Tobacco sucrose ester synthesis related gene NtBMVSE and application thereof

Abstract

The invention provides a tobacco sucrose ester synthesis related gene NtBMVSE and application thereof, and belongs to the technical field of functional genes. The invention relates to a related gene NtBMVSE for synthesizing tobacco sucrose ester. The nucleotide sequence of the related gene NtBMVSE is shown as SEQ ID NO: 1. According to the invention, NtBMVSE is mined based on combination of whole genome sequencing, transcriptome sequencing and localized clone phase, and it is proved that NtBMVSE is subjected to specific up-regulation expression in tobacco glandular hair tissue. The tobacco sucrose ester synthesis related gene NtBMVSE has a biological function of regulating and controlling synthesis of tobacco sucrose ester, and synthesis of sucrose ester in tobacco is promoted by up-regulating expression of NtBMVSE. Therefore, the related gene for regulating the synthesis of the sucrose ester in the tobacco is defined, and a new means is provided for cultivating a new tobacco variety rich in the sucrose ester and promoting the synthesis of the sucrose ester in the tobacco.

Description

Technical Field

[0001] The invention belongs to the technical field of functional genes, and in particular relates to a tobacco sucrose ester synthesis-related gene NtBMVSE and an application thereof. Background Art

[0002] Sugar esters are a class of compounds produced by the esterification reaction of one or more hydroxyl groups in glucose or sucrose with fatty acids. Sugar esters are the main components of glandular hair secretions of Solanaceae plants, mainly in the form of sucrose esters (SE), glucose esters (GE) or their mixtures. Studies have shown that sugar esters play an important role in resisting biotic and abiotic stresses in the external environment. As an important economic crop of Solanaceae, tobacco has a very rich content of sugar esters on the surface of its leaves, accounting for about 20% of the leaf secretions. The sugar esters on the surface of tobacco leaves are mainly composed of sucrose esters, which are an ideal latent aroma substance and are related to the fullness of tobacco flavor. Tobacco sucrose esters include 6 different types from I to VI. Among them, type III to VI sucrose esters release isobutyric acid, 3-methylbutyric acid and 3-methylvaleric acid, which can give cigarettes a special flavored tobacco smoke and contribute significantly to enhancing the characteristic aroma of smoke. However, there is no report on the genes related to the synthesis of type III to VI sucrose esters in tobacco. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a tobacco sucrose ester synthesis-related gene NtBMVSE, and to screen a tobacco sucrose ester synthesis gene through a combination of whole genome sequencing, transcriptome sequencing, and positional cloning techniques, and to positively regulate the biosynthesis of sucrose esters in tobacco glandular hairs.

[0004] The present invention provides a tobacco sucrose ester synthesis-related gene NtBMVSE, whose nucleotide sequence is shown in SEQ ID NO:1.

[0005] The present invention provides a protein encoded by the tobacco sucrose ester synthesis-related gene NtBMVSE, and the amino acid sequence is shown in SEQ ID NO:2.

[0006] The present invention provides application of the tobacco sucrose ester synthesis-related gene NtBMVSE in regulating tobacco sucrose ester synthesis.

[0007] Preferably, the method for regulating tobacco sucrose ester synthesis is to overexpress the tobacco sucrose ester synthesis-related gene NtBMVSE in tobacco plants.

[0008] The invention provides a recombinant overexpression vector comprising the tobacco sucrose ester synthesis-related gene NtBMVSE.

[0009] Preferably, the recombinant overexpression vector further comprises the promoter of tobacco sucrose ester synthesis-related gene NtBMVSE;

[0010] The nucleotide sequence of the promoter of the tobacco sucrose ester synthesis-related gene NtBMVSE is shown in SEQ ID NO: 7;

[0011] The backbone vector of the recombinant overexpression vector includes pBWA(V)HII.

[0012] The present invention provides a reagent for promoting the expression of the tobacco sucrose ester synthesis-related gene NtBMVSE or the protein, or use of the recombinant overexpression vector in promoting the synthesis of sucrose ester in tobacco.

[0013] Preferably, the sucrose ester comprises at least one of the following: type III sucrose ester, type IV sucrose ester, type V sucrose ester and type VI sucrose ester.

[0014] The present invention provides the use of the tobacco sucrose ester synthesis-related gene NtBMVSE or the recombinant overexpression vector in creating a transgenic tobacco variety rich in sucrose ester.

[0015] The invention provides a method for promoting tobacco to secrete sucrose ester, comprising the following steps: overexpressing the tobacco sucrose ester synthesis-related gene NtBMVSE in tobacco.

