Tobacco ntmyb61 gene, protein thereof, and use thereof

Abstract

The present invention relates to a tobacco NtMyB61 gene, a protein thereof, and a use thereof. An NtMyB61 gene capable of affecting the resistance of tobacco to drought stress has a CDS sequence as shown in SEQ ID No. 2, and a genome sequence as shown in SEQ ID No. 1. By means of conventional gene overexpression and transgenic techniques, the NtMyB61 gene is constructed into a plant expression vector and transferred into tobacco for overexpression, and an overexpressing plant material exhibits significantly reduced drought resistance compared with a wild-type control; and by means of gene editing techniques, the NtMyB61 gene in a tobacco genome is knocked out, and the obtained Ntmyb61 mutant material exhibits significantly enhanced drought stress resistance compared with the wild-type control.

Description

Tobacco NtMYB61 gene, its protein, and its applications Technical Field

[0001] This invention relates to the application of the gene NtMYB61, which regulates the drought resistance of tobacco, and its encoded protein, belonging to the fields of molecular biology and genetic engineering. Background Technology

[0002] In the natural environment, plant growth often faces various abiotic stresses. With global warming, limited water resources have become one of the main constraints affecting the growth, development, and yield of crops worldwide. Studies have shown that drought stress mainly causes physical damage, physiological and biochemical disruption, and molecular changes in plants, thus having a severe negative impact on plant growth and productivity, leading to abnormal metabolism, slowed growth, and ultimately plant death. Under drought stress, the plant height of most plants gradually decreases as soil moisture deficit intensifies, and chlorophyll in the leaves degrades, causing the leaves to slowly turn yellow. Especially under severe drought stress, plant height is significantly inhibited, intracellular turgor pressure decreases, and physiological and biochemical metabolic processes such as water use efficiency, photosynthetic system, respiration system, osmotic regulation capacity, cell membrane stability, antioxidant defense capacity, and hormone levels are severely disrupted. These changes have irreversible effects on the external morphological characteristics, biomass, and yield of plants.

[0003] Tobacco is one of my country's important economic crops and a crucial model plant in botanical research, playing a vital role in natural science research and socio-economic development. During tobacco production, drought stress is the primary abiotic stress leading to reduced yield and quality, severely impacting tobacco farmers' income and the quality of tobacco products. Throughout the entire growth cycle of tobacco, drought stress significantly affects plant growth, with the most critical effect being the inhibition of leaf growth. When drought stress intensifies, leaf growth is hindered, plants become stunted, and leaves are smaller and fail to unfold, thus affecting the accumulation and quality of dry matter in tobacco leaves. Insufficient water supply causes flue-cured tobacco leaves to prematurely transition to carbon metabolism, resulting in early yellowing, incomplete development, and insufficient matter accumulation, ultimately leading to a decline in leaf quality.

[0004] Currently, drought control in tobacco mainly relies on optimizing agronomic practices and strengthening irrigation. However, manual irrigation is labor-intensive, and the terrain and water resources in most tobacco-growing areas make effective irrigation difficult to guarantee, which significantly limits the high-quality development of tobacco agriculture. Improving tobacco drought resistance and reducing the decline in tobacco yield and quality caused by drought has become a current research hotspot and challenge. In recent years, with the rapid development of molecular breeding and genetic engineering technologies, at the molecular level, discovering, identifying, and utilizing tobacco resistance genes to cultivate new drought-resistant tobacco varieties has become an important measure to address the challenges of climate change and ensure the stable development of the tobacco industry. However, research on gene discovery and utilization for improving tobacco drought resistance is still very limited, severely restricting the breeding process of new drought-resistant tobacco varieties. Summary of the Invention

[0005] To address the above technical problems, the purpose of this invention is to provide an NtMYB61 gene that can influence the resistance of tobacco to drought stress.

[0006] Another objective of this invention is to provide applications of this gene.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The NtMYB61 gene, which can affect the resistance of tobacco to drought stress, has a CDS sequence as shown in SEQ ID No. 2 and a genome sequence as shown in SEQ ID No. 1.

