Gene related to drought resistance of tobacco and application thereof

Abstract

The invention provides a gene related to drought resistance of tobacco and application of the gene. The sequence of the gene is shown as SE Q ID NO.1; an amino acid sequence of a transcription factor translated by the gene is as shown in SEQ ID NO. 2; according to the application of the gene, the gene as shown in SEQ ID NO.1 or a transcription factor as shown in SEQ ID NO.2 can be applied to tobacco cultivation, and the level of the transcription factor as shown in SEQ ID NO.2 in a plant body is improved; the gene participates in regulation and control of tobacco drought stress response, 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 high yield of tobacco.

Description

Technical Field

[0001] The present invention relates to a gene related to tobacco drought resistance and application thereof, belonging to the technical field of biomolecules. Background Art

[0002] If we can study the molecular biological mechanisms of plant response and adaptation to drought stress, it will have great application value for plant breeding. Summary of the Invention

[0003] In order to overcome the shortcomings of the existing technology, the first purpose of the present invention is to provide a gene related to tobacco drought resistance, which is involved in regulating tobacco drought stress response and can be used to cultivate new drought-resistant plant germplasm. It is of great significance for formulating scientific and effective drought-resistant cultivation measures and ensuring stable and high tobacco yields.

[0004] The second purpose of the present invention is to provide an application of the above-mentioned tobacco drought resistance-related genes, which is of great value in breeding new high-quality tobacco varieties.

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

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

[0007] Furthermore, the transcription factor was expressed in roots and leaves of tobacco.

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

[0009] An application of a gene related to tobacco drought resistance includes preparing an expression vector, wherein a fragment of the gene related to tobacco drought resistance is inserted into the expression vector; the fragment is shown in SEQ ID NO.3.

[0010] The second method is to use the gene shown in SEQ ID NO. 1 or the transcription factor shown in SEQ ID NO. 2 to prepare a regulator for tobacco cultivation.

[0011] The third method may be to apply the gene shown in SEQ ID NO.1 or the transcription factor shown in SEQ ID NO.2 to tobacco cultivation.

[0012] Furthermore, tobacco is bred so that the transcription factor shown in SEQ ID NO. 2 interacts with the NtMYB78 protein.

[0013] Furthermore, tobacco is bred to increase the level of the transcription factor shown in SEQ ID NO. 2 in the plant.

[0014] Furthermore, tobacco is bred to improve its drought resistance.

[0015] The third method may be to apply the gene shown in SEQ ID NO.1 or the transcription factor shown in SEQ ID NO.2 to the study of the molecular mechanism of tobacco drought resistance.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The tobacco drought resistance-related genes of the present invention participate in regulating tobacco drought stress response and can be used to cultivate new drought-resistant plant germplasm. This is of great significance for formulating scientific and effective drought-resistant cultivation measures and ensuring stable and high tobacco yields.

[0018] 2. The tobacco drought resistance-related genes of the present invention regulate the tolerance of tobacco plants to drought stress through interaction with NtMYB78; this facilitates in-depth research on the molecular mechanism of tobacco drought stress resistance and provides an important theoretical basis and rich gene resources for the breeding of new drought-tolerant tobacco varieties;

[0019] 3. The tobacco drought resistance-related genes of the present invention are applied to tobacco cultivation. On the one hand, they can alleviate the serious consequences of drought and extreme weather in tobacco-growing areas. On the other hand, they can be promoted for cultivation in more areas, providing a guarantee for high tobacco yields, and are of great value in cultivating new high-quality tobacco varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graphical representation of the relative mRNA expression levels of the gene NtVOZ1 in Example 1 in root and leaf tissues of tobacco plants at the seedling stage;

[0021] Figure 2 This is a graphical representation of the relative expression levels of the gene NtVOZ1 in Example 1 over a day;

[0022] Figure 3 This is a phenotypic diagram of the infected tobacco plant at the seedling stage in Example 2;

