A tobacco axillary bud development-related gene and its application

Abstract

The present invention belongs to the field of plant biotechnology, and discloses a tobacco axillary bud development-related gene and its application. The sequence of the tobacco axillary bud development-related gene has: (1) any one of nucleotide sequences in SEQ ID NO: 1 or 2 or its complementary sequence; (2) a nucleotide sequence that hybridizes with the complementary sequence of the nucleotide sequence of (1) under medium stringency conditions, preferably high stringency hybridization conditions. Overexpression of the tobacco axillary bud development-related gene promotes the growth of tobacco axillary buds and increases branches. The gene can be used to cultivate new ideal tobacco varieties with few branches and high agricultural efficiency that do not require artificial branch removal or the use of bud inhibitors through genetic engineering technology and breeding technology, providing new methods and research directions for reducing labor and increasing efficiency in tobacco leaf production, reducing pesticide residues, and improving tobacco leaf quality.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a tobacco axillary bud development-related gene and an application thereof. Background Art

[0002] Branching is an important component of plant morphology and one of the determining factors affecting crop yield. Tobacco is an economic crop that mainly harvests leaves. Increased branching will lead to a decrease in leaf nutrients, which in turn affects the yield and quality of tobacco leaves. Therefore, reducing tobacco branching is of great significance for tobacco production. During the budding and flowering period of tobacco growth, topping is usually used to terminate the reproductive development process so that nutrients can be concentrated on the leaves; however, after topping, the apical dominance of the plant is lost, and the tobacco axillary buds will grow rapidly into side branches. This process consumes a large amount of nutrients, causing the outflow and reduction of nutrients in the tobacco leaves, resulting in small and thin tobacco leaves, reduced oil content, reduced aroma, and a serious decrease in yield and quality. To address this problem, the current conventional method in production is to control the growth of tobacco axillary buds by artificially removing branches or using bud inhibitors, but these methods have problems such as increased production costs, environmental pollution, and pesticide residues.

[0003] In the current research field of branch development, the interaction of various factors and the complex hormone regulation network make it difficult to analyze the mechanism of branch development, but it also provides more potential and feasible practical means for the regulation of branch development. The functions of RAV family members in flower development and flowering control have been deeply analyzed, but the molecular mechanism of their participation in the process of plant branch development is rarely studied.

[0004] Therefore, identifying and isolating genes related to tobacco axillary bud development and studying their functions are not only conducive to analyzing the mechanism of tobacco axillary bud development at the molecular level, but also have important significance for cultivating less-branched and high-efficiency tobacco by inhibiting axillary bud development genes, providing new methods and research directions for reducing labor and increasing efficiency in tobacco production, reducing pesticide residues, and improving tobacco quality. Summary of the invention

[0005] In view of the problems of high cost, environmental pollution and excessive pesticide residues in the current tobacco variety production using artificial branch removal or bud inhibitors to control axillary buds, the present invention provides a tobacco axillary bud development-related gene NtMAB1 and a recombinant vector and a recombinant host cell containing the gene. The gene NtMAB1 can be used to cultivate agriculturally efficient tobacco varieties with few branches that do not require artificial branch removal or the use of bud inhibitors after topping through genetic engineering methods, providing a new research direction for controlling and increasing tobacco yield.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a tobacco axillary bud development-related gene, the sequence of which has:

[0008] (1) any one of the nucleotide sequences in SEQ ID NO: 1 or 2 or a complementary sequence thereof;

[0009] (2) A nucleotide sequence that hybridizes to a complementary sequence of the nucleotide sequence of (1) under moderately stringent conditions, preferably highly stringent hybridization conditions.

[0010] The present invention utilizes tobacco T-DNA activation tag insertion mutant library, finds a multi-branched, late-flowering mutant plant in the mutant population, names it mab1 (more axillary branches 1), and conducts molecular biology research on this mutant strain. Candidate genes are determined by Tail-PCR, co-segregation analysis of genotype and phenotype, and qRT-PCR analysis, and gene cloning and protein structure analysis are performed on the gene, confirming that the gene is a transcription factor of the RAV family, and through NtMAB1 gene function research, confirming that the NtMAB1 gene is a positive regulatory factor for tobacco axillary bud growth and development, and conducting specific research on the gene to determine its application prospects and application methods.

[0011] The tobacco axillary bud development-related gene genes claimed in the present application include not only the nucleotide sequences of SEQ ID No: 1 and SEQ ID No: 2 (NtMAB1-S1 and NtMAB1-T1) and their complementary sequences, but also other nucleotide sequences that are further optimized or modified on the sequences but can still encode proteins with basically the same functions. Among them, the protein sequence expressed by the sequence NtMAB1-S1 of the present application is as shown in SEQ ID NO: 7.

[0012] Since the above-mentioned gene is prone to mutation during the replication of the plant genome, the mutants of the above-mentioned gene are also within the scope of protection requested in this application. The above-mentioned gene NtMAB1 not only includes a nucleotide sequence as shown in SeqIDNo: 1 or SeqIDNo: 2 or a complementary sequence thereof, but also includes a mutant sequence having a sequence homology higher than 98-99% with SeqIDNo: 1 or SeqIDNo: 2 and encoding a protein with the same activity or function. The above-mentioned mutant sequence may be a point mutation, a deletion mutation or an addition mutation, and relative to the original nucleotide sequence, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides may change.

[0013] The meaning of "having" in this article is: the gene can be a sequence of only the nucleotide sequence shown in Seq ID No: 1 or Seq ID No: 2, or it can be a derivative sequence obtained by modification and processing based on the nucleotide sequence shown in Seq ID No: 1 or Seq ID No: 2, such as adding restriction sites, enhancers and other genetic elements or various tag sequences at both ends of the gene sequence.

[0014] Among them, the protein sequence expressed by the sequence NtMAB1-S1 of the present application is as shown in SEQ ID N0: 7.

[0015] In a second aspect, the present invention provides a recombinant vector comprising the polynucleotide sequence of the gene NtMAB1, wherein the recombinant vector is a cloning vector or an expression vector for expressing the polynucleotide. The recombinant vector includes various existing cloning vectors and expression vectors, and is not limited to the specific vectors used in the embodiments of the present invention.

