CN109234287A - A kind of tobacco myb transcription factor NtMYB4 and its application - Google Patents

A kind of tobacco myb transcription factor NtMYB4 and its application Download PDF

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CN109234287A
CN109234287A CN201811352756.7A CN201811352756A CN109234287A CN 109234287 A CN109234287 A CN 109234287A CN 201811352756 A CN201811352756 A CN 201811352756A CN 109234287 A CN109234287 A CN 109234287A
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ntmyb4
tobacco
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雷波
蔡凯
赵会纳
丁福章
潘文杰
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Guizhou Institute of Tobacco Science
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

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Abstract

The invention belongs to field of plant genetic project technology, a kind of tobacco myb transcription factor NtMYB4 and its application, the sequence of the tobacco myb transcription factor NtMYB4 are as follows: SEQ ID NO:1 are disclosed.The extraction of Tobacco Leaf total serum IgE；The acquisition of Tobacco Leaf total cDNA and genome DNA；Tobacco NtMYB4 gene cloning design of primers；The PCR amplification of tobacco NtMYB4 gene.Over-express vector building and inspection；The culture of aseptic seedling；Blade preculture；Agrobacterium is infected；Blade co-cultures；Differentiation culture；Bud elongation culture；Culture of rootage；The transplanting of transformation seedlings；Transgenic plant identification.The present invention analyzes tobacco K326 and NtMYB4 and is overexpressed transgenic plant, and the regulation of NtMYB4 gene pairs phenylpropyl alcohol alkane metabolic pathway has dosage effect, to having important regulating and controlling effect in the accumulation of tobacco leaf phenolic substances.

Description

A kind of tobacco myb transcription factor NtMYB4 and its application
Technical field
The invention belongs to field of plant genetic project technology more particularly to a kind of tobacco myb transcription factor gene NtMYB4 And its application.
Background technique
Currently, the prior art commonly used in the trade is such that in recent years, transcription factor is increasingly becoming molecular genetics and thin The important content in extracellular molecule biological study field.Myb gene family is maximum one kind transcription factor in plant, except participation is planted Response of the object to environmental factor and hormone, and it is equal to the morphogenesis of cell differentiation, cell cycle, the formation of organ and blade Outside with important adjustment effect, also has important regulating and controlling effect to a variety of secondary metabolism approachs.Myb transcription factor is also referred to as trans- Acting factor, it contains one section of highly conserved sequence-MYB motif combined with DNA, and it is prevalent in plant；And The growth and development of wide participation plant and metabolic regulation.The regulation of myb transcription factor has diversity, such as protein and protein Between interaction, the regulation of polymerase, redox reaction, phosphorylation, ubiquitination effect etc..It is transcribed according to MYB The structure of the factor can be classified as three categories: R1R2R3-MYB, R2R3-MYB and R1-MYB, in plant, the transcription of R2R3-MYB class The number of the factor is most.Myb transcription factor participates in regulating cell form and pattern formation, as seed is sprouted, epidermis hair cell shape At and reproduction cell formed；Participate in hormone signal response, such as ABA, GA and SA；Participate in environmental signal response, such as arid, height Salt, low temperature, light, nutritional deficiency and disease etc. participate in secondary metabolism and adjust --- flavonoids and volatility phenylpropyl alcohol alkane metabolic pathway It adjusts.Flavonoids and volatility phenylpropyl alcohol alkane these two types active material, but this two metabolic pathways are existed simultaneously in petunia Due to using phenylalanine as common precursor substance, being the relationship competed with one another for again.Wherein, the prior art has tune The upstream myb transcription factor of volatility benzene propane materials synthesis is saved, petunia R2R3-MYB transcription factor ODO1, that is, MYB42 can swash The promoter of 5- enolpyrul-shikimate acid -3- phosphate synthase gene living, to adjust the precursor of shikimic acid metabolic pathway Matter.ODO1 silencing can lead to the serious reduction of fragrance matter in petunia, but not influence the amount of flavonols and anthocyanidin substance. Another R2R3-MYB transcription factor EOBII is also adjustable the metabolism of volatility phenylpropyl alcohol alkane, directly activation petunia isoeugenol The promoter of synthase and tobacco phenylalanine lyase B.Arabidopsis AtMYB4 transcription factor is accredited as cinnamic acid hydroxylase Repressor, therefore fine adjusting can be carried out to the volatile materials formed using coumaric acid as substrate, such as eugenol and isobutyl Fragrant phenol.
Polyphenols is one of tobacco leaf aroma component, influences tobacco aroma, jealous.Therefore, how to develop one kind can Polyphenols is those skilled in the art's technical problem urgently to be resolved containing quantity of material in adjusting tobacco leaf.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of tobacco myb transcription factor NtMYB4 and its applications.
The invention is realized in this way a kind of tobacco myb transcription factor NtMYB4, the tobacco myb transcription factor The sequence of NtMYB4 are as follows: SEQ ID NO:1.