[0016] The present invention provides a tobacco sucrose ester synthesis-related gene NtBMVSE, and the nucleotide sequence is shown in SEQ ID NO: 1. The present invention mines the tobacco sucrose ester synthesis gene NtBMVSE based on a method combining whole genome sequencing, transcriptome sequencing and positional cloning. Through qPCR detection and GUS activity analysis, the gene is specifically upregulated in tobacco glandular hair tissue. The present invention transfers the tobacco sucrose ester synthesis-related gene NtBMVSE into tobacco for overexpression, and the total sucrose content of the leaves of the obtained transgenic positive plants is 5.6 to 6.8 times higher than that of the control group (Yunyan 87), and the content of type III to VI sucrose esters is significantly higher than that of the control group (Yunyan 87). This shows that NtBMVSE has the biological function of regulating the synthesis of sucrose esters in tobacco, and upregulating the expression of NtBMVSE is conducive to promoting the synthesis of sucrose esters in tobacco. It can be seen that the present invention clarifies the relevant genes for regulating the synthesis of sucrose esters in tobacco, and provides a new means for cultivating new tobacco varieties rich in sucrose esters and promoting the synthesis of tobacco sucrose esters. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a fine mapping of sucrose ester biosynthesis genes;

[0018] Figure 2 This is the result of the analysis of the GUS activity of the tobacco NtBMVSE gene;

[0019] Figure 3 This is the result of detecting the expression of NtBMVSE gene in transgenic positive plants;

[0020] Figure 4 This is a diagram showing the results of detecting the types and contents of sucrose esters in the transformed strains. DETAILED DESCRIPTION

[0021]

[0022] In the present invention, the sucrose ester synthesis gene NtBMVSE is precisely located by whole genome sequencing data, and then the glandular hair-specific expression or high expression gene in the candidate segment is mined by transcriptome sequencing data. The results show that sequence analysis found that there is a large sequence difference between the BMVSE gene in Beinhart 1000-1 and flue-cured tobacco K326, and the deletion of a large fragment of the K326 genome sequence, specifically the loss of the second half of the second exon and the third, fourth, and 12 exons, resulting in the inability to synthesize type III to VI sucrose esters. The total RNA of the glandular hairs of the leaves of cigar tobacco Beinhart 1000-1 is reverse transcribed to obtain cDNA amplification to obtain the NtBMVSE gene. The expression of NtBMVSE gene in different tissues of cigar BenHart1000-1 was detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the expression of NtBMVSE gene in roots, stems, senescent leaves, mature leaves, young leaves, flowers and axillary buds was at a low level, while the expression in glandular hairs was extremely high, indicating that the gene may be involved in the biosynthesis of sucrose esters in tobacco glandular hairs. The present invention further uses GUS detection to prove that the promoter of NtBMVSE gene is expressed in glandular hair heads and has glandular hair tissue specificity.

[0023] In the present invention, the primers for amplifying the NtBMVSE are preferably NtBMVSES-CDS_F (ATG GCTTCTCTCTCTGTAAATTCTATAATTTCCCTG, SEQ ID NO: 3) and NtBMVSES-CDS_R (TTACGATTGCGCACTGCCTTCAAATTTGTTC, SEQ ID NO: 4).

[0024] The present invention provides a protein encoded by the tobacco sucrose ester synthesis-related gene NtBMVSE, and the amino acid sequence of the protein is shown in SEQ ID NO: 2 (MASLSVNSIISLSTSLSLHSKNPLIHSVF).

[0025] The present invention provides application of the tobacco sucrose ester synthesis-related gene NtBMVSE in regulating tobacco sucrose ester synthesis.

[0026] In the present invention, the method for regulating tobacco sucrose ester synthesis is preferably to overexpress tobacco sucrose ester synthesis related gene NtBMVSE in tobacco plants. The sucrose ester preferably includes at least one of the following: type III sucrose ester, type IV sucrose ester, type V sucrose ester and type VI sucrose ester.

[0027] In the embodiment of the present invention, the NtBMVSE is introduced into tobacco plants to construct tobacco plants that overexpress the NtBMVSE gene. Sucrose esters are detected in tobacco plants that overexpress the NtBMVSE gene. The results show that compared with the control group (flue-cured tobacco Yunyan 87), the leaves of tobacco plants that overexpress the NtBMVSE gene have significantly increased type III to VI sucrose esters. This indicates that NtBMVSE has the function of synthesizing type III to VI sucrose esters.

[0028] The invention provides a recombinant overexpression vector comprising the tobacco sucrose ester synthesis-related gene NtBMVSE.

[0029] In the present invention, the recombinant overexpression vector further comprises the promoter of the tobacco sucrose ester synthesis-related gene NtBMVSE; the nucleotide sequence of the promoter of the tobacco sucrose ester synthesis-related gene NtBMVSE is shown in SEQ ID NO:7.

[0030] In the present invention, the backbone vector of the recombinant overexpression vector preferably includes pBWA(V)HII. In the embodiment of the present invention, the pBWA(V)HII vector is purchased from Wuhan Boyuan Biotechnology Co., Ltd., and the product number is #REC12H. The cloning sites of the tobacco sucrose ester synthesis-related gene NtBMVSE are preferably BsaI and Eco31I.

