CN115820670A - Application of NtTAS14-like1 gene in regulation of drought tolerance of tobacco - Google Patents
Application of NtTAS14-like1 gene in regulation of drought tolerance of tobacco Download PDFInfo
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Abstract
The invention discloses application of an NtTAS14-like1 gene in regulation and control of drought tolerance of tobacco. Belongs to the technical field of plant drought resistance. The invention screens a gene DS3 obviously induced by drought stress through gene expression analysis, and carries out deep functional analysis on the gene DS 3. The gene codes abscisic acid and an environmental stress induced protein TAS14-like, so the gene is named as NtTAS14-like1. The gene promoter region has a variety of cis-acting elements, including drought-induced and abscisic acid-responsive elements. The tobacco drought tolerance can be enhanced by over-expressing the NtTAS14-like1 gene, and the tobacco drought tolerance is reduced by inhibiting the gene expression, which indicates that the gene positively regulates the tobacco drought tolerance. The gene excavation and function identification provide important molecular elements and theoretical reference for tobacco drought-enduring breeding.
Description
Technical Field
The invention relates to the technical field of plant drought tolerance, in particular to application of an NtTAS14-like1 gene in regulation and control of tobacco drought tolerance.
Background
In recent years, the strength, frequency and duration of drought have increased with global warming and environmental deterioration. In addition, precipitation season and regional precipitation distribution in China are extremely uneven, water resource shortage is increasingly serious, and drought becomes an important limiting factor influencing normal growth and development of plants. Researches show that drought stress can inhibit the physiological metabolism of plants, destroy the balance among endogenous hormones, cause the stomata of leaves to be closed, damage the reaction center of a photosystem, damage the structure of chloroplast of the plants and reduce the photosynthesis capability, thereby causing the plants to grow slowly, wither and even die.
The molecular mechanisms of plants responding to drought stress are quite complexWhen a plant suffers drought stress, the plant can generate a large amount of drought stimulating factors, and a sensor positioned on a cell membrane can receive drought signals transmitted by the drought stimulating factors, then transmits the drought signals into cells from the outside of the cells, and transmits the drought signals through various signal transduction ways, so that the expression of drought response genes in the plant body is quickly activated, and the drought resistance of the plant is improved. Ca 2+ The second messengers play an important role in the drought signal transduction pathway, allowing plants to respond rapidly when subjected to drought stress. The expression products of drought response genes are mainly proteins involved in transcriptional regulation and signaling cascades and other proteins (proteins involved in water and ion transport, sugar transporters, proteins maintaining osmotic pressure, antioxidant proteins, sugar transporters, etc.). Drought stress can stimulate plant roots to synthesize a large amount of ABA, and then the ABA is transported to each tissue of the plant to inhibit the growth and development of the plant and improve the drought resistance.
Drought stress-related genes are largely divided into two types of signal pathways, ABA-dependent and ABA-independent, according to whether drought-responsive genes are dependent on ABA signals. Genes participating in drought resistance regulation in ABA-dependent related signal pathways mainly comprise AREB/ABF, MYB, MYC and the like. Research shows that the overexpression of AtMYC2 and AtMYB2 genes leads to the increase of the sensitivity of transgenic arabidopsis to ABA, and the drought resistance of plants is obviously improved. Drought-resistant factors in an ABA independent signal pathway are mainly transcription regulation and control with CBF/DREB as a center. The DREB2 gene is obviously induced by drought stress, and the drought resistance of transgenic arabidopsis is obviously improved by over-expressing the DREB 2. Similarly, the over-expression of OsDREB1 in the rice obviously improves the cold resistance and the drought resistance of the rice. In addition, NAC family genes involved in drought stress resistance in plants have members that are dependent both by ABA and ABA.
Tobacco originates from tropical areas with abundant rainfall and has high requirements for moisture. Most tobacco areas in China are often lack of necessary irrigation facilities, so that the lack of water becomes a main factor restricting the tobacco planting and quality formation. Therefore, the method has important theoretical significance for breeding and cultivating drought-resistant varieties by utilizing the biological technology to excavate drought-resistant genes and exploring gene functions, and has important practical significance for promoting the income increase of tobacco growers and realizing the sustainable development of tobacco regions. However, compared with model plants such as arabidopsis thaliana and rice, the research on the drought-resistant molecular regulation mechanism of tobacco is very deficient, and only a few genes are reported to participate in the drought-resistant regulation of tobacco. For example, xu et al report that the transcription factor NtNAC2 plays a positive regulatory role in the drought resistance of tobacco, and overexpression of NtNAC2 significantly improves the antioxidant enzyme activity of transgenic tobacco. In addition, xu and the like find that the over-expression of the lipid transfer protein gene NtLTP4 can enhance the activity of antioxidant enzyme, thereby improving the drought resistance of tobacco. Therefore, the drought-resistant gene in tobacco is to be further explored and identified.