Another object of the present invention is to provide a kind of eggs with the tobacco myb transcription factor NtMYB4 sequential coding White matter, the protein have the amino acid sequence of SEQ ID NO:1.
It is more in adjusting tobacco leaf that another object of the present invention is to provide a kind of tobacco myb transcription factor NtMYB4 Application in aldehydes matter content.
In conclusion advantages of the present invention and good effect are as follows: the present invention is overexpressed using tobacco K326 and NtMYB4 to be turned Gene plant analyzes plant polyphenol content and phenylpropyl alcohol alkane metabolic pathway key enzyme activity.Through NtMYB4 gene pairs benzene known to analysis The regulation of propane metabolic pathway has dosage effect, and NtMYB4 gene belongs to R2R3-MYB transcription factor G4 subtype member, can lead to The negative regulation that it is realized to the approach to the transcriptional control of public phenylpropyl alcohol alkane metabolic pathway gene is crossed, major regulatory site is C4H。
Detailed description of the invention
Fig. 1 is the cloning process flow chart of tobacco myb transcription factor NtMYB4 provided in an embodiment of the present invention.
Fig. 2 is over-express vector building provided in an embodiment of the present invention and transgenic plant acquisition methods flow chart.
Fig. 3 is NtMYB4 genomic dna sequence structure chart provided in an embodiment of the present invention.
Fig. 4 is the amino acid sequence figure of NtMYB4 nucleotide sequence provided in an embodiment of the present invention and its derivation.
Fig. 5 is the organizing specific expression ideograph of NtMYB4 gene provided in an embodiment of the present invention.
Fig. 6 is NtMYB4 transgenic plant PCR qualification figure provided in an embodiment of the present invention.
Fig. 7 is the expression detection figure of NtMYB4 in transgenic plant provided in an embodiment of the present invention.
Fig. 8 is polyphenol content detection figure in transgenic plant provided in an embodiment of the present invention.
Fig. 9 be transgenic plant provided in an embodiment of the present invention with compare in NtC4H, NtPAL1 and Nt4CL1 expression quantity Histogram.
Figure 10 be transgenic plant provided in an embodiment of the present invention with compare in C4H determination of activity histogram.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
NtMYB4 is not cloned and expressed for the prior art, it is unclear that the specific regulating and controlling effect of NtMYB4 gene Problem；The present invention is cloned into NtMYB4 gene from tobacco K326, constructs the over-express vector of NtMYB4 gene and carries out heredity Conversion obtains NtMYB4 and is overexpressed transgenic plant, is overexpressed transgenic plant using tobacco K326 and NtMYB4 and analyzes plant Strain polyphenol content and phenylpropyl alcohol alkane metabolic pathway key enzyme activity, the tune through NtMYB4 gene pairs phenylpropyl alcohol alkane metabolic pathway known to analysis Control has dosage effect, and NtMYB4 gene belongs to R2R3-MYB transcription factor G4 subtype member, can be by public phenylpropyl alcohol alkane generation Thank to the negative regulation that the transcriptional control of pathway gene realizes it to the approach, major regulatory site is C4H.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The sequence of tobacco myb transcription factor NtMYB4 provided in an embodiment of the present invention is SEQ ID NO:1.
As shown in Figure 1, the cloning process of tobacco myb transcription factor NtMYB4 provided in an embodiment of the present invention includes following step It is rapid:
S101: the extraction of Tobacco Leaf total serum IgE；
S102: the acquisition of Tobacco Leaf total cDNA and genome DNA；
S103: tobacco NtMYB4 gene cloning design of primers；
S104: the PCR amplification of tobacco NtMYB4 gene；
The building of S105:NtMYB4 over-express vector and genetic transformation；
S106:NtMYB4 is overexpressed plant phenotype analysis and Function Identification.
As shown in Fig. 2, over-express vector provided in an embodiment of the present invention building and transgenic plant acquisition methods include with Lower step:
S201: over-express vector building and inspection；
S202: the culture of aseptic seedling；
S203: blade preculture；
S204: Agrobacterium is infected；
S205: blade co-cultures；
S206: differentiation culture；
S207: bud elongation culture；
S208: culture of rootage；
S209: the transplanting of transformation seedlings；
S210: transgenic plant identification.
The cloning process of tobacco myb transcription factor NtMYB4 provided in an embodiment of the present invention specifically includes:
Step 1: the extraction of Tobacco Leaf total serum IgE.It is limited according to Tiangeng biochemical technology (Beijing) using K326 spire as material The method that the plant total RNA extraction reagent box of company provides extracts total serum IgE.