[0031] In the present invention, the method for constructing the recombinant overexpression vector preferably uses recombinant primers to amplify the NtBMVSE gene and the promoter of the NtBMVSE gene to obtain the NtBMVSE gene amplification product and the promoter amplification product;

[0032] The backbone vector was double-digested with BsaI and Eco31I to obtain a linear backbone vector;

[0033] Connecting the NtBMVSE gene amplification product, the promoter amplification product and the linear backbone vector to obtain a connection product;

[0034] The ligation product is screened and tested to obtain a recombinant overexpression vector cloned with the NtBMVSE gene and the promoter of the NtBMVSE gene.

[0035] In the present invention, the recombinant primers for amplifying the NtBMVSE gene preferably include BM_cds(+)(GAAAAGAATAatggcttctctctctgtaaattctataatttccctg, SEQ ID NO: 10) and BM_cds(-)(TGAAGACAGAGCTAGTTACAttacgattgcgcactgccttcaaatttgttc, SEQ ID NO: 11). The recombinant primers for amplifying the promoter of the NtBMVSE gene preferably include BM_promoter(+)(CGCCTGCAGGTCTAGACAACcgacaaggtttcttcctaaatgagatcaacaatc, SEQ ID NO: 8) and BM_promoter(-)(GAGAAGCCATtattcttttccctaagaaaagagagtggatcgg, SEQ ID NO: 9). Before ligation, the NtBMVSE gene amplification product and the promoter amplification product are purified, preferably using a DNA gel recovery kit (Axygen).

[0036] In the present invention, the reaction system of the backbone vector using BsaI and Eco31I double digestion is preferably 20 μL, specifically including 13 μL of Nuclease-free Water, 2 μL of 10×Buffer, 1 μL of BsaI / Eco31I, and 4 μL of pBWA(V)HII-CCDB-TNOS. The reaction procedure of BsaI and Eco31I double digestion is preferably carried out at 37°C for 1 hour.

[0037] In the present invention, the reaction system of the ligation is preferably 20 μL, specifically including 10 μL of Biorun2×EasyCloneMix, 25 μL of rDNABB and 5 μL of pBWA(V)HII-CCDB-TNOS(D). The reaction procedure of the ligation is preferably to react at 37°C for 30 min.

[0038] In the present invention, the screening and detection method preferably comprises transforming the ligation product into competent E. coli, culturing, and identifying by plaque PCR to obtain positive transformants; and extracting the recombinant overexpression vector from the positive transformants. The transformation method is preferably a calcium chloride transformation method. The culture is preferably screened and cultured using a resistance medium containing Kan. The culture time is preferably 10 to 14 hours, and may be 12 hours. The culture temperature is preferably 36 to 38° C., and may be 37° C. The plaque PCR identification method preferably comprises PCR amplification detection using pBWA(V)HII-CCDB-TNOS identification primers. The pBWA(V)HII-CCDB-TNOS identification primers preferably include HG-F (gctccaccatgttggcaagc, SEQ ID NO: 12) and BM(635C) (gaagttcaagaaactctgaacgttctt, SEQ ID NO: 13).

[0039] The present invention provides a reagent for promoting the expression of the tobacco sucrose ester synthesis-related gene NtBMVSE or the protein, or use of the recombinant overexpression vector in promoting the synthesis of sucrose ester in tobacco.

[0040] In the present invention, the reagents for promoting the expression of the tobacco sucrose ester synthesis-related gene NtBMVSE preferably include high-strength gene promoters, enhancers or gene elements (Kozak sequences, STRs, TATAbox, CAATbox). The reagents for promoting the protein expression preferably include codon-optimized gene sequences to achieve overexpression or use molecular fusion partner technology to increase the solubility of the protein and increase the expression of the protein.

[0041] In the present invention, the sucrose ester preferably includes at least one of the following: type III sucrose ester, type IV sucrose ester, type V sucrose ester and type VI sucrose ester.

[0042] The present invention provides the use of the tobacco sucrose ester synthesis-related gene NtBMVSE or the overexpression vector in creating a transgenic tobacco variety rich in sucrose ester.

[0043] In the present invention, the method for creating a transgenic tobacco variety rich in sucrose esters preferably involves transferring the tobacco sucrose ester synthesis-related gene NtBMVSE into tobacco, verifying and screening plants that overexpress NtBMVSE to obtain a transgenic tobacco variety rich in sucrose esters. The method for transferring into tobacco preferably includes direct gene transfer technology or biologically mediated transformation methods. The direct gene transfer technology includes gene gun method, protoplast method, liposome method, pollen tube channel method, electroporation transformation method, PEG-mediated transformation method, etc., and gene gun transformation method is a representative technology. The biologically mediated transformation method preferably includes two transformation methods: Agrobacterium-mediated and virus-mediated.

[0044] The invention provides a method for promoting tobacco to secrete sucrose ester, comprising the following steps: overexpressing the tobacco sucrose ester synthesis-related gene NtBMVSE in tobacco.

[0045] In the present invention, the tobacco sucrose ester synthesis-related gene NtBMVSE is preferably transferred into tobacco in the form of a recombinant overexpression vector. The construction method of the recombinant overexpression vector in the present invention is the same as the above technical scheme, and will not be repeated here. The tobacco preferably includes cigar tobacco and / or flue-cured tobacco.

