A gene related to drought resistance of tobacco and application thereof

Abstract

The application provides a gene related to drought resistance of tobacco and application thereof, the sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of a transcription factor translated by the gene is shown in SEQ ID NO. 2; for the application of the gene, the gene shown in SEQ ID NO. 1 or the transcription factor shown in SEQ ID NO. 2 can be applied to tobacco cultivation, and the level of the transcription factor shown in SEQ ID NO. 2 in a plant body is improved; the gene is involved in regulating drought stress response of tobacco, and can be used for cultivating new germplasm of drought-resistant plants, and has important significance for formulating scientific and effective drought-resistant cultivation measures and guaranteeing stable and high yield of tobacco.

Description

TECHNICAL FIELD

[0001] The present application relates to a gene related to tobacco drought resistance and application thereof, and belongs to the technical field of biomolecules. BACKGROUND

[0002] If the molecular biological mechanism of plant response and adaptation to drought stress can be studied, it has great application value for plant breeding. SUMMARY

[0003] In order to overcome the deficiencies of the prior art, the first object of the present application is to provide a gene related to tobacco drought resistance, which is involved in the regulation of tobacco drought stress response, and can be used for cultivating new germplasm of drought-resistant plants, which has important significance for formulating scientific and effective drought-resistant cultivation measures and ensuring stable and high yield of tobacco.

[0004] The second object of the present application is to provide an application of the gene related to tobacco drought resistance, which has important value for cultivating new varieties of high-quality tobacco.

[0005] The first object of the present application can be achieved by adopting the following technical solution: A gene related to tobacco drought resistance, the sequence of the gene is shown as SEQ ID NO. 1.

[0006] Further, the amino acid sequence of the transcription factor translated by the gene is shown as SEQ ID NO. 2.

[0007] Further, the transcription factor is expressed in the root and leaf of tobacco.

[0008] The second object of the present application can be achieved by adopting the following technical solution:

[0009] An application of the gene related to tobacco drought resistance, one is to prepare an expression vector, the expression vector is inserted with a fragment of the gene related to tobacco drought resistance; the fragment is shown as SEQ ID NO. 3.

[0010] Two is to apply the gene shown as SEQ ID NO. 1 or the transcription factor shown as SEQ ID NO. 2 to prepare a regulator for tobacco cultivation.

[0011] Three is to apply the gene shown as SEQ ID NO. 1 or the transcription factor shown as SEQ ID NO. 2 to tobacco cultivation.

[0012] Further, the tobacco cultivation is to interact the transcription factor shown as SEQ ID NO. 2 with NtMYB78 protein.

[0013] Further, the tobacco is cultivated to improve the level of the transcription factor as shown in SEQ ID NO. 2 in the plant body.

[0014] Further, the tobacco is cultivated to improve the drought resistance of the tobacco.

[0015] The third aspect can be that the gene as shown in SEQ ID NO. 1 or the transcription factor as shown in SEQ ID NO. 2 is applied to the molecular mechanism research of the drought resistance of the tobacco.

[0016] Compared with the prior art, the beneficial effects of the present application are that:

[0017] 1. The gene related to the drought resistance of the tobacco participates in the regulation of the drought stress response of the tobacco, and can be used for cultivating new germplasm of drought-resistant plants, and has important significance for formulating scientific and effective drought-resistant cultivation measures and guaranteeing stable and high yield of the tobacco.

[0018] 2. The gene related to the drought resistance of the tobacco regulates the tolerance of the tobacco plant to drought stress through the interaction with NtMYB78, and is beneficial to the in-depth research of the molecular mechanism of the drought stress tolerance of the tobacco, and provides important theoretical basis and rich gene resources for cultivating new tobacco varieties with drought resistance.

[0019] 3. The gene related to the drought resistance of the tobacco is applied to the cultivation of the tobacco, which can alleviate the serious consequences caused by drought extreme weather in the tobacco area, can promote the planting in more areas, guarantees the high yield of the tobacco, and has important value for cultivating new tobacco varieties with high quality. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a graph showing the relative expression level of the mRNA of the gene NtVOZ1 in the root tissue and the leaf tissue of the tobacco plant at the seedling stage in Example 1.

