CN104388431A - Genes capable of regulating and controlling nicotine content of tobacco and application of genes - Google Patents
Genes capable of regulating and controlling nicotine content of tobacco and application of genes Download PDFInfo
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- CN104388431A CN104388431A CN201410601193.6A CN201410601193A CN104388431A CN 104388431 A CN104388431 A CN 104388431A CN 201410601193 A CN201410601193 A CN 201410601193A CN 104388431 A CN104388431 A CN 104388431A
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Abstract
The invention discloses genes capable of regulating and controlling nicotine content of a tobacco and application of the genes. A sequence of the genes capable of regulating and controlling the nicotine content of the tobacco is any one as shown in SEQ ID: No.1-21. The application of the genes is as follows: the genes capable of regulating and controlling the nicotine content of the tobacco are used for regulating synthesis and translocation amount of nicotine of the tobacco, so as to realize regulating and controlling of nicotine content in the tobacco. 21 genes obtained in the invention can form a stable hairpin structure, and analysis on the obtained tobacco non-coding small RNA sequence shows that part of small RNAs are from a sequence of a stem of the tobacco; and the small RNAs predict that target genes of the small RNAs are respectively targeted to a tobacco nicotine synthesis and transportation key protein coding gene. An miRNA regulating and controlling mechanism obtained in the invention can provide a deeper understanding for a role played by a key gene for a synthesis and translocation route of nicotine in tobacco, so that nicotine content character of the tobacco can be better improved.
Description
Technical field
The invention belongs to phytology technical field, be specifically related to one and grow tobacco nicotine content regulatory gene and application.
Background technology
The synthesis of tobacco Nicotine and transhipment are subject to the regulation and control of multiple factor, at present also completely thorough from the research of molecule angle to the metabolic pathway of synthesizing of Nicotine and transhipment, although differentiated and cloned out the key gene in some Nicotine route of synthesis, as (Dwey and Xie such as QPT, PMT, MPO, 2013, Phytochemistry; Sierro, 2014, Nature Communication), but regulate and control Nicotine synthesis and transhipment key gene thereof by miRNA, thus the research affecting nicotine content has no report.Therefore, this patent, as the important ring in Nicotine synthesis and transporting pathway, has vital role for improvement tobacco nicotine content etc.
Summary of the invention
The first object of the present invention is to provide one to grow tobacco nicotine content regulatory gene; Second object is to provide the application of described tobacco nicotine content regulatory gene.
The first object of the present invention is achieved in that described tobacco nicotine content regulatory gene sequence is any one in SEQ ID:No.1 ~ 21.
The second object of the present invention be achieved in that described tobacco nicotine content regulatory gene for conditioning tobacco Nicotine synthesis and transhipment amount with realizes regulation and control Determination of Nicotine Content of Tobacco in application.
21 genes that the present invention obtains can form stable hairpin structure, and the tobacco non-coding tiny RNA sequential analysis of acquisition shows that part tiny RNA is from its stem's sequence.These tiny RNA predict its target gene synthesis of target tobacco Nicotine and transhipment key protein matter encoding gene respectively.The miRNA regulatory mechanism that the present invention obtains, provides a more deep understanding, better can improve tobacco nicotine content proterties by for the synthesis of tobacco Nicotine and transporting pathway key gene institute role.