Step 2: the acquisition of Tobacco Leaf total cDNA and genome DNA.Using 1 μ g of K326 spire total serum IgE sample as template, Reverse transcription is carried out using Oligo dT-Adaptor Primer, product is total cDNA after reverse transcription；Tobacco gene group total DNA Extraction using the EasyPure Plant Genomic DNA Kit of Beijing Quanshijin Biotechnology Co., Ltd referring to explanation Book extracts；
In a preferred embodiment of the invention, reverse transcription condition are as follows: 42 DEG C of 40min, 50 DEG C of 30min, 99 DEG C of 5min, 5 DEG C 5min。
Step 3: tobacco NtMYB4 gene cloning design of primers.It is obtained by the phylogenetic analysis of tobacco myb gene family The coding region sequence and genomic dna sequence of NtMYB4 gene are obtained, the blast program for utilizing 4.85 software of Geneious to integrate Multiple sequence comparison is carried out to a gene of all tobacco myb genes family more than 160, the site of NtMYB4 gene specific is selected to carry out Design of primers designs the primer of NtMYB4 full length gene cDNA and genomic dna sequence amplification and construction of eukaryotic expression vector SEQ ID NO:3
NtMYB4-F1(5′-CACCGCTAATTAAGAGAAATATTTCTTTGAAAATTGGCTCA-3′)/NtMYB4-R1 (5′-CCTCATGCGGCCGCTAACTAATTTTA-3′)；
In a preferred embodiment of the invention, carrier for expression of eukaryon is pEarley100 and pEarley301.
Step 4: the PCR amplification of tobacco NtMYB4 gene.With the first chain of tobacco spire cDNA and each 1 μ L of genome DNA Fidelity dna polymeric enzymatic amplification NtMYB4 full length gene is surpassed with Phusion using 50 μ L PCR reaction systems of standard for template CDNA and genomic dna sequence；
In a preferred embodiment of the invention, the parameter of PCR amplification cDNA is as follows: 94 DEG C of initial denaturation 4min, 35 circulations, 72 DEG C of heat preservation 10min.When genomic DNA amplification, the time extended increases to 3min；
In a preferred embodiment of the invention, the process of circulation is 94 DEG C of denaturation 1min, and 58 DEG C of annealing 1min, 72 DEG C extend 2min。
In a preferred embodiment of the invention, the assembling of sequencing sequence, multiple sequence comparison, ORF are searched and are translated, albumen Matter fundamental property is analyzed on 4.85 software of Geneious.The BLAST of nucleic acid and protein analysis and and protein sequence Conserved structure domain search carried out on the website NCBI, the prediction of the posttranscriptional modification of protein, secondary structure prediction and three-level knot The main bioinformatics on-line analysis software provided by the website Expasy of structure prediction carries out.In order to obtain more comprehensive life Object bioinformatics analysis by the website http://www.cbs.dtu.dk/services/ and SoftBerry as a result, also mended Sufficiently analysis.The building of phylogenetic tree first carries out sequence alignment with Clustal X 1.83, is then introduced into MEGA4.0 software, adopts It is constructed with adjacent method, the certificate authenticity parameter bootstrap of tree is set as 1000.
Over-express vector building provided in an embodiment of the present invention and transgenic plant acquisition methods specifically include:
Step 1: over-express vector building and inspection.After the PCR product glue recycling that clone is obtained, pENTR-D- is connected TOPO carrier adds the mixing of 1 μ L 5 × LR recombinase with carrier for expression of eukaryon pEarley100 and pEarley301 mixed in equal amounts LR recombinant products are added 37 degree of warm bath after 0.5 μ L Proteinase K Solution in 37 degree of digestion 1h with MluI by room temperature warm bath 2-4h afterwards 10min；All final products are converted into E. coli competent TOP10, will be applied to after the bacterium solution recovery after conversion containing 50 μ On the LB plate of g/ml kanamycins, after 16h is cultivated in 37 degree of inversions, selects positive colony and carry out bacterium solution PCR identification, and send sun Property clone sequencing, checking carrier building whether succeed；
Step 2: the culture of aseptic seedling.Tobacco K326 seed is put into 1.5ml centrifuge tube, then pours into 75% alcohol and disappears Malicious 30s adds 2% hypochlorite disinfectant 10min, is then cleaned 3-4 times with aqua sterilisa, is inoculated in after the agar aqueous suspension that sterilizes In MS solid medium；It is transferred in triangular flask after lateral root is formed, one plant every bottle；Tender terminal bud or axillary bud is taken to move to culture Squamous subculture is carried out in base；
In a preferred embodiment of the invention, media environment is 4.43g/L MS powder, 30g/L sucrose, 8g/L agar, pH It is 5.8.
Step 3: blade preculture.Prepare pre-culture medium, spread sterilizing filter paper, pours into a small amount of sterilizing in sterilizing disk Water soaks filter paper, and middle and upper part blade is taken to be cut into the leaf dish of 1cm × 1cm size, is placed in 25 degree of illumination precultures one on filter paper It；
In a preferred embodiment of the invention, pre-culture medium environment is 4.43g/LMS powder, 1mg/L 6-BA, 0.1mg/L NAA, 30g/L sucrose, 8g/L agar, pH 5.8.
