CN109180791B - Gene related to plant drought tolerance, and coding protein and application thereof - Google Patents
Gene related to plant drought tolerance, and coding protein and application thereof Download PDFInfo
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- CN109180791B CN109180791B CN201811107003.XA CN201811107003A CN109180791B CN 109180791 B CN109180791 B CN 109180791B CN 201811107003 A CN201811107003 A CN 201811107003A CN 109180791 B CN109180791 B CN 109180791B
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
The invention relates to a gene related to plant drought tolerance, and a coded protein and application thereof, belonging to the technical field of biology. The gene related to plant drought resistance is named as NtDSR1 and is derived from tobacco cultivation, and the gene sequence is shown in SEQ ID NO. 1. The NtDSR1 is a new gene for regulating the drought tolerance of tobacco, and is highly expressed by drought and high-salt induction. The constructed over-expression plasmid PDT1-NtDSR1 is introduced into a plant, and the drought tolerance of an obtained NtDSR1 over-expression strain is obviously enhanced compared with that of an empty vector control strain, namely, the NtDSR1 gene can improve the survival rate of the plant in the seedling stage and the vegetative growth stage under drought conditions, obviously improve the drought tolerance of the plant and provide excellent candidate genes for tobacco drought-tolerant molecular breeding.
Description
Technical Field
The invention relates to a gene related to plant drought tolerance, and a coded protein and application thereof, belonging to the technical field of biology.
Background
Plants are subjected to a number of abiotic stress factors throughout their growth and development, such as drought, high salinity, low temperature, etc. Drought stress is one of the most common factors in various adversity stresses, seriously affects the growth and development of plants and the planting distribution of crops, causes the yield reduction of the crops, and also causes the increasingly worsened ecological environment. Therefore, the improvement of the drought resistance of crops has important significance for agricultural production and ecological environment protection.
Tobacco is an important economic crop, the growth period of the tobacco has high requirement on water, and the yield and the quality of the tobacco leaves are seriously influenced if the tobacco is subjected to drought stress. In recent years, partial tobacco areas in China frequently suffer from severe drought, the drought occurrence range is continuously enlarged, the duration and the loss are increased, and the production of tobacco leaves in China is greatly influenced. The drought problem suffered by tobacco in the growing season is solved by making and repairing irrigation of water conservancy facilities, although the drought problem has good effect, the cost is too high, the complete irrigation of all tobacco fields is difficult to realize in a short time, and the method is not suitable for some tobacco fields. Therefore, the new variety of the tobacco with drought resistance is obtained, and the method has important significance for the high quality and the high yield of the tobacco. Traditional crossbreeding is limited by the lack of resistant parent resources, and the process of obtaining excellent varieties is slow. Tobacco completes whole genome sequencing as the tobacco genome project is implemented. The drought-resistant related gene is separated from the tobacco and applied to drought-resistant tobacco molecular breeding, and has important practical significance for ensuring the quality and the yield of the tobacco.
So far, although the Chinese patent application with application publication No. CN107177602A discloses a NtDR1 gene related to plant drought tolerance and application thereof, and experiments prove that the NtDR1 gene can obviously enhance the drought tolerance of plants, the research on the plant drought tolerance of the NtDSR1 gene is not reported at present.
Disclosure of Invention
The invention aims to provide a gene related to drought tolerance of a plant, which can improve the survival rate of the plant under drought conditions.
The invention also provides a protein coded by the gene related to plant drought tolerance.
In addition, the invention provides a recombinant vector containing a gene related to plant drought tolerance and a construction method thereof.
The invention provides a recombinant bacterium containing a gene related to plant drought resistance and a construction method thereof.
In addition, the invention provides a transgenic cell line containing a gene related to plant drought tolerance and a construction method thereof.
The invention also provides a gene related to plant drought tolerance, a recombinant vector containing the gene related to plant drought tolerance, a recombinant bacterium containing the gene related to plant drought tolerance or application of a transgenic cell line containing the gene related to plant drought tolerance in cultivating drought-tolerant plants.
