Tobacco NtATAF1 gene and application thereof

Abstract

The invention discloses a tobacco NtATAF1 gene and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the NtATAF1 gene is as shown in SEQ ID NO. 1. The NtATAF1 gene explored by the invention is closely related to the drought tolerance of the tobacco, and the drought resistance of a tobacco plant edited with the NtATAF1 gene is obviously better than that of a wild type under a drought stress condition. The NtATAF1 gene explored by the invention has important theoretical and practical significance on tobacco drought-resistant breeding work.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a tobacco NtATAF1 gene and an application thereof. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an important leaf-producing economic crop, currently cultivated extensively in over 120 countries and primarily distributed in the Yunnan and Guizhou regions of my country. Tobacco is a key economic crop primarily harvested for its leaves, which are thick and plump, representing the part of the tobacco plant with the highest water content. Water is a crucial buffer in plants, playing a vital role in tobacco growth, development, and physiological and biochemical metabolism. Appropriate water content is crucial for ensuring high tobacco quality and yield. Studies have shown that tobacco has a high water requirement throughout its growth period. During tobacco growth and development, drought stress is the primary abiotic stress leading to reduced yield and quality. Field water holding capacity below 50% reduces tobacco yield and quality.

[0003] In recent years, with global warming, the uneven temporal and spatial distribution of rainfall has intensified, and drought events have become more frequent. Some tobacco-growing areas in Yunnan and Guizhou often suffer from adverse stresses such as "seedling drought" and "summer drought", which pose an increasing threat to tobacco production. Most areas in Yunnan and Guizhou provinces in my country have typical karst landforms. Guizhou Province, in particular, is the heart of the karst region in southwest my country, with a karst area of ​​109,000 km. 2 , accounting for 61.92% of the total land area of ​​Guizhou Province. There are many mountains and little water, and the water resources are unevenly distributed in time and space, resulting in drought disasters occurring every year, and traditional irrigation and drought relief measures are difficult to implement in this area.

[0004] Therefore, it is urgent to explore important functional genes through biotechnology and develop relevant molecular markers for application in tobacco breeding practice, promote the selection and breeding of drought-resistant tobacco varieties, and solve practical production problems. Summary of the Invention

[0005] In view of this, the present invention provides tobacco NtATAF1 gene and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The tobacco NtATAF1 gene has a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, or a degenerate sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Another object of the present invention is to provide tobacco NtATAF1 protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] Another object of the present invention is to provide an sgRNA, which is used to edit the above-mentioned NtATAF1 gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID No. 6 and / or SEQ ID No. 9.

[0010] Another object of the present invention is to provide a biomaterial, which comprises the above-mentioned NtATAF1 gene or the above-mentioned sgRNA, and the biomaterial is one or more of a vector, an engineered bacterium, or an expression cassette.

[0011] Another object of the present invention is to provide an application of the NtATAF1 gene or its encoded protein or the sgRNA or the biomaterial, wherein the application is any one of the following:

[0012] A. Regulate tobacco drought resistance,

[0013] B. Regulate tobacco superoxide dismutase activity,

[0014] C. Regulate tobacco peroxidase activity,

[0015] D. Regulate the content of malondialdehyde in tobacco,

[0016] E. Regulate the proline content in tobacco,

[0017] F. Tobacco genetics and breeding,

[0018] G. Preparation of transgenic tobacco.

[0019] Another object of the present invention is to provide a method for improving the drought resistance of tobacco by reducing the expression level of the NtATAF1 gene in tobacco or knocking out the NtATAF1 gene to cultivate drought-resistant strains.

[0020] Another object of the present invention is to provide a tobacco mutant encoding gene, wherein the protein encoded by the encoding gene has a mutation at position 32 of SEQ ID NO. 3, from leucine L to phenylalanine F.

[0021] Preferably, the coding gene undergoes a single base mutation at position 94 in the forward direction of SEQ ID NO.1 or SEQ ID NO.2, from C to T.

