Tobacco alanine aminotransferase gene NtALAAT2 and application thereof

Abstract

This invention discloses a tobacco alanine aminotransferase gene, NtALAAT2, and its applications, belonging to the fields of plant genetic engineering and tobacco genetic breeding technology. The nucleotide sequence of the NtALAAT2 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This invention reveals for the first time that the expression level of the endogenous NtALAAT2 gene in tobacco is negatively correlated with proline content. By reducing the expression level of this gene, the proline content in tobacco can be effectively increased, thereby enhancing the tobacco's tolerance to drought and / or soil salinization stress, without significantly adversely affecting the normal growth and development of the plant. This invention employs an endogenous gene regulation strategy, avoiding the biosafety risks of introducing exogenous genes, and provides a new gene resource and efficient technical pathway for breeding new tobacco varieties with high stress resistance.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and tobacco genetic breeding technology, specifically relating to a tobacco alanine aminotransferase gene NtALAAT2 and its application. Background Technology

[0002] Tobacco is an important leaf crop in my country, and its yield and quality directly affect the supply of high-quality raw materials for the cigarette industry and the income of tobacco farmers. Tobacco has relatively strict requirements for water and soil conditions throughout its growth cycle, especially during the vigorous growth and maturity stages. Abiotic stresses, such as drought and soil salinization, can easily lead to stunted growth and development, premature leaf senescence, and decreased photosynthetic efficiency, ultimately resulting in yield losses and deterioration of tobacco quality. In recent years, seasonal drought and secondary soil salinization have become increasingly prominent problems in my country's major tobacco-producing areas, becoming key environmental factors restricting the stable and high-quality yield of tobacco. Therefore, cultivating new tobacco varieties with high stress resistance is an urgent need to ensure the sustainable development of the tobacco industry. Research shows that proline, as one of the most important osmotic regulators in plants, has a significantly positive correlation between its accumulation level and the plant's tolerance to abiotic stresses such as drought and high salinity. Therefore, improving the proline accumulation capacity of tobacco through genetic engineering, and thus cultivating new tobacco varieties with high stress resistance, is an effective technical path to ensure the sustainable development of the tobacco industry.

[0003] Traditional tobacco stress resistance breeding mainly relies on conventional hybridization and phenotypic screening, which has inherent limitations such as long cycles, low efficiency, and difficulty in breaking the linkage between desirable and undesirable traits. With the development of molecular biology, the targeted improvement of tobacco stress resistance through genetic engineering has become an important technical approach. Currently, genetic engineering research on tobacco stress resistance largely relies on the introduction of exogenous stress resistance genes, such as transcription factor genes and osmotic regulator synthase genes. However, such strategies may raise biosafety controversies, and the expression stability of exogenous genes in tobacco and their compatibility with the endogenous metabolic network are highly uncertain, making it difficult to adapt to the complex genomic characteristics of tobacco allotetraploids.

[0004] More importantly, cultivated tobacco is an allotetraploid plant formed through natural hybridization, and its genome has undergone multiple expansions, with almost all genes existing in multiple copies. This complex genomic structure endows tobacco with a unique lineage of secondary metabolites and a highly redundant metabolic regulatory network. Stress-resistance genes and regulatory strategies validated in diploid crops or other model plants cannot be directly transferred or simply applied to tobacco. Therefore, identifying tobacco's own endogenous stress-resistance functional genes and achieving targeted improvement of stress resistance through the regulation of endogenous gene expression is the optimal technical approach to avoid the biosafety risks of heterologous genes and adapt to the unique genomic characteristics of tobacco. It is also one of the core directions of current tobacco stress-resistance genetic engineering research.

[0005] To address the above problems, this invention is proposed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a technical solution that enhances tobacco stress resistance (drought resistance, salinity tolerance) by regulating the expression of an endogenous gene to increase the proline content in tobacco. This avoids the biosafety risks and compatibility issues associated with the introduction of exogenous genes. Simultaneously, it provides the corresponding gene, protein, vector, host cell, and method for cultivating new tobacco varieties with high stress resistance. This solves the technical problems of low efficiency in traditional breeding and poor compatibility of existing genetic engineering strategies, providing new gene resources and technical pathways for tobacco stress resistance genetic breeding.