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

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

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

[0012] The CDS sequence of the NtMYB61 gene was constructed into a plant expression vector and expressed in tobacco. The transgenic tobacco plants overexpressing NtMYB61 were subjected to drought stress treatment, confirming that overexpression reduced drought tolerance. However, by knocking out the NtMYB61 gene in the tobacco genome using gene editing technology, the resulting Ntmyb61 mutant exhibited significantly improved drought resistance. Therefore, this study indicates that the NtMYB61 gene can serve as an excellent gene resource for improving drought resistance in tobacco. These findings lay the foundation for future applications of this gene to improve drought tolerance in tobacco and other plants. Beneficial effects:

[0013] This invention clones the NtMYB61 gene, which regulates drought resistance in tobacco, from tobacco. Overexpression of this gene through transgenic technology weakens the tobacco's resistance to drought, while mutants of this gene obtained through gene editing technology improve the drought resistance of tobacco. This provides a theoretical basis and molecular target for research on the genetics of tobacco drought resistance (current status of research on the genetics of tobacco drought resistance, application of drought-resistant genes in tobacco, cloning of tobacco drought-resistant genes), research on tobacco germplasm resources under drought stress (collection and preservation of tobacco germplasm resources, tobacco drought-resistant germplasm resources, breeding of tobacco drought-resistant germplasm resources), and breeding research on tobacco drought stress (genetic breeding strategies for tobacco drought stress, application of molecular biotechnology in the genetic improvement of tobacco drought resistance, research on the breeding of tobacco drought-resistant varieties). Attached Figure Description

[0014] Figure 1 shows a schematic diagram of the NtMYB61 gene structure.

[0015] Figure 2 shows the agarose gel electrophoresis results of the NtMYB61 genome and CDS fragment.

[0016] (Note: Maker is 2000DL)

[0017] Figure 3 shows the changes in NtMYB61 gene expression in response to mannitol (Figure A) and ABA (Figure B) treatments.

[0018] Figure 4 shows the NtMYB61 gene expression levels in wild-type K326 control and NtMYB61 overexpression materials.

[0019] Figure 5 shows the base changes and encoded amino acids of the NtMYB61 gene in wild-type K326 control and Ntmyb61 mutant materials.

[0020] Figure 6 shows the water loss rate of detached leaves at different time points for NtMYB61 overexpression material (A) and mutant material (B).

[0021] Note: K326 is a wild-type control material.

[0022] Figure 7 shows the results of 5 days of treatment with normal irrigation (control group) and drought stress (treatment group) for NtMYB61 overexpression materials.

[0023] Note: K326 is a wild-type control material.

[0024] Figure 8 shows the results of 7 days of treatment with normal irrigation (control group) and drought stress (treatment group) on the Ntmyb61 mutant material.

[0025] Note: K326 is a wild-type control material.

[0026] Figure 9 shows the fresh weight biomass (A) and relative water content (B) of NtMYB61 overexpression materials after 5 days of normal irrigation (control group) and drought stress (treatment group).

[0027] Note: K326 is a wild-type control material.

[0028] Figure 10 shows the results of fresh weight biomass (A) and relative water content (B) of Ntmyb61 mutant materials after 7 days of normal irrigation (control group) and drought stress (treatment group).

[0029] Note: K326 is a wild-type control material. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments and data.

[0031] Note: Unless otherwise specified, the actual products, instruments and materials involved in the following examples are all commonly available products in the laboratory and can be obtained through legitimate commercial means; similarly, the experimental techniques and methods involved in the following examples, including detection methods, are all common techniques and methods in the prior art unless otherwise specified, or may be slightly adjusted and modified according to all laboratory conditions, without affecting the final experimental results.

[0032] Example 1: Tobacco seedling culture and NtMYB61 gene amplification

[0033] Seedling culture: The wild-type tobacco seeds K326 were disinfected with 75% anhydrous ethanol for about 30 seconds, then soaked in 15% H2O2 for about 8-10 minutes, and finally rinsed three times with sterile water. After drying to semi-dryness, they were cultured on 1 / 2MS solid medium and germinated and grew in a sterile tissue culture room with culture conditions of (25±1)℃ and 16h / d light.