[0046] The tobacco sucrose ester synthesis-related gene NtBMVSE and its application provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1

[0048] Obtaining the tobacco sucrose ester biosynthesis gene NtBMVSE

[0049] 1. The tobacco sucrose ester synthesis gene NtBMVSE was discovered by combining whole genome sequencing, transcriptome sequencing and positional cloning.

[0050] Leaves of cigar tobacco Beinhart1000-1 seedlings were collected, genomic DNA was extracted, and a library was constructed. The whole genome of cigar tobacco Beinhart1000-1 was sequenced using the second-generation sequencer Illumina Hiseq. The original data obtained by sequencing was about 90G, and the depth of genome sequencing was 20×. Reads were aligned to the reference genome K326 (http: / / lifenglab.hzau.edu.cn / Nicomics / ) using alignment software. Combining the chromosome sequence information and resequencing data of the reference genome K326, 273 SNP markers were mined in this interval. The progeny obtained by hybridizing the parent Beinhart1000-1 and Honghua Dajinyuan were self-pollinated to construct the F2 segregation population, and the F1 individual was hybridized with Honghua Dajinyuan to obtain the BC1 F1 backcross population. The type and content of sucrose esters in the parents, F1 population individual plants, F2 segregation population and BC1 segregation population were detected by GC-MS. The detection method is as follows:

[0051] Take 3 leaves from each tobacco seedling, divide them into two halves with the middle main vein as the boundary, and use a hole puncher to take 1 small round leaf from each, for a total of 6 leaves. Add 20mL of dichloromethane to extract the surface material of the fresh tobacco leaves. After taking out the leaves, add anhydrous sodium sulfate to dry. Transfer 10ml of supernatant to a 30mL centrifuge tube, add 0.1mL of 17decanol, and vortex for 1min. Dry with nitrogen in a 40-degree water bath. Add 0.5mL of derivatization reagent, vortex and mix well, transfer to a 2ml injection vial, seal, heat in a 75℃ water bath for 30min, cool to room temperature, and analyze by GC-MS.

[0052] GC conditions: DB-5MS (30m×0.25mm×0.25μm) capillary column was used for separation; the injection port temperature was set to 250℃, and the injection volume was 1μL; the carrier gas was helium, and the flow rate was 1mL / min; the injection method was splitless injection; the temperature rise program of the column oven: the starting temperature was set to 150℃, maintained for 2min, then increased to 270℃ at a heating rate of 15℃ / min, and then increased to 300℃ at a heating rate of 5℃ / min, and maintained for 20min.

[0053] MS conditions: transfer line temperature was 250 °C; ion source temperature was set to 280 °C; ionization source was EI, ionization energy was 70 eV, and solvent delay was 5 min.

[0054] Scanning mode of mass spectrometry: Full scan mode is used to detect the target components, and the scanning range is 33-800 amu. The internal standard semi-quantitative method is used to quantify the target components. For each target substance, one quantitative ion and two qualitative ions are selected from all its ion fragments.

[0055] The results showed that type III-VI sucrose esters were not detected in any single plant of the P1 parent "K326", while type III-VI sucrose esters were detected in the P2 parent "Beinhart1000-1" and all the single plants of the F1 population. This proves that the presence of sucrose esters in tobacco is dominant over the absence of sucrose esters, and this trait is controlled by nuclear genes. The phenotypic identification results of the F2 segregation population showed that among the 210 F2 single plants, type III-VI sucrose esters were detected in 167 plants, and type III-VI sucrose esters were not detected in 43 plants. The chi-square test results showed that the segregation ratio of the F2 population was consistent with 3:1, which indicates that the gene controlling the synthesis of type III-VI sucrose esters is controlled by a dominant single gene.

[0056] Table 1 Results of genetic law analysis

[0057] generation total Number of strains with sucrose esters Number of strains without sucrose ester Separation χ2 χ2(0.05,1) P1(K326) 20 20 - - - - P2(Beinhart1000-1) 20 - 20 - - - F1 40 - 40 - - - F2 210 167 43 3:1 2.29 3.84

[0058] After polymorphism screening and genotyping, the III-VI type sucrose ester synthesis gene NtBMVSE was located in the 0.72Mb genomic interval ( Figure 1 ).

[0059] 2. In order to further explore the genes for sucrose ester synthesis, transcriptome sequencing was performed on the glandular hairs of cigar tobacco Beinhart 1000-1 and flue-cured tobacco K326, and the BMVSE gene in the candidate segment was specifically highly expressed in the glandular hairs. Sequence analysis revealed that there were sequence differences between the BMVSE gene in Beinhart 1000-1 and K326, and the deletion of a large fragment of the K326 genome sequence led to the loss of the second half of exon 2 and exons 3, 4, and 12. The deletion of part of the sequence of this mutant gene in flue-cured tobacco may be the reason why type III to VI sucrose esters cannot be synthesized in flue-cured tobacco.