[0007] The present invention solves the technical problem by adopting the following technical solution:

[0008] The first aspect of this invention provides a tobacco alanine aminotransferase gene NtALAAT2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] A second aspect of the present invention provides a tobacco alanine aminotransferase NtALAAT2 protein encoded by the gene described in the first aspect, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising the tobacco alanine aminotransferase gene NtALAAT2 described in the first aspect.

[0011] A fourth aspect of the present invention provides a recombinant host cell comprising the recombinant expression vector described in the third aspect.

[0012] A fifth aspect of this invention provides the application of the gene described in the first aspect, the protein described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect in increasing the proline content in tobacco. Further, when the application involves the gene or the protein, the application increases the proline content in tobacco by reducing the expression level of the gene or protein in tobacco.

[0013] A sixth aspect of the present invention provides the application of the gene described in the first aspect, the protein described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect in improving the stress resistance of tobacco. Further, the stress resistance refers to tolerance to drought stress and / or soil salinization stress; when the application involves the gene or the protein, the application is achieved by reducing the expression level of the gene or protein in tobacco.

[0014] The seventh aspect of this invention provides a method for cultivating new tobacco varieties with high stress resistance, comprising: reducing the expression level of the tobacco alanine aminotransferase gene NtALAAT2 as described in claim 1 in tobacco through gene silencing technology, and screening for tobacco plants with improved stress resistance.

[0015] The gene silencing technology refers to techniques that reduce the expression level of a target gene by specifically degrading its mRNA or inhibiting its transcription. This includes, but is not limited to, virus-induced gene silencing, RNA interference, and genome editing. In this invention, by reducing the expression level of the NtALAAT2 gene, its negative regulatory effect on proline synthesis is relieved, thereby increasing the proline content in tobacco and enhancing its stress resistance.

[0016] Furthermore, the gene silencing technology is a virus-induced gene silencing technology; the virus-induced gene silencing technology uses a tobacco brittle virus vector, the vector carrying a specific silencing fragment of the gene according to claim 1, the nucleotide sequence of the specific silencing fragment being shown as bases 172 to 526 in SEQ ID NO.1.

[0017] The present invention has the following beneficial effects:

[0018] 1. This invention uses the tobacco's own endogenous gene NtALAAT2 as the core, and improves the stress resistance of tobacco by regulating the expression of this endogenous gene. It does not require the introduction of exogenous stress resistance genes, fundamentally solving the biosafety controversies that may be caused by existing exogenous gene introduction strategies. At the same time, it is adapted to the complex genomic structure and redundant metabolic regulatory network of tobacco allotetraploids, avoiding the problems of unstable expression of exogenous genes in tobacco and poor compatibility with endogenous metabolic networks, and significantly improving the stability of the technology.

[0019] 2. This invention is the first to clearly define the direct association between the endogenous NtALAAT2 gene in tobacco and the proline content and stress resistance (drought resistance and salinity tolerance) in tobacco. It was found that by reducing the expression level of this gene and its encoded protein, the proline content in tobacco can be effectively increased, thereby enhancing the tolerance of tobacco to drought stress and soil salinity stress. The specific regulatory methods and technical pathways were also clarified. All technical solutions have clear parameter limitations (such as the silenced fragment sequence and the type of gene silencing technology). Those skilled in the art can repeat the implementation according to the description of this invention, and the technical effects are stable and controllable.

[0020] 3. This invention not only provides the NtALAAT2 gene with a clearly defined function and its encoded protein, but also provides a recombinant expression vector containing the gene, a recombinant host cell, and application methods and high-stress-resistant tobacco cultivation methods based on the gene. This forms a complete technical system from gene resources to practical applications, which can be directly applied to tobacco stress resistance genetic breeding practices. It effectively solves the technical problems of long cycle, low efficiency, and difficulty in breaking the linkage between good and bad traits in traditional hybridization breeding, and provides new gene resources and efficient technical paths for tobacco stress resistance breeding.