In conclusion, the problem to be solved by those skilled in the art is how to provide a gene for regulating drought tolerance of tobacco.
Disclosure of Invention
In view of the above, the invention provides an application of the NtTAS14-like1 gene in regulating and controlling the drought tolerance of tobacco.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the NtTAS14-like1 gene in regulating and controlling the drought tolerance of tobacco, wherein the nucleotide sequence of the NtTAS14-like1 gene is shown as SEQ ID NO:1 is shown in the specification;
ATGGCACAATACAACGAGGGATACGGTAGCCAGGGGCAAATGCGCCAGACTGATGAATATGGAAACCGGGTCCAAGAAAGTGGGGGCATGGGCAGTACTGGTGCCTATGGAACTCAGCAAGGTATGGGGGGCATAGGTGGTGGAGAATATGGAACCCAAGGCCAAGGTACTGGTATGGGGACCACTGGTGGTGGAGCCTATGGAACTCAGGGTGGTACTGGAATGGGGGCTATGGGTGGAGATCAGTATGGAACCCAAGGTACTGGAATGGGTATGGGTACTGGTGGTATGCATACTCAGCACCATGAGGGCCAACAACAGCTTCGTCGATCCGACAGCTCTAGCTCTTCTGAGGATGATGGAGAAGGTGGAAGAAGGAAGAAGGGAATGAAGGAGAAGATAATGGAGAAGATGCCAGGAGGACATGCCCAACAGGAAGGTGAGTATAACCAACATGCGCAAACAACTTATACTAGTACTGAAGGAGGAGAGAAGAAGGGAATGATGGACAAAATCAAGGACAAGATCCCTGGGATGCACTGA，SEQ ID NO.1。
further, the application of the NtTAS14-like1 gene in the positive regulation of the drought tolerance of tobacco.
Further, the promoter region of the NtTAS14-like1 gene has various cis-acting elements, including drought-induced and abscisic acid response elements.
The application of protein or substance for regulating and controlling the expression of protein coding gene in the drought tolerance of tobacco, wherein the amino acid sequence of the protein is shown as SEQ ID NO:2 is shown in the specification;
MAQYNEGYGSQGQMRQTDEYGNRVQESGGMGSTGAYGTQQGMGGIGGGEYGTQGQGTGMGTTGGGAYGTQGGTGMGAMGGDQYGTQGTGMGMGTGGMHTQHHEGQQQLRRSDSSSSSEDDGEGGRRKKGMKEKIMEKMPGGHAQQEGEYNQHAQTTYTSTEGGEKKGMMDKIKDKIPGMH，SEQ IDNO.2。
the NtTAS14-like1 gene or the protein-related biological material is applied to drought tolerance of tobacco, and the biological material is any one of the following materials:
a: a nucleotide sequence such as the nucleic acid molecule described above or a nucleic acid molecule encoding the protein described above;
b: an expression cassette comprising the nucleic acid molecule of a;
c: an expression vector containing the nucleic acid molecule of A or a recombinant vector containing the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, or a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
A method for improving the drought tolerance of tobacco, which improves the drought tolerance of tobacco by over-expressing or improving the expression of the NtTAS14-like1 gene or the activity of the protein in tobacco.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the NtTAS14-like1 gene is obviously induced by drought stress, the length of a CDS sequence of the gene is 543bp, 180 amino acids are coded, and the gene codes abscisic acid and an environmental stress induced protein TAS14-like. The promoter region of the NtTAS14-like1 gene has a variety of cis-acting elements, including drought-inducing and abscisic acid-responsive elements. The tobacco drought tolerance can be enhanced by over-expressing the NtTAS14-like1 gene, and the tobacco drought tolerance is reduced by inhibiting the gene expression, which indicates that the gene positively regulates the tobacco drought tolerance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is the analysis of the expression pattern of drought-tolerant candidate genes under drought stress according to the present invention;
FIG. 2 is a schematic diagram of the sequence signature analysis of the NtTAS14-like1 of the present invention;
FIG. 3 is a phylogenetic tree analysis of the NtTAS14-like1 of the present invention;
FIG. 4 is a drawing showing the NtTAS14-like1 promoter element of the present invention;