Accompanying drawing explanation
Fig. 1 is the hairpin structure figure (MFE=-70.10) that SEQ ID NO:1 is formed;
Fig. 2 is the hairpin structure figure (MFE=-49.57) that SEQ ID NO:2 is formed;
Fig. 3 is the hairpin structure figure (MFE=-21.60) that SEQ ID NO:3 is formed;
Fig. 4 is the hairpin structure figure (MFE=-23.50) that SEQ ID NO:4 is formed;
Fig. 5 is the hairpin structure figure (MFE=-28.50) that SEQ ID NO:5 is formed;
Fig. 6 is the hairpin structure figure (MFE=-40.60) that SEQ ID NO:6 is formed;
Fig. 7 is the hairpin structure figure (MFE=-76.40) that SEQ ID NO:7 is formed;
Fig. 8 is the hairpin structure figure (MFE=-64.80) that SEQ ID NO:8 is formed;
Fig. 9 is the hairpin structure figure (MFE=-70.10) that SEQ ID NO:9 is formed;
Figure 10 is the hairpin structure figure (MFE=-59.80) that SEQ ID NO:10 is formed;
Figure 11 is the hairpin structure figure (MFE=-65.50) that SEQ ID NO:11 is formed;
Figure 12 is the hairpin structure figure (MFE=-105.10) that SEQ ID NO:12 is formed;
Figure 13 is the hairpin structure figure (MFE=-51.60) that SEQ ID NO:13 is formed;
Figure 14 is the hairpin structure figure (MFE=-44.40) that SEQ ID NO:14 is formed;
Figure 15 is the hairpin structure figure (MFE=-67.70) that SEQ ID NO:15 is formed;
Figure 16 is the hairpin structure figure (MFE=-22.10) that SEQ ID NO:16 is formed;
Figure 17 is the hairpin structure figure (MFE=-40.10) that SEQ ID NO:17 is formed;
Figure 18 is the hairpin structure figure (MFE=-50.50) that SEQ ID NO:18 is formed;
Figure 19 is the hairpin structure figure (MFE=-53.80) that SEQ ID NO:19 is formed;
Figure 20 is the hairpin structure figure (MFE=-49.90) that SEQ ID NO:20 is formed;
Figure 21 is the hairpin structure figure (MFE=-53.30) that SEQ ID NO:21 is formed;
In figure, the base portion that hairpin structure stem wire frame marks is its mature sequence.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or replacement, all belong to protection scope of the present invention.
Tobacco nicotine content regulatory gene of the present invention, its sequence is any one in SEQ ID:No.1 ~ 21.
Described tobacco nicotine content regulatory gene is any one replacement through 1 ~ 2 base in SEQ ID:No.1 ~ 21, disappearance or interpolation form;
Described base is at least one in A, T, C, G, N or U;
Described replacement, disappearance or interpolation refer to: at least one in A, T, C, G, N or U, replace, lack or add.
Of the present invention be applied as described tobacco nicotine content regulatory gene for conditioning tobacco Nicotine synthesis and transhipment amount with realizes regulation and control Determination of Nicotine Content of Tobacco in application.
The present invention contains the miRNA finding regulation and control Nicotine route of synthesis key gene from tobacco, provides a well basis for further studying this gene institute's role in tobacco growing growth course.The research that the present invention carries out can help us better to understand this gene, and is applied to control tobacco synthesis nicotine content etc. further.
Concrete operations of the present invention are as follows:
Materials and methods:
1, vegetable material and preparation of samples
Plant tissue materials all in the present invention takes from flue-cured tobacco (Nicotiania tabacum) kind " seedleaf " (" Hicks Broad Leaf ").G and D stage of tobacco plant be all in the controlled environment chamber in, and keep growth temperature between 22-25 ° of C, to reduce outside environmental elements to the impact in tobacco Nicotine building-up process as far as possible.Test the tobacco material chosen be three strain vegetative period at about 40 days, the plant that growing height is close with plant type.Choose a strain wherein, the complete and fully extended blade of its growth carries out mechanical blade injury process by hole knockout; To it, top is carried out for another strain and extracts process; Last strain tobacco plant is then as the blank of this experiment.After tobacco plant having been carried out to injure process, all plant have all been put back in its original growing environment, and cultivate 48 hours so that the Induced synthesis of fully its defensive physiological response produced for environmental damage mechanism of induction and Nicotine.Afterwards, from pinch injury, blade injury and contrast, be extracted the tobacco plant root tissue in these three groups of process respectively, and from blade injury and blank, be extracted plant leaf tissue respectively, for the experiment carrying out next step is got ready.