[0034] NtMYB61 gene amplification:

[0035] (1) When the tobacco plant has grown to three leaves and one bud or larger, take about 0.1g of sample with sterile scissors and obtain tobacco genomic DNA using the SLS DNA extraction method. Design specific primers upstream and downstream of the NtMYB61 gene. The primer sequences are shown in the table below, and the amplified fragment length is 1805bp. Using the genomic DNA as a template, perform PCR amplification with the upstream and downstream primer sequences in the table below to obtain the NtMYB61 gene sequence.

[0036] The 1% agarose gel electrophoresis results are shown in Figure 2. The results show that the target band size is consistent with the prediction. Further sequencing of the PCR product revealed that the gene sequence is shown in SEQ ID No. 1, and the full-length NtMYB61 gene is 1409 bp. A schematic diagram of the gene structure is shown in Figure 1. Further sequence analysis revealed that the NtMYB61 gene contains three exons and two introns. The three exons were spliced ​​together to obtain the full-length NtMYB61 CDS sequence of 876 bp, as shown in SEQ ID No. 2.

[0037] The detailed steps for DNA extraction are as follows:

[0038] a. Grind the sample with liquid nitrogen and transfer it into a 2.0 ml centrifuge tube. Add 800 μL of SLS extraction buffer (pH = 8.0).

[0039] b. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix thoroughly.

[0040] c. Centrifuge at 12000 rpm for 10 minutes, then transfer the supernatant to a new 1.5 ml centrifuge tube. Add 0.6 times the volume of pre-chilled isopropanol and mix well.

[0041] d. Centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and rinse with 75% ethanol for a longer period of time.

[0042] e. Dissolve in ddH2O after drying.

[0043] (2) Simultaneously, the above-mentioned tobacco seedlings were used for RNA extraction. The RNA extraction procedure for tobacco leaves was performed according to the instructions of the RNA extraction kit: The sample was placed in liquid nitrogen and ground in a mortar and pestle, then quickly transferred to a centrifuge tube containing 1 ml of Buffer RZ and vortexed vigorously to mix; 200 μl of chloroform was added, vortexed to mix, and centrifuged at 13000 rpm for 1 min at 4℃; 400-500 μl of supernatant was transferred to a new RNase-free centrifuge tube, 0.5 times the volume of the supernatant was added, and the mixture was inverted and mixed, then transferred together to the adsorption column 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, centrifuged at 13000 rpm for 30 s, and the waste liquid was discarded, and this step was repeated once; after 2 min of air centrifugation, the adsorption column was transferred to a new RNase-free centrifuge tube, and 30-100 μl of RNase-free ether was added to the adsorption membrane. Incubate with H2O at room temperature for 2 minutes, then centrifuge at 12000 rpm for 1 minute to obtain the RNA solution.

[0044] According to the instructions of the RNA reverse transcription kit, using the RNA obtained in the above steps as a template, take about 1 μg of RNA, 4 μl of 4xg DNA wiper Mix, and add RNase-free ddH2O to make up to 16 μl. Mix well and incubate at 42°C for 2 min. Add 4 μl of 5x HiScriptⅢqRT SuperMix to the system and mix well. Incubate at 37°C for 15 min and 85°C for 5 s to obtain the desired cDNA.

[0045] Using the cDNA obtained in the above process as a template, and the Primer 3 & 4 sequences in the table above as upstream and downstream primers for amplification, an 876bp PCR product of the NtMYB61 gene was obtained as its full-length CDS. The subsequent agarose gel electrophoresis results are shown in Figure 2. The results show that the size of the PCR band of this product is consistent with the size of the target product, and the sequencing results are consistent with those shown in SEQ ID No. 2. The RNA extraction kit used in this process was purchased from Beijing Novell Biotechnology Co., Ltd.; the reverse transcription kit was purchased from Novizan Biotechnology Co., Ltd.; and all pipette tips, centrifuge tubes, and other equipment used were RNase-free products purchased from Axygen Biotechnology Co., Ltd.