In order to achieve the purpose, the invention adopts the technical scheme that:
a gene related to plant drought resistance is derived from common tobacco and is named as NtDSR1, and the gene sequence is as follows:
(1) a nucleotide sequence shown as SEQ ID NO. 1; or
(2) A nucleotide sequence which can be hybridized with the sequence defined by SEQ ID NO.1 under high-stringency conditions and encodes the same functional protein; or
(3) A nucleotide sequence with more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1.
In the above (1), SEQ ID NO.1 is a coding region of gene NtDSR1, which consists of 2643 bases and encodes the NtDSR1 protein of the amino acid sequence of SEQ ID NO. 2. In the coding region, base a accounted for 30.76% (813), base C accounted for 16.99% (449), base G accounted for 21% (555), base T accounted for 31.26% (826), with a + T accounting for 62.01% (1639) and C + G accounting for 37.99% (1004).
The specific operation steps of hybridizing with the sequence defined by SEQ ID NO.1 under the high-stringency conditions are as follows:
placing the hybrid membrane in a prehybridization solution (0.25mol/L sodium phosphate buffer solution, pH7.2, 7% SDS), and prehybridization at 65 deg.C for 30 min; discarding the pre-hybridization solution, adding hybridization solution (0.25mol/L sodium phosphate buffer solution, pH7.2, 7% SDS, isotope labeled nucleotide fragment), and hybridizing at 65 deg.C for 12 h; discarding the hybridization solution, adding membrane washing solution I (20mmol/L sodium phosphate buffer solution, pH7.2, 5% SDS), washing membrane at 65 deg.C for 2 times, each time for 30 min; adding washing solution II (20mmol/L sodium phosphate buffer solution, pH7.2, 1% SDS), and washing at 65 deg.C for 30 min.
The protein coded by the gene related to plant drought tolerance is one of the following amino acid sequences:
(1) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; or
(2) And (b) a derivative protein which is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO.2 and is related to plant drought tolerance.
In order to facilitate purification of the NtDSR1 protein encoded by the NtDSR1 gene, a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO. 2.
TABLE 1 sequences of tags
| Label (R) | Residue of | Sequence of |
| Poly-Arg | 5-6 (typically 5) | RRRRR |
| Poly-His | 2-10 (generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
A recombinant vector containing the gene related to plant drought resistance.
The construction method of the recombinant vector comprises the following steps: inserting the gene related to plant drought tolerance into the enzyme cutting site of the corresponding expression vector, and connecting through ligase to obtain the recombinant vector. For example, the gene related to drought tolerance of a plant is inserted into the SpeI cleavage site of pDT1 to obtain a recombinant vector.
When constructing the recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin (Ubiquitin) gene promoter (pUbi), Actin promoter, etc. may be added in front of the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters.
In addition, in constructing plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be either the ATG initiation codon or the initiation codon of adjacent regions, but must be in the same reading frame as the coding sequence, to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.
In order to facilitate the identification and selection of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding antibiotic markers (gentamicin marker, kanamycin marker, etc.) which are expressed in plants and can develop resistance or chemical agent resistance marker genes (e.g., herbicide resistance gene), etc.
A recombinant bacterium containing the gene related to plant drought resistance.
The construction method of the recombinant bacterium comprises the following steps: the gene related to plant drought resistance is introduced into the thallus to obtain the recombinant bacteria. Specifically, the recombinant vector can be transferred into a thallus and screened to obtain a recombinant bacterium.
A transgenic cell line containing the gene related to plant drought tolerance.
The construction method of the transgenic cell line comprises the following steps: the gene related to plant drought tolerance is introduced into plant cell to obtain transgenic cell line. Specifically, the recombinant bacteria can be transferred into plant cells to be cultivated to obtain a transgenic cell line.