2, the extraction of tobacco RNA and the order-checking of degraded group
The fresh sample of plant tissue extracted to be stored in liquid nitrogen and through grinding to form fine powder, to extract test kit TRIZOL (Invitrogen company) extract respectively and obtain the total serum IgE of tobacco plant by RNA.Being further purified and processing through tiny RNA sample preparation reagents box (Illumina company), obtaining the library of four groups of tobacco tiny RNA, is 2 libraries of the root tissue of the root tissue of blade injury process and 2 tiny RNA libraries of leaf tissue and pinch injury process and blank respectively.Total serum IgE 15% polyacrylamide, 8 M urea gels are separated, in 18-25 nt gel section by excising and having carried out wash-out and purifying by Nucleic acid purification kits (Axygen company) to tiny RNA.Tiny RNA sample after the purifying obtained is connected to RNA adapter: 5'RNA adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3 ') and 3'RNA adapter (5 '-pUCGUAUGCCGUCUUCUGCUUGUidT-3 ').Reacted by the PCR of Reverse Transcription box SuperScript III (Invitrogen company) and 15 circulations, build the cDNA library of these four groups of tiny RNA respectively.The sample obtained by carrying out purifying (PureLink PCR purification kit, Invitrogen company) to final PCR primer will carry out high-flux sequence through SOLEXA genomic analysis system.The primer sequence wherein related to comprises: 5'-CAAGCAGAAGACGGCATACGA-3 ' (RT primer), 5'-CAAGCAGAAGACGGCATACGA-3 ' (small RNA PCR primer 1), 5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3 ' (small RNA PCR primer 2) and 5'-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3 ' (small RNA sequencing primer).
The RNA target gene that the people (2009) such as the method Primary Reference German of degraded group library construction use and the research of degraded group.Extract test kit by the RNA of Oligotex mRNA mini kit (Qiagen company), the total serum IgE sample containing poly (A) tail is separated from the tobacco leaf tissue of blade injury process.Total serum IgE containing poly (A) tail is connected to 3 ' the 5'RNA adapter of end containing MmeI restriction enzyme site by ligase enzyme, by the reverse transcription reaction of oligo d (T) and PCR, product is connected to a double-stranded DNA adapter after being cut by purifying and MmeI enzyme.React through further purification and amplification, product carries out degraded group order-checking (Invitrogen company) on Illumina GA II platform platform.
3, the database information summary of tobacco sequence analysis
Involved in the present invention to reference genomic information be mainly derived from tobacco sequence information known in current GenBank, Rfam 9.1(http: //rfam.janelia.org/) and RepBase 14.03(http: //www.girinst.org) database is for filtering known RNA and tumor-necrosis factor glycoproteins.Participate in the research of the key gene Primary Reference Nicotine synthesis in the past in tobacco Nicotine metabolic pathway of synthesizing.MiRBase 20.0 (http://www.mirbase.org/) is used to identify and detect conservative miRNA in tobacco.
4, the discriminating of miRNA
The present invention uses Perl language scripts to analyze the tiny RNA sequence information that high-flux sequence obtains.In order to identify the miRNA of candidate, the tiny RNA sequence information obtained is screened, choose length in tobacco gene group sequence and be no more than the tiny RNA sequence of one group of complementation of 400-nt, predict its loop-stem structure (Hofacker et al., 1994) by the analysis of Vienna RNA package software.Utilize this fragment and linked sequence design primer thereof to increase from seedleaf and obtain the homologous sequence fragment of Nicotiana tabacum.This fragment has a desirable hairpin structure usually, includes the tiny RNA sequence of a pair complementation in the structure shown here, and this group sequence can form the complex body (can comprise the mispairing being less than 4 bases) of a kind of miRNA::miRNA*.For a specific miRNA family, if contain any 1 base mispairing or contain more than 3 base mispairings in pre-miRNA sequence between the miRNA of candidate, this miRNA is then considered to the different members belonging to same family, and the pre-miRNA sequence of redundancy is then eliminated.