[0046] Example 2: Response of NtMYB61 to Mannitol and ABA

[0047] Following the method described in Example 1, tobacco seedlings were transplanted into substrate nutrient soil. After 3-4 weeks of cultivation, intact seedlings were removed from the substrate, the rhizosphere soil was washed, and the seedlings were allowed to acclimate in water for 12-24 hours. Then, they were transferred to solutions of 200 mM mannitol or 100 μM ABA for cultivation, respectively. Samples were collected at 0, 1, 3, 6, 12, and 24 hours according to the method described in Example 1. RNA was extracted from the corresponding samples, and cDNA was obtained through reverse transcription. The mixture was prepared according to the following system:

[0048] Perform the qPCR reaction according to the following procedure:

[0049] Finally according to ΔΔ The expression levels of NtMYB61 were calculated using the Ct method, and the results shown in Figure 3 are the expression levels of NtMYB61 in response to mannitol and ABA, indicating that NtMYB61 expression is upregulated by mannitol and ABA. This is the first indication that NtMYB61 may respond to drought stress.

[0050] The real-time quantitative PCR enzyme used in this process was purchased from Novizan Biotechnology Co., Ltd.; the primer sequences used are as follows:

[0051] Example 3: Obtaining tobacco materials with NtMYB61 gene overexpression and knockout

[0052] The product obtained by PCR using cDNA as a template in Example 1 was detected and purified by 1% agarose gel electrophoresis, ligated into the pEASY-Blunt Zero Cloning kit vector, and transformed into E. coli DH5α competent cells. Transformants were screened and cultured in LB solid medium containing kanamycin (50 mg / L) for 12 h. Single clones were picked and verified by bacterial PCR using universal primers for the vector. Positive clones were subjected to bidirectional sequencing to obtain the positive plasmid Blunt-NtMYB61 containing the NtMYB61 gene sequence.

[0053] Using the cloned Blunt-NtMYB61 plasmid as a template, Primer 7 & 8 were used for amplification PCR. The fragment was then inserted into the plant binary expression vector pBWA(V)HS-ccdb-GLosgfp according to the standard vector construction method. The bacteria used for screening were resistant to kanamycin, resulting in the overexpression vector plasmid pBWA(V)HS-NtMYB61 of the NtMYB61 gene.

[0054] The NtMYB61 gene overexpression material was obtained using standard procedures in tobacco transgenic technology.

[0055] RNA was extracted and reverse transcribed from NtMYB61 gene-overexpressing plants as described in Example 1, and the corresponding cDNA was obtained. The cDNA of wild-type tobacco K326, grown at the same time, was used as a control to detect the NtMYB61 overexpression effect. The expression effect of the NtMYB61 overexpression materials was calculated in the same manner as in Example 2. As shown in Figure 4, 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.

[0056] Based on the sequence information of the NtMYB61 gene in the tobacco genome and the CDS sequence, a specific sgRNA target sequence for knocking out NtMYB61 was designed: GAGGCCAGGAATCAAAA. The sgRNA target sequence fragment was synthesized, and NtMYB61 mutant material was obtained using standard CRISPR / Cas9 technology and named Ntmyb61.

[0057] Genomic DNA was extracted from K326 and Ntmyb61 mutant tobacco using the SLS method. High-fidelity PCR amplification was performed using Primer 9 & 10 primers, followed by 1% agarose gel electrophoresis and sequencing by a sequencing company. Homozygous mutant materials Ntmyb61-2 and Ntmyb61-4 were screened and identified. The mutation sites of the two homozygous Ntmyb61 mutants and their affected protein amino acids are shown in Figure 5. The Ntmyb61-2 line showed a homozygous deletion of 4 bp at +276 bp after the start codon of the NtMYB61 gene; the Ntmyb61-4 line showed a homozygous insertion of base A at +281 bp after the start codon of the NtMYB61 gene. The amino acids encoded by the mutated NtMYB61 gene are significantly different from those encoded by the normal NtMYB61 gene (Figure 5), which can lead to the loss or inhibition of activity of the tobacco NtMYB61 gene-encoded protein.