FIG. 5 is a graph showing the identification of drought tolerant phenotypes of VIGS plants of the present invention, wherein A is an analysis of the expression levels of NtTAS14-like1 in three lines, and represents a P value <0.05; b, performing phenotype analysis on the three strains after drought stress treatment; c is the relative conductivity (EL) of the three strains after drought stress treatment, and represents the P value is less than 0.05; d is the Malondialdehyde (MDA) content in the three strains after drought stress treatment, wherein the value of P represents 0.01; WT is wild tobacco K326, BSMV:00 is empty vector control, BMSV: DS3 is NtTS14-like1 gene silencing strain;
FIG. 6 is a graph showing the identification of drought tolerant phenotype of over-expressed plants of the present invention, wherein A is the expression level analysis of NtTAS14-like1 in three lines, and represents P value <0.01; b, performing phenotype analysis on the three strains after drought stress treatment; c is the relative conductivity (EL) of the three strains after drought stress treatment, and represents the P value is less than 0.05; d is the Malondialdehyde (MDA) content in the three strains after drought stress treatment, represents the P value <0.05, represents the P value <0.01; WT was wild-type tobacco K326, and OE1 and OE2 were two NtTAS14-like1 overexpression lines.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The required medicament is a conventional experimental medicament purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.
Example 1
1 seed Germination
(1) Putting a proper amount of tobacco seeds into a 2mL centrifuge tube, washing with sterile water for 1-2 times, and washing off dust on the surfaces of the seeds;
(2) Adding 2mL 75% ethanol (containing 0.02% (wt)% Txiton-100), soaking for 1min, and shaking repeatedly;
(3) Pouring off the ethanol, and repeating the step (2) for 3-5 times;
(4) Pouring off the ethanol, washing with sterile water for 3-5 times, pouring the seeds on sterile filter paper, and drying;
(5) Uniformly spreading the seeds on an MS solid culture medium, vernalizing the seeds for 48 to 72 hours at 4 ℃ in the dark, and then transferring the culture dish to a constant-temperature incubator for germination at the culture temperature of 25 ℃ (day/night);
(6) After the seeds germinate for 2 weeks, the seedlings are moved into a plastic basin filled with nutrient soil, placed in an artificial growth chamber at 25 ℃ (day/night) for culture, 1/2Hoagland nutrient solution is poured 2-3 times per week, and the test treatment is carried out after the culture is carried out for 1 month.
2 drought treatment
Mannitol was used to simulate drought stress in this study. Preparing 200mM mannitol solution for treating tobacco seedlings, pouring 100mL solution in each pot, sampling at 0h,2h,4h,6h and 12h, and quickly placing in liquid nitrogen after sample collection for later use at-80 ℃.
3 Gene expression analysis
3.1RNA extraction
The Trizol method is used for extracting the total RNA of the plant, and comprises the following specific steps:
(1) Placing 0.1g tobacco leaf in a precooled mortar, adding liquid nitrogen, rapidly grinding into fine powder, transferring the powder into a centrifuge tube of 1.5mL RNAase-free, adding 1mL TriansZol Up, violently shaking and mixing uniformly, standing for 5min at room temperature, and immediately placing on ice;
(2) Adding 200 μ L chloroform, shaking vigorously for 30s, standing at room temperature for 10min, centrifuging at 12000rpm at 4 deg.C for 15min, collecting supernatant, and transferring into new 1.5mL RNAase-free centrifuge tube;
(3) Adding 500 μ L isopropanol, turning upside down, mixing, standing at room temperature for 10min, centrifuging at 12000rpm for 10min at 4 deg.C;
(4) Discarding the supernatant, adding 1mL of 75% ethanol, reversing the upper part and the lower part, uniformly mixing, and washing the precipitate;
(5) Centrifuging at high speed of 12000rpm for 5min at 4 ℃, discarding the supernatant, reversely buckling the centrifuge tube on absorbent paper, and standing for 3-5 min;
(6) Adding 30 μ L of RNAase-free ddH 2 Dissolving the precipitate with O, incubating at 55 deg.C for 10min, and slightly centrifuging to obtain RNA, which can be directly reverse transcribed or stored at-80 deg.C.