[0022] Beneficial Effects: This study used 300 core tobacco germplasm resources as materials, measured drought-resistance-related traits through soil-culture drought tests, and combined them with genome-wide association analysis using over 3 million high-quality SNP markers screened through resequencing. This analysis identified key genetic loci regulating tobacco drought tolerance and discovered the tobacco drought-resistance gene NtATAF1. Based on this gene's function, the tobacco drought-resistance gene NtATAF1 of this invention has important application value. Editing the tobacco NtATAF1 gene improves tobacco drought resistance and can be used to improve tobacco breeding efficiency. It also has important application value in improving plant drought resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 The Manhattan plot (A) and QQ plot (B) of tobacco drought resistance phenotype and genome-wide association analysis in the examples of the present invention are shown.

[0025] Figure 2 Schematic diagram of the tobacco NtATAF1 gene overexpression and gene editing vector structure in the embodiments of the present invention.

[0026] Figure 3 This is the gene expression situation of tobacco NtATAF1 gene overexpression and gene editing strains in the examples of the present invention.

[0027] Figure 4 The growth conditions of the gene-overexpressing plants, edited plants and wild-type K326 under natural drought stress in the examples of the present invention are shown.

[0028] Figure 5 These are the test results of superoxide dismutase activity, a drought resistance physiological indicator, in the gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in the examples of the present invention.

[0029] Figure 6 These are the test results of peroxidase activity, a physiological indicator of drought resistance, in the gene-overexpressed plants, edited plants, and wild-type K326 under natural drought stress in the examples of the present invention.

[0030] Figure 7 These are the test results of malondialdehyde content, a drought resistance physiological indicator, in the gene-overexpressed plants, edited plants, and wild-type K326 under natural drought stress in the examples of the present invention.

[0031] Figure 8 These are the test results of proline content, a physiological indicator of drought resistance, in the gene-overexpressed plants, edited plants, and wild-type K326 under natural drought stress in the examples of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Whole-genome resequencing

[0035] Whole-genome resequencing variant data analysis was performed on the 300 tobacco accessions listed in Table 1 (published in Shizhou Yu et al. "A high-resolution 20K SNP array for comprehensive genotyping and genetic mapping in Nicotiana tabacum L." The Crop Journal 13(2025):630-635). Genotype data analysis results were obtained using the same method as described in the comparison of SNP extraction software combinations based on tobacco genome resequencing data (Yu Shizhou et al., "Comparison of SNP extraction software combinations based on tobacco genome resequencing data," Tobacco Science & Technology 50.10(2017):1-7). The publicly available genome sequence of cultivated tobacco (NataSR1 V1.0, http: / / lifenglab.hzau.edu.cn / Nicomics / index.php) was used as the reference genome.

[0036] Table 1

[0037]

[0038]

[0039]

[0040]

[0041] 2. Identification of drought resistance phenotypes and genome-wide association analysis

[0042] The drought stress test was carried out in the artificial climate chamber of Guizhou Tobacco Science Research Institute in Guiyang. Each material was planted in two 12-hole planting boxes, with one tobacco seedling sown and retained in each hole. The experimental medium was tobacco seedling medium.

[0043] Tobacco seedlings were subjected to drought stress and a control treatment at the three-leaf, one-heart stage. One seedling was subjected to drought stress, with watering stopped until the soil relative moisture content reached approximately 60%. This was marked as drought stress day 0, after which watering was stopped and the seedlings remained in a natural state for 7 days (at which point significant phenotypic differences emerged between accessions). The other seedling served as a control, with normal watering throughout to maintain a soil relative moisture content of 60%-70%. Tobacco seedling traits were recorded according to tobacco industry standard YC / T142-2010. Leaf chlorophyll content (SPAD) and leaf temperature were measured for each accession using a chlorophyll meter. The study found that the SPAD value was highly correlated with the drought resistance phenotype of tobacco. Based on this phenotype, referring to the relevant research methods of Yu Shizhou et al. (Yu, Shizhou, et al. "Haplotype variations in QTL for salt tolerance in Chinese wheat accessions identified by marker-based and pedigree-based kinship analyses." The Crop Journal 8.6(2020):1011-1024.), a genome-wide association analysis was performed with the obtained genotype data. The Manhattan plot and QQ-plot of the genome-wide association analysis results are attached. Figure 1 The significant SNP sites associated with this trait are mainly located on chromosomes 7 and 14 of the NtaSR1 genome. There are hundreds of genes in the two significantly associated regions. Further combining genome annotation information and gene expression data, 20 important candidate genes were screened and obtained, and the NtATAF1 gene was one of the important candidate genes.

