Tobacco NtMYB61 gene and protein and application thereof

Abstract

The invention discloses a tobacco NtMYB61 gene as well as a protein and application thereof. The CDS sequence of the NtMYB61 gene capable of influencing the drought stress resistance of the tobacco is shown as SEQ ID No.2, and the genome sequence of the NtMYB61 gene is shown as SEQ ID No.1. According to the invention, the NtMYB61 gene is constructed on a plant expression vector and is transferred into tobacco through a traditional gene overexpression and transgenic technology, so that the tobacco is overexpressed, and an overexpressed material plant shows significantly reduced drought resistance compared with a wild type plant; the NtMYB61 gene in the tobacco genome is knocked out through a gene editing technology, and the obtained NtMYB61 mutant material shows significantly enhanced drought stress resistance compared with a wild type control.

Description

Technical Field

[0001] The invention relates to an application of a gene NtMYB61 for regulating tobacco drought resistance and a protein encoded therein, and belongs to the fields of molecular biology and genetic engineering. Background Art

[0002] In the natural environment, plants often face a variety of abiotic stresses. Studies have shown that drought stress has a serious negative impact on plant growth and productivity mainly by causing physical damage, physiological and biochemical damage and molecular changes to plants, leading to abnormal metabolism in plants, slowed growth, and ultimately leading to plant death. Under drought stress, the plant height of most plants will gradually decrease as the degree of soil water deficit increases, and the chlorophyll in the leaves will degrade, causing the leaves to slowly turn yellow. Especially under severe drought stress, the plant height will be significantly inhibited, the intracellular turgor pressure will be reduced, and the physiological and biochemical metabolic processes such as water use efficiency, photosynthetic system, respiratory system, osmotic regulation ability, cell membrane stability, antioxidant system defense ability and hormone levels will be seriously disturbed. These changes will have irreversible effects on the external morphological characteristics, biomass and yield of plants.

[0003] Tobacco is one of the important model plants in the field of botanical research and plays an important role in natural science research. In the process of tobacco leaf production, drought stress is the main abiotic stress that leads to reduced yield and quality, seriously affecting the economic income of tobacco farmers and the quality of tobacco products. During the entire growth cycle of tobacco growth, the impact of drought stress on plant growth is particularly important, the most critical of which is to inhibit leaf growth. When drought stress worsens, leaf growth is hindered, plants are dwarfed, leaves are smaller and do not unfold, which in turn affects the dry matter accumulation and quality of tobacco leaves. Insufficient water supply will cause flue-cured tobacco leaves to enter carbon metabolism prematurely, resulting in premature yellowing of leaves, incomplete development and insufficient material accumulation, which ultimately leads to a decline in leaf quality.

[0004] At present, the main focus of tobacco drought prevention and control is to optimize agronomic measures and strengthen irrigation. However, the labor cost of artificial irrigation is high, and factors such as topography and water resources in most tobacco-growing areas make it difficult to ensure effective irrigation, which to a large extent limits the high-quality development of tobacco agriculture. How to improve tobacco drought resistance and reduce the reduction in tobacco leaf yield and quality caused by drought has become a current research hotspot and difficulty. In recent years, with the rapid development of molecular breeding and genetic engineering technology, at the molecular level, by mining, identifying and utilizing tobacco resistance genes, cultivating new drought-resistant tobacco varieties has become an important measure to solve and respond to the challenges brought by climate change and ensure the stable development of the tobacco industry. However, at present, research on gene mining and utilization that can be used to improve tobacco drought resistance is still very limited, which seriously restricts the selection and breeding process of new tobacco drought-resistant varieties. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a NtMYB61 gene that can affect the ability of tobacco to resist drought stress.

[0006] Another object of the present invention is to provide application of the gene.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A NtMYB61 gene capable of affecting tobacco's ability to resist drought stress, the CDS sequence of which is shown as SEQ ID No.2, and the genome sequence of which is shown as SEQ ID No.1.

[0009] The amino acid sequence of the protein encoded by the NtMYB61 gene is shown in SEQ ID No.3.

[0010] The application of the NtMYB61 gene of the present invention in improving the drought resistance of tobacco is to improve the drought resistance of tobacco by mutating the NtMYB61 gene of claim 1.