[0021] 4. The virus-induced gene silencing technology used in this invention is a mature technology in the field of plant genetic engineering. The operation process is standardized and does not require complex equipment and processes. The tobacco brittle virus vector used is easy to obtain, simple to construct, and has controllable cultivation costs. It is suitable for large-scale application and promotion, and can quickly cultivate new tobacco varieties with high stress resistance, helping the tobacco industry to cope with environmental stresses such as drought and salinization, ensuring high-quality and stable tobacco production, and increasing tobacco farmers' income.

[0022] 5. This invention is the first to discover and apply the tobacco endogenous NtALAAT2 gene to stress resistance breeding, breaking the existing technology's dependence on stress resistance genes in diploid crops, filling the technological gap in applying the tobacco endogenous alanine aminotransferase gene to stress resistance improvement, enriching the gene resources for tobacco stress resistance breeding, and providing reference and guidance for the subsequent discovery and application of tobacco stress resistance-related genes. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a phenotypic comparison diagram of tobacco plants transformed with TRV2-PDS, TRV2-GFP and TRV2-NtALAAT2 vectors in the embodiments of the present invention.

[0025] Figure 2 This is a comparison diagram of the relative expression levels of the NtALAAT2 gene in plants with silenced NtALAAT2 and control plants in an embodiment of the present invention.

[0026] Figure 3 This is a comparison chart of proline content in tobacco leaves with NtALAAT2 gene silencing and control tobacco leaves in an embodiment of the present invention. Detailed Implementation

[0027] The present invention will be further described below through embodiments, but is not limited to these embodiments. Experimental methods not specifically described in the embodiments generally use conventional conditions and conditions described in manuals, or conditions recommended by the manufacturer. The general equipment, materials, reagents, etc., used are all commercially available unless otherwise specified.

[0028] Biomaterials:

[0029] Nicotiana benthamiana, a commonly used tobacco model plant, was used in the VIGS silencing experiment described below. Nicotiana benthamiana was cultivated in the greenhouse of Zhengzhou Tobacco Research Institute, with seedlings raised in seedling pots. Two weeks after germination, the seedlings were transplanted and planted in plastic pots (10cm × 10cm). Routine fertilization and watering were carried out at 22℃ under 16h light / 8h darkness conditions. The tobacco variety K326 was used for total RNA extraction and cloning of the NtALAAT2 gene.

[0030] The VIGS vector used in the following examples is a viral vector derived from tobacco brittle virus (TRV). The specific TRV2 used was preserved by the Zhengzhou Tobacco Research Institute Gene Center. It carries a kanamycin selection marker and a 35S promoter. TRV2 also carries multiple cloning sites such as EcoRI and BamHI, which can be used to carry and transform exogenous genes.

[0031] The Agrobacterium strain was GV3101.

[0032] Main reagents:

[0033] LB liquid medium, 1L contains: 10g bacterial peptone; 10g sodium chloride (NaCl); 5g yeast extract, autoclaved;

[0034] YEB liquid culture medium, 1L contains: 5g beef extract; 5g bacterial peptone; 5g sucrose; 1g yeast extract; 2mL 1M magnesium sulfate (MgSO4), autoclaved;

[0035] 1M 2-(N-morpholine) ethanesulfonic acid (MES) stock solution: Dissolve in ddH2O, filter and sterilize, store at -20℃ for later use;

[0036] 200mM Acetosyringone (As) stock solution: Dissolve in dimethyl sulfoxide (DMSO) and store at -20°C for later use;

[0037] MMA buffer (100 mL): 1 mL (1 M) MgCl2; 1 mL (1 M, pH 5.6) MES; 75 μL (200 mM) As.

[0038] Example 1

[0039] This embodiment describes the cloning of the tobacco NtALAAT2 gene and the construction of a silencing vector. The specific steps are as follows:

[0040] 1. Cloning of a tobacco NtALAAT2 gene-specific fragment

[0041] PCR amplification primers were designed based on the tobacco genome sequence. The primer sequences are as follows:

[0042] NtALAAT2-F: 5'-TCCGATTATTCATCTACCCC-3' (e.g., SEQ ID NO. 3)

[0043] (as shown)

[0044] NtALAAT2-R: 5'-CGCACTGAACAGACCTTGTG-3' (e.g., SEQ ID NO.4)

[0045] (As shown).