[0044] NtATAF1 gene sequence (lowercase letters represent introns of the gene):

[0045]

[0046] NtATAF1 gene CDS sequence:

[0047] ATGATCAAAGGCGTACACAGAAATCAGCAGCAATTGGAATTACCAGCGGGATTTAGATTCCATCCAACGGATGAGGAATTAGTTCAACATTATCTCTGCCGTAAATGCGCTGGGCAGCCTATATCTGTTTCAATTATAGCTGAAATTGATCTTTACAAGTTTGATCCTTGGCAATTACCTGAGAAGGCTTTGCACGGTGAAAAGGAATGGTATTTTTTCTCACCAAGAGATAGAAAATATCCGAACGGCTCACGGCCAAACAGAGCGGCCGGAGCCGGTTACTGGAAGGCCACCGGAGCTGATAAGCCAGTGGGAAAGCCCAAGACTTTAGGGATAAAGAAGGCGTTAGTGTTTTACGCTGGAAAAGCACCCAGAGGAATCAAAACCAATTGGATAATGCACGAATATCGACTCGCTAATGTGGACCGGTCTGCTGGCAAGAGCAATAATAACTTGAGGCTTGACGATTGGGTATTGTGTCGAATATACAACAAGAAGGGCACACTTGAGAAGTATTACAATGTGGATAACAAGGAACATTTGGGCTTCGATGAAGTTGAGGAAGAAGAGGAAAAACCAAAAATTTTACCATTTTCACAAAGTAATGAGTTGATAGCACAATTAGCACCAGCGCCGATGCCACCGCGGCCACAATCGATGCCGGGCTACTTTCACTTTGAAACGTCCGAGTCGATGACTAGGATGCATACAACAAACTCGAGCTCTGGGTCAGAGCATGTTTTGTCGTCGTGTAATAAGGAGGTCCAAAGTGCTCCCAAATGGGACGACCTCGACAATGCCCTTGATTTCCAGCTAAATTATTTGGACAGCTTCCAATATGACCCTTTTGAGCCCCAAATGCAGCAGCAAAATTGCAACATTGATCAGTTCAATTCTTTCCAAGACATGTTCGTATACATGCAGAAACCTTACTAA, SEQ ID NO.2。

[0048] NtATAF1 protein sequence:

[0049] MIKGVHRNQQQLELPAGFRFHPTDEELVQHYLCRKCAGQPISVSIIAEIDLYKFDPWQLPEKALHGEKEWYFFSPRDRKYPNGSRPNRAAGAGYWKATGADKPVGKPKTLGIKKALVFYAGKAPRGIKTNWIMHEYRLANVDRSAGKSNNNLRLDDW SEQ ID NO.3.

[0050] Example 2

[0051] Construction of tobacco NtATAF1 gene overexpression and gene knockout genetically transformed plants

[0052] Tobacco variety K326 was used as the test material and grown into seedlings. Total RNA was extracted using a total RNA miniprep kit (AxyPrep) and reverse transcribed using an EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen). The RNA was used as a template for PCR amplification.