The gene related to plant drought tolerance, the recombinant vector containing the gene related to plant drought tolerance, the recombinant bacterium containing the gene related to plant drought tolerance or the application of the transgenic cell line containing the gene related to plant drought tolerance in cultivating drought-tolerant plants.
The application of the gene related to plant drought tolerance in cultivating drought tolerant transgenic plants comprises the following steps: the gene related to plant drought tolerance is introduced into target plant to obtain drought tolerant transgenic plant.
The gene related to drought tolerance of the plant is introduced into a target plant by the recombinant vector. The plant expression vector carrying the gene related to plant drought tolerance can be transformed into target plant cells or tissues by Ti plasmid, Ri plasmid, plant virus vector and conventional biological methods such as gene gun method, pollen tube channel, microinjection, conductance, Agrobacterium mediation and the like.
The target plant is a dicotyledonous plant or a monocotyledonous plant.
The drought tolerance of the transgenic plant is higher than that of a wild plant, and the transgenic plant is a mannitol-tolerant or drought-tolerant transgenic plant.
The invention has the beneficial effects that:
the invention provides a new gene NtDSR1 related to plant drought tolerance, which is derived from tobacco and is induced by drought and high salt to be highly expressed. The invention clones NtDSR1, constructs an expression vector, and introduces the expression vector into a target plant for expression, thereby proving that the drought resistance of the NtDSR1 over-expression strain is stronger than that of an empty vector control strain in the seedling stage and the vegetative growth stage, namely, the NtDSR1 gene can improve the survival rate of plants in the seedling stage and the vegetative growth stage under drought conditions, obviously improve the drought resistance of the plants, and provide excellent candidate genes for the drought-resistant molecular breeding of tobacco.
Drawings
FIG. 1 shows the results of in vitro amplification electrophoresis of the NtDSR1 gene of the present invention;
FIG. 2 shows the expression levels of the NtDSR1 gene in different transgenic lines;
FIG. 3 is a drought tolerance analysis of the transgenic plants of the invention NtDSR 1; wherein, FIG. 3A is a comparison of control and NtDSR1 transgenic plant phenotypes at simulated drought; FIG. 3B is the cotyledon greening rate of control and NtDSR1 transgenic plants when drought was simulated; FIG. 3C is a comparison of control and NtDSR1 transgenic plant phenotypes under drought conditions; figure 3D is the survival of control and NtDSR1 transgenic plants at simulated drought.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings.
In the following examples, unless otherwise specified, all methods are conventional.
The reagents and materials in the following examples are commercially available unless otherwise specified.
Example 1
This example is the cloning of the NtDSR1 gene, comprising the following steps:
(1) extraction of Total RNA from NtDSR1
K326 tobacco leaves grown under normal growth conditions for 4 weeks were taken, immediately ground into powder in liquid nitrogen, and about 100mg of the powdered material was placed in a 1.5mL centrifuge tube containing 1.0mL TRIzol (Invitrogen), and stored at-80 ℃ for later use.
Extracting total RNA by using a TRIzol reagent extraction method: taking out the sample stored at-80 ℃, thawing at room temperature, adding 200 mu L chloroform, shaking and mixing uniformly, centrifuging, carefully taking out the upper aqueous phase, transferring into another centrifuge tube, adding 500 mu L isopropanol, precipitating, centrifuging to separate RNA, washing with 75% alcohol, slightly drying at room temperature, adding a proper volume of RNase-free water, and fully dissolving. Treating the extracted RNA with DNase (Fermentas) to obtain total RNA.
(2) Reverse transcription reaction
Taking 2 mu g of total RNA for reverse transcription, adding 1 mu L of Oligo (dT) with 0.5 mu g/mu L, then adding deionized water to complement to 12 mu L, preserving heat at 70 ℃ for 5min, then sequentially adding 4 mu L of 5 x RTbuffer, 1 mu L of 20U/mu L RNase inhibitor and 2 mu L of 10mmol/LdNTP, preserving heat at 37 ℃ for 5min, then adding 1 mu L of 200U/mu L M-MLV reverse transcriptase, and the final reaction volume is 20 mu L. Reacting at 42 deg.C for 60min, heating at 70 deg.C for 10min to terminate the reaction and obtain reverse transcription product cDNA.