Step 4: Agrobacterium is infected.MS fluid nutrient medium suspension Agrobacterium thallus adjusts OD600 to 0.5, infects tobacco Blade 10min closes the fluorescent lamp of workbench, sops up extra bacterium solution after infecting with sterilizing filter paper when infecting；
In a preferred embodiment of the invention, MS fluid nutrient medium environment is 4.43g/L MS powder, 30g/L sucrose, and pH is 5.8。
Step 5: blade co-cultures.Prepare co-culture medium, the blade after infecting is transferred to and is placed with sterilizing filter paper It co-cultures on base, is co-cultured 3 days under 25 degree of dark conditions；
In a preferred embodiment of the invention, co-culture medium environment be 4.43g/L MS powder, 1mg/L 6-BA, 0.1mg/L NAA, 3.7g/L MES, 30g/L sucrose, 8g/L agar, pH 5.4.
Step 6: differentiation culture.The blade of co-cultivation is transferred on differential medium, differentiation culture is carried out；
In a preferred embodiment of the invention, differential medium environment is 4.43g/LMS powder, 1mg/L 6-BA, 0.1mg/L NAA, 400mg/L Cef, 10mg/L Hyg, 30g/L sucrose, 8g/L agar, pH 5.8.
Step 7: bud elongation culture.By differentiation culture in 20 days or so, green bud point will form at paddle cutout, it will Blade is transferred in growth promotion culture medium, promotes bud elongation growth；
In a preferred embodiment of the invention, growth promotion culture medium environment be 4.43g/L MS powder, 400mg/L Cef, 10mg/L Hyg, 30g/L sucrose, 8g/L agar, pH 5.8.
Step 8: culture of rootage.Will the long bud to 2-3cm be transferred to environment be 4.43g/L MS powder, 0.1mg/L IBA, 200mg/L Cef, 10mg/L Hyg, 30g/L sucrose, 8g/L agar root culture medium in, PH 5.8.
Step 9: the transplanting of transformation seedlings.Root is cleaned, first transplants into vermiculite, covers disposal plastic cup moisturizing, 7-10 It can take away plastic cup, be transferred in Nutrition Soil after slow seedling 2 days or so, during which pay attention to pouring and apply nutrient solution.
Step 10: transgenic plant is identified.The real-time quantitative PCR for designing NtMYB4 gene expression amount identifies primer SEQ ID NO:5NtMYB4-qF (GCTTAGAATTAGTCCTCCTCATGACC) and SEQ ID NO:6NtMYB4-qR (TTCTATAGTCCAAAACTAGACCATTAGCC), the 35S promoter primer SEQ ID NO on pEarley100 carrier is utilized: 7 35S-F (CCCCCACCCACGAGGAGCAT) and NtMYB4-qR are matched, the primer SEQ ID on OCS terminator on carrier Whether NO:8OCR-R (GGTAAGGATCTGAGCTACACATGCTC) and NtMYB4-qF pairing identification NtMYB4 gene correctly arrives In tobacco gene group；
In a preferred embodiment of the invention, real-time quantitative PCR detection primer, SEQ ID NO:9 are devised
NtPAL1-qF(GTGGATTTTTCGAGTTGCAGCCT)/NtPAL-qR (TGTTCCATAATAGCAGCAGCCTCA)；
SEQ ID NO:10
NtC4H-qF(AGTCCCACACATGAACCTTCACG)/NtC4H-qR(ATTCCAGGGCAGCTCCTCCT)；
SEQ ID NO:11
Nt4CL1-qF (TTACGGCCGCTGAGGTTGTG)/Nt4CL1-qR (CGGCCTGAGTTAGCACGGAG), is used for Detect the expression quantity variation of tri- kinds of genes of NtC4H, NtPAL1 and Nt4CL1 in transgenic plant.
Application principle of the invention is further described with reference to the accompanying drawing.
1, the nucleic acid extraction of tobacco K326 and transgenic plant.
The present invention using tobacco K326 and NtMYB4 be overexpressed transgenic plant carried out Total RNAs extraction, obtain 28S and The complete high quality RNA of 18S can be used for downstream gene clone and real-time quantitative PCR detection.Light is divided by NanoDrop2000 Its quality of the ratio in judgement of degree meter measurement 0D260/0D280,00260/0D230,0D260/0D280 is in 1.8- as the result is shown Between 2.0,0D260/00230 is equal > and 2.0, it is also demonstrated that obtained RNA is high-quality.
2, the clone and nucleotide sequence Parameter analysis of NtMYB4 and genomic DNA.
Using the overall length primer of design, Successful amplification of the present invention goes out the full-length cDNA and corresponding genome of NtMYB4 gene DNA, and clip size is consistent with expection.TOPO is carried out with pENTR-D-TOPO carrier after glue recycling at once to react, and is converted big Enterobacteria competence TOP10, chooses positive colony sequencing.