[0053] The NtATAF1 gene was amplified by PCR using primers NtATAF_0S1_F (cagtGGTCTCacaacatgatcaaaggcgtacac ag, SEQ ID NO. 4) and NtATAF_0S1_R (cagtGGTCTCatacattagtaaggt ttctgcatgt, SEQ ID NO. 5). The reaction conditions were: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 15 seconds, 60°C annealing for 20 seconds, and 72°C extension for 3 minutes, for 35 cycles. Correct banding was observed by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel extraction kit. The recovered NtATAF1 gene coding region fragment was ligated with the pBWA(V)HS vector fragment containing the 35S promoter using T4 ligase to construct an overexpression vector, which was then sequenced to verify the correctness of the constructed vector.

[0054] CRISPR / Cas9 knockout sgRNA was designed in the first exon of the NtATAF1 gene using the CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) online website. The synthesized oligonucleotides were annealed and inserted into the CRISPR / Cas9 vector (pKSE401) as a gene editing vector. The target sites and detection primers are shown in Table 2.

[0055] Table 2

[0056]

[0057] Schematic diagram of gene overexpression and gene editing vectors is attached Figure 2 .

[0058] The constructed gene overexpression and gene editing vectors were respectively transferred into Agrobacterium (EHA105), and the leaf callus of tobacco K326 was infected by the leaf disc method. After screening and differentiation culture, the formation of buds and roots was induced to obtain genetically transformed plants.

[0059] The vector construction and genetic transformation of the present invention were commissioned to Wuhan Boyuan Biotechnology Co., Ltd.

[0060] The T0 generation positive plants were continuously multiplied in the artificial climate chamber of Guizhou Tobacco Science Research Institute to obtain T2 generation homozygous materials. Overexpression plants with higher gene expression levels and knockout plants with lower gene expression levels were selected as experimental materials for gene function verification (see Appendix Figure 3 ).

[0061] The primers used to identify the expression level of tobacco NtATAF1 gene are as follows:

[0062] NtATAF1-F:ACGACCTCGACAATGCCC, SEQ ID NO.10,

[0063] NtATAF1-R:TGCTGCATTTGGGGCTCA, SEQ ID NO.11;

[0064] Actin-F:TGGTTAAGGCTGGATTTGCT, SEQ ID NO.12,

[0065] Actin-R: TGCATCCTTTGACCCATAC, SEQ ID NO. 13.

[0066] Example 3

[0067] Identification of drought resistance in transgenic tobacco plants

[0068] The NtATAF1 gene overexpression T2 generation homozygous strain (OE-NtATAF1-4), gene knockout T2 generation homozygous strain (gE-ntafaf1-2) and K326 wild type (WT) seeds obtained in Example 2 were used as experimental materials. The responses of different strains to drought stress were observed by seedling drought stress treatment, and some physiological indicators such as superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), and proline (PRO) were detected.

[0069] Compared with the wild type, the gene sequence of the T2 generation homozygous knockout strain (gE-ntafaf1-2) showed no change in target 1, but a single base mutation occurred in target 2, namely, a single base mutation occurred at position 94 in the forward direction of SEQ ID NO.1 or SEQ ID NO.2, from C to T. As a result, the leucine (L) encoded by the codon was changed to phenylalanine (F), namely, the leucine (L) at position 32 in SEQ ID NO.3 was mutated to phenylalanine (F).

[0070] Seedling stage test treatment process: Place the seeds in a culture dish containing Hoagland nutrient solution for germination, and transplant them into peat soil for cultivation after germination. Grow them in an incubator with 16h light / 8h dark and 28℃ / 23℃. After about 45 days, when the material grows to three leaves and one heart, transplant them into a 9.5cm square culture box for growth. After transplanting, water the soil thoroughly (soil moisture is above 95%), and then maintain a natural growth state. Check the soil moisture at any time. When the soil moisture is 60%, it is recorded as dry day 0 (Dry 0d). Then stop watering and maintain natural drought. Dry for 7 days (Dry 7d) and 14 days (Dry 14d) in turn. After surveying and recording data on the day of dryness 14 days, rewater treatment is carried out, that is, water the soil thoroughly. Then, on the 7th day, that is, rewater 7d, record the growth of the material (see Appendix). Figure 4 ) and determination of enzymatic reactions (see Appendix Figure 5 -Attached Figure 8 ).