[0023] Figure 4 This is a graphic representation of the expression level of the gene NtVOZ1 in seedlings detected by reverse transcription in Example 2;

[0024] Figure 5 This is a graph showing the measurement data of relative conductivity of Example 2;

[0025] Figure 6 This is the measurement data diagram of the relative water content of Example 2;

[0026] Figure 7This is the subcellular localization map of the gene NtVOZ1 in Example 3;

[0027] Figure 8 This is a diagram showing the growth of yeast in Example 3;

[0028] Figure 9 This is a diagram illustrating the interaction between NtVOZ1 and NtMYB78 verified by the Co-IP experiment in Example 4;

[0029] Figure 10 This is a diagram illustrating the pull-down experiment in Example 5 verifying the interaction between NtVOZ1 and NtMYB78;

[0030] Figure 11 This is the phenotypic diagram of the drought stress resistance test in Example 6;

[0031] Figure 12 This is a graphical representation of the relative conductivity in Example 6. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0033] A gene Ntvoz1 related to tobacco drought resistance has a gene sequence shown in SEQ ID NO.1. The amino acid sequence of the transcription factor Ntvoz1 translated from the gene is shown in SEQ ID NO.2. The transcription factor Ntvoz1 is expressed in the roots and leaves of tobacco.

[0034] When amplifying genes, the primer pairs used are:

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

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

[0037] During tobacco breeding, the drought resistance of tobacco can be improved by overexpressing the transcription factor shown in SEQ ID NO.2 and causing it to interact with the NtMYB78 protein.

[0038] Example 1:

[0039] Under normal culture conditions, wild-type tobacco (K326 tobacco seedlings) plants were grown to seven or eight leaves, and then the expression levels of wild-type tobacco (K326 tobacco seedlings) plants were extracted for determination, with three biological replicates for each tissue location. The gene Ntvoz1 was mainly expressed in roots and leaves, and was a root and leaf tissue expression gene. Figure 1 shown.

[0040] Under normal culture conditions, wild-type tobacco (K326 tobacco seedlings) plants were grown to seven or eight leaves, and the expression levels of wild-type tobacco (K326 tobacco seedlings) plants were measured at 0h, 3h, 6h, and 24h in a day, with three biological replicates for each tissue site. The gene Ntvoz1 was mainly expressed at night and was a gene that was mainly expressed at night. Figure 2 This provides a biochemical basis for the regulation of gene function and application of Ntvoz1 in tobacco plants.

[0041] Example 2:

[0042] Cloning the target gene fragment: Use PCR to amplify the target gene fragment. Select a coding region approximately 300-500 bp in length. Based on the coding region sequence analysis, design primers F and R to amplify the coding region of the gene. The upstream primer NtVOZ1-F is shown in SEQ ID NO. 4; the downstream primer NtVOZ1-R is shown in SEQ ID NO. 5.

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

[0044] Construction of VIGS vector: Using restriction endonucleases EcoR1 and Kpn1, pTRV2 vector, SEQ ID NO.4 and SEQ ID NO.5 to amplify gene fragment SEQ ID NO.3, the amplified gene fragment was then inserted into the TRV2 vector using DNA recombination technology to obtain the VIGS vector, which was then sequenced to ensure that the vector was constructed correctly.

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

[0046] Virus propagation and infection: Transformation into Agrobacterium: pTRV1 vector and VIG S vector were extracted from Escherichia coli and transformed into Agrobacterium GV3101.

[0047] Virus propagation: Resuspend the Agrobacterium solution containing pTRV1 in VIGS buffer to obtain mixture 1, and resuspend the Agrobacterium solution containing the VIGS vector in VIGS buffer to obtain mixture 2. Mixture 1 and mixture 2 are mixed in equal proportions and 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 with a pH of 5.6.