[0060] 3. Obtaining the full-length CDS and promoter sequence of NtBMVSES gene

[0061] 3.1 Total RNA extraction from cigar Beinhart 1000-1

[0062] Total RNA was extracted from the glandular hairs of Beinhart 1000-1 leaves using TRIzon Reagent TRIZOL method. The steps are as described in the instruction manual. The extracted RNA was analyzed for RNA quality by electrophoresis and ultra-micro spectrophotometer.

[0063] 3.2 Reverse transcription

[0064] The RNA after the above reaction was reverse transcribed using Conway HiFi-MmLV cDNA Kit to obtain cDNA. Specific amplification primers were designed, and the specific sequences are as follows:

[0065] NtBMVSES-CDS_F:atggcttctctctctgtaaattctataatttccctg (SEQ ID NO: 3);

[0066] NtBMVSES-CDS_R:ttacgattgcgcactgccttcaaatttgttc (SEQ ID NO: 4);

[0067] The cDNA obtained by reverse transcription was used as a template to amplify the NtBMVSES gene, and the above primers were used for PCR amplification (using Vazyme's phantamax super-fidelity DNA Polymerase). The PCR reaction system (total volume 50μl) was as follows: ddH2O 17μl, 2×PhantaMax Buffer 25μl, dNTPMix (10mM each) 1μl, template cDNA 2μl, primer 1 (10μM) 2μl, primer 2 (10μM) 2μl, Phanta Max Super-Fidelity DNA Polymease (1U / μl) 1μl. The PCR reaction program was: 95℃30s; (95℃15s, annealing temperature 55℃15s, extension 72℃1min)×39 cycles; final extension 72℃, 5min. The target fragment was purified and recovered and connected to the TA cloning vector, and a single clone was picked for sequencing.

[0068] The results showed that the full-length CDS sequence of the NtBMVSES gene is 1809 bp, and the specific sequence is as follows: ATGGCTTCTCTCTCTGTAAATTCTATAATTTCCCTGAGCACTTCCCTTTCATTACATTCTAAAAACCCACTTATTCACAGTGTCTTCAGTTTCACGCCTTCAACCACAAGGCATTCAGCTATATGCTGCTCAAATATTCGTCGGCGGCCGGAGTATAAACATGGAAAATTCTCTGACCCTGATTATGTTGGTATTTTTGACACCAGTCTTCGCGATGGCGAACAGGCCGCTGGTGCTACCATGACTAGTAAAGAAAAACTGGACATTGCACGTCAGTTGGCTAAGCTTGGTGTTGATGTTATTGAGGCCGGTTTTCCTTTTGCCTCTGAAGCTGAGTTCGAGCTTGTAAAGTTGATAGCACAGGAAATTGGTAATAACGTAGACAAAGAAGGATACGTGCCGATGATATGTGCCTTAGCTAGGTCTAGTAAGAAGGATATTGAAAGAGCTTGGGATGCTTTAAAGTATGCAAAGAAACCAATGCTTCATATGTTTATTGCGACGAGTGATATACATATGAAGTACAAGTTAAAGATGAGTAGAGAAGAAATTGTGGAGACAGCTAGGAGTACGGTGGCTTATGCAAAAACCCTATTTGAGGATGTTCGGTTTAGCGCTGAAGATGCTGCAAGATCTGATAGGGAGTTCCTTTATCATATTATTGGAGAAGTTATCAAAGCTGGTGCAACAGTGATTGGCCTCCCTGATACAGTTGGATGCAATTTGCCCAGTGAATATGCACAACTGATTTCTGATATAAAAGCCAATACCCCAGGAATACAAGATGCAAACATTTCAACACACTGTCACAACGATCTTG(SEQ IDNO: 1).

[0069] The encoded protein is 602 amino acids in length, and the specific sequence is as follows:

[0070] MASLSVNSIISLSTSLSLHSKNPLIHSVFSFTPSTTRHSAICCSNIRRRPEYKHGK

[0071] FSDPDYVGIFDTSLRDGEQAAGATMTSKEKLDIARQLAKLGVDVIEAGFPFAS

[0072] EAEFELVKLIAQEIGNNVDKEGYVPMICALARSSKKDIERAWDALKYAKKPM

[0073] LHMFIATSDIHMKYKLKMSREEIVETARSTVAYAKTLFEDVRFSAEDAARSDR

[0074] EFLYHIIGEVIKAGATVIGLPDTVGCNLPSEYAQLISDIKANTPGIQDANISTHC

[0075] HNDLGLATANSLAGICAGARLVDVTINGIGERAGNASLEEIVMALKYRGEQV

[0076] LGGIYTGINTKHIFMTSKMVEEYSGLKLQPHKAIVGANAFSHESGIHQDGVLK

[0077] NRDTYEFVSHEDVGYRRANENGISLGKLSGRHALKAKMAELGYDFDGKELD

[0078] DLFRRFKSLAERKKKITDDDLRALVSDDVFQPQVSWQLGDVQITCGNVGRST

[0079] ANVKLIDSDGQEHTAFSVGTGPVDAAYKAVDLIVKVPVTLVEYSVNAITKRIN

[0080] STASTRVLVRGNDDYASFNTSNGQTVNRTVSGTGAHMDIVVSSVQAYVEALN

[0081] KIFSYKKTGLVNKFEGSAQS (SEQ ID NO: 2).