[0016] In a third aspect, the present invention provides a recombinant host cell, comprising the polynucleotide sequence of the gene NtMAB1 or the above-mentioned recombinant vector, or the polynucleotide sequence of the gene NtMAB1 integrated into its genome. The host cell can be selected from plant cells or microbial cells, such as Escherichia coli cells, Agrobacterium cells, preferably plant cells, most preferably tobacco cells. The cell can be isolated, in vitro, cultured, or a part of a plant. Preferably, the cell is an Escherichia coli or Agrobacterium cell.

[0017] In a fourth aspect, the present invention also provides a specific primer pair for identifying genes related to tobacco axillary bud development, the specific primer pair comprising NtMAB1S1-F1 and NtMAB1S1-R1, whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and NtMAB1T1-F1 and NtMAB1T1-R1, whose sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0018] In a fifth aspect, the present invention provides a method for identifying a gene related to tobacco axillary bud development, comprising the following steps:

[0019] The extracted genome of the tobacco to be identified is used as a template, and the above-mentioned specific primer pair is used as a primer for PCR amplification. The amplified product is detected by electrophoresis, and the amplified product is sequenced. The sequencing result is compared with the sequence of the tobacco axillary bud development-related gene to obtain the identification result.

[0020] In a sixth aspect, the present invention also provides an application of the above tobacco axillary bud development-related gene in cultivating high-efficiency tobacco varieties with inhibited axillary bud growth.

[0021] Preferably, the application includes:

[0022] (1) Using RNA silencing technology, genetic engineering operations are performed on the nucleotide sequences of the above tobacco axillary bud development-related genes to suppress and silence their expression, thereby cultivating new tobacco varieties with fewer branches and higher agricultural efficiency;

[0023] or (2) using gene editing technology to edit the target nucleotide sequence of the tobacco axillary bud development-related gene, knock out and block its expression, and cultivate a new tobacco variety with less branches and agricultural high efficiency;

[0024] or (3) using TILLING technology, screening the EMS mutant library for the sequence fragments of the aforementioned tobacco axillary bud development-related genes to obtain the expression termination mutants thereof, and cultivating new tobacco varieties with less branches and agricultural high efficiency;

[0025] or (4) using PCR technology to screen a library of deletion mutants of sequence fragments of the aforementioned tobacco axillary bud development-related genes, obtain deletion mutants thereof, and cultivate new tobacco varieties with fewer branches and higher agricultural efficiency.

[0026] In a seventh aspect, the present invention further provides a method for preparing a tobacco NtMAB1 gene knockout plant, the method comprising the following steps:

[0027] In a fifth aspect, the present invention further provides a method for preparing a tobacco NtMAB1 gene knockout plant, the method comprising the following steps:

[0028] (1) Sequence analysis of the aforementioned NtMAB1 gene was performed and target sites were designed in the conserved regions;

[0029] (2) Using the sequence of the NtMAB1 gene as a template, M1Cas9-1F and M1Cas9-1R were amplified using the target primers shown in SEQ ID NO: 8 and SEQ ID NO: 9 to obtain a double-stranded insert fragment; the fragment was connected with the purified BsaI-digested pHSE401 vector fragment, and the ligation product was transformed into Escherichia coli competent cells, cultured overnight in a resistant medium, and the positive clones were sequenced, and the NtMAB1 knockout vector was extracted from the positive clones with correct sequencing;

[0030] (3) The successfully constructed NtMAB1 knockout vector was transformed into Agrobacterium and tobacco was infected through the leaf disc method to obtain tobacco NtMAB1 gene knockout plants.

[0031] Preferably, the above step (2) is specifically as follows: based on the target primer pair M1Cas9-1F and M1Cas9-1R as shown in SEQ ID NO: 8 and SEQ ID NO: 9, 10 ul of each of the above primers with a concentration of 100 uM are mixed in equal volumes, denatured at 95°C for 3 min, and cooled in a 0.3% gradient to 25°C. After 10°C, a double-stranded insertion fragment is generated; the fragment is mixed with the purified BsaI-cut pHSE401 vector fragment in equal proportions, and the competent Escherichia coli is transformed after ligation with ligase, and cultured overnight on Kana-resistant LB medium, and the positive clones are identified and sent for sequencing.

[0032] In an eighth aspect, the present invention further provides a use of the above-mentioned tobacco NtMAB1 gene knockout plant in cultivating high-efficiency tobacco varieties with inhibited axillary bud growth.

[0033] In a ninth aspect, the present invention further provides a method for identifying the above-mentioned tobacco NtMAB1 gene knockout plant, the method comprising: using the extracted genome of the tobacco to be identified as a template, performing PCR amplification on it with identification primers, sequencing the amplified product, and comparing the sequencing result with the sequence of the NtMAB1 gene to obtain the identification result;

[0034] The identification primers are: M1C1-SF1 and M1C1-SR1 as shown in SEQ ID NO: 10 and SEQ ID NO: 11, or M1C1-TF2 and M1C1-TR2 as shown in SEQ ID NO: 12 and SEQ ID NO: 13.

[0035] The technical solution of the present invention has the following beneficial effects:

[0036] 1. The present invention provides a multi-branched, late-flowering mutant plant screened out, which provides good material for the study of the molecular mechanism of tobacco axillary bud development. More importantly, a new gene NtMAB1 that regulates axillary bud development in tobacco was identified, and it was determined that the gene belongs to a transcription factor of the RAV family.

[0037] 2. The present invention cloned and verified the function of the gene NtMAB1. The mutation or overexpression of the NtMAB1 gene promoted the growth of tobacco axillary buds and increased the number of branches. The gene can be used through genetic engineering technology and breeding technology to cultivate new ideal tobacco varieties with few branches and agricultural high efficiency that do not require artificial branch removal or the use of bud inhibitors, providing new methods and research directions for reducing labor and increasing efficiency in tobacco production, reducing pesticide residues, and improving tobacco quality.

[0038] 3. The present invention also provides a gene knockout plant of the NtMAB1 gene. Compared with the wild type, the growth trend of axillary buds and branches at each leaf position of this plant before topping is weaker. After topping, the growth of the lower axillary buds is significantly inhibited, which can reduce or avoid the use of bud inhibitors and manual branch removal, thereby achieving the effects of reducing labor and increasing efficiency in tobacco production and reducing pesticide residues. It has important theoretical reference value and application value.