[0011] Application of CRISPR / Cas9 gene editing system of NtMYB61 gene in improving tobacco drought resistance.

[0012] The CDS sequence of the gene NtMYB61 was constructed into a plant expression vector and introduced into tobacco for expression. The transgenic tobacco with overexpression of NtMYB61 was subjected to drought stress treatment, and it was confirmed that the drought stress resistance of this gene was reduced after overexpression. The NtMYB61 gene in the tobacco genome was knocked out through gene editing technology, and the drought stress resistance of the Ntmyb61 mutant was significantly improved. This shows that the NtMYB61 gene can be used as an excellent gene resource for improving the drought resistance of tobacco. The research results lay the foundation for the later use of this gene to improve the drought resistance of tobacco and other plants.

[0013] Beneficial effects:

[0014] The invention regulates the gene NtMYB61 affecting tobacco drought resistance and is cloned from tobacco. The gene is overexpressed by transgenic technology to weaken the tobacco's resistance to drought, while the mutant of the gene obtained by gene editing technology improves the tobacco's drought resistance. This provides a theoretical basis and molecular targets for genetic research on tobacco drought resistance (the current status of tobacco drought resistance genetic research, application research of drought resistance-related genes in tobacco, and cloning of tobacco drought resistance genes), research on tobacco drought stress resistance germplasm resources (collection and preservation of tobacco germplasm resources, tobacco drought resistance germplasm resources, and tobacco drought resistance germplasm resource variety selection), and research on tobacco drought stress resistance breeding (tobacco drought stress resistance genetic breeding strategy, application of molecular biotechnology in tobacco drought resistance genetic improvement, and research on tobacco drought stress resistance variety selection). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram showing the structure of the NtMYB61 gene

[0016] Figure 2 Agarose gel electrophoresis results showing the genome and CDS fragments of NtMYB61

[0017] (Note: Maker is 2000DL)

[0018] Figure 3 The results show the expression changes of NtMYB61 gene in response to mannitol (Figure A) and ABA (Figure B) treatments.

[0019] Figure 4 Showing the expression levels of NtMYB61 gene in wild-type K326 control and NtMYB61 overexpressing materials

[0020] Figure 5 Showing the base changes and encoded amino acids of NtMYB61 gene in wild-type K326 control and Ntmyb61 mutant materials

[0021] Figure 6 Showing the water loss rate of detached leaves of NtMYB61 overexpression materials (A) and mutant materials (B) at different time points

[0022] Note: K326 is the wild-type control material

[0023] Figure 7 Shows the results of 5-day treatment of NtMYB61 overexpressing materials with normal irrigation (control group) and drought stress (treatment group)

[0024] Note: K326 is the wild-type control material

[0025] Figure 8Showing the results of Ntmyb61 mutant materials under normal irrigation (control group) and drought stress (treatment group) for 7 days

[0026] Note: K326 is the wild-type control material

[0027] Fig. 9 Showing the results of fresh biomass (A) and relative water content (B) of NtMYB61 overexpressing materials after 5 days of normal irrigation (control group) and drought stress (treatment group) treatment

[0028] Note: K326 is the wild-type control material

[0029] Fig.10 Showing the results of fresh biomass (A) and relative water content (B) of Ntmyb61 mutant materials after 7 days of normal irrigation (control group) and drought stress (treatment group) treatment

[0030] Note: K326 is the wild-type control material DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to examples and data.

[0032] Note: Unless otherwise specified, the actual objects, instruments, and materials involved in the following embodiments are all visible products commonly found in laboratories and can be obtained through regular commercial means; similarly, the experimental techniques, methods, including detection methods, etc. involved in the following embodiments, unless otherwise specified, are all common techniques and methods in the prior art, or are slightly modified under all laboratory conditions without affecting the final experimental results.

[0033] Example 1 Tobacco seedling cultivation and NtMYB61 gene amplification

[0034] Seedling culture: Tobacco wild-type seeds K326 were sterilized with 75% anhydrous ethanol for about 30 seconds, then soaked in 15% H2O2 for about 8-10 minutes, and finally washed three times with sterile water. After being half-dried, they were cultured on 1 / 2MS solid culture medium and germinated and grown in a sterile tissue culture room with culture conditions of (25±1)℃ and light intensity of 16h / d.