[0046] PCR amplification was performed using cDNA from tobacco K326 leaves as a template. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for 34 cycles, followed by a final extension at 72℃ for 5 min. The PCR amplification products were detected by agarose gel electrophoresis, and the target fragment was recovered. Sequencing confirmed that the amplified fragment was a specific fragment of the NtALAAT2 gene, and its nucleotide sequence is shown as bases 172 to 526 in SEQ ID NO.1.

[0047] 2. Construction of the recombinant TRV2-NtALAAT2 silencing vector

[0048] The PCR amplification products were digested with EcoRI and BamHI, and the empty vector TRV2 was also digested with EcoRI and BamHI. The digested products were recovered and ligated using T4 DNA ligase. The ligation products were transformed into competent E. coli DH5α cells, plated on LB agar containing 50 mg / L kanamycin, and incubated overnight at 37°C. Positive single colonies were selected for PCR identification and sequencing verification to ensure the correct recombinant vector TRV2-NtALAAT2 was constructed. This vector carries a specific silenced fragment of the NtALAAT2 gene, the nucleotide sequence of which is shown at bases 172 to 526 in SEQ ID NO. 1.

[0049] Meanwhile, TRV2-GFP vector control and TRV2-PDS VIGS efficiency control recombinant vectors were constructed according to the above method.

[0050] The TRV2-GFP vector, carrying a fragment of the green fluorescent protein (GFP) gene, served as a negative control. GFP emits green fluorescence under ultraviolet or blue light excitation. By observing the fluorescence signal, one can directly determine whether the TRV virus has successfully infected tobacco plants and the extent of viral spread within the plant, thus verifying the infection efficiency. Simultaneously, GFP is an exogenous reporter gene, unrelated to the function of endogenous genes in tobacco; using it as a control eliminates the non-specific influence of the TRV virus vector itself and the Agrobacterium infection process on the experimental results.

[0051] The TRV2-PDS vector carries a fragment of the phytopene dehydrogenase (PDS) gene and serves as a positive control to verify the effectiveness of the VIGS silencing system. When the PDS gene is silenced, the phytopene dehydrogenase it encodes cannot be synthesized normally, resulting in the plant's inability to accumulate carotenoids. New leaves exhibit photobleaching due to the lack of photoprotective pigments, thus providing a direct indication of whether the silencing system is functioning properly.

[0052] Example 2

[0053] This embodiment performs Agrobacterium-mediated transient transformation of VIGS and phenotypic analysis, as detailed below:

[0054] 1. Agrobacterium-mediated transformation

[0055] Recombinant plasmids TRV2-GFP, TRV2-PDS, and TRV2-NtALAAT2 were introduced into Agrobacterium GV3101 competent cells via electroporation. The cells were then plated on YEB plates containing 50 mg / L kanamycin and 50 mg / L rifampin and incubated upside down at 28°C for 2 days. Positive clones were screened by colony PCR.

[0056] 2. Preparation of bacterial suspension for infection

[0057] Select a single positive Agrobacterium colony and inoculate it into 5 mL of YEB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin), and incubate overnight at 28°C and 250 rpm. Take 50 μL of the overnight culture and inoculate it into 50 mL of YEB liquid medium (containing 50 mg / L kanamycin), and incubate until OD500. 600 =1.0~1.5. Centrifuge at 4000g for 5 min to collect bacterial cells, resuspend in MMA buffer, and adjust OD. 600 ≈1.0. After standing at room temperature for 3 hours, it is used as the bacterial solution for infection.

[0058] 3. Instantaneous transformation

[0059] Using 3-4 week old Nicotiana benthamiana seedlings as material, the infecting bacterial solution was injected into the underside of tobacco leaves using a 1 mL syringe. The injected tobacco plants were then cultured in an artificial incubator, and phenotypic changes were observed.

[0060] 4. Phenotypic observation

[0061] The phenotype of tobacco plants was observed approximately 3 weeks after injection.

[0062] Under natural light conditions, the newly emerging leaves of TRV2-PDS-infected plants showed obvious bleaching, indicating that the VIGS silencing system was functioning normally; neither TRV2-GFP-infected nor TRV2-NtALAAT2-infected plants showed obvious phenotypic abnormalities under natural light conditions. Figure 1 ).