5, based on the tiny RNA target site identification of degraded group order-checking
The data that the order-checking of degraded group obtains are through processing and filter, sequence information reading is mapped and mate some noncoding house-keeping genes, as tRNAs, rRNAs, snRNAs and snoRNAs, wherein length is retained at the high quality sequence reads of 20-21 Nucleotide and does to analyze further.The microRNA target prediction of miRNA by the method for psRNATarget, the tiny RNA of 20 ~ 24 length of nucleotides that we are screened by psRNATarget program maps in tobacco Nicotine regulation and control genes involved.This sequence fragment of further analysis whether bind by tiny RNA, wherein the gene fragment that result threshold value is less than 7 is joined for connection and will be considered as tiny RNA target gene candidate target, transcript profile according to degraded is mapped to the relative abundance of prediction cleavage site, the target gene identified is classified as five large classes.
6, sequence information
By the tobacco tiny RNA sequence data analytical results that obtains high-flux sequence and pcr amplification order-checking, obtain 6 gene orders, this sequence can form stable hairpin structure, its stem can produce tiny RNA sequence (mature sequence), and through microRNA target prediction, the synthesis of its tiny RNA target tobacco Nicotine and transporting pathway key protein matter encoding gene.Above-mentioned mature sequence information is shown in SEQ ID:NO. 1 ~ 21.
Embodiment 1
Obtain through to pinch and the tobacco root of blade injury and blade tiny RNA sequence and tobacco two ancestors plant genome sequence based on high-flux sequence, a large amount of tobacco gene sequence can be found, this sequence can form stable hairpin structure, and its stem can produce or express tiny RNA sequence (mature sequence).Wherein the hairpin structure of 21 gene order formation is shown in Fig. 1-2 1.The hairpin structure stable (MEF value is all less than-21.60) of these predictions.These genes all detect its expressed sequence, particularly obviously increase pinching and express in the tobacco root of blade injury and blade, show what these genes were likely by these process abduction deliverings.
Embodiment 2
The tiny RNA sequence that above-mentioned 21 genetic expressions produce is through microRNA target prediction, equal target tobacco Nicotine synthesis and transporting pathway key protein matter encoding gene, comprising QPT(quinolinate phosphirobosyltransferase), PMT(putrescine N-methyltransferase), MATE(multi antimicrobial extrusion family protein) etc. gene (table 1).Existing large quantity research shows, these genes are respectively Nicotine synthesis (QPT and PMT) and transhipment (MATE) key function gene (Dwey and Xie, 2013, Phytochemistry).21 genes that the present invention finds and its microRNA target prediction value all between 0-3.0 (generally closing on value is 3.0, exceedes this value and shows that binding result is unreliable), and are shearing (cleavage) mode and regulate and control target gene.Meanwhile, the degraded group data results that experiment obtains shows, portion gene has detected that 14-21 bar shears fragment, confirms above-mentioned control methods (table 2).
21 gene mature sequences that table 1 the present invention finds and its target gene (tobacco Nicotine synthesizes and transporting pathway key protein matter encoding gene) bindings bit Point matching result
In table 2 the present invention, 2 gene mature sequences and target gene data unit sequence of degrading is binding Point matching result.This data provides degraded group evidence that SEQ ID NO:4 and SEQ ID NO:13 shears in target gene binding site (target gene that detects in degraded group data is sheared fragment sequence number and to be listed in after its sequence in bracket)
| SEQ_ID_NO-4 | UUGGGUAUUUGAAAUUUAGGA |
| .: :::::::: : | |
| t0222900 | GAGUUUAAAUCUUUGCAG（14） |
| SEQ_ID_NO-13 | AGAAAGGCGGUUCUCUAGGC |
| ::::: | |
| t0107027 | ATCGGAAATATCCGAACGG（21） |
SEQUENCE LISTING
<110> Yunnan Academy of Tobacco Agricultural Science
<120> mono-grows tobacco nicotine content regulatory gene and application
<130> 2014
<160> 21
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:NO.1
<400> 1
aagagttcga gcaacatagg t 21
<210> 2
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.2
<400> 2
agaagagaag tggagaagaa a 21
<210> 3
<211> 23
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.3
