CN104962565A - Purple bud tea tree R2R3-MYB gene CsMYB2 and application thereof - Google Patents
Purple bud tea tree R2R3-MYB gene CsMYB2 and application thereof Download PDFInfo
- Publication number
- CN104962565A CN104962565A CN201510419264.5A CN201510419264A CN104962565A CN 104962565 A CN104962565 A CN 104962565A CN 201510419264 A CN201510419264 A CN 201510419264A CN 104962565 A CN104962565 A CN 104962565A
- Authority
- CN
- China
- Prior art keywords
- csmyb2
- gene
- purple
- tea leaves
- smyb2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a purple bud tea tree R2R3-MYB gene CsMYB2 and application thereof, belonging to the field of biological genes. A CsMYB2 segment, a RACE segment and full-length primers are designed, amplification and sequencing are performed to obtain the CsMYB2 gene of which the nucleotide sequence is disclosed as SEQ NO ID:1, and the gene structure is analyzed after translating the CsMYB2 gene into amino acid sequences. The analysis on the amino acid structure of the CsMYB2 indicates that the CsMYB2 can possibly have the function of regulating the biosynthesis of the purple bud tea tree bud leaf anthocyanin, and the transformation of Arabidopsis thaliana proves that the CsMYB2 has the function of regulating the biosynthesis of anthocyanin. The invention lays foundation for application of CsMYB2 in the aspect of improving plant pigment accumulation.
Description
Technical field
The invention belongs to field of biological genes, particularly a kind of purple young tea leaves tree R2R3-MYB gene C sMYB2 and application thereof.
Background technology
Anthocyanidin is also called cyanidin(e) (anthocyanidin), is one of topmost meta-bolites in flavonoid biosynthesis pathway, is the important water-soluble flavone class pigment of occurring in nature.Anthocyanidin in plant is unstable, and normal combination with sugar exists with the form of glycoside, forms anthocyanin (anthocyanin).Wherein glycosyl donor is except glucose, can also replace by the different sugar of semi-lactosi, rhamnosyl and pectinose and wood sugar etc. (Panagiotis Arapitsas, 2008).There is the difference of glycosylation site and number due to cyanidin(e) and methylate, the difference of acidylate degree, thus in natural plant, define various anthocyanin, be accumulated in the vacuole of vegetable cell with the form of glucosides.Anthocyanin belongs to flavonoid compound, its basic structure has double bond to exist, 465 ~ 560nm is respectively and 270 ~ 280nm has maximum light absorption in wavelength region, impart the tissue such as the flower of plant, fruit, stem, leaf and root and produce redness, pink colour, blueness and the purple even multicoloured color (Koes such as black, R., 2005; Winkel-Shirley, B., 2001).
In plant, the biosynthesizing of anthocyanidin regulates and controls by the regulatory gene of several different family usually, as MYB family, MYC family (coding Bhlh albumen) and WD40 proteinoid family, this three classes transcription factor also to play a crucial role (He in the synthesis of other flavonoid meta-bolites of regulation and control (pycnogenols and flavonol), F., 2008; Dixon, R.A., 2005).At present, by the research of model plant Arabidopsis Mutants, isolation identification goes out most regulatory gene, as the transcription factor such as TTG1 of PAP1 and the PAP2 of MYB family, GL3 and the EGL3 of MYC family and WD40 family.This three classes transcription regulatory protein forms a ternary complex and regulates the expression of structure gene (Chi, Chs, F3h, with Dfr etc.) in flavonoid approach and then regulate biosynthesizing (He, F., 2008 of Arabidopsis anthocyanin; Gonzalez, A., 2008; Ramsay, N.A., 2003; PAYNE CT., 2000).But, also there are a series of Myb and Myc transcription factor such as MYBL2, MYB4 and BHLH32 in the biosynthetic pathway of Arabidopsis anthocyanin, play a part negative regulation (Dubos C., 2008; Chen, Z.-H., 2007; Jin, H., 2000).In the anthocyanidin route of synthesis of monocotyledonous plant Zea mays, its key structure enzyme gene (CHS, CHI, F3H, DFR, ANS, LDOX and UFGT) is mainly by the adjustment of MYB and MYC family associated transcription factor, and then the biosynthesizing of regulation and control anthocyanidin.