[0035] NtMYB61 gene amplification:

[0036] (1) When tobacco grows to three leaves and one heart or larger, use sterile scissors to sample about 0.1g, and use the SLS DNA extraction method to obtain tobacco genomic DNA. Design specific primers upstream and downstream of the NtMYB61 gene. The primer sequences are shown in the table below, and the length of the amplified fragment is 1805bp. Use genomic DNA as a template and the sequences in the table below as upstream and downstream primers for PCR amplification to obtain the NtMYB61 gene sequence.

[0037]

[0038] The results of 1% agarose gel electrophoresis were as follows Figure 2 As shown, the results show that the target band size is consistent with the prediction. The PCR product was further sequenced, and the sequencing results showed that the gene sequence is as shown in SEQ ID No.1, and the full length of the NtMYB61 gene is 1409bp. Figure 1 As shown; further sequence analysis found that the NtMYB61 gene contains three exons and two introns. The three exons were spliced ​​to obtain the NtMYB61 CDS full-length sequence of 876 bp as shown in SEQ ID No. 2.

[0039] The detailed steps of DNA extraction are as follows:

[0040] a. Grind the sample with liquid nitrogen into a 2.0ml centrifuge tube and add 800ul of SLS extract (PH=8.0)

[0041] b. Add equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) and mix thoroughly

[0042] c. Centrifuge at 12000r / m for 10 minutes, pipette the supernatant into a new 1.5ml centrifuge tube, add 0.6 times the volume of pre-cooled isopropanol and mix well.

[0043] d. Centrifuge at 12000r / m for 10 minutes, discard the supernatant, and rinse with 75% ethanol. The rinsing time can be longer.

[0044] e. Dissolve in ddH2O after drying

[0045] (2) At the same time, the above tobacco seedlings were used for RNA extraction. The steps of RNA extraction from tobacco leaves were carried out according to the instructions of the RNA extraction kit: the sample was placed in liquid nitrogen and ground with a mortar, and then quickly transferred to a centrifuge tube with 1 ml of Buffer RZ added, and vigorously vortexed to mix; 200 μl of chloroform was added, vortexed to mix, and centrifuged at 13000 rpm at 4°C for 1 min; 400-500 μl of supernatant was transferred to a new RNase-free centrifuge tube, 0.5 times the volume of anhydrous ethanol of the supernatant was added, and the mixture was inverted and mixed, and then transferred to the adsorption column loaded in the collection tube, centrifuged at 13000 rpm for 30 s, and the waste liquid was discarded; 600 μl of Buffer RW2 was added to the adsorption column, and the mixture was centrifuged at 13000 rpm for 30 s, and the waste liquid was discarded. This step was repeated once; the adsorption column was left vacant for 2 min, and then transferred to a new RNase-free centrifuge tube, and 30-100 μl of RNase-free was added to the adsorption film. H2O, place at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute to obtain the RNA solution.

[0046] According to the instructions of the RNA reverse transcription kit, use the RNA obtained in the above step as a template, absorb about 1μg RNA, 4μl 4xg DNA wiper Mix, add RNase-free ddH2O to 16μl, mix well in an RNase-free centrifuge tube, and incubate at 42℃ for 2min; add 4μl 5x HiScriptⅢqRT SuperMix to the system and mix well, incubate at 37℃ for 15min, and 85℃ for 5s to obtain the required cDNA.

[0047]

[0048] The cDNA obtained in the above process was used as a template, and the Primer 3&4 sequences in the above table were used as upstream and downstream primers for amplification. The PCR product of 876 bp in size of the NtMYB61 gene was obtained, which was the full length of the CDS. According to the subsequent agarose gel electrophoresis results, Figure 2 As shown, the results show that the PCR band size of the product is consistent with the size of the target product, and the sequencing result is consistent with the result shown in SEQ ID No. 2. The RNA extraction kit used in the process was purchased from Beijing Nobel Biotechnology Co., Ltd.; the reverse transcription kit was purchased from Novazon Biotechnology Co., Ltd.; the gun tips, centrifuge tubes and other items used were all RNase-free products purchased from Axygen Biotechnology Co., Ltd.