[0063] Under ultraviolet light (or blue light excitation source), the new leaves and infected parts of TRV2-GFP infected plants showed green fluorescence, indicating that the TRV virus had successfully infected and spread systematically in the plant, with good infection efficiency; TRV2-NtALAAT2 infected plants also did not show abnormal fluorescence phenotype under fluorescence.

[0064] The above results indicate that silencing the NtALAAT2 gene has no significant adverse effects on the normal growth, development, and basic physiological state of tobacco plants.

[0065] Example 3

[0066] In this embodiment, the expression level of the NtALAAT2 gene and the proline content were detected, as detailed below:

[0067] 1. Detection of NtALAAT2 gene expression level

[0068] Leaves were collected from TRV2-NtALAAT2-infected plants and TRV2-GFP control plants. Total RNA was extracted and reverse transcribed into cDNA. Using a tobacco internal reference gene as a control, the relative expression level of the NtALAAT2 gene was detected by qRT-PCR. Results are as follows: Figure 2 As shown, compared with the control plants, the expression level of the NtALAAT2 gene in TRV2-NtALAAT2-infected plants was significantly reduced, by about 60%.

[0069] 2. Proline content detection

[0070] Leaves were collected from the above-mentioned plants, and the free proline content was determined using the ninhydrin colorimetric method. The results are as follows: Figure 3 As shown, compared with the control plants, the proline content in NtALAAT2 gene-silenced plants decreased by 70%.

[0071] The above results indicate that the expression level of the NtALAAT2 gene is negatively correlated with the proline content in tobacco, and that reducing the expression level of the NtALAAT2 gene can effectively increase the proline content in tobacco.