(3) PCR in vitro amplification
PCR amplification A pair of primers was artificially synthesized and 15-20bp of vector complementary sequences was added to the 5' ends thereof, respectively, with reference to the In-fisuon method of Clontech. The upstream and downstream primer sequences are as follows, the underlined regions are the vector linker sequences:
NtDSR1P-F：
5ˊ-TGACCTCGAGACTAGTATGAATGGTACATTACCTGA-3ˊ；
NtDSR1P-R：
5ˊ-TACCGTCGCACCATACTAGTTCAAATAAGTGATGTAAAAC-3ˊ。
the cDNA was used as a template, NtDSR1P-F and NtDSR1P-R were used as primers, and high fidelity was used
GXL DNA Polymerase (TAKARA) was subjected to PCR amplification. Establishment of reactions in sterilized PCR tubesThe units are shown in Table 2 below: μ L.
TABLE 2 PCR amplification reaction System
Reaction procedure: pre-denaturation at 98 ℃ for 5 min; at 95 ℃ for 10s, at 52 ℃ for 15s, at 68 ℃ for 3min, for 35 cycles, and at last at 72 ℃ for 10min, after the reaction is finished, the PCR result is detected by electrophoresis (as shown in lanes 1 and 2 in FIG. 1, and lane 3 is marker).
(4) Purification of PCR products
After electrophoresis, the gel was recovered by cutting under irradiation of ultraviolet light, and the desired gene fragment was recovered by using a gel recovery kit (TAKARA).
Sequencing and identifying to obtain the sequence shown in SEQ ID NO. 1.
Example 2
This example is the construction of a recombinant expression vector comprising the following steps:
(1) recombinant vector construction
The ligation reaction system was set up as per kit requirements, as shown in Table 3 below, in units, with reference to the in-fusion seamless ligation instructions from Clontech: μ L.
TABLE 3 ligation reaction System
After 15min of ligation at 50 ℃ the mixture was placed on ice and used for the next transformation.
(2) Transformation of competent cells of Escherichia coli by ligation products by heat shock method
Adding 5 mu L of the ligation product into competent cells under aseptic conditions, gently mixing, performing ice bath for 30min, performing heat shock at 42 ℃ for 90s, quickly transferring the centrifuge tube into the ice bath, standing for 2min (2-3 min), adding 800 mu L of LB culture medium without antibiotics, shaking gently at 37 ℃ for 1h, taking 200 mu L of culture solution, smearing on LB solid culture medium containing 50 mu g/mL kanamycin, and performing inversion culture at 37 ℃ for 14h (12-16 h).
(3) Identification of expression vectors
White bacterial plaques growing in the culture medium are picked up and respectively inoculated into LB liquid culture medium containing 50 mu g/mL kanamycin to be cultured for 14h (12-16 h), plasmid DNA is extracted in a small amount by an alkaline lysis method, the obtained plasmid is subjected to double enzyme digestion by Bam HI and Hind III, and the positive clone with correct result is sent to a company for sequencing to obtain pDT1-NtDSR1 recombinant expression vector.
In the construction process of the above recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin (Ubiquitin) gene promoter (pUbi), Actin promoter, etc. may be added in front of the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters.
In addition, when constructing plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.
Example 3
This example is an expression vector for transforming Agrobacterium comprising the following steps:
the expression vector pDT1-NtDSR1 is transformed into agrobacterium GV3101 by a heat shock method (the agrobacterium GV3101 is purchased from Tiangen Biochemical technology Co., Ltd.), plasmids are extracted and enzyme digestion identification is carried out, the method is the same as the steps for identifying the expression vector, and the result shows that the expression vector pDT1-NtDSR1 has successfully transformed agrobacterium.