[0049] Example 2 Response of NtMYB61 to mannitol and ABA

[0050] According to the method described in Example 1, the tobacco seedlings were transplanted into the matrix nutrient soil. After culturing for 3-4 weeks, the complete seedlings were taken out from the matrix, the rhizosphere soil was cleaned, and the seedlings were slowed down in clean water for 12-24 hours, and then transferred to 200mM mannitol or 100μM ABA solution for cultivation respectively. Samples were taken at 0, 1, 3, 6, 12, and 24 hours according to the method described in Example 1, and the corresponding sample RNA was extracted and reverse transcribed to obtain cDNA. The mixed solution was configured according to the following system:

[0051]

[0052] Perform qPCR reaction as follows:

[0053]

[0054] Finally follow ΔΔ The expression level was calculated by Ct method, and the Figure 3 The expression results of NtMYB61 in response to mannitol and ABA show that NtMYB61 is upregulated by mannitol and ABA, which is the first time that NtMYB61 may respond to drought stress.

[0055] The real-time fluorescence quantitative PCR enzyme used in this process was purchased from Novozyme Biotechnology Co., Ltd.; the primer sequences used are as follows:

[0056]

[0057] Example 3 Obtaining tobacco materials with NtMYB61 gene overexpression and knockout

[0058] The product obtained by PCR using cDNA as a template in Example 1 was detected and purified by 1% agarose gel electrophoresis, then connected to the pEASY-Blunt Zero Cloning kit vector and transformed into Escherichia coli DH5α competent cells. The transformants were screened and cultured for 12 hours using LB solid culture medium containing kanamycin (50 mg / L). Single clones were picked and verified by bacterial liquid PCR using universal primers for the vector. Positive clones were bidirectionally sequenced to obtain the positive plasmid Blunt-NtMYB61 containing the NtMYB61 gene sequence.

[0059] The cloned Blunt-NtMYB61 plasmid was used as a template and PCR was amplified using Primer 7&8. The fragment was inserted into the plant binary expression vector pBWA(V)HS-ccdb-GLosgfp according to the common vector construction method. The bacteria used for screening resistance was kanamycin, and the NtMYB61 gene overexpression vector plasmid pBWA(V)HS-NtMYB61 was obtained.

[0060] The NtMYB61 gene overexpression material was obtained by conventional tobacco transgenic technology.

[0061]

[0062] RNA extraction and reverse transcription were performed according to Example 1 to obtain RNA and corresponding cDNA of NtMYB61 gene overexpression plants, and the cDNA of common wild-type tobacco K326 grown in the same period was used as a control to detect the NtMYB61 overexpression effect. The expression effect of NtMYB61 overexpression material was calculated in the same manner as in Example 2. Figure 4 As shown, compared with the wild-type control K326, the expression level of the NtMYB61 gene in the transgenic plants overexpressing the NtMYB61 gene was significantly higher than that in the control.

[0063] According to the sequence information and CDS sequence of the NtMYB61 gene in the tobacco genome, the sgRNA target sequence for specific knockout of NtMYB61 was designed: GAGGCCAGGAATCAAAA. The sgRNA target sequence fragment was synthesized, and the NtMYB61 mutant material was obtained by conventional operation of CRISPR / Cas9 technology and named Ntmyb61.

[0064] The SLS method was used to extract the genomic DNA of K326 and Ntmyb61 mutant tobacco. After high-fidelity PCR amplification using Primer 9 & 10 primers, 1% agarose gel electrophoresis was performed and the sequencing company was commissioned to sequence. The homozygous mutant materials Ntmyb61-2 and Ntmyb61-4 were screened and identified. The mutation sites of the two Ntmyb61 homozygous mutants obtained and the protein amino acids affected are as follows Figure 5 As shown. The Ntmyb61-2 strain homozygous deleted 4bp at +276bp after the start codon of the NtMYB61 gene; the Ntmyb61-4 strain homozygous inserted base A at +281bp after the start codon of the NtMYB61 gene. The amino acids encoded by the NtMYB61 gene after the mutation are completely different from the amino acids normally encoded by NtMYB61 ( Figure 5 ), which can lead to the loss or inhibition of the activity of the protein encoded by the tobacco NtMYB61 gene.

[0065]

[0066] Example 5 Analysis of drought resistance in tobacco plants with overexpression and knockout of NtMYB61

[0067] After harvesting the seeds of the NtMYB61 overexpressing tobacco materials and Ntmyb61 homozygous mutant plants constructed in Screening Example 4, they were sown in nutrient soil together with wild-type tobacco K326. When they had three leaves and one heart, they were transplanted into plug trays for normal culture. The weight of the substrate in each plug tray was kept consistent to keep the soil moisture sufficient and uniform.