[0082] Example 2

[0083] Verification of tissue-specific expression of NtBMVSE gene

[0084] Real-time fluorescence quantitative PCR was used to analyze the expression pattern of NtBMVSE gene in roots, stems, senescent leaves, mature leaves, young leaves, flowers, axillary buds and glandular hairs of cigar BenHart1000-1. The specific steps are as follows: the above RNA samples were transcribed into cDNA and diluted to a concentration of 50 ng / μL for qRT-PCR. The reaction system is shown in Table 2 and the reaction procedure is shown in Table 3. When preparing the reaction system, each sample should be repeated 3 times technically and 3 times biologically. After the experiment, 2 -ΔΔCt The algorithm was used to analyze the difference in gene expression.

[0085] Table 2 qRT-PCR reaction system

[0086] Components Addition amount 2×SYBR Green Pro Taq HS 10μL template 200ng Forward Primer 0.4μL Reverse primer 0.4μL RNase-free water Add to 20 μL

[0087] The primers are as follows: BM_RT-1F: 5'-ACAGTGTCTTCAGTTTCACGC-3' (SEQ ID NO: 5); BM_RT-1R: 5'-CTTACTAGACCTAGCTAAGGCACAT-3' (SEQ ID NO: 6).

[0088] Table 3qRT-PCR reaction procedure

[0089]

[0090] The results showed that the expression levels of NtBMVSE gene in roots, stems, senescent leaves, mature leaves, young leaves, flowers and axillary buds were all at low levels, while the expression level in glandular hairs was extremely high. The above results indicate that NtBMVSE gene may be involved in the biosynthesis of sucrose esters in glandular hairs of tobacco.

[0091] Example 3

[0092] Analysis of candidate gene GUS activity

[0093] In order to further analyze the tissue expression pattern of the NtBMVSE gene, the promoter of 2000 bp upstream of the NtBMVSE gene was cloned by specific PCR amplification technology using the genomic DNA of the leaves of cigar tobacco Beinhart 1000-1 as a template. The specific sequence is as follows:

[0094] cgacaaggtttcttcctaaatgagatcaacaatcattcttttaaggatttttatttttatttttatcacaaatatattactcaat

[0095] atctaagtaatctttaaaaatctgtatactcattactataaagcatacatgcaaaacgcgtacaataaaaactaatttttaaaaaa

[0096] tagggggaaggagaagggatgaacagaaccctcattaataaagcaaaaatttaaatatccaacaaattgaactgttaagatt

[0097] ataaaaaaaaaagaaaaaaaaagaacttcattgtcgaagtaagtcaaagtatcactagccgtaaaaatttcggcaccttttaaaa

[0098] gaaaaaaatactagggaaagcctaaaacctcttggtattaataactatagtttagaaattaggcaaagccgctactagg

[0099] gtagttaattattggttctgtttatatttatatcattcgttttgttgataatgatgagcaacgttccattacatgaagaaaccacgtca

[0100] atgttttccctcattcgttttctaaatttattcgagatgtgagtaaacctccccccaccccccacacaccccttttttttttgctgtt

[0101] aaagtttcgacaataagaacgttcagagtttcttgaacttctgttttatctgcagaaattataaattggagtcaaccactgcatcc

[0102] caaattgatgcagactttatctgaataattaatcagtatataattataatatctattttctatgtttctaatttatgtgacacatttcttat

[0103] ccgttagtttaaaaaaaatgataaatttatatatttcagaataatttaactttaaatttttttattttatccattttacctttaatgagaaa

[0104] cttttgtaaccatacaaatgttatagaatatatttagaacaacaagtttcaaatattttatagtcacataaatttaacacacaaatat

[0105] tagagcaaatttcaaactacttcacataaattgaaacagagggaaaaataatactccctccgttcccttttacttggcacgtttt

[0106] gactttttacgccccttaagaaataataaatgaagtgcataatttaccatgatacccatattaattgatgtatattttattggatttg

[0107] aaaaatgatttgaaatgagtaataaatatcgtgggtataacaaaaaaaaaaattatcttctcttgatatgcgtaaagtgacaag

[0108] caaaaatgaaaatctatttttagtatacatgccaaataaaagtgaacggtaaaaatgaacggagggagtatatattattactac

[0109] taaaacgggaaactgggcatgctaagtttttcattgctatttcaagttccgattaatagccataatataatgttttctagaaacaa

[0110] agaacaacgccaagataaattttcccaaaaagtagcacaaatgtaacaaattcttacctcaaattttaaatctaaacacataaatt

[0111] ttctttataattgtttcttaaaaacttaaactaaattcaaatgccaacaagtttaaatgagcaaggaagtattttcagtttctatttctt

[0112] gaatttaataagaatggaaatattttccgtttgtttactgggttctgtctagtctagcttaagacgaaaattgtcttgttttaagtttt

[0113] acaaaccgtaaggggtggtaattcaattttcaattgtagtatgagcaattaattccaacacaaaatctcacgtgaaataatata

[0114] ctctagtgtttgtttggccgtacgaaaaagaaagtctacataattaatgcagcgttccatagttgatattaatataatacccgtat

[0115] taaattatataaaactttatgtgttattcttttgataaaacagcaaccagacacaccaggggctactttggtcattttttctccaata

[0116] aaaacccttgtttcaccagctttgtaggtgttcgaattcaccaaagcgaacattccattaactccgatccactctcttttcttagg

[0117] gaaaagaata (SEQ ID NO: 7).