NtMYB4 cDNA and genomic DNA sample presentation sequencing result analysis are had found, the cDNA overall length of NtMYB4 is 918bp, Wherein 5 ' UTR length are 42bp, G/C content 23.8%, 3 ' UTR long 93bp, G/C content 31.2%；Code area (the packet of gene Include terminator codon) total 783bp, G/C content is significantly higher than UTR region, reaches 37.9%, such as Fig. 3, Fig. 4.NtMYB4 genome The total 1325bp of sequence includes 2 exons and 1 introne, length of intron 407bp altogether.
3, the physicochemical property of NtMYB4 albumen and posttranscriptional modification Locus Analysis in Shoots
The physicochemical property of NtMYB4 albumen is analyzed using 4.85 software of Geneious and the website Expasy.As a result Show that NtMYB4 gene is separately encoded 260 amino acid residues, molecular weight of the encoded protein 29.49kD, theoretical isoelectric point pI are 8.21 in all amino acid residues of NtMYB4, and leucine (Leu) accounting weight is maximum, there is 28, account for entire coding albumen 10.8%, followed by serine (Ser) encode residue 21, account for the 8.1% of entire protein sequence；NtMYB4 encodes albumen Atom group becomes C1270H2047N381O397S15, extinction coefficient is 35575 in 280nm, and unstability index is 43.57, is shown to be One labile protein, fat coefficient 76.96, overall average hydrophilicity are only -0.732, illustrate the inclined without hydrophily of the albumen It is good.
Glycosylation analysis is carried out to NtMYB4 protein sequence with NetGly 1.0 and finds that there are 2 possible N- for the albumen Glycosylation modified site (N69And N236), using NetPhos to serine kinase, threonine kinase and tyrosine-kinase in NtMYB4 The phosphorylation site of enzyme is analyzed, and discovery NtMYB4 contains 8 serine kinase phosphorylation site (S83、S118、S130、 S164、S185、S202、S220And S238) and 3 threonine kinase phosphorylation site (T18、T125And Y136), but without tyrosine kinase phosphorus Polyadenylation sites.Result explanation, NtMYB4 albumen can be by kinases institute phosphorylation, to realize the regulation of its function.
4, trans-membrane region and topological structure are predicted.
It is predicted using transmembrane structure of TMHMM, TMpred and the Geneious 4.85 to NtMYB4.The result shows that Transmembrane structure is not present in NtMYB4 transcription factor, this albumen is prompted to be possible for typical transcription factor in nucleus expert Make function.
Secondary structure prediction is analyzed with SOPMA software.According to calculating, random coils are most in NtMYB4 albumen It is more, 58.85%, followed by alpha-helix are accounted for, accounts for 26.92%；The extended chain and beta sheet of NtMYB4 only has 10.32% He respectively 3.91%.Since the main MYB functional domain of NtMYB4 albumen is located at the N-terminal of albumen, it can be seen that α-helixstructure pair The correct binding function for exercising myb transcription factor is of great significance, and the mutation of this moiety site may cause function difference Allele occurs, and plays a significant role to regulation downstream secondary metabolism approach gene, and potential tobacco volatile fragrance The Probability Area of agent modulates.
5, protein sequence homologies and functional structure domain analysis.
For the function and its evolutionary relationship for clearly analyzing NtMYB4, NtMYB4 protein sequence is committed to NCBI and is carried out BLASTp is compared, and downloads the homologous MYB4 of other plant and other MYB protein sequences progress multiple alignment and phylogenetic tree Building.The result shows that the SlMYB4 albumen (NP_001233975) of NtMYB4 and tomato (Solanum lycopersicum) With highest consistency 77.5%, followed by hybridization petunia (Petunia x hybrida) PhMYB4 (ADX33331), one Cause property is 73.8%.The consistency of NtMYB4 and the GhMYB9 albumen (AF336286) of cotton (Gossypium hirsutum) is 70.6%, the consistency with arabidopsis (Arabidopsis thaliana) AtMYB4 (NP_195574) is 62.1%.Other are more The MYB4 albumen of a species and the consistency of NtMYB4 are also all larger than 60%, such as: Chinese cabbage (Brassica rapa Var.purpuraria) BrMYB4 (ABQ81931), shepherd's purse (Capsella rubella) CrMYB4 (EOA17158), chick-pea (Cicer arietinum) CaMYB4 (XP_004505789) and dahlia (Dahlia pinnata) DpMYB4 (BAJ33514)。
Tree is developed from the plant MYB4 protein system of building it is also seen that tobacco NtMYB4 and tomato SlMYB4 albumen position In the same branch, and other plant R2R3-MYB transcription factors, including MYB4 times is gathered in another branch, prompts Certain function variation may has occurred in NtMYB4 in plant of Solanaceae.
6, tissue specificity is analyzed.