[0072] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

[0073] sequence list

[0074] SEQ ID NO.1

[0075] atgcggagat tcgtagctga aaaagccaaa aatctcatca ctaaaaccag gaccacctat 60

[0076] tcttctccta cttctactta cgtgttacaa cgccacacct cttccccatt accctcacct 120

[0077] tctcaatccg cttctgttct tcgttttttg tccactcttc acacagctcc ttctgattca 180

[0078] atggcttccg attattcatc taccccggtg actcttaaca acgttaatcc aaaggtattc 240

[0079] cagctgctgt tatacttttg aatttgtttt ttctgttttt gcccttattc acttgcgttt 300

[0080] atttttttta gctcgaccca atttaatgat tttcaattgt ttcgtagtaa ttgatatctt 360

[0081] cgcgaataca tttaattagt tttcagtgta aaaataatgg tttatccttt ttgcttttca 420

[0082] ctaatgctat atattcggtt cttggttata attcctctat tttggtccaa ctaccaattc 480

[0083] aatgttttat ttgtctgttt ttttaatggt taatatcact agttctagac cactttaatt 540

[0084] aacctgatag taactctatg tgaacctatt tctatttctt ttttgatctg ttccatagag 600

[0085] aatgatccct ttttaaattt gaaaacaatt tagtttaaac ttataatttt accttgagaa 660

[0086] acttttataa tggttaacat aaattggaac tgagggagta gctagtaatg tcaaaaaatt 720

[0087] tctactgcta gtgaatgtga cgtctttttt aatatctttc caaaagtagg atctttttat 780

[0088] gtatttgccc tgtctgatgt aggattttaa tgttgtttct agagtcaaat cacttgatgt 840

[0089] agttcaactt attgttggtt tcaggttttg aaatgtgagt atgctgttcg tggagaaatt 900

[0090] gtcaaccttg ctcaggtagt tccggaactc gatttcacac actagctaga attaacaatc 960

[0091] ggaatttcag aatcttgtta gttgcacttg cacaattagt ttttgggcat gcttaatctt1020

[0092] acaaattaaa taaaaattgt attgatggtg tcctgtgcga tgcatcatca tgcagaaatt1080

[0093] acagcaagac ctcaaggaga atccaggctc tcatcccttt gacgaggtac caactcttgt1140

[0094] ggacttggtt ttttattttc atttaagttg taatattgca tatcataacg aatttatctt1200

[0095] ctttcccctt gtaacaaaga gtaatatagg tgacctggag tgttattgtt gtcttgatct1260

[0096] tttctttact cattcctcta atttatgcag atcttatact gcaatattgg aaatcctcaa1320

[0097] tccctggccc agcagcctat tactttcttt agggaggtag gataaattgt ttgattctag1380

[0098] attatgttac ttgtctggtc gagcaatgtc taaggtggat tcctgtattt aggtccttgc1440

[0099] attatgtgac catccagtta ttttggacaa aagtgaaaca caaggtctgt tcaggtatga1500

[0100] agtttgtcta tcaacctcac tgcaccttgt agcgcaatga aacaattcgc gtgccttagt1560

[0101] ttccagagat gtctgaagaa gtggaattaa catgactaat tttcataaaa caaatgtttt1620

[0102] cccctgtatt tttcaaacat ggcacagtta aattttctgc tactgatggg agctttcgtc1680

[0103] cttgctgttg tatagtaat aatttgcatc agaattttaa tgatattat ctttttaatg1740

[0104] ttttgaagtc caagtccaac atataaaacc aagtgattgt tattgtaatt gatgcaagtt1800

[0105] agaaaaagtg aactatgaga gtgatgactt tattctattt catgtcatct tgacatatca1860

[0106] agtaatgttt ttgaggcctg agaaaagtga aatgtattac tttaagctaa aagatctata1920

[0107] gaatgtggaa ggttgtgatg attgagaagc tctaagttaa tatatttgat ggtgaacact1980

[0108] ttgagataat tgatgactct gttttattag cacctactgc ctgtgggtat tgtttcagtt2040

[0109] ttagcttcaa actgtataca tatcatctgg tatttctaaa aaatgcaagt ggctatgatc2100

[0110] aaactcttac tttttcttta ttctgtgaaa tcagtgcaat gcagtgttat tattgaatta2160

[0111] tagtcattca cttttgttt tcaaaatctt gtatgtttta gttgataggt tctttctttg2220

[0112] agcttttcct tttcctaaat accatggtat tgattatgac tttacaccca agtagaagca2280

[0113] aaaacatttt gcccggatat ttgttggaaa catcggtcac caatgtaaag gagtatggtg2340

[0114] aagaatttga atcaacttca gccatgttct acaggatttt atggtctaaa tggcattgtc2400

[0115] ctgctaccta agataaggaa aagaagattt ctgaactacg tctcatagaa acaatcagaa2460

[0116] actacgagaa attgttatt gatgtctttc attctctctt tgctttgtag tgcggattca2520

[0117] atagaacgag ctttccagat cctcgaccaa attcctggca gagcgacagg tgcatacagc2580

[0118] cacagtcagg tgactatatc ttctattgca atgtcctaga tttgcagcca gtatactcat2640

[0119] agaacttatt tttgtgtttg ctaaatctag ggcatcaaag gattacgtga tacaattgct2700

[0120] tctggtatcg aagctcgtga tggcttccct gctgatccaa atgatatttt cttgactgat2760