[0068] (1) Analysis of water loss rate of detached leaves: When tobacco seedlings grow to 3-4 weeks old, use scissors to cut 7-10 leaves of each tobacco strain with the same growth and the same position, count and weigh them, and keep them in the dark after weighing to reduce the influence of photorespiration; then weigh them at different time points to analyze the water loss rate of each tobacco strain in a continuous period of time. Figure 6 As shown: Within the same treatment period, it can be seen that the average water loss rate of the overexpression strain is significantly higher than that of the wild type K326, while the average water loss rate of the Ntmyb61 mutant is significantly lower than that of the control K326, which preliminarily proves that the protein encoded by the NtMYB61 gene can affect the response of tobacco to drought environment.

[0069] (2) Analysis of drought resistance under natural drought treatment: Wild-type tobacco K326, NtMYB61 gene overexpression and knockout tobacco materials with a seedling age of about 30 days and normal irrigation growth in the soil were selected and subjected to natural drought treatment in an artificial climate chamber. During the treatment, the light, temperature and humidity in the climate chamber were kept normal. After several days, the adaptability of NtMYB61 gene overexpression and knockout transgenic materials to drought stress was obtained. Figure 7-8 As shown in the figure: after 5 days of drought treatment, the overexpression material of NtMYB61 showed obvious wilting, while the leaf growth of the wild-type control plant remained normal; after 7 days of drought treatment, the control K326 material showed wilting, while the mutant material of NtMYB61 still maintained normal growth, and the leaf state showed obvious differences from the control. This result is consistent with the results of the water loss rate of detached leaves, indicating that the overexpression of the NtMYB61 gene can accelerate the loss of water from tobacco leaves, thereby reducing the ability of tobacco to resist drought stress; the knockout of the NtMYB61 gene can alleviate the loss of water from tobacco leaves, resulting in a significant improvement in the drought resistance of tobacco.

[0070] The fresh weight biomass and relative water content of wild-type, NtMYB61 gene overexpression and knockout tobacco materials under normal irrigation (control group) and drought stress (treatment group) were further measured and analyzed. The aboveground individual plants of each strain were weighed and recorded and individually packed in kraft paper envelopes and dried at 60°C to constant weight. The weight of each tobacco plant was recorded again, and the relative water content of each strain was calculated according to the formula: relative water content (%) = (fresh weight - dry weight) / fresh weight * 100%. The results are shown in the figure. Fig. 9 and 10 : Under normal irrigation culture (control group), the fresh biomass and relative water content of each strain were not significantly different from those of wild-type tobacco K326. Under drought treatment (treatment group), the fresh biomass and relative water content of NtMYB61 overexpression materials were significantly lower than those of K326; on the contrary, the fresh biomass and relative water content of mutant Ntmyb61 were significantly higher than those of control K326. In summary, the data show that the three materials of NtMYB61 overexpression, K326, and Ntmyb61 mutant show successively enhanced ability to resist natural drought.

[0071] From all the above data, we can see that the protein encoded by NtMYB61 induced by mannitol and ABA plays an important role in plants' response to natural drought stress. The exploration of the protein's ability to respond to drought will provide an important theoretical basis and breeding molecular targets for the research on crop drought-resistant germplasm resources and crop drought-resistant breeding.

Claims

1. A NtMYB61 gene capable of affecting tobacco's ability to resist drought stress, characterized in that: The genome sequence is shown in SEQ ID No.

1.

2. A NtMYB61 gene capable of affecting tobacco's ability to resist drought stress, characterized in that: The CDS sequence is shown as SEQ ID No.

2.

3. The protein encoded by the NtMYB61 gene according to claim 1, characterized in that The amino acid sequence is shown in SEQ ID No.

3.

4. The use of the NtMYB61 gene according to claims 1, 2 and 3 in improving tobacco drought resistance, characterized in that: The drought resistance of tobacco is improved by mutating the NtMYB61 gene described in claim 1.

5. Application of CRISPR / Cas9 gene editing system of NtMYB61 gene in improving tobacco drought resistance.

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