Tobacco ntataf1 gene and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the tobacco NtATAF1 gene and its applications. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an important leaf crop, widely cultivated in over 120 countries, and mainly distributed in the Yunnan-Guizhou region of my country. Tobacco is a crucial economic crop primarily harvested for its leaves, which are thick and large, containing the highest water content of the tobacco plant. Water is an important buffer within plants, playing a vital role in tobacco growth, development, and physiological and biochemical metabolism. Adequate water availability is essential for ensuring high quality and yield. Studies have shown that tobacco has high water requirements throughout its entire growth cycle. Drought stress is the main abiotic stress leading to reduced yield and quality during tobacco growth and development; field water holding capacity below 50% will decrease both yield and quality.

[0003] In recent years, with global warming, the uneven distribution of rainfall in time and space has intensified, and drought events have become more frequent. The "seedling drought" and "summer drought" that tobacco-growing areas in Yunnan and Guizhou provinces frequently suffer from are increasingly threatening tobacco production. Furthermore, most areas of Yunnan and Guizhou provinces in my country have typical karst landforms. Guizhou Province, in particular, is located in the heart of the southwestern karst region of my country, with a karst area of ​​109,000 km². 2 It accounts for 61.92% of the total land area of ​​Guizhou Province. It is mountainous with little water, and the uneven distribution of water resources in time and space causes drought disasters to occur every year. Moreover, traditional irrigation and drought relief measures are difficult to implement in this region.

[0004] Therefore, it is urgent to use biotechnology to discover important functional genes and develop related molecular markers for application in tobacco breeding practices, so as to promote the breeding of drought-resistant tobacco varieties and solve practical production problems. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The tobacco NtATAF1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2, or the degenerate sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Another object of the present invention is to provide a 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 for editing the NtATAF1 gene described above, wherein 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 biological material comprising the above-mentioned NtATAF1 gene or the above-mentioned sgRNA, wherein the biological material is one or more of a vector, engineered bacteria, or expression cassette.

[0011] Another object of the present invention is to provide the application of the above-mentioned NtATAF1 gene or its encoded protein, or the above-mentioned sgRNA or the above-mentioned biological material, wherein the application is any one of the following:

[0012] A. Regulating the drought resistance of tobacco,

[0013] B. Regulating the activity of tobacco superoxide dismutase

[0014] C. Regulates tobacco peroxidase activity.

[0015] D. Regulating the malondialdehyde content in tobacco.

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

[0017] F. Tobacco genetics and breeding,

[0018] G. Preparation of genetically modified tobacco.

[0019] Another objective of this 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 by knocking out the NtATAF1 gene, and cultivating 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 is mutated at position 32 of SEQ ID NO.3, changing 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, changing from C to T.

[0022] Beneficial Effects: This invention uses 300 core tobacco germplasm resources as materials. Drought-related traits were determined through soil-culture drought experiments. Genome-wide association analysis was then performed on over 3 million high-quality SNP markers selected through resequencing to identify key genetic loci regulating tobacco drought resistance, and the tobacco drought resistance gene NtATAF1 was discovered. Based on the function of this gene, the tobacco drought resistance gene NtATAF1 of this invention has significant application value. Editing the tobacco NtATAF1 gene improves the drought resistance of tobacco and can be used to improve tobacco breeding efficiency. It also has important application value in improving plant drought resistance breeding. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

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

[0025] Figure 2 This is a schematic diagram of the structure of the gene editing vector for overexpressing the tobacco NtATAF1 gene in an embodiment of the present invention.

[0026] Figure 3 This document illustrates the overexpression of the NtATAF1 gene in tobacco and the gene expression in gene-edited lines in embodiments of the present invention.

[0027] Figure 4 This illustrates the growth of gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in embodiments of the present invention.

[0028] Figure 5 The results of the detection of superoxide dismutase activity, a physiological indicator of drought resistance, in gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in the embodiments of this invention.

[0029] Figure 6 The results of the detection of peroxidase activity, a physiological indicator of drought resistance, in gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in the embodiments of this invention.

[0030] Figure 7 The results of the detection of malondialdehyde (MDA) content, a physiological indicator of drought resistance, in gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in the embodiments of this invention.

[0031] Figure 8 The results of the detection of proline content, a physiological indicator of drought resistance, in gene-overexpressing plants, edited plants, and wild-type K326 under natural drought stress in the embodiments of this invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Whole genome resequencing

[0035] Whole-genome resequencing variation data were analyzed for 300 tobacco germplasms listed in Table 1 (published in Shizhou Yu et al. "A high-resolution 20K SNP array for comprehensive genotyping and genetic mapping in Nicotianatabacum L." The Crop Journal 13(2025):630-635). The analysis method referred to 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.), and genotypic data analysis results were obtained. The publicly available Havana cultivated tobacco genome sequence was selected as the reference genome (NtaSR1 V1.0, http: / / lifenglab.hzau.edu.cn / Nicomics / index.php).

[0036] Table 1

[0037]

[0038]

[0039]

[0040]

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

[0042] The drought stress experiment was conducted in the Guiyang artificial climate chamber of the Guizhou Provincial Tobacco Science Research Institute. Each material was planted in two 12-well planting boxes, with one tobacco seedling planted in each well. The experimental substrate was tobacco seedling substrate.