[0021] Figure 2 It is a graph showing the relative expression level of the gene NtVOZ1 in one day in Example 1.

[0022] Figure 3 It is a phenotype graph of the tobacco plant after infection at the seedling stage in Example 2.

[0023] Figure 4 It is a graph showing the expression amount of the gene NtVOZ1 in the seedling detected by reverse transcription in Example 2.

[0024] Figure 5 It is a graph showing the determination data of the relative conductivity in Example 2.

[0025] Figure 6 It is a graph showing the determination data of the relative water content in Example 2.

[0026] Figure 7Figure for subcellular localization of gene NtVOZ1 in Example 3;

[0027] Figure 8 Figure for growth of yeast in Example 3;

[0028] Figure 9 Figure for Co-IP experiment to verify interaction between NtVOZ1 and NtMYB78 in Example 4;

[0029] Figure 10 Figure for Pull-down experiment to verify interaction between NtVOZ1 and NtMYB78 in Example 5;

[0030] Figure 11 Figure for phenotype of drought stress resistance test in Example 6;

[0031] Figure 12 Figure for relative conductivity in Example 6. DETAILED DESCRIPTION

[0032] The present application is further described below in conjunction with the accompanying drawings and specific embodiments:

[0033] A gene Ntvoz1 related to drought resistance of tobacco, the sequence of the gene is shown as SEQ ID NO. 1, the amino acid sequence of the transcription factor Ntvoz1 translated from the gene is shown as SEQ ID NO. 2, and the transcription factor Ntvoz1 is expressed in the root and leaf of tobacco.

[0034] The primer pair used when amplifying the gene is:

[0035] The upstream primer NtVOZ1 F: SEQ ID NO. 4

[0036] The downstream primer NtVOZ1 R: SEQ ID NO. 5.

[0037] When breeding tobacco, overexpression of the transcription factor shown as SEQ ID NO. 2 to interact with the NtMYB78 protein can improve the drought resistance of tobacco.

[0038] Example 1:

[0039] Under normal culture conditions, wild-type tobacco (K326 tobacco seedlings) plants were cultivated to seven or eight leaves, and then the wild-type tobacco (K326 tobacco seedlings) plants were extracted for expression level determination, with three biological replicates for each tissue site; the gene Ntvoz1 is mainly expressed in the root and leaf, and is a root tissue and leaf tissue expressed gene, as shown in Figure 1

[0040] ​Under normal culture conditions, wild-type tobacco (K326 seedlings) plants were cultivated to seven or eight leaves, and then the wild-type tobacco (K326 seedlings) plants were extracted for expression level determination at a fixed point within one day at 0 h, 3 h, 6 h, and 24 h, with three biological repeats for one tissue site; the gene Ntvoz1 is mainly expressed at night and belongs to a mainly night-expressed gene, as shown in Table 1. Figure 2 The present application provides a biochemical basis for regulating the gene function of tobacco plants and the application of the gene Ntvoz1.

[0041] Example 2

[0042] Cloning of the target gene fragment: The target gene fragment was amplified using PCR technology. A sequence with a coding region length of about 300-500 bp was selected, and primers F and R were designed according to the coding region sequence analysis to amplify the coding region of the gene. The used upstream primer NtVOZ1-F is shown in SEQ ID NO. 4; the downstream primer NtVOZ 1-R is shown in SEQ ID NO. 5.

[0043] The amplification reaction program is as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 sec, 58°C annealing for 30 sec, 72°C extension for 30 sec, 34 cycles; and finally 72°C extension for 5 min.

[0044] Construction of the VIGS vector: The restriction endonuclease EcoR1 and Kpn1, the pTRV2 vector, the amplified gene fragment SEQ ID NO. 3 of SEQ ID NO. 4 and SEQ ID NO. 5 were used, and then the amplified gene fragment was inserted into the TRV2 vector by DNA recombination technology to obtain the VIGS vector, which was sequenced and verified to ensure correct vector construction.