3.2 reverse transcription
MonScript using reverse transcription kit TM The cDNA is synthesized, the reverse transcription method refers to the instruction book, and the specific operation steps are as follows:
(1) RNA and RNAase-free ddH as shown in Table 1 2 Adding O into a PCR tube of RNAase-free, mixing uniformly on ice, centrifuging briefly, reacting at 70 ℃ for 10min to denature RNA, and then placing on ice for 5min;
(2) Respectively adding 5 XRT Mix and dsDNase into a PCR tube according to the table 1, uniformly mixing, reacting at 37 ℃ for 2min,55 ℃ for 30min and 85 ℃ for 1min in a PCR instrument in sequence, immediately placing on ice after the reaction is finished, and directly using the reverse transcription cDNA or storing at-20 ℃.
TABLE 1 reverse transcription System
3.3 Gene expression analysis
(1) Quantitative analysis of gene expression by Real-time fluorescent quantitative PCR (qRT-PCR) method, the reaction system is shown in Table 2;
TABLE 2qRT-PCR reaction System
(2) The reverse transcribed cDNA was used as template for qRT-PCR reaction on the Step One Plus One Real-Time PCR Systems system, each sample was repeated at least 3 times, and the amplification procedure is shown in Table 3.
TABLE 3qRT-PCR amplification procedure
To study gene expression of tobacco after drought stress treatment, we screened 17 differentially expressed genes (named DS1-DS 17) significantly affected by drought from transcriptome sequencing results. Further, we simulated drought stress with 200mM mannitol solution and investigated the gene expression pattern of these 17 genes after mannitol treatment by qRT-PCR technique (results are shown in FIG. 1). The qRT-PCR analysis result shows that most of the differentially expressed genes can be expressed by drought induction, and few of the differentially expressed genes are expressed by drought inhibition. According to the results of gene expression analysis, the DS3 gene with the highest drought stress induced multiple is screened as a target gene, and deep functional research is carried out.
4 sequence feature analysis
We characterized the protein sequence of the DS3 gene. The result shows that the CDS sequence of DS3 gene is 543bp in length, encodes 180 amino acids, encodes abscisic acid and environmental stress-induced protein TAS14-like, and has a typical Dehydranmotif (as shown in FIG. 2).
4.1CDS sequence analysis
ATGGCACAATACAACGAGGGATACGGTAGCCAGGGGCAAATGCGCCAGACTGATGAATATGGAAACCGGGTCCAAGAAAGTGGGGGCATGGGCAGTACTGGTGCCTATGGAACTCAGCAAGGTATGGGGGGCATAGGTGGTGGAGAATATGGAACCCAAGGCCAAGGTACTGGTATGGGGACCACTGGTGGTGGAGCCTATGGAACTCAGGGTGGTACTGGAATGGGGGCTATGGGTGGAGATCAGTATGGAACCCAAGGTACTGGAATGGGTATGGGTACTGGTGGTATGCATACTCAGCACCATGAGGGCCAACAACAGCTTCGTCGATCCGACAGCTCTAGCTCTTCTGAGGATGATGGAGAAGGTGGAAGAAGGAAGAAGGGAATGAAGGAGAAGATAATGGAGAAGATGCCAGGAGGACATGCCCAACAGGAAGGTGAGTATAACCAACATGCGCAAACAACTTATACTAGTACTGAAGGAGGAGAGAAGAAGGGAATGATGGACAAAATCAAGGACAAGATCCCTGGGATGCACTGA，SEQ ID NO.1。
4.2 amino acid sequence
MAQYNEGYGSQGQMRQTDEYGNRVQESGGMGSTGAYGTQQGMGGIGGGEYGTQGQGTGMGTTGGGAYGTQGGTGMGAMGGDQYGTQGTGMGMGTGGMHTQHHEGQQQLRRSDSSSSSEDDGEGGRRKKGMKEKIMEKMPGGHAQQEGEYNQHAQTTYTSTEGGEKKGMMDKIKDKIPGMH，SEQ IDNO.2。
4.3 phylogenetic Tree analysis
Phylogenetic tree analysis results showed that DS3 encodes TAS14 dehydrin, which has a close relationship to TAS14 (XM _ 009626277.3) in the diploid progenitor Nicotiana villosa (TT) (see fig. 3).