Purple young tea leaves tree is a kind of rare characteristic Resources of Tea Plant, and its bud-leaf is purple, redness or red-violet colour, has the anthocyanidin of high level.Tealeaves, as a kind of natural health drink, world's beverage history has very important status.Research shows, anthocyanidin has antioxidant (Bae & Suh, 2007), anticancer (Leeet al., 2009), angiogenesis inhibitor (Bagchi, Sen, Bagchi, & Atalay, 2004), antibacterial (Viskeliset al., 2009), anti-apoptotic (Elisia & Kitts, 2008) with the functional performance such as short apoptosis (Lo et al., 2007).
Chu Shilin investigates discovery, and in tea tree local sexual colony kind, heliotrope new shoot accounts for significant proportion, but Various Seasonal has very big-difference, as ordinary group kind has the bud-leaf of about 30% to present micro-red-violet colour or red-violet colour spring, then has 88.7% in summer; In the sexual colony in Anhua kind tea tree that Liu Fuzhi etc. plant in Agricultural University Of Hunan, there is deep mixed heliotrope new shoot, to some extent containing anthocyanidin in the tender tip of plant of 82.6% in the plant finding that there is 76%; Zhou Zhigao etc. also find, the tender tip that purple bamboo shoot tea strain is extracted out be brilliant violet look or coppery and proterties can be hereditary, is not the cause due to temperature and edaphic condition; Tea tree bud-leaf is divided into green, lilac, middle purple, intense violet color and special purple 5 grades according to the colour-difference meter measured color value of Purple tea shoots and the contents level of bud-leaf anthocyanidin by Xiao Lizheng etc., comprises free 9803 of special purple and free 9809 of lilac; Kerio etc.
[10], Rashid etc. then carried out correlative study report to Kenya's purple young tea leaves tree extraction and identification of anthocyanidin, the physiological function of tea tree anthocyanidin respectively.The material that Liu Fuzhi reaches 4 according to purple bud-leaf dense in colony kind tea tree and acidic ethanol index of Response accounts for 4.35% this ratio, inference can filter out the high tea tree type of anthocyanidin content in some local sexual colony kind, but above research does not all have further breed breeding to report.2005, " purple beautiful " tea tree with purple bud, purple leaf, purple stem that hereditary property is stablized, varietal character is single, nothing makes a variation of tealeaves institute of Yunnan Province seed selection, obtained the Plant new variety protection power that the State Administration of Forestry authorizes." purple beautiful " is by the national improved tea variety of Yunnan large-leaf seed colony---Menhai daye tea individual plant is cultivated and formed, and young sprout bud-leaf is the tender tip of purple bud, purple leaf, purple stem, and made dry tea and millet paste are all purple, and anthocyanidin content is about 3 times of general red young tea leaves
[1-2].Academy of agricultural sciences of Guangdong Province drinks Institute of Zoology (former tealeaves institute) and from Yunnan large-leaf seed and phoenix narcissus colony kind, selected about 20 purple bud kind excellent strains in 2003, new lines young sprout 2 ~ 4 leaves are red-violet colour, along with the growth of blade is with ripe, the red-violet colour of blade is taken off, and changes green into.Except the minority strains such as phoenix and No. 4, red autumnal leaves, the red-violet colour of other purple bud strain bud-leafs is all the darkest in summer, bud-leaf color and luster is described simultaneously by external environment and genic control.The formation of tea tree purple bud-leaf and the anabolism of anthocyanidin and accumulate closely related, anthocyanidin content determines the bud-leaf purple depth.Compare green bud-leaf, in purple bud, anthocyanidin content significantly increases, as anthocyanidin content in Yunnan " purple beautiful " kind young sprout two leaves and a bud can up to 29mg/g, far above contrast; And Guangdong purple young tea leaves anthocyanidin content is 3.6 ~ 12.8 times of the large pale green kind of contrast cloud, the content of the purpureal depth of bud-leaf and anthocyanidin is proportionate.