[0043] Two treatments were applied to tobacco seedlings at the three-leaf stage: drought stress and a control. One planting box was subjected to drought stress, with watering stopped until the relative soil moisture content reached approximately 60%, initially recorded as day 0 of the drought stress treatment. Watering was then stopped, and the seedlings were left to rest for 7 days (at which point significant phenotypic differences between germplasms became apparent). The other planting box served as a control, receiving normal watering throughout the process to maintain a relative soil moisture content of 60%-70%. Relevant traits of the tobacco seedlings were investigated and recorded according to the tobacco industry standard YC / T142-2010. The relative chlorophyll content (SPAD value) and leaf temperature of each material were measured using a chlorophyll meter. The study found a high correlation between SPAD values ​​and drought resistance phenotype in tobacco. Based on this phenotype, and following the methods used by 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.), genome-wide association analysis (GWIA) was performed with the obtained genotype data. The Manhattan plot and QQ-plot of the GWIA 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. By further combining genome annotation information and gene expression data, 20 important candidate genes were screened, and the NtATAF1 gene was one of the important candidate genes.

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

[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 genetically transformed plants with overexpression and knockout of the NtATAF1 gene in tobacco

[0052] Using tobacco variety K326 as the test material, seedlings were cultivated. Total RNA was extracted from tobacco using a total RNA mini-preparation kit (AxyPrep), and reverse transcription of RNA was performed using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen) to serve as a PCR amplification template.

[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 min, 95°C denaturation for 15 sec, 60°C annealing for 20 sec, and 72°C extension for 3 min, for 35 cycles. The bands were checked 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 to the pBWA(V)HS vector fragment with a 35S promoter using T4 ligase to construct an overexpression vector. Sequencing was then used to verify the correctness of the constructed vector.

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

[0055] Table 2

[0056]

[0057] See attached diagrams for gene overexpression and gene editing vectors. Figure 2 .

[0058] The constructed gene overexpression and gene editing vectors were transferred into Agrobacterium (EHA105) and infected with tobacco K326 leaf callus tissue by 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 work of this invention were entrusted to Wuhan Boyuan Biotechnology Co., Ltd.

[0060] The obtained T0 generation positive plants were continuously passaged in the artificial climate chamber of the Guizhou Provincial Tobacco Research Institute to obtain T2 generation homozygous materials. Overexpression plants with high gene expression levels and knockout plants with low 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 the 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] Using seeds of the NtATAF1 gene overexpression T2 generation homozygous line (OE-NtATAF1-4), the gene knockout T2 generation homozygous line (gE-ntafaf1-2), and the K326 wild-type (WT) obtained in Example 2 as experimental materials, the response of different lines to drought stress was observed by treating them with drought stress during the seedling stage, and the levels of some physiological indicators such as superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), and proline (PRO) were detected.

[0069] Compared to the wild type, the gene sequence of the gene knockout T2 generation homozygous line (gE-ntafaf1-2) showed no change at target site 1, but a single-base mutation at target site 2. Specifically, a single-base mutation occurred at position 94 of SEQ ID NO.1 or SEQ ID NO.2, changing from C to T. This resulted in the codon encoding leucine (L) being replaced by phenylalanine (F), specifically at position 32 of SEQ ID NO.3.

[0070] Seedling stage treatment procedure: Seeds were placed in petri dishes containing Hoagland nutrient solution for germination. After germination, they were transplanted into peat moss for cultivation in an incubator with 16h light / 8h darkness and 28℃ / 23℃. After approximately 45 days, when the seedlings reached the three-leaf stage, they were transplanted into 9.5cm square culture boxes. After transplanting, the soil was thoroughly watered (soil moisture above 95%). Natural growth was then maintained, with soil moisture monitored regularly. A dry day (Dry 0d) was recorded when soil moisture reached 60%. Watering was then stopped, and natural drought was maintained for 7 days (Dry 7d) and 14 days (Dry 14d). Data was recorded on the 14th day of drought, followed by a rewatering treatment (thorough watering). Growth was recorded on the 7th day after rewatering (Rewater 7d) (see appendix). Figure 4 And the determination of enzyme-catalyzed reactions (see Appendix) Figure 5 - Appendix Figure 8 ).

[0071] Enzyme-catalyzed reactions were performed using appropriate assay kits (Suzhou Greelife Biotechnology Co., Ltd.) to determine the enzyme activity and content of each component in different materials. Three biological replicates were set up.

[0072] Superoxide dismutase (SOD) can catalyze the free radical O2 of superoxide anion. 2- Disproportionation reaction occurs to clear 0 -Generally, when plants are subjected to drought stress, superoxide dismutase (SOD) activity increases, with drought-resistant varieties showing a greater increase. Simultaneously, the activity of peroxidase (POD), a reactive oxygen species scavenger, increases, thereby clearing low concentrations of peroxides. Malondialdehyde (MDA) is one of the most important products of plant membrane lipid peroxidation, and its production exacerbates membrane damage. The higher the MDA content in plants under drought and other stresses, the more severe the damage to the membrane system. Conversely, under drought and other stresses, plants accumulate large amounts of proline (PRO) to regulate cell osmotic potential, stabilize biomolecular structures, regulate cell pH, detoxify ammonia, and act as an energy reservoir to regulate cell redox potential, thereby enhancing the plant's ability to resist drought.

[0073] Comparison of growth and antioxidant enzyme activity between wild-type and transgenic plants under drought stress revealed that, on day 14 of treatment, the superoxide dismutase activity in the knockout plants was significantly higher than that in the recipient plants. At days 0, 7, 14, and 7 after rehydration, the peroxidase activity and proline content in the knockout plants were all higher than those in the recipient plants. Meanwhile, the malondialdehyde content in the knockout plants was lower than that in the recipient plants, and the difference was significant at day 14 of treatment.

[0074] The experimental results above show that the tobacco NtATAF1 gene is significantly associated with tobacco drought resistance, and it is a negatively regulated gene.

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

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of tobacco mutant encoding genes in improving the drought resistance of tobacco, characterized in that, The protein encoded by the gene encoding the tobacco mutant has a mutation at position 32 of SEQ ID NO.3, changing from leucine L to phenylalanine F.

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

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