[0118] The 2000 bp NtBMVSES gene promoter was connected to the GUS gene using the NtBMVSES gene promoter sequence recombination primers (BM_promoter(+): CCTCTAGA GTCGACCTGCAGcgacaaggtttcttcctaaatgaga, SEQ ID NO:8); BM_promoter(-): CTCAGATCTACCATGGtattcttttccctaagaaa, SEQ ID NO:9) to construct the plant expression vector pCAMBIA1301-NtB MVSE-GUS, and transgenic tobacco was transformed using the Agrobacterium-mediated method.

[0119] The results of GUS activity assay in transgenic tobacco plants showed that the NtBMVSE gene was expressed in the glandular hair heads and had glandular hair tissue specificity ( Figure 2 ).

[0120] Example 4

[0121] Construction of recombinant vector containing NtBMVSES gene and creation of transgenic plants

[0122] 1. The NtBMVSES gene promoter sequence (2000 bp) and CDS sequence (1809 bp) were recombined with the tobacco expression vector pBWA(V)HII using homologous recombination method. The specific steps are as follows.

[0123] NtBMVSES gene promoter sequence recombination primers:

[0124] BM_promoter(+): CGCCTGCAGGTCTAGACAACcgacaaggtttcttcctaaatgagatcaacaatc (SEQ ID NO: 8);

[0125] BM_promoter(-):GAGAAGCCATtattcttttccctaagaaaagagagtggatcgg (SEQ ID N0: 9);

[0126] Primers for recombination amplification of NtBMVSES gene CDS sequence:

[0127] BM_cds(+):GAAAAGAATAatggcttctctctctgtaaattctataatttccctg (SEQ ID NO: 10);

[0128] BM_cds(-): TGAAGACAGAGCTAGTTACAttacgattgcgcactgccttcaaatttgttc (SEQ ID NO: 11).

[0129] Prepare 2 50 μL systems, refer to Table 4 for details. Perform amplification reaction according to the reaction procedure in Table 5.

[0130] Table 4 PCR system

[0131] Element volume Nuclease-free water 20μL Biorun Pfu PCR Mix 25μL Forward primer (100 μM) 2μL Reverse primer (100 μM) 2μL DNA template 1 μL Total volume 50μL

[0132] Table 5 PCR reaction program

[0133]

[0134]

[0135] Use 1% agarose gel electrophoresis at 5 V / cm for 20 min, cut out the electrophoretic fragment of BMpromoter.(2000 bp)BM CDS(1809 bp) under ultraviolet light, and place it in a system for sol recovery. The recovery procedure is shown in the instructions of the DNA gel recovery kit (Axygen). Use a total volume of 40 μL of water to dissolve and recover the DNA (the recovered product is marked as: rDNABB2). After testing, recombinate with the vector.

[0136] 2. Vector digestion and purification

[0137] The enzyme digestion and ligation system is shown in Table 6, and the digestion was carried out at 37°C for 1 h.

[0138] Table 6 Enzyme Digestion and Linking System

[0139] Nuclease-free Water 13μL 10×Buffer 2μL BsaI / Eco31I 1μL pBWA(V)HII-CCDB-TNOS 4μL total 20μL

[0140] The vector digestion product was purified using a PCR purification kit and the purified product was recorded as pBWA(V)HII-CCDB-TNOS(D) for the next in vitro or in vivo recombination reaction.

[0141] 3. Recombination reaction

[0142] The recombination reaction system was configured according to Table 7, and the reaction was carried out at 37° C. for 30 min to obtain the recombination product.

[0143] Table 7 Recombination reaction system

[0144] Reagents volume Nuclease-free Water 0μL Biorun 2×EasyClone Mix 10μL rDNABB2 5μL pBWA(V)HII-CCDB-TNOS(D) 5μL total 20μL

[0145] 4. Conversion

[0146] Transform 5-10 μL of the ligation product into competent E. coli. For details, refer to the standard method for competent E. coli transformation. Transform Kan-resistant plates and culture at 37°C for 12 hours to obtain bacterial plaques.

[0147] 5. Plaque PCR Identification

[0148] Pick 10 plaques and perform 1.5ml EP tube inoculation and PCR identification at the same time. The primer sequences are as follows:

[0149] HG-F: gctccaccatgttggcaagc (SEQ ID N0: 12);

[0150] BM (635C): gaagttcaagaaactctgaacgttctt (SEQ ID NO: 13).

[0151] Configure 10 25 μL PCR reactions, see Table 8 for details.