For the tissue specificity of clear NtMYB4 gene, the present invention utilizes the corresponding est sequence SGN- of NtMYB4 gene U430855, analytical sequence utilize the cigarette of Affymetrix company at Solanaceae genome alliance (Sol Genomics Network) Careless chip to the TobEA data constructed on the basis of the expression quantity analysis on change of each gene of tobacco different development stage, thus The tissue specificity of NtMYB4 gene expression is obtained, as shown in Figure 5.Analysis the result shows that, NtMYB4 gene is in cotyledon Expression quantity highest, followed by young leaflet tablet, seed and young tender stem stalk, the expression quantity in ageing leaves and mature leaf is relatively It is weak.
7, the Molecular Identification of transgenic plant.
It is overexpressed transgenic positive plant in order to obtain, the present invention has carried out heredity using agrobacterium tumefaciens-mediated transformation and turned Change, and successfully screens the transgenic plant for obtaining NtMYB4 with Basta.Utilize 35S-F/NtMYB4-qR and NtMYB4-qF/ Whether OCS-R primer identification NtMYB4 over-express vector frame is properly inserted on the genome of transgenic plant.As shown in fig. 6, PCR qualification result shows that multiple transgenic plants energy Successful amplification goes out the 35S-F/NtMYB4-qR segment peace treaty of about 750bp The NtMYB4-qF/OCS-R segment of 250bp, wherein 19,21,24 and 29 transgenic lines can expand two segments, table simultaneously The bright over-express vector with 35S promoter driving NtMYB4 gene has successfully been gone on the genome of tobacco K326, can be used for Subsequent Functional identification of genes.
8, transgenic plant NtMYB4 expression analysis and functional analysis.
Real-time quantitative PCR detects NtMYB4 gene expression dose in transgenic plant, as shown in Figure 7, the results showed that selecting The expression quantity of NtMYB4 has significant up-regulation in the positive strain of 4 selected, illustrates successfully to be transferred in the transgenic plant obtained It is overexpressed the expression cassette of NtMYB4.For being transferred to for the transgene negative plant OX-VC for being overexpressed box not successfully, NtMYB4's Significant change does not occur for expression quantity.Due to the difference of insertion point, the up-regulated expression level of different strains is not identical, 4 The expression quantity of OX-24 is highest in the transgenic line of a analysis, has reached 6 times or more of control, and OX-19 and OX-29 Relatively low, up-regulated expression level is only 4 times.
In order to further analyze the biological function of NtMYB4 in transgenic plant, analyze whether it is metabolized way to phenylpropyl alcohol alkane Diameter has an impact, and the present invention determines the polyphenol content in plant mature leaf, as shown in Figure 8.Control and transgene negative plant The polyphenol content of per dry wt is above 60mg g-1, but in being overexpressed transgenic plant, the total phenol content of all strains is aobvious Decline is write, shows that the synthesis of NtMYB4 gene pairs total phenol has negative regulation effect, likely via inhibition phenylpropyl alcohol alkane metabolism The transcription of pathway gene or the activity of enzyme realize its regulatory function.Further, it is also possible to find out, between different transgenic lines there is also Significant difference, the NtMYB4 expression quantity for being overexpressed transgenic line OX-19 and OX-29 are lower than OX-21 and OX-24, led to table NtMYB4 up to transgenic line OX-19 and OX-29 is also relatively small to the inhibition of phenylpropyl alcohol alkane metabolic pathway, therefore synthesizes total Phenol is relatively high, this regulation for also further demonstrating NtMYB4 gene pairs phenylpropyl alcohol alkane metabolic pathway has dosage effect.
Aldehydes matter content decline may be with NtMYB4 gene overexpression to phenylpropyl alcohol alkane metabolic pathway base in transgenic plant The negative regulation of cause is related, thus the present invention have detected control, in transgene negative and positive plant NtC4H, NtPAL1 with The expression quantity of Nt4CL1 gene, as shown in Figure 9.The result shows that NtC4H, NtPAL1 and Nt4CL1 gene are by NtMYB4 negative gene To regulation, because said gene is inhibited by different degrees of in being overexpressed transgenic plant, in comparison, NtC4H is suppressed Processing procedure degree highest, prompting the site is the major site by NtMYB4 negative regulation.Although NtPAL1 and Nt4CL1 gene expression Level decline is not highly significant, but decline level also basically reaches 20% or so, is had very to the metabolism of common phenylpropyl alcohol alkane Important influence.
Public phenylpropyl alcohol alkane metabolic pathway key gene is by different journeys in the transgenic plant of overexpression NtMYB4 gene The inhibition of degree, therefore the present invention has detected the cinnamic acid -4- hydroxylase of the highest NtC4H gene coding of wherein suppressed degree (C4H) enzyme activity variation, as shown in Figure 10.The activity of C4H with its gene expression dose with similar trend, i.e., and transgenosis Control is compared, and the C4H enzyme activity of 4 transgenic positive strains is inhibited by NtMYB4 albumen, and expression quantity, which is remarkably decreased, to be caused Its enzymatic activity is also remarkably decreased, and activity only reaches half of normal plant or so.