Example 4
This example is a method for breeding drought-tolerant transgenic Arabidopsis thaliana, comprising the following steps:
integrating an expression vector into an arabidopsis thaliana (Col-0) genome by adopting an agrobacterium-disseminated inflorescence method, spraying a herbicide (0.1%) to seedlings by screening, continuously screening seedlings of T2 generations by using the herbicide (0.1%), selecting a strain with the separation ratio of 3:1 to continuously screen in T3 generations, and considering that the strain is a homozygous strain if 100% of seedlings can continuously and normally grow after the herbicide is sprayed in T3 generations.
Two homozygous lines are randomly selected for molecular detection. Arabidopsis total RNA was extracted using Trizol reagent extraction (Invitrogen). Carrying out reverse transcription on the total RNA of the transgenic arabidopsis thaliana to obtain cDNA; using the cDNA obtained by the reverse transcription as a template (the reverse transcription step was the same as in example 1), real-time quantitative PCR was carried out using primers specific to NtDSR1 (amplifiable gene itself). The results show that the NtDSR1 gene can be expressed in different transgenic lines, but the expression level is different, as shown in FIG. 2.
The above results indicate that the NtDSR1 gene has been integrated into the arabidopsis genome and is stably expressed in transgenic arabidopsis.
The obtained amino acid sequence is determined to be the sequence shown in SEQ ID NO.2 through sequencing identification.
Test examples
The test example is the identification of drought tolerance of arabidopsis thaliana, and comprises the following steps:
the transgenic arabidopsis thaliana transferred with pDT1-NtDSR1 is transferred to a greenhouse for culture, after selfing, the seeds of the transgenic arabidopsis thaliana are collected, meanwhile, the transgenic arabidopsis thaliana transferred with pDT1 is used as an empty vector control strain, and two transgenic strains with relatively high expression levels, namely a strain 4 and a strain 9, are selected for subsequent drought tolerance identification.
Seeds of pDT1-NtDSR1 transgenic Arabidopsis thaliana and pDT1 transgenic Arabidopsis thaliana harvested at the same time were sterilized and spotted on normal MS medium or MS medium containing 300mM mannitol, respectively. Placing the culture dish in the dark at 4 deg.C, taking out the culture dish after 2 days, placing in a light incubator, culturing under conventional growth conditions (16 h in light phase, 8h in dark phase; 22 deg.C), and counting germination rate after 4 days. As a result, the germination rate of the transgenic line of the NtDSR1 under simulated drought conditions is obviously higher than that of the control. In addition, the sterilized and purified seeds were respectively spotted on MS medium containing 300mM mannitol and normal MS medium under conventional growth conditions (16 h in light phase, 8h in dark phase; 22 ℃ C.). Under high mannitol concentration conditions, both control and transgenic plants can germinate, but most of the controls die white after cotyledon formation, while NtDSR1 transgenic plants are less inhibited and show a higher cotyledon greening rate than the controls (as shown in a and B in fig. 3). Transgenic Arabidopsis thaliana transformed with pDT1-NtDSR1 and transgenic Arabidopsis thaliana transformed with pDT1 are germinated and transferred into nutrient soil, and water cut-off is started after 3 weeks of growth under conventional conditions (16 h of light-phase culture and 8h of dark-phase culture; the culture temperature is 22 ℃). Rehydration was started 14 days after water cut off, and pictures were taken one week after rehydration. As a result, as shown in FIG. 3, the survival rate of the pDT1-NtDSR 1-transfected Arabidopsis thaliana (line 4, line 9) after undergoing water-break-before-rehydration was significantly higher than that of the empty vector control line (as shown in C, D in FIG. 3).
In summary, the following steps: under drought/simulated drought stress, the tolerance of the transgenic arabidopsis thaliana transformed with pDT1-NtDSR1 is obviously stronger than that of the control strain with an empty vector.