[0039] Specifically, after studying the branch development of gene knockout plants, mutants and wild types under topping measures, it was found that the length of axillary buds and branches at each leaf position of different tobacco strains before topping showed a trend of mab1≥HD>KO-1, among which mab1 was a gain-of-function mutant, HD was a wild type, and KO-1 was a NtMAB1 gene knockout strain. We measured and statistically analyzed the length of axillary buds and branches three days and seven days after topping. The results showed that within three days of topping, the growth length trend of axillary buds at each leaf position was mab1>KO-1≥HD, while within seven days of topping, the growth trend of axillary buds in the middle and upper parts was mab1>KO-1>HD, and the growth trend of axillary buds in the lower parts was mab1>HD>KO-1.

[0040] 4. The present invention also provides a specific primer pair for identifying tobacco axillary bud development-related genes, which facilitates rapid and accurate identification of recombinant vectors, recombinant host cells and transgenic plants carrying the axillary bud development-related genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The phenotypic characteristics of mab1 mutants at different growth stages and quantitative analysis of axillary bud traits; a: Phenotypic characteristics of branches during flowering (leaves removed): HD-fp, wild-type Honghua Dajinyuan (HD) in flowering period; mab1, mutant in the same period as HD-fp; mab1-fp, mab1 mutant in flowering period, flowering period 20 days later than wild-type under greenhouse conditions; b and c are the axillary branch phenotypes of wild-type and mutants in cluster stage, respectively; d: Comparison of branch lengths at corresponding leaf positions between wild-type and mutants in cluster stage; e and f are the axillary branch phenotypes of wild-type and mutants in vigorous growth stage, respectively; g: Comparison of branch lengths at corresponding leaf positions between wild-type and mutants in vigorous growth stage;

[0042] Figure 2Determination of target insertion sites and candidate genes; A: T-DNA activation tag insertion site and possible activated genes upstream and downstream of the insertion site, U (Upstream) and D (Downstream) are respectively upstream (opposite to the direction of the 35S enhancer sequence) and downstream (consistent with the direction of the 35S enhancer sequence) of the insertion site; BE: Expression of candidate genes in different tissues of HD and mab1 mutants, 1S, stem samples at the seedling stage; 1L, leaf samples at the seedling stage; 3AB1U, mixed samples of the three upper axillary buds before topping in the vigorous growth stage; 3AB1D, mixed samples of the three lower axillary buds before topping in the vigorous growth stage;

[0043] Figure 3 Phenotypic characteristics of the F1 generation heterozygous line F1001 and the expression analysis of the NtMAB1 gene in F1001 plants; A: Phenotypic characteristics of F1001; B: Quantitative analysis of axillary bud traits in F1001; C, D: Expression analysis of NtMAB1 and NtBRC1 genes in upper and lower axillary buds in F1001;

[0044] Figure 4 PCR amplification of NtMAB1 gene in genomic DNA and cDNA of Nicotiana velutipes and Nicotiana tabacum; T: Nicotiana velutipes; S: Nicotiana tabacum; M: Marker;

[0045] Figure 5 This is the structural analysis of NtMAB1-S1 / T1 protein and RAV family member proteins; the black line marks the AP2 functional domain; the red line marks the B3 functional domain shared by the 9 members; the blue line marks the B3 inhibitory functional domain that is highly conserved between tobacco and Arabidopsis;

[0046] Figure 6 Phenotypic analysis of NtMAB1 overexpressing plants; A, B: Photos of NtMAB1 overexpressing plants; C: Quantitative analysis of axillary bud traits of NtMAB1 overexpressing plants; D: Photos of leaves of NtMAB1 overexpressing plants; E, F: Expression levels of NtMAB1 gene in upper and lower axillary buds of overexpressing plants;

[0047] OE: over expression; U: upper axillary bud or branch; D: lower axillary bud or branch;

[0048] Figure 7 is the expression analysis of NtBRC1 gene in the overexpression line; AB-U: upper axillary bud; AB-D: lower axillary bud;

[0049] Figure 8The transgenic positive plants edited by CRISPR-Cas9 in tobacco NtMAB1 gene were obtained; A: The target site of CRISPR-Cas9 editing of NtMAB1 gene in tobacco; B: The PCR test results of the transgenic positive seedlings edited by CRISPR-Cas9 in tobacco;

[0050] Fig. 9 CRISPR-Cas9 gene editing method of NtMAB1 gene in M1CR1-102 plants; A: Sequencing results of gene editing sites in M1CR1-102 strain; B: NtMAB1-S1 gene editing causes premature codon termination;

[0051] Fig.10 Analysis of the phenotypic characteristics and NtMAB1 expression levels of different types of tobacco plants before topping; A: Phenotypic characteristics of axillary buds of KO-1 and control plants; B: Expression levels of the NtMAB1 gene in KO-1 and control strains, IU is the upper axillary buds of the tobacco plant before topping; ID is the lower axillary buds of the tobacco plant before topping. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0053] Example 1 Screening of tobacco branching mutant mab1

[0054] 1. Plant materials:

[0055] The donor parent Honghua Dajinyuan (abbreviated as Hongda, HD) and the T-DNA activation tag T1 generation mutant strain mab1 were selected as experimental materials. The wild-type material HD and the T-DNA activation tag insertion mutant mab1 seeds were provided by the tobacco germplasm resource sub-platform of the National Crop Germplasm Resource Platform and the tobacco mutant library of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences. The mutant library was constructed by inserting the T-DNA activation tag vector pSKI015. Other experimental materials were obtained by vector construction and tobacco genetic transformation with HD as the background.

[0056] Table 1 Strains and vectors used in the experiment

[0057]

[0058] 2. Instruments and equipment: flower pots, seedling medium, Canon 5Ds camera, ruler

[0059] 3. Test methods

[0060] The axillary bud phenotypes of Honghua Dajinyuan and T1 generation mab1 mutant were observed throughout the growth period, and the growth of the plants was photographed using a Canon 5Ds camera. During the clustering and vigorous growth period, five lines of mab1 homozygous mutant plants were counted, and the length of axillary buds or branches of Honghua Dajinyuan and mab1 mutants were measured and recorded with a ruler.