<400> 3
agaaggaaaa aagtgctttt gag 23
<210> 4
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.4
<400> 4
aggatttaaa gtttatgggt t 21
<210> 5
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.5
<400> 5
cagaagttca tcaaggaaga t 21
<210> 6
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.6
<400> 6
cggatctctt ggcggaaaga c 21
<210> 7
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.7
<400> 7
cgttgaagtg gcaaggatga c 21
<210> 8
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.8
<400> 8
ctatgttgcg cggactttcc a 21
<210> 9
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.9
<400> 9
ctatgttgtg cggactctcc a 21
<210> 10
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.10
<400> 10
ggagagtcca agcaacatag a 21
<210> 11
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.11
<400> 11
ggagagtccg agcaacatag a 21
<210> 12
<211> 23
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.12
<400> 12
gggattatac tgggttgttg tta 23
<210> 13
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.13
<400> 13
tatttttgga gagttcgagc a 21
<210> 14
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.14
<400> 14
tctatgttgc tcgggctctc c 21
<210> 15
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.15
<400> 15
ttactttttc ttttttcttt g 21
<210> 16
<211> 23
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.16
<400> 16
ttagaagaaa acggattact gat 23
<210> 17
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.17
<400> 17
ttcaatataa ggtcaaatgg a 21
<210> 18
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.18
<400> 18
tttggaggat ccgagcaaca t 21
<210> 19
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.19
<400> 19
ttttgaaggg tccgcgcaat a 21
<210> 20
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.20
<400> 20
tttttgaaga gtgcgagcaa c 21
<210> 21
<211> 21
<212> DNA
<213> tobacco (Nicotiana tabacum) SEQ ID:No.21
<400> 21
tttttgcaga gtccgagcaa c 21
Claims (3)
1. one to grow tobacco nicotine content regulatory gene, it is characterized in that described tobacco nicotine content regulatory gene sequence is any one in SEQ ID:No.1 ~ 21.
2. tobacco nicotine content regulatory gene according to claim 1, is characterized in that described tobacco nicotine content regulatory gene is any one replacement through 1 ~ 2 base in SEQ ID:No.1 ~ 21, disappearance or interpolation forms;
Described base is at least one in A, T, C, G, N or U;
Described replacement, disappearance or interpolation refer to: at least one in A, T, C, G, N or U, replace, lack or add.
3. an application for the tobacco nicotine content regulatory gene described in claim 1 or 2, it is characterized in that described tobacco nicotine content regulatory gene for conditioning tobacco Nicotine synthesis and transhipment amount with realizes regulation and control Determination of Nicotine Content of Tobacco in application.
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| CN201410601193.6A CN104388431A (en) | 2014-11-01 | 2014-11-01 | Genes capable of regulating and controlling nicotine content of tobacco and application of genes |
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| CN104388431A true CN104388431A (en) | 2015-03-04 |
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| CN107488223A (en) * | 2017-09-08 | 2017-12-19 | 云南省烟草农业科学研究院 | One grows tobacco nicotine content controlling gene Ribosomal L4/L1 and its cloning process and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107488223A (en) * | 2017-09-08 | 2017-12-19 | 云南省烟草农业科学研究院 | One grows tobacco nicotine content controlling gene Ribosomal L4/L1 and its cloning process and application |
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| CN109295072A (en) * | 2018-10-17 | 2019-02-01 | 云南省烟草农业科学研究院 | A kind of tobacco smoke alkaloid synthesis regulation gene NtERF115 and its cloning process and application |
| CN111748554A (en) * | 2020-07-17 | 2020-10-09 | 中国烟草总公司郑州烟草研究院 | Application of tobacco Nt-miRNA203 in nicotine content regulation |
| CN111849982A (en) * | 2020-07-17 | 2020-10-30 | 中国烟草总公司郑州烟草研究院 | Application of tobacco Nt-miRNA2635 in nicotine content regulation |
| CN111748554B (en) * | 2020-07-17 | 2022-07-19 | 中国烟草总公司郑州烟草研究院 | Application of tobacco Nt-miRNA203 in nicotine content regulation |
| WO2023082780A1 (en) * | 2021-11-10 | 2023-05-19 | 云南省烟草农业科学研究院 | Molecular marker nicotine associated snp 1 for identifying content of nicotine in tobacco and kit and application thereof |
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