Sequence listing
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> gene related to plant drought tolerance, and coding protein and application thereof
<160>4
<170>PatentIn version 3.5
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ttagtgttaa gtcaaaatgg ttttgctcca caagaaatac catcaagttt tactcagtta 240
aaaaagctga ggaatttttt gatgacagaa gcaaatttag ttggagaaat acctgaaaat 300
attggtaata tgacaagtct tgaactttta gacttgtctt taaatggctt aagtggtaat 360
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aataacagct tgactggaaa aatacctgaa gattttggaa agttgacaaa gttgactggt 540
ttgtctttgt tttacaacca attatcagga gaaatacctt tgagcatagg caaattacca 600
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cctaaggata tatgtaacaa caaggtgttg tcaaaaatga ttgtttttga gaacaatctt 780
actggtgtac tacctgattc acttggaaat tgtgacagtt tgaagtccgt tcgcgttgag 840
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tttatgatca atgataattt gttcagtggt caacttccag ataaactggg atcaaattta 960
tccagagttg atatcagaaa caacaagttt tcgggcgagt taccaactgg aataggtagt 1020
tggtacagtt taattgtatt cagggcaagt aataatctct taattggtga aatccctcag 1080
gaattgactg ttttaccaga attaactgaa cttttattgg atggtaattt actttctggt 1140
aattttccat ctaatattac ctcatggaaa tcacttgtta ctttaagttg tagcaagaat 1200
cagatatcag gtcaaatccc atctgcactt ggcctattac caaaactcag tgttttggac 1260
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tcacttaacc tctcttcaaa ccgcctatcg ggtaaaatcc cagctcagtt agagaatgca 1380
gctttcgata agagcttctt gaataatact ggactttgtg cgagtaatcc attagttgga 1440
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aagttggact tcacagaatc agacatttta tcttatctga ctgataacaa cgcgatagga 1680
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gcagaagttc agatattagg cacgattcga cattccaata tagtaaaact tctttgttgc 1860
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ggagataaca cagtgacaac agtagctggc tcttttgggt atattgctcc agagtatgcg 2220
cgaaaaacta gagtgactga gaaggttgat gtgtatagct tcggagtcat ccttttagaa 2280
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<213>Nicotiana tabacum
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MNGTLPDDIN RLSANLQVLN LTSNNFTGDI PSTIGDLSDQ LKELVLAANL FGGNLLDLES 60
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IPSGLFQIKN LTIVYLYNNR LSGEIPRSIM SLNLDVIDLC NNSLTGKIPE DFGKLTKLTG 180
LSLFYNQLSG EIPLSIGKLP SLIVFKLFGN KLSGEIPPDF GRFSKLEDFQ VSQNQLVGNI 240
PKDICNNKVL SKMIVFENNL TGVLPDSLGN CDSLKSVRVE NNHLSGEIPD GLWTAEKLST 300
FMINDNLFSG QLPDKLGSNL SRVDIRNNKF SGELPTGIGS WYSLIVFRAS NNLLIGEIPQ 360
ELTVLPELTE LLLDGNLLSG NFPSNITSWK SLVTLSCSKN QISGQIPSAL GLLPKLSVLD 420
LSSNQFSGEI PTELGKLILT SLNLSSNRLS GKIPAQLENA AFDKSFLNNT GLCASNPLVG 480
LSSCKGKKQS DKFPAKLVAV LGSVAAVAFL VAVLYSVFVL RSHRKRKQEL VSTWKQTSFH 540
KLDFTESDIL SYLTDNNAIG SGGSGQVYLV PLSRSGNCVA VKRIWSSNQR LDQKLEKEFL 600
AEVQILGTIR HSNIVKLLCC ISSEESKLLV YEYMENRSLD IWLHSKKRLN NVSGSAPHLV 660
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GDNTVTTVAG SFGYIAPEYA RKTRVTEKVD VYSFGVILLE LVTRKEANFG DEDSCLADWA 780
WRQLQKGHPI VDVLDENIKK SRYLDEICTV FKLGIFCTST FPSSRPTMKE VLQILLQCNN 840
NSPTSGEKKD ETEHDVSPLL KNSRSERIAE NDDVGFTSLI 880
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<212>DNA
<213> Artificial sequence
<221> upstream primer sequence of NtDSR1
<400>3
tgacctcgag actagtatga atggtacatt acctga 36
<211>40
<212>DNA
<213> Artificial sequence
<221> downstream primer sequence of NtDSR1
<400>4
taccgtcgca ccatactagt tcaaataagt gatgtaaaac 40
Claims (9)
1. A gene associated with drought tolerance in plants, characterized by: the gene sequence is shown as the nucleotide sequence shown in SEQ ID NO. 1.