[0061] 4. Results and Analysis

[0062] The laboratory screened a multi-branched late-flowering mutant strain from the T1 generation activation tag mutant library. At the cluster stage, axillary branches were clearly visible at each leaf position of the entire plant, and the length of branches at each leaf position of the mutant was significantly longer than that of the wild type ( Figure 1 b,c,d), we named this gain-of-function mutant mab1.

[0063] In the vigorous growth period, axillary branches at all leaf positions of mab1 mutant plants were still clearly visible ( Figure 1 e, f), the branch lengths at each leaf position of the mutant were still significantly higher than those of the wild type ( Figure 1 g). The mutant axillary buds continued to grow and later grew into branches; they also flowered late. Under greenhouse conditions, the mutant's flowering period was 20 days later than that of the wild type ( Figure 1 a), and the flowering process is slow, with small and few flowers; inflorescences appear on the top 2-3 branches, but they are all smaller than the main stem inflorescences; in terms of plant height, the mutant is slightly shorter than the wild type HD during the clustering and vigorous growth stages, but in the reproductive growth period, the mutant's plant height is consistent with HD, or even higher than HD ( Figure 1 a).

[0064] Example 2 Determination of target insertion site and candidate gene NtMAB1

[0065] Reagents and kits:

[0066] CWBIO plant genomic DNA extraction kit, plant RNA extraction kit, reverse transcription kit, rTaq enzyme, KOD enzyme, TAKARA fluorescent quantitative reaction polymerase kit.

[0067] 1. Experimental methods

[0068] (1) Flanking sequence amplification

[0069] This experiment uses the TAIL-PCR (thermal asymmetric interlaced PCR) method to amplify the fragments on both sides of the T-DNA activation tag. Using the DNA of the T1 generation positive plant as a template, three PCR amplifications were performed based on the pSKI015 specific nested primers SP1, SP2, SP3, and random primers AD. The reaction system for each time is as follows:

[0070] Set up the PCR reaction program:

[0071]

[0072] The products obtained by three-step PCR amplification were subjected to electrophoresis test. Because fragments smaller than 500 bp are mostly vector sequences, it is difficult to analyze effective flanking sequence information. Therefore, products with bands larger than 500 bp were excised and recovered and sent for sequencing. The sequencing results were subjected to Blast comparison analysis in the tobacco genome database to determine the T-DNA insertion position.

[0073] (2) Co-segregation analysis

[0074] A pair of genome-specific primers GF / GR were designed based on the 1000bp sequence of the genome upstream and downstream of the insertion position. A specific primer TP was designed based on the T-DNA insertion sequence and combined with GR to form a pair of primers (see Table 2). Two PCR reactions were performed to determine whether the plant was a homozygous mutation or a heterozygous mutation and to prove the accuracy of the insertion position. The PCR reaction system and procedure refer to the conventional PCR reaction.

[0075] Table 2 Primers for co-segregation experiments

[0076]

[0077] (3) Identification of candidate genes

[0078] The target insertion position was determined by flanking sequence analysis and co-segregation, and the Gbrows tool was used to search for genes within 100 kb upstream and downstream of the insertion site to obtain candidate genes flanking the insertion site.

[0079] (4) Expression of candidate genes

[0080] RNA extraction: Homozygous mutants and safflower Dajinyuan were planted in the greenhouse under the same cultivation method and management conditions. Fresh leaves and axillary buds of the mutants and safflower Dajinyuan were taken during the vigorous growth period, wrapped in tin foil and quickly stored in liquid nitrogen. Total RNA was extracted using the TAKARA Plant RNA Mini Extraction Kit, and the specific steps were carried out according to the instructions.

[0081] Synthesis of cDNA chain: This experiment uses Takara PrimeScript TM II 1st Strand cDNA Synthesis Kit was used to reverse transcribe the extracted RNA. The system steps were referred to the instruction manual. The reverse transcribed cDNA was stored in a -20℃ refrigerator.

[0082] Fluorescence quantitative PCR to determine the target gene: refer to the design principle of real-time fluorescence quantitative PCR primers, and design primers according to the candidate gene sequence (Table 3). The cDNA concentration was diluted 4 times as a template for qRT-PCR (real-time quantitative reverse transcription-PCR) reaction, and the tobacco housekeeping gene Ubiquitin-conjugating enzyme E2 (Ntubc2) was used as an internal reference. TAKARA's fluorescence quantitative kit (SYBR Premix Ex Taq TM) was used for qRT-PCR amplification. Three technical replicates were designed for each reaction, and the system and reaction procedures were completely referred to the instructions. The experimental data were analyzed using ABI7500 fluorescence quantitative analysis software.

[0083] Table 3 Fluorescence quantitative PCR primers

[0084]

[0085] (5) Analysis of the first generation of hybrids between homozygous mab1 mutants and HD

[0086] 2. Experimental results and analysis

[0087] (1) The mutant DNA was amplified using the TAIL-PCR method to obtain two flanking sequences, indicating that the mutant contains at least two insertion sites. According to the gene sequences on both sides of the insertion site and the activation tag vector sequence, a pair of genome-specific primers and a vector-specific primer were designed respectively. The three primers formed two pairs of primer combinations, and the mutant segregation generations were genotyped to obtain the genotype of each individual in the isolated strain, and then matched with the individual phenotype. If both the homozygous and heterozygous genotypes of the mutant correspond to the mutant phenotype, and the wild-type genotype corresponds to the wild-type phenotype, then the insertion site is the target insertion site, that is, the genotype and phenotype are co-segregated. The co-segregation analysis of genotype and phenotype showed that only insertion site 1 (C110) met the co-segregation rule of genotype and phenotype, so C110 is the target insertion site.

[0088] (2) Since the effect of the 35S enhancer is not directional, after obtaining the target insertion site, we analyzed the genes within 100 Kb upstream and downstream of the target insertion site ( Figure 2 A) If Figure 2As shown, there are 6 genes within 100kb upstream and downstream of the insertion site, including 4 upstream genes named U1-U4 and 2 downstream genes named D1-D2. All 6 genes within this range may be activated for expression.