The starting of tea tree anthocyanidin functional gene separating clone is more late, compares the progress of tea tree anthocyanidin composite structure enzyme gene, the researches of associated regulatory genes.Chen Linbo etc. are by analyzing the genetic expression of " purple beautiful " tender leaf and climax leaves, screen 59 difference expression genes, wherein in purple young leaflet tablet, obtain 26 and raise fragment, comprise the transcription factor containing AP2 structure, metabolism associated protein, signal protein etc., and point out that these genes may participate in the regulation and control of leaf of tea tree look.Wang Hongxue has cloned 3 myb genes, respectively called after CsMYB4-5, CsMYB4-6 and CsMYB4-7, and arabidopsis thaliana transformation papl-D mutant plants.Horse spring thunder etc. utilizes biochip technology to analyze purple bud and green bud, screen difference expression gene 43, comprise 2 and may synthesize relevant transcription factor MYB albumen and WD40 protein gene with anthocyanidin, myb gene is respectively CsMYB1 and CsMYB2, total length is respectively 1 132 and 1 020bp, is respectively HQ660373 and HQ660374 in the accession number of GenBank.Quantitative analysis finds that the relative expression quantity of CsMYB2 in blade is more than 100 times of root, and point out that Shading treatment obviously can reduce the anthocyanidin content in blade, improve the expression of CsMYB1, but little on the impact of transcription factor CsMYB2, the dependency that itself and tea tree anthocyanidin synthesize needs to be studied further.
Summary of the invention
For overcoming the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is to provide a kind of purple young tea leaves to set the nucleotide sequence of R2R3-MYB gene C sMYB2.
Another object of the present invention is to provide above-mentioned purple young tea leaves to set the aminoacid sequence of R2R3-MYB gene C sMYB2.
Another object of the present invention is to provide the application of above-mentioned purple young tea leaves tree R2R3-MYB gene C sMYB2.
Object of the present invention is achieved through the following technical solutions: a kind of purple young tea leaves tree R2R3-MYB gene C sMYB2, and its nucleotide sequence is as follows:
GGAAGGGCTCCATGTTGTTCCAAGGTTGGGTTGCATAGAGGTCCATGGACTGCTAGAGAGGATGCATTGCTCACCAAGTATATTCAGCTTCATGGTGAAGGCAATTGGAGATCTTTGCCTAAAAAAGCTGGTCTACTAAGATGCGGAAAGAGTTGCAGGCTAAGATGGATGAACTATCTAAGACCTGATATCAAGAGAGGGAACATCTCCCCTGATGAGGATGACCTAATTATCAAAATGCATGCTCTTCTTGGCAACCGATGGTCTCTTATCGCAGGAAGACTACCGGGTCGAACTGATAACGAGATTAAGAACTATTGGAACACCCATCTCAGCAAAAGACTCTTAAGCCAAGGAACCGACCCGAATACCCACAAAAAAATATCAGATCCTCACCTAAAAGAACCAAAGAAGAGAAGGAGCAACAGCAAAAAGCAAAAAAACAAGTCCAAATCCAATGTTGTAGTGATTGAAAAGCTCAAAGTTCATAACCCAAAGCCCACTAGGATCAAGTCCTTGAGTTCCTTCTCAATGTCAAGGAATGGTAGTTTTGGTTGGACAATTAGTGGCGGTTCTTCATCAAGTCATGAGGGAGACCACAGAGGATCATTACTTGTTGGTACAGAAGTCGTGCATGTTCCATGGTCTGATTTCAAAGATGATGCGGATCATGAAAATAGGGTTGGCTTTCTTGTTGGTGATGATCATCAAGATCGCGATCTAGTCAACGGTTCGGACCTCGAATGTCATTCTCAATTGTTGCCAATGTCTGATCACACTCTAGAGAAGCTCTATGAGGAGTACCTACAACTACTCAAGGCAGAAGATGATGTCCAAGTTCAGTTTGATTCCTTTGCTGAATCTTTGCTGATA
Wherein, ATG and TGA in frame is respectively initiator codon and the terminator codon of CsMYB2 nucleotide sequence.