[0045] Transformation of host cells: The universal vector pTRV1 and the constructed VIGS vector were transformed into E. coli and identified, respectively.

[0046] Virus propagation and infection: Transformation of Agrobacterium: The pTRV1 vector and the VIGS vector were extracted from E. coli and transformed into Agrobacterium GV3101.

[0047] Virus propagation: The Agrobacterium solution containing pTRV1 was resuspended in VIGS buffer to obtain mixture one, the Agrobacterium solution of the VIGS vector was resuspended in VIGS buffer to obtain mixture two, mixture one and mixture two were mixed in equal proportions, and were activated at 28°C for 3 h to obtain a virus suspension. The VIGS buffer contains 10 mmol / L MgCl2, 10 mmol / L MES, and 200 μmol / L AS, the solvent is deionized water, and the pH is 5.6.

[0048] Equal amounts of soil were weighed for seedling cultivation. One week later, the virus suspension obtained from propagation was injected into the cotyledons of two-week-old seedlings. *Nicotiana benthamiana* seedlings were used as recipient materials for gene silencing. TRV:00 (empty vector) infection of recipient materials served as a negative control, while PDS infection of recipient materials served as a reporter gene control.

[0049] After infection, the soil was allowed to fully absorb water. Ten days after infection, the plants with gene silencing (TRV:NtVOZ1) and the control group (TRV:00) were observed. Figure 3 As shown, under artificial drought stress for 7 days, it was found that the leaves of the gene-silenced material were more prone to wilting compared to the uninfected material. RNA was extracted from seedlings and reverse transcribed to detect the expression level of NtVOZ1 in the seedlings. Figure 4 As shown. Then, the relative conductivity and relative water content were measured, as follows. Figures 5-6 As shown.

[0050] Experimental results show that, under the same drought stress, plants with silenced NtVOZ1 genes exhibit more wilting behavior, and NtVOZ1-silenced plants affect the relative water content of tobacco plants, indicating that NtVOZ1 can significantly improve the drought resistance of tobacco plants.

[0051] Example 3:

[0052] Subcellular localization determined that NtVOZ1 is primarily located in the cell nucleus, such as... Figure 7 As shown.

[0053] Yeast double hybridization: The stop codons were removed from the coding sequences of the NtMYB78 and NtVOZ1 genes, and NdeⅠ and EcoRⅠ were ligated into the pGBKT7 and pGADT7 vectors, respectively, to construct the recombinant vectors pGBKT7-NtMYB78 and pGADT7. pGBKT7-53-+pGADT7-T was set as a positive control, and pGBKT7-La m+pGADT7-T as a negative control. Another negative control, pGBKT7-NtMYB78+pGADT7, was also set up. The experimental groups pGBKT7-NtMYB78+NtVOZ1 were co-transformed into Y2HGold yeast strains and plated on SD / -Leu / -Trp incubators, which were then inverted at 30℃. Colony growth was observed after 2-4 days. Single colonies were selected for shake culture until the OD600 concentration reached approximately 0.5. Then, 0.5 μL of the culture was spot-clustered onto SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade / X-α-gal+AbA+3-AT solid media, respectively, and incubated at 30℃ for 2-4 days. The growth of the yeast was then observed. Figure 8 As shown.

[0054] Example 4:

[0055] Co-IP: NtVOZ1 and NtMYB78 were amplified and cloned into modified FLAG and GFP vectors, respectively; small shake: the Agrobacterium containing different bait and capture plasmids of tagged proteins and P19 Agrobacterium were inoculated into 3-5 mL LB medium containing appropriate antibiotics, 28°C, 200 rpm, overnight culture; large shake: the bacterial liquid of overnight culture was inoculated into 10-20 mL of new LB medium containing appropriate antibiotics, 10 mmol·L –1 MES and 40 μmol·L –1 Acetyl-syringone (AS) in LB medium, 28°C, 200 rpm, overnight culture; collect bacterial liquid and discard supernatant, resuspend bacterial liquid, stand bacterial liquid, prepare bacterial liquid into injection liquid, inject tobacco, collect labeled leaves 3 days after transformation, grind tobacco leaves into powder in a pre-cooled mortar in liquid nitrogen to prepare protein crude extract, use two kinds of tagged antibodies for Western blotting detection, as shown in Figure 9 The experiment shows the interaction of tobacco genes NtVOZ1 and NtMYB78.