[0089] The upper and lower axillary buds of HD and mab1 mutant plants at the vigorous stage, and the stems and leaves of HD and mab1 at the seedling stage were used as samples to perform qRT-PCR analysis on the above six flanking genes ( Figure 2 BE), the results show that:

[0090] The expression level of gene D1 in the stem and leaf of the mutant mab1 in the seedling stage was 45 and 32 times higher than that in HD, respectively; the expression level of gene D1 in the upper axillary buds AB1U and the lower axillary buds AB1D in the vigorous growth stage was 32 and 116 times higher than that in the upper and lower axillary buds of HD, respectively; the expression level of gene D1 in the lower axillary buds was 3-4 times higher than that in the upper axillary buds, while the expression levels of the other five genes had no significant differences compared with HD, indicating that the D1 gene is the candidate gene of this study and it was named NtMAB1.

[0091] To further confirm that the phenotype of the mab1 mutant was caused by the activation of the NtMAB1 gene, we crossed the homozygous mab1 mutant with HD and found that the phenotype of axillary branches in the F1 generation (named F1001) was between the wild type and the homozygous mab1 mutant, and the expression level of the NtMAB1 gene in the upper and lower axillary buds of the F1001 plants was also between that of HD and the homozygous mab1 ( Figure 3 A, B). NtBRC1 is a gene that maintains axillary bud dormancy in many species. The higher the expression of NtBRC1, the stronger the inhibitory effect on the activity of axillary buds, and the slower the growth of axillary buds. The expression of the NtBRC1 gene that maintains axillary bud dormancy in tobacco was detected. The results showed that the expression of the NtBRC1 gene in the upper and lower axillary buds of F1001 plants was between HD and mab1, and the expression level of the NtBRC1 gene in mab1 was the lowest. The expression of NtBRC1 and NtMAB1 in the three types of plants showed opposite trends ( Figure 3 C, D).

[0092] Example 3 Cloning and bioinformatics analysis of NtMAB1 gene

[0093] 1. Experimental methods

[0094] (1) Amplification and purification of target gene fragments

[0095] Common tobacco is an allotetraploid, and its two ancestral species are forest tobacco (N. sylvestris) and hairy tobacco (N. tomentosiformis). Similarly, the NtMAB1 gene has two copies from forest tobacco and hairy tobacco, respectively. A pair of primers (Table 4) were designed according to their specificity, and the genomic DNA (gDNA) and cDNA sequences of NtMAB1-S1 and NtMAB1-T1 were amplified from the genomic DNA and cDNA of common tobacco, respectively. The PCR reaction products were purified using the Takara DNA fragment purification kit.

[0096] Table 4 Primers for NtMAB1 gene amplification

[0097]

[0098] (2) TA cloning and transformation of E. coli

[0099] After the purified DNA fragments were subjected to terminal addition "A" reaction, they were mixed with pMD TM Add the 19T vector to the centrifuge tube at a ratio of 3:1 and mix evenly. Add an equal volume of SolutionI and mix evenly. Connect at 16℃ for 1-2h. Transform the connected vector into E. coli. When E. coli grows on LB medium, add 1ml liquid LB medium and 1μl ampicillin to a 1.5ml centrifuge tube. Pick a single colony in the clean bench, place it in this centrifuge tube, number it in sequence, and shake it on a 37℃ shaker for 5-6h. Perform bacterial liquid PCR to detect the target fragment. The PCR system and procedure refer to the rTaq enzyme reaction system. Select the positive strain and send it to BGI for sequencing.

[0100] 2. Experimental results

[0101] like Figure 4 As shown, the gDNA sequence of NtMAB1-S1 is 2294bp in length, and the gDNA fragment length of NtMAB1-T1 is 2368bp. At the same time, the cDNA sequences of NtMAB1-S1 and NtMAB1-T1 were amplified from the cDNA of common tobacco, with full lengths of 1002bp and 1187bp, respectively. When analyzing the gene sequence, it was found that the gene copy of the villous tobacco had a stop codon, and the gene copy was a pseudogene.

[0102] The NtMAB1-S1 gene was subjected to Blast in NCBI, and it was found that the gene had a high similarity with the NGAL1 gene in the RAV subfamily of the B3 superfamily in Arabidopsis thaliana, and belonged to the RAV family.

[0103] In addition to the B3 domain, RAV family members may also contain an AP2 domain. Sequence alignment of NtMAB1-S1 with several RAV proteins reported in Arabidopsis revealed that NtMAB1-S1 does not contain the AP2 domain and the nuclear localization signal NLS; however, RAV family members are highly conserved in the B3 domain; tobacco and Arabidopsis contain a highly conserved transcriptional repression domain (BRD, B3 repression domain), and its key repression region sequence is "R / KLFGV" (e.g. Figure 5 This suggests that the NtMAB1 gene likely functions as a transcriptional repressor.

[0104] Example 4 Construction of tobacco NtMAB1 gene overexpression vector and genetic transformation

[0105] 1. Experimental methods

[0106] (1) Overexpression vector construction

[0107] The positive strain prepared in the previous example with the correct sequencing result was inoculated into a 15 mL sterile centrifuge tube for propagation, and the plasmid was extracted using the Tiangen Company's high-purity plasmid extraction kit. The extracted plasmid was double-digested. TM 19T restriction site and the restriction site on the overexpression vector pCAMBIA1300-35s. The restriction sites were determined to be Sal I and BamH I. The pMD TM The 19T vector and the overexpression vector pCAMBIA1300-35s were double-digested, and the reaction system was based on the NEBCutSmart reaction system. The digestion products were analyzed by electrophoresis, the correct bands were recovered, and their concentrations were measured.

[0108] The obtained double-enzyme-cut DNA fragment containing the target gene and the double-enzyme-cut DNA fragment of the overexpression vector pCAMBIA1300-35s were subjected to enzyme ligation reaction. After the reaction was completed, the reaction product was transformed into Escherichia coli competent cells, and the transformed Escherichia coli was positively tested and sequenced to determine whether the transformation was successful.

[0109] (2) Cultivation of sterile tobacco seedlings

[0110] Agrobacterium-mediated genetic transformation of tobacco leaf discs requires sterile tobacco leaves, so it is necessary to culture tobacco sterile seedlings before transformation. In the clean bench, the seeds of Honghua Dajinyuan were sterilized and cultured at a constant temperature of 25°C on MS medium for about 40 days, and sterile seedlings with dark green leaves and strong growth were selected for use.

[0111] (3) Agrobacterium-mediated tobacco leaf disc transformation

[0112] The recombinant vector was transformed into tobacco using the leaf disc transformation method. Hygromycin should be added to the differentiation medium for resistance screening. The operation steps were completely based on the existing tobacco leaf disc transformation method.