[0048] An equal weight of soil was weighed and seedlings were cultured. One week later, the viral suspension obtained from the propagation was injected into the cotyledons of two-week-old seedlings. Nicotiana benthamiana seedlings served as the recipient material for gene silencing. TRV:00 (empty vector)-infected recipient material served as a negative control, while PDS-infected recipient material served as a reporter gene control.

[0049] After infection, the soil was allowed to absorb water fully. Ten days after infection, the gene-silenced plants (TRV:NtVOZ1) and the control plants (TRV:00) were observed. Figure 3 As shown in Figure 2, artificial drought stress was applied for 7 days, and it was found that the leaves of the gene-silenced materials were more likely to wilt than those of the uninfected materials. RNA was extracted from the seedlings and reverse transcribed to detect the expression level of NtVOZ1 in the seedlings, as shown in Figure 2. Figure 4 Then the relative conductivity and relative water content are measured, as shown in Figure 5-6 shown.

[0050] The experimental results show that when facing the same drought stress, plants with NtVOZ1 silenced will show more wilting, and plants with NtVOZ1 silenced will 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 confirmed that NtVOZ1 was mainly located in the cell nucleus. Figure 7 shown.

[0053] Yeast two-hybrid: The stop codons of the coding regions of the NtMYB78 and NtVOZ1 genes were removed and ligated with NdeI and EcoRI into the pGBKT7 and pGADT7 vectors, respectively, to construct the recombinant vectors pGBKT7-NtMYB78 and pGADT7. pGBKT7-53-+pGADT7-T was used as a positive control, and pGBKT7-La m+pGADT7-T was used as a negative control. Another negative control, pGBKT7-NtMYB78+pGADT7, was also established. The experimental group, pGBKT7-NtMYB78+NtVOZ1, was co-transformed into the Y2HGold yeast strain, plated on SD / -Leu / -Trp plates, and inverted, incubated at 30°C. Colony growth was observed after 2-4 days. Select a single colony for shake culture. When the bacterial solution concentration OD600 is about 0.5, take 0.5 μL of bacterial solution and culture it on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade / X-α-gal+AbA+3-AT solid culture medium respectively. After culturing in a 30℃ constant temperature box for 2-4 days, observe the growth of yeast. Figure 8 shown.

[0054] Example 4:

[0055] Co-IP: Ntvoz1 and the NtMYB78 genes were amplified and cloned into modified FLAG and GFP vectors, respectively. Agrobacterium containing different tagged bait proteins and capture protein plasmids and P19 Agrobacterium were inoculated into 3-5 mL LB medium containing appropriate antibiotics, and cultured overnight at 28°C and 200 rpm. The overnight culture solution was transferred to 10-20 mL of new medium containing appropriate antibiotics and 10 mmol·L –1 MES and 40 μmol·L –1 Incubate in LB medium containing acetosyringone (AS) at 28°C and 200 rpm overnight; collect the bacterial solution and discard the supernatant, resuspend the bacterial solution, let the bacteria stand, and prepare the injection solution. Collect the labeled leaves of tobacco plants 3 days after transformation, grind them into powder in a mortar pre-cooled with liquid nitrogen to prepare the crude protein extract, and perform Western blotting detection with two labeled antibodies, such as Figure 9 The experiment showed the interaction between tobacco genes NtVOZ1 and NtMYB78.

[0056] Example 5:

[0057] Pull-down: Induced expression of soluble GST and MBP-tagged proteins in E. coli: Pick out single clones expressing GST and MBP-tagged proteins, take the overnight culture solution at a ratio of 1:20, incubate for 3 hours, detect the OD value, add IPTG to a final concentration of 1mM, induce expression at 37℃ for 2-4 hours, collect the culture solution into a centrifuge tube, break up and purify the GST and MBP-tagged proteins, and finally perform GST pull-down verification. The proteins obtained from the GST pull-down experiment were detected by Western blotting, such as Figure 10 The experiment showed the interaction between tobacco genes NtVOZ1 and NtMYB78.