[0056] Example 5:

[0057] pull-down: induction and expression of soluble GST and MBP tagged proteins in E. coli: single colonies expressing GST and MBP tagged proteins were picked, 1:20 ratio of overnight culture liquid was taken, 3h detection of OD value, then IPTG was added to a final concentration of 1 mM, 37°C induction and expression for 2-4h, the bacterial liquid was collected into a centrifuge tube, the GST and MBP tagged proteins were broken and purified, and finally GST pull-down verification was performed. Western blotting detection was performed on the proteins obtained by GST pull-down experiment, as shown in Figure 10 The experiment shows the interaction of tobacco genes NtVOZ1 and NtMYB78.

[0058] Example 6:

[0059] Wild-type tobacco, i.e. Chinese tobacco main cultivar K326 (Nicotiana tobacum L. cv. K326), was used to construct NtVOZ1 overexpression strain (OE), and the specific steps were as follows:

[0060] The coding region sequence of NtVOZ1 gene is removed from the stop codon, and is connected to the plant expression vector pRI101-AN-eGFP (purchased from Beijing Bomeide Gene Technology Co., Ltd., item number CL499-01) through Nde I and EcoR I to construct the recombinant vector pRI101-AN-eGFP-NtVOZ1, and then the pRI101-AN-eGFP-NtVOZ1 recombinant plasmid is transformed into Agrobacterium LBA4404 by freeze-thaw method, and the leaf disc transformation method is used for genetic transformation.

[0061] Kan + Continue to screen by PCR method, obtain T2 generation stable strain material, select three independent overexpression transgenic lines (OE#8, #10 and #11) for subsequent experiments.

[0062] The overexpression transgenic lines (OE#8, #10 and #11) tobacco and wild type tobacco are subjected to drought stress resistance test, and when the seedlings grow to six leaf stage, the tobacco seedlings grown for about 30d are subjected to natural drought treatment for 5d. The control condition of drought stress resistance test is the overexpression transgenic lines (OE#8, #10 and #11) tobacco and wild type tobacco under normal planting condition (normal watering), and the results are shown in Figure 11 As shown in the figure, under normal planting condition, there is no obvious difference between the overexpression transgenic lines tobacco and wild type tobacco; after natural drought treatment, the overexpression transgenic lines tobacco is obviously more drought resistant than the wild type tobacco. In addition, the relative conductivity experiment is also carried out to determine the drought resistance performance between each line, and the results are shown in Figure 12 The experimental results show that overexpression of NtVOZ1 can improve the drought resistance of tobacco, indicating that NtVOZ1 plays an important role in the drought stress response of tobacco.

[0063] For those skilled in the art, other various corresponding changes and modifications can be made according to the above described technical solutions and concepts, and all of these changes and modifications should belong to the protection scope of the claims of the present application.

Claims

1. The application of a gene related to drought resistance in tobacco, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.1; the application is to prepare a reagent to improve the drought resistance of tobacco; the reagent is a reagent to overexpress the gene in tobacco, or a reagent to increase the level of the transcription factor of the gene in tobacco; the amino acid sequence of the transcription factor is shown in SEQ ID NO.

2.

2. The application of a gene related to drought resistance in tobacco, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.1; the application is to overexpress the gene in tobacco or increase the level of the transcription factor of the gene in tobacco to improve the drought resistance of tobacco; the amino acid sequence of the transcription factor is shown in SEQ ID NO.2.

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