf gene CeDREB1 as claimed in claim 2 for improving the anti-oxidative stress capability of Saccharomyces cerevisiae cells.
4. An casuarina equisetifolia gene CeDREB1 recombinant expression vector, which is characterized in that the casuarina equisetifolia gene CeDREB1 of claim 1 is inserted on the recombinant expression vector.
5. The casuarina gene CeDREB1 recombinant expression vector of claim 4, wherein the recombinant expression vector is pCAMBIA1300-CeDREB 1.
6. The use of the casuarina equisetifolia gene CeDREB1 recombinant expression vector of claim 4 or 5 in improving the anti-oxidative stress capability of a saccharomyces cerevisiae strain.
7. An engineered strain of saccharomyces cerevisiae, wherein the casuarina equisetifolia gene CeDREB1 recombinant expression vector of claim 4 or 5 is transferred.
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