[0058] Example 6:

[0059] Wild-type tobacco, namely the main cultivated tobacco variety in China, K326 (Nicotiana tobacum L.cv. K326), was used to construct an NtVOZ1 overexpression strain (OE). The specific steps are as follows:

[0060] The stop codon was removed from the coding region sequence of the NtVOZ1 gene and the recombinant vector pRI101-AN-eGFP (purchased from Beijing Bomade Gene Technology Co., Ltd., catalog number CL499-01) was constructed by connecting it to the plant expression vector pRI101-AN-eGFP via NdeⅠ and EcoRⅠ. The pRI101-AN-eGFP-NtVOZ1 recombinant plasmid was then transformed into Agrobacterium LBA4404 using the freeze-thaw method, and genetic transformation was performed using the leaf disc transformation method.

[0061] By Kan + The PCR method was used for further screening to obtain T2 stable strain materials, and three independent overexpression transgenic strains (OE#8, #10 and #11) were selected for subsequent experiments.

[0062] Overexpression transgenic tobacco lines (OE#8, #10, and #11) and wild-type tobacco were tested for drought stress resistance. When the seedlings reached the six-leaf stage, the seedlings, about 30 days old, were subjected to natural drought treatment for 5 days. The control conditions for the drought stress resistance test were the overexpression transgenic tobacco lines (OE#8, #10, and #11) and wild-type tobacco under normal planting conditions (normal watering). The results are shown in Figure 2. Figure 11 As shown in the figure, under normal planting conditions, there was no significant difference between the overexpression transgenic tobacco lines and wild-type tobacco; after natural drought treatment, the overexpression transgenic tobacco lines were significantly more drought-resistant than wild-type tobacco. In addition, a relative conductivity experiment was conducted to determine the drought resistance of each line. The results are shown in the figure. Figure 12 The experimental results showed that overexpression of NtVOZ1 can improve tobacco drought resistance, indicating that NtVOZ1 plays an important role in tobacco's response to drought stress.

[0063] Those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all of these changes and modifications should fall within the scope of protection of the claims of the present invention.

Claims

1. A gene related to tobacco drought resistance, characterized in that: The sequence of the gene is shown in SEQ ID NO.

1.

2. The gene related to tobacco drought resistance according to claim 1, wherein The amino acid sequence of the transcription factor translated from the gene is shown in SEQ ID NO.

2.

3. The gene related to tobacco drought resistance according to claim 2, wherein The transcription factor is expressed in roots and leaves of tobacco.

4. An expression vector, characterized in that The expression vector is inserted with the gene fragment related to tobacco drought resistance as claimed in claim 1; the fragment is shown as SEQ ID NO.

3.

5. An application of a gene related to tobacco drought resistance, characterized in that: The gene shown in SEQ ID NO. 1 or the transcription factor shown in SEQ ID NO. 2 is used to prepare a regulator for tobacco cultivation.

6. An application of a gene related to tobacco drought resistance, characterized in that: The gene shown in SEQ ID NO. 1 or the transcription factor shown in SEQ ID NO. 2 is applied to tobacco cultivation.

7. The use of the gene related to tobacco drought resistance according to claim 5 or 6, characterized in that: The tobacco is cultivated so that the transcription factor shown in SEQ ID NO. 2 interacts with the NtMYB78 protein.

8. The use of the gene related to tobacco drought resistance according to claim 5 or 6, characterized in that: The tobacco cultivation is to increase the level of the transcription factor shown in SEQ ID NO. 2 in the plant body.

9. The use of the gene related to tobacco drought resistance according to claim 5 or 6, characterized in that: The tobacco cultivation is to improve the drought resistance of tobacco.

10. An application of a gene related to tobacco drought resistance, characterized in that: The gene shown in SEQ ID NO.1 or the transcription factor shown in SEQ ID NO.2 is applied to the study of the molecular mechanism of tobacco drought resistance.

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