[0071] The enzyme activity and content of each component in different materials were determined using corresponding assay kits (Suzhou Gres Biotechnology Co., Ltd.) for the enzymatic reactions. Three biological replicates were set up.

[0072] Superoxide dismutase (SOD) catalyzes the production of superoxide anion free radicals (O 2- Disproportionation reaction occurs to remove 0 -. Generally speaking, when plants are subjected to drought stress, the activity of SOD will increase, and the increase in SOD of drought-resistant varieties will be greater. At the same time, the activity of peroxidase (POD), one of the active oxygen scavengers, will increase, thereby clearing low concentrations of peroxides. Malondialdehyde (MDA) is one of the most important products of plant membrane lipid peroxidation, and its production aggravates membrane damage. When plants are subjected to stress such as drought, the higher the MDA content in the plant, the more serious the damage to the membrane system. In contrast, when subjected to stress such as drought, a large amount of proline (PRO) will accumulate in the plant body to regulate cell osmotic potential, stabilize the structure of biological macromolecules, regulate cell pH, eliminate ammonia toxicity, and act as an energy reservoir to regulate cell redox potential, thereby improving the plant's ability to resist drought.

[0073] By comparing the growth and antioxidant enzyme activity of wild-type plants and transgenic plants under drought stress, it was found that on the 14th day of treatment, the superoxide dismutase activity in the knockout plants was significantly higher than that in the recipient plants; on 0 days, 7 days, 14 days and 7 days after rehydration, the peroxidase activity and proline content of the knockout plants were higher than those of the recipient plants; at the same time, the malondialdehyde content of the knockout plants was lower than that of the recipient plants, and showed significant differences on the 14th day of treatment.

[0074] From the above experimental results, it can be seen that the tobacco NtATAF1 gene is significantly correlated with tobacco drought resistance, and it is a negative regulatory gene.

[0075] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0076] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Tobacco NtATAF1 gene, characterized in that The nucleotide sequence of the NtATAF1 gene is shown as SEQ ID NO.1 or SEQ ID NO.2, or a degenerate sequence shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. Tobacco NtATAF1 protein, characterized in that The amino acid sequence of the NtATAF1 protein is shown in SEQ ID NO.

3.

3. An sgRNA, characterized in that The sgRNA is used to edit the NtATAF1 gene according to claim 1, and the sgRNA nucleotide sequence is shown as SEQ ID No. 6 and / or SEQ ID No.

9.

4. A biomaterial, characterized in that The biological material comprises the NtATAF1 gene according to claim 1 or the sgRNA according to claim 3, and the biological material is one or more of a vector, an engineered bacterium, or an expression cassette.

5. Use of the NtATAF1 gene or its encoded protein according to claim 1, the sgRNA according to claim 3, or the biomaterial according to claim 4, characterized in that: The application is any of the following: A. Regulate tobacco drought resistance, B. Regulate tobacco superoxide dismutase activity, C. Regulate tobacco peroxidase activity, D. Regulate the content of malondialdehyde in tobacco, E. Regulate the proline content in tobacco, F. Tobacco genetics and breeding, G. Preparation of transgenic tobacco.

6. A method for improving tobacco drought resistance, characterized in that: Drought-resistant strains can be cultivated by reducing the expression level of the NtATAF1 gene in tobacco or knocking out the NtATAF1 gene.

7. A tobacco mutant encoding gene, characterized in that The protein encoded by the coding gene is mutated at position 32 of SEQ ID NO. 3, from leucine L to phenylalanine F.

8. The tobacco mutant encoding gene according to claim 7, characterized in that The coding gene has a single base mutation at position 94 in the forward direction of SEQ ID NO.1 or SEQ ID NO.2, from C to T.

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