[0113] (4) Positive identification and phenotypic analysis of transgenic tobacco seedlings

[0114] Use the cut gun tip as a punch to take a 5-7mm diameter leaf tissue into a 100μ0 centrifuge tube. Add 50 μl of Buffer P1 to the centrifuge tube. Cover the centrifuge tube cap and place it in a PCR instrument to lyse at 95℃ for 10 minutes. Add 50 μl of Buffer P2 and mix with a pipette. The resulting lysate mixture can be directly used as a template for PCR reaction. Positive identification primers JC-F and JC-R were designed based on the vector fragment and the connected gene fragment (see Table 5). The positive transgenic seedlings identified are the 35Spro:NtMAB1 overexpression lines.

[0115] Table 5 Primers for positive identification of transgenic seedlings

[0116]

[0117] PCR reaction system:

[0118]

[0119] The PCR reaction procedure is the same as the conventional PCR procedure.

[0120] 2. Experimental results and analysis

[0121] (1) Compared with wild-type HD, 35Spro:NtMAB1 overexpression lines showed phenotypes such as many and long axillary branches, narrow leaves, short plants and late flowering. Among them, the OE-1 type overexpression plants were very consistent with the phenotype of the activation tag insertion mutant mab1. In order to quantitatively analyze the axillary bud phenotypic characteristics of NtMAB1 overexpression plants, the length of axillary buds or branches at the axils of each leaf position of HD and 35Spro:NtMAB1 was measured. The results showed that the phenotypes of 35Spro:NtMAB1 overexpression OE-1 and mab1 in branching were very similar, with a large number of branches and long axillary branches, while the axillary buds at each leaf position of the control group HD were shorter and dormant ( Figure 6 AC). The height and leaf width of different types of tobacco plants were observed, and it was found that the NtMAB1 overexpression plant OE-1 had a similar phenotype to the mab1 activation tag mutant, with shorter plants and narrower leaves than the control HD. In the 35Spro:NtMAB1 overexpression line, some phenotypes similar to OE-2 type plants were found. The plants showed extreme dwarfing, with longer axillary branches at each leaf position than OE-1, narrower leaves, and remained in a dwarfed and multi-branched state during subsequent growth ( Figure 6 C).

[0122] (2) At the transcriptional level, the expression level of NtMAB1 in the upper and lower axillary branches (or axillary buds) of HD and two 35Spro:NtMAB1 overexpressing lines was detected. It was found that in the upper axillary buds of the two overexpressing lines OE-1 and OE-2, the expression level of NtMAB1 was 120 times higher than that of the control HD, and in the lower axillary buds of OE-1 and OE-2, the expression level of NtMAB1 was 200 and 120 times higher than that of the control HD, respectively ( Figure 6 E, F), and it was also found that in the OE-1 overexpression strain with the phenotype of mab1 activation tag mutant, the expression level of NtMAB1 in the lower axillary buds was twice that in the upper axillary buds, which was consistent with the expression trend of NtMAB1 in the upper and lower axillary buds of mab1. In the OE-2 strain, the expression levels of NtMAB1 gene in the upper and lower axillary buds were close, and there was no situation where the expression level of NtMAB1 gene in the lower axillary buds was higher than that in the upper axillary buds ( Figure 6 E, F).

[0123] BRC1 is a key gene for maintaining axillary bud dormancy. During the phenotypic investigation of HD and overexpression strains, it was found that the axillary buds in the overexpression strains were very active, and axillary buds began to appear in the seedling stage of the plants, and the axillary buds also grew rapidly during the growth of the tobacco plants. This may be due to the inhibition of BRC1 gene expression, which weakened its role in maintaining dormancy. Therefore, the expression level of the BRC1 gene in the axillary buds of the above three strains was detected, and it was found that in the NtMAB1 overexpression tobacco plants, the expression level of the NtBRC1 gene was significantly lower than that of the control HD, and in the OE-2 strain with the longest axillary bud length, the expression of the NtBRC1 gene was the lowest ( Figure 7 ).

[0124] Example 5 Construction of NtMAB1 gene knockout plants and the effect of gene knockout on axillary bud growth

[0125] 1. Experimental methods

[0126] The CRISPR-Cas9 technology was used to perform genome editing on the NtMAB1 gene in wild-type red pigs. The two copies of the NtMAB1 gene were analyzed for sequence conservation, and a target site was designed in the more conservative region of the two NtMAB1 gene sequences through the target site design website (http: / / www.multicrispr.net / index.html). The location of the target site in the exon region of the gene is as follows Figure 8 As shown in A.

[0127] Using the sequence of the NtMAB1 gene as a template, 100uM concentrations of two target primers M1Cas9-1F and M1Cas9-1F1R (Table 6) were mixed in equal volumes, denatured at 95°C for 3min, and cooled by 0.3% gradient to 25°C. After 10°C, a double-stranded insertion fragment was generated. The fragment was mixed with the purified BsaI-digested pHSE401 vector fragment in equal proportions, and transformed into competent Escherichia coli after ligation with ligase, cultured overnight on Kana-resistant LB medium, and positive clones were identified and sent for sequencing. The successfully constructed NtMAB1 knockout vector was transformed into Agrobacterium, and transgenic plants were obtained by leaf disc infection.

[0128] After obtaining transgenic plants, the M1C1-SF1 / SR1 and M1C1-TF2 / TR2 primer pairs were used to sequence and identify the NtMAB1 gene target sites to determine the editing effect.

[0129] Table 6 CRISPR-Cas9 knockout fragment sequences and identification primers

[0130]

[0131]

[0132] 2. Experimental results and analysis

[0133] The transgenic plants were screened with hygromycin and the gene sequences were amplified and sequenced ( Figure 8 B). Since both copies of the NtMAB1 gene in each transgenic positive plant may be edited, target amplification and sequencing were performed on the two copies of NtMAB1-S1 and NtMAB1-T1 of each transgenic individual plant. The sequencing results were compared with the normal NtMAB1-S1 and NtMAB1-T1 gene sequences to find the editing deletion or editing insertion sites. The target sites of the NtMAB1-S1 gene in the transgenic positive plants were detected, and a total of 9 gene editing sites were obtained, including 8 editing deletions and 1 editing insertion; the target sites of the NtMAB1-T1 gene in the transgenic positive plants were detected, and 6 gene editing sites were also obtained, including 5 editing deletions and 1 editing insertion (Table 7).