The above results show that NtMYB4 gene belongs to R2R3-MYB transcription factor G4 subtype member, can be by public benzene The transcriptional control of propane metabolic pathway gene realizes its negative regulation to the approach, and NtMYB4 gene major regulatory site is C4H.To increase the accumulation of phenolic substances, then need to inhibit NtMYB4 gene；Conversely, to the content for reducing phenolic substances, It can then be realized by being overexpressed NtMYB4 gene.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Guizhou Province Tabacco Science and Technology Institute
<120>a kind of tobacco myb transcription factor NtMYB4 and its application
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1325
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aaagctaatt aagagaaata tttctttgaa aattggctca aaatgggaag gtcaccatgt 60
tgtgaaaagg ctcatacaaa caaaggagca tggactaaag aagaagatga aaggcttatt 120
gcttacatta aagctcatgg cgaaggttgt tggaggtctc ttcctaaagc tgctggcctt 180
ctcagatgtg gtaaaagctg ccgtcttcgt tggattaatt acttacgacc tgatcttaaa 240
cgtggtaact tcactgatga agaagatgaa ctcattatca aactccatag cctcctcggt 300
aacaagtaag tatttactgt cggaagtaat taaaagtatg tctgctctga aggagagctt 360
tgcggtcaaa ttttctctct atgacctata ggtcacgggt tcgaccttga agtaatcact 420
aatattgcat cccttgggtg caaccctacc tcaaacgcag gatgacttgt gcgccggact 480
acacaatagt ctgtgtgctc tctctaagtt gttagtacta attatagtca aacctctata 540
taacagactc atttgttcag atattttttg gctgttataa ttaagtgttg ttatacttag 600
aagatacata ttataacaat aacataatcg attccgaaaa aaaaaatgac tttgttatat 660
aaaaatgcta ttatagagag tctgaccgta ttttctttgt ttcttgtaat aggtggtcac 720
ttatagcggg aagattacca ggaagaacag ataatgagat aaagaattat tggaacacac 780
atataagaag gaagcttttg agtaggggta ttgatccaac aacacatagg ccaatgagtg 840
agcctactct tggtacgcaa aaagtgacaa ccatttcttt tgctgctgat gatcaagatc 900
agaagattaa gatcaaatcc gaattaattg agacgatgag caaagaagaa gatcatgaaa 960
ttcaagaacg gtgtcctgac ttgaatcttg agcttagaat tagtcctcct catgaccaac 1020
aaaaccaact tgatcataat caaagagcaa actctttgtg ttttacatgt agtttgggta 1080
tacaaaatag taaagattgc agttgcagta ctaaaagtag taatggaaat ggctgtagta 1140
atattataag tatgaatatt tctggttatg attttttagg gttgaaggct aatggtctag 1200
ttttggacta tagaaccttg gaaactaagt gatcagatct gtattatgat ctttaatttt 1260
ctgtatgtag aaaaaaatat aagaatgtca agagagaagt aaaattagtt agcggccgca 1320
tgagg 1325
<210> 2
<211> 260
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Gly Arg Ser Pro Cys Cys Glu Lys Ala His Thr Asn Lys Gly Ala
1 5 10 15
Trp Thr Lys Glu Glu Asp Glu Arg Leu Ile Ala Tyr Ile Lys Ala His
20 25 30
Gly Glu Gly Cys Trp Arg Ser Leu Pro Lys Ala Ala Gly Leu Leu Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp
50 55 60
Leu Lys Arg Gly Asn Phe Thr Asp Glu Glu Asp Glu Leu Ile Ile Lys
65 70 75 80
Leu His Ser Leu Leu Gly Asn Lys Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Ile
100 105 110
Arg Arg Lys Leu Leu Ser Arg Gly Ile Asp Pro Thr Thr His Arg Pro
115 120 125
Met Ser Glu Pro Thr Leu Gly Thr Gln Lys Val Thr Thr Ile Ser Phe
130 135 140
Ala Ala Asp Asp Gln Asp Gln Lys Ile Lys Ile Lys Ser Glu Leu Ile
145 150 155 160
Glu Thr Met Ser Lys Glu Glu Asp His Glu Ile Gln Glu Arg Cys Pro
165 170 175
Asp Leu Asn Leu Glu Leu Arg Ile Ser Pro Pro His Asp Gln Gln Asn
180 185 190
Gln Leu Asp His Asn Gln Arg Ala Asn Ser Leu Cys Phe Thr Cys Ser
195 200 205
Leu Gly Ile Gln Asn Ser Lys Asp Cys Ser Cys Ser Thr Lys Ser Ser
210 215 220
Asn Gly Asn Gly Cys Ser Asn Ile Ile Ser Met Asn Ile Ser Gly Tyr
225 230 235 240
Asp Phe Leu Gly Leu Lys Ala Asn Gly Leu Val Leu Asp Tyr Arg Thr
245 250 255
Leu Glu Thr Lys
260
<210> 3
<211> 67
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caccgctaat taagagaaat atttctttga aaattggctc acctcatgcg gccgctaact 60
aatttta 67
<210> 4
<211> 65
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
caccgagaaa agtgagaact ttacaagaaa agttgcttcc tagtactata gacatgatac 60
atttg 65
<210> 5
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gcttagaatt agtcctcctc atgacc 26
<210> 6
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ttctatagtc caaaactaga ccattagcc 29
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cccccaccca cgaggagcat 20
<210> 8
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ggtaaggatc tgagctacac atgctc 26
<210> 9
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtggattttt cgagttgcag ccttgttcca taatagcagc agcctca 47
<210> 10
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
agtcccacac atgaaccttc acgattccag ggcagctcct cct 43
<210> 11
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ttacggccgc tgaggttgtg cggcctgagt tagcacggag 40

Claims (3)

1. a kind of tobacco myb transcription factor NtMYB4, which is characterized in that the sequence of the tobacco myb transcription factor NtMYB4 are as follows: SEQ ID NO:1。
2. a kind of protein with tobacco myb transcription factor NtMYB4 sequential coding described in claim 1, which is characterized in that The protein has the amino acid sequence of SEQ ID NO:1.