[0121] ggtgcaagcc cagcggtgaa ttattttgat ctcaaatagt tattcgtaca gctttgagag2820

[0122] aagtaactaa aagaatagca aggtgtagat aatgttggtt cttcatctct attcaggtc2880

[0123] acatgatgat gcagttgctc attgggtcgg agaatgatgg aattctctgt cccatcccccc2940

[0124] aatatccact ttatccgct tcaattgcc tccatggtgg cactcttgta cgtgtctttg3000

[0125] cttcaattta aagctgatca gtgctagttg tatagttgac cttgaatgca actattagat3060

[0126] cttttaatta aataattaat taataggcta ggttaaccta cttctggaag agattatgt3120

[0127] aaacttttat taatgagagc ttacctgatt gacttactat cagttgaagg tgatccacac3180

[0128] aggagcctct tatgtatgct tttatctggt attttaggtt ccctattatc ttgatgaaga3240

[0129] aacaggatgg ggactggaga tctcagagct tgagaatcag ctgaaaactg caaaatccaa3300

[0130] gggtattaaa gttagggctt tggttgtgat caatccaggc aacccaactg ggcaggtaat3360

[0131] ggtttattgg tttattagat tttggaatct catcattgat ttacgtcatt taggaagaac3420

[0132] ctactgtggt ggaaaatgct ggtcttccct ttcccaataa tatttcagaa tgtttacatg3480

[0133] tagcatgaat agcttctatg tataccttag gttcttgctg aggccaacca acgagatatt3540

[0134] gtggaattct gcaagaaaga aggcctcgtt cttctggcgg atgaagtgag tttatttccc3600

[0135] tctagatgct cttctttgtt tgattctggc aaattatttt ccttccacgg gaatttacca3660

[0136] gtgattctgc acttgtttga ggggggtcat aattttttca ttcctgagta ttgtgcaact3720

[0137] aattctaaaa ttgtaagtcc tctcatatct tgtggaactc atgcaggtgt atcaggaaaa3780

[0138] tgtttatgtg cctgagaagc agttccactc gtttaagaaa attgctcgct ctatgggatt3840

[0139] tggagaaaag gacatctctt tagtttcttt tcagtctgtg tcaaaaggta gattgtacta3900

[0140] cctccatcca tccctctctt gttggtggtc tttctcattc gtttgtctgc atcttacaaa3960

[0141] gttgtggggg agaaggggga aaggggcgag ttttcattt tcacaaaagc ttctgattta4020

[0142] ttatatcttt taatccttgg cttttgctta cttcccttac tggctttagg attttatgga4080

[0143] gagtgtggaa agcgaggagg ttacatggag gtcactggat ttagccctga gataagggaa4140

[0144] cagatataca aagtggcatc tgtcaatctg tgttccaaca tttctggtca gattcttgca4200

[0145] agcctggtca tgagccctcc aaaggttggt aatgctgttt ctgtaataca tggcattgca4260

[0146] ttccccaaaa ggagtaaaat tgatattgag acgaccccag atagcttggg attgaggtgc4320

[0147] agctattaat aggagtgggg tttccaccct cattggtcga atattttgg tagtggtagt4380

[0148] tatagaatct ataaccagaa cgagtacctc aatggtttaa tttatgtagg tgtatcatac4440

[0149] tctttaatgt ggtgagctaa acattagatg tcacaacaat gatatgcact cagctctgac4500

[0150] tataaactta gaagcttgtt gtgaggttcc ttgtatctgt gttattgatg cttgactaac4560

[0151] aggactgctc tattgttttc tgcaggtggg tgatgaatca tacgagtctt ttagtgctga4620

[0152] gaaagaagct atactctcat ccttggcaag gcgtgcaaag gtcagtagca tcagcacata4680

[0153] caactagtgg tgactaaata ataaactagt ttgttttggt gaagtagtgg gagttgtttt4740

[0154] tgtatcatga accttgcatc tgaaaaaaga agagccggtt aaagtaggaa gcagaaatgg4800

[0155] tgcctgaaat tttctccaaa aaaaggttgt cacaaaataa tatcctctag agtttcattt4860

[0156] cagcttttca aaaatggtat atccaaattt tttgcttcct gttgctgaat tttatctgta4920

[0157] caaatttggt gaattttatc tgtccaattt gtacattttc tattcttctg tgtttggcaa4980

[0158] ttttttttga ctaatttgtc aacagacact agaagatgca ttcaacagtt tggagggaat5040

[0159] aacatgcaat aaagcggagg gcgcaatgta tctatttcca cgtattaact tacctgctaa5100

[0160] agcaataaa gcagcagaag aagctaaaac tgcaccagat gccttttatg ctcgacgcct5

[0161] cctcaatgcc actggaattg ttgttgtccc aggctctgga tttggccagg tgattcttat5220

[0162] atttttgaca acttgcttca tgctattcct agtattaa tagttggtaa ctgacaccac5

[0163] taatttcgaa aaatgcacat ggcttttacc tggatgagta aaatgtcaaca gacagaccta5

[0164] aatagcatt tcctgaacag tttgaattc tctaccttca gaggaaaa aaaggtctct5

[0165] accttcagaa gggttcaagg aaaatgaat tgtaaaaaga aattgggga acaaatttg5

[0166] gaggattgtt tcaatagaaa agaagcaact atatttgctg tcaaggttag tctgattcaa5

[0167] gaaacgttcc ttttgtcaag atgctgatta gtttgcatac ccggtaggca ttaaaatttc5

[0168] attctattat attgcaatct gagatgctag atgccgacat aggagaga aggatgcagt5640