[0134] Table 7 Summary of CRISPR / Cas9 editing events of tobacco NtMAB1 gene

[0135]

[0136] Among them, the gene editing type of the gene editing T1 homozygous strain M1CR1-102 in the NtMAB1-S1 gene copy was a gene editing mismatch of 2 bases, and the insertion of 12 bases TACATGAACACT, and the TGA termination codon appeared in the inserted 12 codons. The protein translation results showed that the editing insertion sequence caused the protein translation to terminate prematurely ( Fig. 9 A, B). NtMAB1-T1 was edited to insert two bases GT and a mismatched A base ( Fig. 9 A), and the strain was named KO-1, and this strain was used for subsequent experiments ( Fig. 9 ).

[0137] The length of axillary buds at each leaf position of KO-1 strains in the vigorous growth period was measured. The results showed that the length of several axillary buds in the upper part of tobacco plants was not significantly different from that of axillary buds at the corresponding leaf position in HD, while the axillary buds in the middle and lower parts were shorter than those in HD, and the difference in the length of the lower axillary buds reached an extremely significant level ( Fig.10 A). Analysis of the expression level of the NtMAB1 gene in KO-1 plants revealed that the expression level of the NtMAB1 gene in the lower axillary buds of this line was slightly higher than that in WT, and the expression level in the upper axillary buds was lower than that in WT, with no significant difference in expression level ( Fig.10 B) This reaction: After the NtMAB1 gene was knocked out, although it had little effect on the transcription of the gene, its protein translation was inhibited.

[0138] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention. Sequence Listing <110> Tobacco Research Institute, Chinese Academy of Agricultural Sciences <120> A tobacco axillary bud development-related gene and its application <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1002 <212> DNA <213> NtMAB1-S1 cDNA(NtMAB1-S1 cDNA) <400> 1 atgtccataa accattgtcc ttcacaactt acagaaccat tctggtgtaa aacaaaacaa 60 cattatatca tggaatcaac tagaaatcc tcatctccaa atcaccaaaa ccaactaca 120 cctactctc tctgtcctaa tagcaaccaa ttagaagaaa atgaatccc aaagaacac 180 ttgtttgaaa agccattaac accaagcgat gtagggaagc tcacagatt agtcatccca 240 aaacaacacg cggaaaata cttccctatt aatggtaccc aaaatgactc gggcgaaaag 300 ggatttttac tgaatttcga ggacgagtta gggaaattat ggacttttag atattcatat 360 tggaatagta gtcaaagtta tgttattact aaaggatgga gccgttttgt gaaggagaaa 420 aaattagacg ctggtgattt tattgttt gagcgccacc ggttagacgg cgaccggaca 480 tttattggggt ggaggaggag gataccgcc gctggagcca ccgttttgga gcaagaaagt 540 ggggttgctc cgccgggagg cggtggtggt gggtggggcc aggtgtacta cggtgggtct 600 gggcatcctt atccgtcggc aggtgttcca taccacctg actgtcttca tgcaggaagt 660 aagtaatgc ahaac agcaagcaat ggaaaaac ahagaa agtaggttg 720 tttgggtga acctagcaga gtgcgaggta gatgagtctt cctggtccga accatcgacc 780 tcagatggtt cgtcgacacc gagccaccac cacagagtc atgagtatca gggtcaagct 840 ggccagccgt attaccaata tcaagtccat tgtcccatc ctcctgctgc ctcttcctat 900 gctaaaaata accatcaca taacatgta cgctctttat tttatttt aaactttaac 960 tttctatgg gattataga tatgactaat ttcccactt ga 1002 <210> 2 <211> 1208 <212> DNA <213> NtMAB1-T1 cDNA(NtMAB1-T1 cDNA) <400> 2 atgtccataa accattgttc ttcacaactt acagaaccat tctagtgtac aaaaaacaa 60 cactatatca tggaatcaac aagaaaatcc tcatctccaa cttacaaaaa ccaactaca 120 cctactctc tctgtcctaa tagcaccga ttagaagaa atgaatccc aaagaacgt 180 ttgtttgaga aaccattaac accaagcgat gtagggaagc tcaatagatt agtcatccca 240 aaacaacacg ccgaaaaaca cttccctt aacgggacac aaaatgactc gggcgaaag 300 ggatttttac tgaatttcga ggacgagtta gggaaattat ggacttttag atattcatat 360 tggatagta gtcaagtta tgtattaca aaaggatgga gccgtttcgt gaaggagaaa 420 aaattagatg ctggtgattt cgttttgttc gagcgccacc ggttagacgg cgaccggaca 480 tttattgggt gaggaggag gaacgccgcc gctggagcca ccgttttgga gcaagaagt 540 ggtattgctc cgccgggagg cggtggtggt gggtggggcc aggtgtacta cggtgggtct 600 gggcatcctt atccgtcggc aggtgttcca taccacctg actgtcttca tgcagtaaga 660 agaacaatgc ahaacaaac agcaagcaat gggaacaa acggagaca agtagattg 720 tttgggtga acctagcaga gtgcgaagta gacgagtctt tgtggtccga accatcgacc 780 tcagatggtt cgtcgacacc gagccaccac cacagagtc atgagtatca gggtcaagct 840 ggccagctac attaccaata tcaagtccat tgttccatc cttctgcgcc tgcctctcc 900 catgctaaaa ataccatca cataacatg gtacgctacg ctctttatt tattatta 960 aactttaatt actttcttat gcactaa aattacagg aattagcaac aaacgctaaa 1020 taaaaaaatc cgttgctaat tctatttagt gataggttag tgatagatta tcaaaaatt 1080 ctgttaataa tttagcgaca aatcaacgac gaaattcata agtaatttca gtttttttta 1140 atagtgatgg gaattataga tatgactagt ccgatttcca actttgattt tcttgtttcc 1200 tttggtga 1208 <210> 3 <211> 23 <212> DNA <213> NtMAB1S1-F1(NtMAB1S1-F1) <400> 3 cccattgctc tctctctttc page 23 <210> 4 <211> 25 <212> DNA <213> NtMAB1S1-R1(NtMAB1S1-R1) <400> 4 cctttactat gacctatttc cgatt 25 <210> 5 <211> 25 <212> DNA <213> NtMAB1T1-F1(NtMAB1T1-F1) <400> 5 gctccatta taacccattg ctctc 25 <210> 6 <211> 24 <212> DNA <213> NtMAB1T1-R1(NtMAB1T1-R1) <400> 6 tgtagggcga ctgtaggtt gtgt 24 <210> 7 <211> 333 <212> PRT <213> NtMAB1-S1(protein of NtMAB1-S1) <400> 7 Met Ser Ile Asn His Cys Pro Ser Gln Leu Thr Glu Pro Phe Trp Cys 1 5 10 15 Lys Thr Lys Gln His Tyr Ile Met Glu Ser Thr Arg Lys Ser Ser Ser 20 25 30 Pro Asn His Gln Asn Gln Thr Thr Pro Thr Ser Leu Cys Pro Asn Ser 35 40 45 Asn Gln Leu Glu Glu Asn Glu Ile Pro Lys Glu His Leu Phe Glu Lys 50 55 60 Pro Leu Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro 65 70 75 80 Lys Gln His Ala Glu Lys Tyr Phe Pro Ile Asn Gly Thr Gln Asn Asp 85 90 95 Ser Gly Glu Lys Gly Phe Leu Leu Asn Phe Glu Asp Glu Leu Gly Lys 100 105 110 Leu Trp Thr Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val 115 120 125 Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Lys Leu Asp Ala 130 135 140 Gly Asp Phe Ile Leu Phe Glu Arg His Arg Leu Asp Gly Asp Arg Thr 145 150 155 160 Phe Ile Gly Trp Arg Arg Arg Asn Thr Ala Ala Gly Ala Thr Val Leu 165 170 175 Glu Gln Glu Ser Gly Val Ala Pro Pro Gly Gly Gly Gly Gly Gly Trp 180 185 190 Gly Gln Val Tyr Tyr Gly Gly Ser Gly His Pro Tyr Pro Ser Ala Gly 195 200 205 Val Pro Tyr Gln Pro Asp Cys Leu His Ala Gly Ser Arg Val Met His 210 215 220 Lys Gln Thr Ala Ala Asn Gly Asn Asn Thr Arg Arg Gln Val Arg Leu 225 230 235 240 Phe Gly Val Asn Leu Ala Glu Cys Glu Val Asp Glu Ser Ser Trp Ser 245 250 255 Glu Pro Ser Thr Ser Asp Gly Ser Ser Thr Pro Ser His His Gln Gln 260 265 270 Ser His Glu Tyr Gln Gly Gln Ala Gly Gln Pro Tyr Tyr Gln Tyr Gln 275 280 285 Val His Cys Pro Asn Pro Pro Ala Ala Ser Ser Tyr Ala Lys Asn Asn 290 295 300 His His Asn Asn Met Val Arg Ser Leu Phe Tyr Tyr Leu Asn Phe Asn 305 310 315 320<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Phe Ser Met Gly Ile Ile Asp Met Thr Asn Phe Pro Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 325 330<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> M1Cas9-1F (M1Cas9-1F)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> attgcattaa caccaagcga tgt 23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> M1Cas9-1R (M1Cas9-1R)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaacacatcg cttggtgtta atg 23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> M1C1-SF1(M1C1-SF1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctagaaaatc ctcatctcca aatc 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> M1C1-SR1(M1C1-SR1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggctccatcc tttagttaat acata 25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> M1C1-TF2(M1C1-TF2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aacactatat catggaatca acaag 25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <213> M1C1- TR2(M1C1- TR2) <400> 13 cattttgtgt cccgttaaga g 21