3. a kind of tobacco myb transcription factor NtMYB4 as described in claim 1 is in adjusting tobacco leaf polyphenol content Application.
CN201811352756.7A 2018-11-14 2018-11-14 A kind of tobacco myb transcription factor NtMYB4 and its application Pending CN109234287A (en)

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CN110964740A (en) * 2019-12-10 2020-04-07 河南农业大学 Preparation method and application of tobacco with high flavonol content
CN111548399A (en) * 2020-05-15 2020-08-18 贵州省烟草科学研究院 MYB transcription factor for regulating and controlling accumulation of tobacco cembratriene diol, coding gene and application
CN111778258A (en) * 2020-01-18 2020-10-16 西南科技大学 MYB140 gene, constructed vector and expressed transgenic tobacco plant
CN112322654A (en) * 2020-10-16 2021-02-05 山东大学 Application of corn transcription factor ZmMYB42 gene in plant drought-resistant breeding
CN112724213A (en) * 2021-01-13 2021-04-30 中国农业大学 Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof
CN112779272A (en) * 2021-03-31 2021-05-11 合肥工业大学 Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application
CN113234726A (en) * 2021-06-21 2021-08-10 贵州省烟草科学研究院 Tobacco glandular hair specific promoter pNtTCP9a and application thereof
CN114645055A (en) * 2020-12-21 2022-06-21 中国农业科学院烟草研究所 Tobacco NtMYB1 gene and coding protein and application thereof

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109762830B (en) * 2019-03-21 2020-09-11 安徽农业大学 MYB transcription factor FvMYB330 gene for regulating and controlling eugenol accumulation in strawberry fruits and application thereof
CN109762830A (en) * 2019-03-21 2019-05-17 安徽农业大学 Regulate and control the myb transcription factor FvMYB330 gene of eugenol accumulation and its application in strawberry fruit
CN110964740B (en) * 2019-12-10 2021-11-23 河南农业大学 Preparation method and application of tobacco with high flavonol content
CN110964740A (en) * 2019-12-10 2020-04-07 河南农业大学 Preparation method and application of tobacco with high flavonol content
CN111778258A (en) * 2020-01-18 2020-10-16 西南科技大学 MYB140 gene, constructed vector and expressed transgenic tobacco plant
CN111778258B (en) * 2020-01-18 2022-09-16 西南科技大学 MYB140 gene, constructed vector and expressed transgenic tobacco plant
CN111548399A (en) * 2020-05-15 2020-08-18 贵州省烟草科学研究院 MYB transcription factor for regulating and controlling accumulation of tobacco cembratriene diol, coding gene and application
CN112322654A (en) * 2020-10-16 2021-02-05 山东大学 Application of corn transcription factor ZmMYB42 gene in plant drought-resistant breeding
CN114645055A (en) * 2020-12-21 2022-06-21 中国农业科学院烟草研究所 Tobacco NtMYB1 gene and coding protein and application thereof
CN114645055B (en) * 2020-12-21 2022-12-20 中国农业科学院烟草研究所 Tobacco NtMYB1 gene and coding protein and application thereof
CN112724213A (en) * 2021-01-13 2021-04-30 中国农业大学 Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof
CN112779272A (en) * 2021-03-31 2021-05-11 合肥工业大学 Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application
CN112779272B (en) * 2021-03-31 2022-03-18 合肥工业大学 Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application
CN113234726A (en) * 2021-06-21 2021-08-10 贵州省烟草科学研究院 Tobacco glandular hair specific promoter pNtTCP9a and application thereof
CN113234726B (en) * 2021-06-21 2022-07-22 贵州省烟草科学研究院 Tobacco glandular hair specific promoter pNtTCP9a and application thereof

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Application publication date: 20190118