[0058] Example 5: Analysis of drought resistance in tobacco plants with NtMYB61 overexpression and knockout

[0059] After harvesting the seeds of the NtMYB61 overexpressing tobacco material constructed in Example 4 and the Ntmyb61 homozygous mutant plants, they were sown together with wild-type tobacco K326 in nutrient soil. When the plants had three leaves and one heart, they were transplanted into seedling trays for normal cultivation. The substrate weight in each seedling tray was kept consistent, and the soil moisture was kept sufficient and uniform.

[0060] (1) Analysis of water loss rate of detached leaves: When tobacco seedlings grew to 3-4 weeks old, 7-10 leaves from the same part of each line with uniform growth were cut with scissors, and weighed. After weighing, the leaves were kept in the dark to reduce the influence of photorespiration. Subsequently, the leaves were weighed at different time points to analyze the water loss rate of each line over a continuous period of time. As shown in Figure 6, within the same treatment period, the average water loss rate of the overexpression line was significantly higher than that of the wild type K326, while the average water loss rate of the Ntmyb61 mutant was significantly lower than that of the control K326. This preliminarily proves that the protein encoded by the NtMYB61 gene can affect the tobacco response to drought.

[0061] (2) Analysis of drought resistance under natural drought treatment: Wild-type tobacco materials with K326 and NtMYB61 gene overexpression and knockout, which were grown under normal irrigation in the soil and were about 30 days old, 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 transgenic materials with NtMYB61 gene overexpression and knockout to drought stress was shown in Figure 7-8: After 5 days of drought treatment, the NtMYB61 overexpression material showed obvious wilting, while the wild-type control plants maintained normal leaf growth; after 7 days of drought treatment, the control K326 material showed wilting, while the NtMYB61 mutant material still maintained normal growth, and the leaf state showed obvious differences from the control. This result is consistent with the results of water loss rate from detached leaves, indicating that overexpression of the NtMYB61 gene can accelerate water loss from tobacco leaves, thereby reducing the drought resistance of tobacco; knockout of the NtMYB61 gene can alleviate water loss from tobacco leaves, leading to a significant improvement in the drought resistance of tobacco.

[0062] Further analysis was conducted on the fresh weight biomass and relative water content of wild-type, NtMYB61 gene-overexpressing, and knockout tobacco materials under normal irrigation (control group) and drought stress (treatment group). Individual aboveground plants from each line were weighed and recorded, then individually placed in kraft paper envelopes and dried at 60℃ to constant weight. The weight of each plant was recorded again, and the relative water content of each line was calculated using the formula: Relative water content (%) = (fresh weight - dry weight) / fresh weight * 100%. The results are shown in Figures 9 and 10. Under normal irrigation (control group), there was no significant difference in fresh weight biomass and relative water content between the lines and wild-type tobacco K326. However, under drought treatment (treatment group), the fresh weight biomass and relative water content of the NtMYB61 overexpressing material were significantly lower than those of K326; conversely, the fresh weight biomass and relative water content of the mutant Ntmyb61 were significantly higher than those of the control K326. In summary, the data indicate that the three materials—NtMYB61 overexpression, K326, and Ntmyb61 mutant—exhibit progressively increasing resistance to natural drought.

[0063] In summary, all the data indicate that the protein encoded by NtMYB61, induced by mannitol and ABA, plays an important role in plant response to natural drought stress. Exploring the drought response capabilities of this protein will provide an important theoretical basis and molecular target for research on drought-resistant crop germplasm resources and crop drought-resistant breeding.

Claims

1. An NtMYB61 gene that can affect the resistance of tobacco to drought stress, characterized in that, The genome sequence is shown in SEQ ID No.

1.

2. An NtMYB61 gene that can affect the resistance of tobacco to drought stress, characterized in that, The CDS sequence is shown in 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 application of the NtMYB61 gene according to claims 1, 2 and 3 in improving the drought resistance of tobacco, characterized in that, The drought resistance of tobacco can be improved by mutating the NtMYB61 gene as described in claim 1.

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

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