Claims

1. A tobacco axillary bud development-related gene, characterized in that: Its sequence is: the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence.

2. A recombinant vector, characterized in that: A polynucleotide sequence comprising the tobacco axillary bud development-related gene according to claim 1, wherein the recombinant vector is a cloning vector or an expression vector for expressing the polynucleotide.

3. A recombinant host cell, characterized in that The invention comprises the polynucleotide sequence of the tobacco axillary bud development-related gene according to claim 1 or the recombinant vector according to claim 2, or the polynucleotide sequence of the tobacco axillary bud development-related gene according to claim 1 is integrated into its genome, and the recombinant host cell is Escherichia coli or Agrobacterium cell.

4. A specific primer pair for identifying the tobacco axillary bud development-related gene according to claim 1, characterized in that: The specific primer pair includes NtMAB1S1-F1 and NtMAB1S1-R1, whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. Use of the tobacco axillary bud development-related gene according to claim 1 in cultivating high-efficiency tobacco varieties with inhibited axillary bud growth.

6. A method for preparing tobacco NtMAB1 gene knockout plants, characterized in that: The method comprises the following steps: (1) performing sequence analysis on the tobacco axillary bud development-related gene as described in claim 1 and designing target sites in the conserved regions; (2) Using the sequence of the NtMAB1 gene as a template, the target primers shown in SEQ ID NO: 8 and SEQ ID NO: 9 were used to amplify M1Cas9-1F and M1Cas9-1R to obtain a double-stranded insert fragment; the fragment was ligated with the purified BsaI-digested pHSE401 vector fragment, and the ligation product was transformed into competent Escherichia coli, cultured overnight in a resistant medium, and the positive clones were sequenced, and the NtMAB1 knockout vector was extracted from the positive clones with correct sequencing; (3) The successfully constructed NtMAB1 knockout vector was transformed into Agrobacterium and infected with tobacco through the leaf disc method to obtain tobacco NtMAB1 gene knockout plants.

7. Use of the tobacco NtMAB1 gene knockout plant according to claim 6 in cultivating high-efficiency tobacco varieties with inhibited axillary bud growth.

8. A method for identifying tobacco NtMAB1 gene knockout plants according to claim 6, characterized in that: The extracted genome of the tobacco to be identified is used as a template, PCR amplified with identification primers, the amplified product is sequenced, and the sequencing result is compared with the sequence of the NtMAB1 gene to obtain the identification result; The identification primers are: M1C1-SF1 and M1C1-SR1 as shown in SEQ ID NO: 10 and SEQ ID NO: 11.

Images