The 5NtTAS14-like1 promoter sequence contains drought induction and abscisic acid response elements
The cis-acting elements in the promoter region can provide reference for understanding the induced expression and gene function of the gene, therefore, the DS3 promoter sequence (shown in figure 4) is analyzed by using a Plantcare website (http:// bioinformatics. Psb. Content. Be/webtools/plantarce/html /), and the promoter region 2000bp upstream of the initiation codon is found to have various cis-acting elements and comprises drought-induced and abscisic acid response elements, so that the drought-resistant function of DS3 is presumed to be possibly related to an ABA signal path.
Example 2 Virus-induced Gene silencing (VIGS) experiment
1 candidate Gene VIGS vector construction
The VIGS technique used in this study was mainly improved by referring to the method of Yuan et al (2011) [ Yuan et al. A high throughput strip magnetic virus vector for virus induced gene cloning in monomers and dicots. Plos ONE,2011, 6.
(1) Designing a specific primer with a joint by taking cDNA of tobacco K326 as a template:
DS3-VIGS F:AAGGAAGTTTAATCTGAGGATGATGGAGAAGG，SEQ ID NO.3；
DS3-VIGS R:AACCACCACCACCGTTCAGTGCATCCCAGGGATCT，SEQ ID NO.4；
amplifying a specific fragment of about 300bp, and recovering a target fragment by using an Omega gel recovery kit;
(2) Carrying out enzyme digestion on a pCa-gamma bLIC vector (present by a Lidamei teacher team of China agricultural university) by using ApaI restriction enzyme (NEB, UK), wherein the reaction condition is incubation for 30min at a constant temperature of 25 ℃, and the reaction system is shown in Table 4;
TABLE 4 reaction System for enzymatic cleavage of pCa-gamma bLIC vector by ApaI endonuclease
(3) T4 DNA polymerase (NEB, UK) digests the target fragment to form a sticky end, the reaction condition is incubation for 30min at a constant temperature of 25 ℃, then reaction is carried out for 10min at a constant temperature of 75 ℃ to inactivate the enzyme, and the reaction system is shown in Table 5;
TABLE 5T4 DNA polymerase digestion of target fragment to form viscous end reaction system
(4) T4 DNA polymerase digests linearized pCa-gamma bLIC vector to form a sticky end, the reaction conditions are the same as (3), and the reaction system is shown in Table 6;
TABLE 6T4 DNA polymerase digestion of linearized pCa-gamma bLIC vector to form cohesive end reaction system
(5) Adding the digested pCa-gamma bLIC vector and the target fragment into a PCR tube according to the volume ratio of 1.
TABLE 7PCR identification reaction System
TABLE 8PCR amplification procedure
2 candidate gene VIGS vector infected tobacco K326
(1) Respectively selecting positive single colonies to be cultured in 5mL LB liquid culture medium containing 50mg/L kana and 100mg/L rif by shaking overnight at 28 ℃;
(2) Inoculating all the bacterial liquid in the step (1) to 50mL of LB liquid culture medium containing the same antibiotics in the next morning, and performing shake culture at 28 ℃ until OD is reached 600 Centrifuging at 6000r/min for 6min at about 0.6, discarding the supernatant, collecting the thallus, and suspending in 50mL of suspension, wherein the formula of the suspension is shown in Table 9;
TABLE 9 heavy suspension formula
(3) Uniformly mixing equivalent pCaBS-alpha, pCaBS-beta and pCa-gamma bLIC-target fragments (recorded as BSMV: target fragment), and taking a mixed bacterial solution of pCaBS-alpha, pCaBS-beta and pCa-gamma bLIC as a negative control (recorded as BSMV: 00);
(4) Incubating the mixed bacterial liquid at room temperature for 2-3 h, uniformly coating the mixed bacterial liquid on tobacco leaves, and vacuumizing to fully inoculate the mixed bacterial liquid on the tobacco leaves;
(5) And (3) placing the tobacco in an artificial growth chamber for culturing for 7d, extracting leaf RNA for positive identification, and using the positive material for subsequent resistance analysis.
The cultivated tobacco material is subjected to drought stress simulation by using 20% PEG6000, and after 7d, phenotype is observed, and sampling is carried out to measure physiological indexes.