2. A protein encoded by the plant drought tolerance-associated gene of claim 1, wherein: the protein is a protein consisting of an amino acid sequence shown as SEQ ID NO. 2.
3. A recombinant vector comprising the plant drought-tolerance-associated gene of claim 1.
4. The method of constructing a recombinant vector according to claim 3, wherein: the method comprises the following steps: inserting the gene related to plant drought tolerance into the enzyme cutting site of the corresponding expression vector, and connecting through ligase to obtain the recombinant vector.
5. A recombinant bacterium comprising the gene related to drought tolerance of a plant according to claim 1.
6. The method for constructing a recombinant bacterium according to claim 5, wherein: the method comprises the following steps: the gene related to plant drought resistance is introduced into the thallus to obtain the recombinant bacteria.
7. A method of constructing a transgenic cell line, comprising: a transgenic cell line obtained by introducing the gene related to drought tolerance of a plant according to claim 1 into a plant cell.
8. The use of the gene related to drought tolerance of plants as claimed in claim 1, the recombinant vector as claimed in claim 3, the recombinant bacterium as claimed in claim 5 for the cultivation of drought tolerant transgenic plants.
9. Use according to claim 8, characterized in that: the method comprises the following steps: a drought-tolerant transgenic plant obtained by introducing the plant drought-tolerance-associated gene of claim 1 into a plant of interest.
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| CN115704036B (en) * | 2021-08-13 | 2024-03-12 | 湖南中烟工业有限责任公司 | Tobacco NtDSR1 gene and application thereof |
| CN115704035B (en) * | 2021-08-13 | 2024-03-12 | 湖南中烟工业有限责任公司 | Tobacco NtDSR2 gene and application thereof |
| CN115820670A (en) * | 2022-12-13 | 2023-03-21 | 中国烟草中南农业试验站 | Application of NtTAS14-like1 gene in regulation of drought tolerance of tobacco |
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| CN102899296A (en) * | 2011-07-28 | 2013-01-30 | 中国科学院遗传与发育生物学研究所 | Rice stress tolerance-related receptor-like protein OsSIK3, its coding gene and application |
| CN106232620A (en) * | 2014-02-07 | 2016-12-14 | 先锋国际良种公司 | Insecticidal protein and using method thereof |
| CN107177602A (en) * | 2017-07-06 | 2017-09-19 | 中国烟草总公司郑州烟草研究院 | The NtDR1 gene related to drought tolerance in plants and its application |
| KR20180039211A (en) * | 2016-10-07 | 2018-04-18 | 중앙대학교 산학협력단 | Method for improving the resistance to the drought stress using pepper E3 ligase CaDSR1 in plants |
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| CN106232620A (en) * | 2014-02-07 | 2016-12-14 | 先锋国际良种公司 | Insecticidal protein and using method thereof |
| KR20180039211A (en) * | 2016-10-07 | 2018-04-18 | 중앙대학교 산학협력단 | Method for improving the resistance to the drought stress using pepper E3 ligase CaDSR1 in plants |
| CN107177602A (en) * | 2017-07-06 | 2017-09-19 | 中国烟草总公司郑州烟草研究院 | The NtDR1 gene related to drought tolerance in plants and its application |
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