[0152] Table 8 Plaque PCR reaction system

[0153] Reagents volume Nuclease-free water 9.5μL Biorun Magic PCR Mix 12.5μL HG-F (100 μM) 1μL E3472(635C)(100μM) 1μL template 1μL total 25μL

[0154] Table 9 Plaque PCR reaction procedure

[0155]

[0156] PCR amplification results showed that the recombinant vector was successfully cloned with NtBMVSES gene and its promoter.

[0157] Example 5

[0158] The recombinant vector constructed in Example 4 was transferred into Yunyan 87 plants by Agrobacterium-mediated method, and the transgenic plants were detected by using specific primers developed for NtBMVSE gene (BM_RT-1F: ACAGTGTCTTCAGTTTCACGC, SEQ ID NO: 14; BM_RT-1R: CTTACTAGACCTAGCTAAGGCACAT, SEQ ID NO: 15). A total of 12 transgenic positive plants were obtained. During the vigorous growth period of tobacco plants, the gene expression levels of the restored positive plants were detected.

[0159] Figure 3 The results showed that the NtBMVSE gene of all positive plants was normally expressed in the glandular hairs of the restored expression positive plants, among which the expression levels of OE-1, OE-6, OE-7, OE-8 and OE-12 strains were higher. Subsequently, individual plants of the above five strains were selected to detect the content and type of sucrose esters.

[0160] Example 6

[0161] GC-MS was used to detect the types and contents of sucrose esters on the leaves of plants with restored expression (OE-1, OE-6, OE-7, OE-8 and OE-12 lines), and the control was Yunyan 87.

[0162] Table 10 Comparison of sucrose ester content and types in NtBMVSE gene restored expression plants and Yunyan 87 (μg / g)

[0163] strain Sucrose ester Ⅰ Sucrose ester Ⅱ Sucrose ester Ⅲ Sucrose ester Ⅳ Sucrose ester Ⅴ Clouds and Smoke 87 0.527 2.3715 0 0 0 OE-1 0.137 1.647 5.174 5.824 3.613 OE-6 0.132 2.365 5.123 7.201 5.102 OE-7 0.207 1.003 5.615 6.901 4.532 OE-8 0.285 1.622 5.156 7.116 5.807 OE-12 0.005 1.111 4.678 5.832 4.945

[0164] The results are shown in Table 10 and Figure 4: The types of sucrose esters in Yunyan 87 are mainly type I-II, and the content of type II esters is much higher than that of type I esters. OE-1, OE-6, OE-7 and OE-8OE-12 strains contain type I-V (type VI sucrose lipids are trace amounts), and the content of type III-V sucrose esters is relatively rich, and the total sucrose ester content in tobacco leaves is about 5.6 to 6.8 times higher than that in the control. This shows that the transgenic positive plants have the function of synthesizing type I to VI sucrose esters, especially the function of synthesizing type III-V sucrose esters. This also verifies that due to the deletion of a sequence in the corresponding gene in flue-cured tobacco, it does not have the biological function of synthesizing type I to VI sucrose esters.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tobacco sucrose ester synthesis-related gene NtBMVSE, characterized in that: The nucleotide sequence of the tobacco sucrose ester synthesis-related gene NtBMVSE is shown in SEQ ID NO:

1.

2. A protein encoded by the tobacco sucrose ester synthesis-related gene NtBMVSE according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. Use of the tobacco sucrose ester synthesis-related gene NtBMVSE according to claim 1 in regulating tobacco sucrose ester synthesis.

4. The use according to claim 3, characterized in that: The method for regulating tobacco sucrose ester synthesis is to overexpress the tobacco sucrose ester synthesis related gene NtBMVSE in tobacco plants.

5. A recombinant overexpression vector, characterized in that: It comprises the tobacco sucrose ester synthesis related gene NtBMVSE as described in claim 1.

6. The recombinant overexpression vector according to claim 5, characterized in that: The recombinant overexpression vector also includes the promoter of tobacco sucrose ester synthesis-related gene NtBMVSE; The nucleotide sequence of the promoter of the tobacco sucrose ester synthesis-related gene NtBMVSE is shown in SEQ ID NO: 7; The backbone vector of the recombinant overexpression vector includes pBWA(V)HII.

7. Use of an agent for promoting the expression of the tobacco sucrose ester synthesis-related gene NtBMVSE according to claim 1 or the protein according to claim 2, or the recombinant overexpression vector according to claim 5 or 6 in promoting the synthesis of sucrose esters in tobacco.

8. The use according to claim 3 or 7, characterized in that: The sucrose ester includes at least one of type III sucrose ester, type IV sucrose ester, type V sucrose ester and type VI sucrose ester.

9. Use of the tobacco sucrose ester synthesis-related gene NtBMVSE according to claim 1 or the recombinant overexpression vector according to claim 5 or 6 in creating a transgenic tobacco variety rich in sucrose ester.

10. A method for promoting the secretion of sucrose esters in tobacco, characterized in that: The following steps are involved: The tobacco sucrose ester synthesis-related gene NtBMVSE according to claim 1 is overexpressed in tobacco.

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