We used VIGS technology to silence gene expression of NtTAS14-like1 (DS 3) in tobacco. The results of expression analysis showed that the expression level of DS3 was significantly reduced in VIGS line compared to wild-type and empty vector controls (fig. 5A). The phenotype identification result shows that the leaves of the BMSV: DS3 strain are obviously wilted after drought treatment (figure 5B) and the electric conductivity and the MDA content are both increased (figures 5C-D) compared with wild type and empty vector control, which indicates that the drought tolerance of the tobacco is reduced due to the expression silence of NtTAS14-like1, and further indicates that the gene positively regulates the drought tolerance of the tobacco.
Example 3 Gene overexpression experiments
1 overexpression vector construction
Design of primers with homology arms:
DS3-OE F:AGCTCGGTACCCGGGATGGCACAATACAACGAG，SEQ IDNO.5；
DS3-OE R:CATGTCGACTCTAGAGTGCATCCCAGGGATCTT，SEQ IDNO.6；
constructing candidate functional gene overexpression vectors and transforming agrobacterium, respectively transforming tobacco K326 by an agrobacterium-mediated method, identifying positive plants at RNA level by adopting qRT-PCR technology, and using positive materials for subsequent resistance analysis.
2 tobacco genetic transformation
(1) Transforming agrobacterium-infected LBA4404 by the vector to be transformed, culturing the monoclonal to OD by LB liquid culture medium containing corresponding antibiotic after the monoclonal grows out 600 About 0.4-0.6, and re-suspending with re-suspension solution for later use;
(2) Cutting leaves of the tobacco K326 aseptic seedlings into small pieces, soaking for 20min by using a heavy suspension liquid, and vacuumizing for 30s;
(3) Placing the infected explants on a co-culture medium (MS +1mg/L6-BA +0.5mg/LIAA +20mg/L AS) for dark culture for 48-72 h;
(4) Transferring the co-cultured explant to a screening culture medium (MS +1mg/L6-BA +0.5mg/L IAA +200mg/L TET) containing corresponding antibiotics to continuously culture and induce the generation of resistant callus, and replacing the culture medium every 15 days until seedlings are differentiated;
(5) Transferring the differentiated plantlets to a 1/2MS solid culture medium for rooting, opening the bottle mouth of a culture bottle for hardening the plantlets for 2d when the plantlets grow to be about 6cm, and then transferring the plantlets to nutrient soil for culture.
The cultivated tobacco material is subjected to drought stress simulation by 20% PEG6000, and the phenotype is observed after 7 days and sampled to measure the physiological index.
We over-expressed the NtTAS14-like1 (DS 3) gene in tobacco and selected two lines with higher expression (FIG. 6A) for drought tolerant phenotype identification. The results show that wild type K326 leaves wilted significantly after drought treatment, while DS3 over-expressed lines had less leaf wilting compared to wild type (fig. 6B). Biochemical results show that the electrical conductivity and MDA content of the leaves of the two overexpression lines are significantly lower than those of the wild type (FIGS. 6C-D), indicating that overexpression of DS3 improves the drought tolerance of tobacco, and further proving that DS3 positively regulates the drought tolerance of tobacco.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
- The application of the NtTAS14-like1 gene in regulating and controlling the drought tolerance of tobacco is characterized in that the nucleotide sequence of the NtTAS14-like1 gene is shown as SEQ ID NO:1 is shown.
- 2. The application of the protein or the substance for regulating the expression of the protein coding gene in the drought tolerance of tobacco is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:2, respectively.
- 3. Use of the NtTAS14-like1 gene of claim 1 or the protein-related biomaterial of claim 2 for drought tolerance in tobacco, wherein the biomaterial is any one of the following:a: a nucleic acid molecule having a nucleotide sequence according to claim 1 or a nucleic acid molecule encoding a protein according to claim 2;b: an expression cassette comprising silencing of the nucleic acid molecule of A;c: an expression vector containing the nucleic acid molecule of A or a recombinant vector containing the expression cassette of B;d: a recombinant microorganism comprising the nucleic acid molecule of A, or a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
- 4. A method for improving drought tolerance in tobacco, comprising increasing the drought tolerance of tobacco by overexpressing or increasing the expression of the NtTAS14-like1 gene of claim 1 or the activity of the protein of claim 2 in tobacco.
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