[0169] actgaatagt atattctaaa taatgagagg gtaatgattt ttggtgcaaa agaatcactt5

[0170] tatcaataga aaaaattaaa taaaaaatga aacactttac catatagagaa gaaactgttt5760

[0171] attgtttgaa aactaagggt atttgttaca ctactgttta ttgagaactg ttggtattta5820

[0172] ctaaatctgt tgtatttgct tgttgcttcc gtgaagcgtc ctggaacgtg gcatttataga5880

[0173] tgcacaatat taccgcaaga agagaagata ccatctattg tatctcgtct tacagatttc5940

[0174] cataagcagt tcatggatga attccgcgac taa 5973

[0175] SEQ ID NO.2

[0176] MRRFVAEKAK NLITKTRTTY SSPTSTYVLQ RHTSSPLPSP SQSASVLRFL STLHTAPSDS 60

[0177] MASDYSSTPV TLNNVNPKVL KCEYAVRGEI VNLAQKLQQD LKENPGSHPF DEILYCNIGN 120

[0178] PQSLAQQPIT FFREVLALCD HPVILDKSET QGLFSADSIE RAFQILDQIP GRATGAYSHS 180

[0179] QGIKGLRDTI ASGIEARDGF PADPNDIFLT DGASPAVHMM MQLLIGSEND GILCPIPQYP 240

[0180] LYSASIALHG GTLVPYYLDE ETGWGLEISE LENQLKTAKS KGIKVRALVV INPGNPTGQV 300

[0181] LAEANQRDIV EFCKKEGLVL LADEVYQENV YVPEKQFHSF KKIARSMGFG EKDISLVSFQ360

[0182] SVSKGFYGEC GKRGGYMEVT GFSPEIREQI YKVASVNLCS NISGQILASL VMSPPKVGDE 420

[0183] SYESFSAEKE AILSSLARRA KTLEDAFNSL EGITCNKAEG AMYLFPRINL PAKAIKAAEE 480

[0184] AKTAPDAFYA RRLLNATGIV VVPGSGFGQR PGTWHFRCTI LPQEEKIPSI VSRLTDFHKQ 540

[0185] FMDEFRD 547

[0186] SEQ ID NO.3

[0187] tccgattatt catctacccc 20

[0188] SEQ ID NO.4

[0189] CGCACTGAAC AGACCTTGTG 20。

Claims

1. A tobacco alanine aminotransferase gene NtALAAT2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A tobacco alanine aminotransferase NtALAAT2 protein encoded by the gene of claim 1, characterized by, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It contains the tobacco alanine aminotransferase gene NtALAAT2 as described in claim 1.

4. A recombinant host cell, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The application of the tobacco alanine aminotransferase gene NtALAAT2 according to claim 1, the protein according to claim 2, the recombinant expression vector according to claim 3, or the recombinant host cell according to claim 4 in increasing the proline content in tobacco.

6. The application according to claim 5, characterized in that, When the application involves the gene of claim 1 or the protein of claim 2, the application increases the proline content in tobacco by reducing the expression level of the gene or protein in tobacco.

7. The application of the tobacco alanine aminotransferase gene NtALAAT2 according to claim 1, the protein according to claim 2, the recombinant expression vector according to claim 3, or the recombinant host cell according to claim 4 in improving tobacco stress resistance.

8. The application according to claim 7, characterized in that, The stress resistance refers to the ability to tolerate drought stress and / or soil salinization stress; when the application involves the gene of claim 1 or the protein of claim 2, the application is achieved by reducing the expression level of the gene or protein in tobacco.

9. A method for breeding new tobacco varieties with high stress resistance, characterized in that, include: By reducing the expression level of the tobacco alanine aminotransferase gene NtALAAT2 as described in claim 1 in tobacco using gene silencing technology, tobacco plants with improved stress resistance were screened.

10. The method according to claim 9, characterized in that, The gene silencing technology is a virus-induced gene silencing technology; the virus-induced gene silencing technology uses a tobacco brittle virus vector, the vector carrying a specific silencing fragment of the gene according to claim 1, the nucleotide sequence of the specific silencing fragment being shown as bases 172 to 526 in SEQ ID NO.1.

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