Above-mentioned purple young tea leaves tree R2R3-MYB gene C sMYB2, its aminoacid sequence is as follows:
MGRAPCCSKVGLHRGPWTAREDALLTKYIQLHGEGNWRSLPKKAGLLRCGKSCRLRWMNYLRPDIKRGNISPDEDDLIIKMHALLGNRWSLIAGRLPGRTDNEIKNYWNTHLSKRLLSQGTDPNTHKKISDPHLKEPKKRRSNSKKQKNKSKSNVVVIEKLKVHNPKPTRIKSLSSFSMSRNGSFGWTISGGSSSSHEGDHRGSLLVGTEVVHVPWSDFKDDADHENRVGFLVGDDHQDRDLVNGSDLECHSQLLPMSDHTLEKLYEEYLQLLKAEDDVQVQFDSFAESLLI。
The application of above-mentioned purple young tea leaves tree R2R3-MYB gene C sMYB2, mainly application in the anthocyanin accumulation improving plant.
The application of above-mentioned purple young tea leaves tree R2R3-MYB gene C sMYB2, mainly application in the anthocyanin accumulation improving transgenic plant.
Described transgenic plant preferably proceed to the plant of CsMYB2 gene, are more preferably the Arabidopis thaliana proceeding to CsMYB2 gene.
Described proceeds to CsMYB2 gene, is connected on pDONR221 plasmid and pB2GW7 carrier preferably by replacement(metathesis)reaction by CsMYB2, obtains recombinant plasmid, by recombinant plasmid transformed Agrobacterium EHA105 bacterial strain competent cell; Complete and proceed to CsMYB2 gene.
Described conversion is preferably freeze-thaw method transform mode.
The present invention has following advantage and effect relative to prior art:
1. the present invention obtains the nucleotide sequence of a kind of tea tree R2R3-MYB gene C sMYB2, and analyzes the constitutional features of its aminoacid sequence and species comparison.
2. the present invention analyzes CsMYB2 from the amino acid structure of CsMYB2 and may have the biosynthetic function of regulation and control purple young tea leaves tree bud-leaf anthocyanin, then confirms that CsMYB2 has the biosynthetic function of regulation and control anthocyanin by arabidopsis thaliana transformation.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of CsMYB2 cloning procedure; Wherein, M, DNA Marker (stripe size is respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); In A, 1 swimming lane is 3 ' the RACE fragment of CsMYB2; In B, 2 swimming lanes are 5 ' the RACE fragment of CsMYB2; In C, 3,4 swimming lanes are respectively ORF and the CsMYB2 full length fragment of CsMYB2.
Fig. 2 is the structural analysis figure of the aminoacid sequence that CsMYB2 derives, wherein, sequence gene title used in figure and GenBank accession number are: DkMYB4 (AB503701), TcTT2_like_MYB (ADD51352), VvMYBA1 (BAD18977), Vbputative MYB transcription factor (AFG28178), VcR2R3_MYB (AEV21970), AtTT2 (AT5G35550), AtMYB111 (NP_199744), LcMYB1, AtPAP2 (AAG42002), MdMYB10 (ABB84753), PmMBF1 (AAA82943), VvMybPA1 (CAJ90831), VvMYBPA2 (EU919682), VvMYBA2 (AB097924), ZmMYB31 (AM156906).
Fig. 3 is the evolutionary analysis of the aminoacid sequence of CsMYB2 and the MYB albumen of other plant.
Fig. 4 is the expression analysis of CsMYB2 between black tea young tea leaves tree development degree blade (red-violet colour leaf/greenery) and between purple bud tea tree breed and green bud kind cloud large pale green.
Fig. 5 is the result figure of overexpression CsMYB2 in wildtype Arabidopsis thaliana (Col-0); Transgenic arabidopsis blade accumulation anthocyanin is shown in figure.
Fig. 6 is transfer-gen plant and the comparing of WT lines anthocyanidin content.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
1. black tea young tea leaves tree is chosen
Purple bud tea leaf used of the present invention is taken from Guangdong Academy of Agricultural Sciences and is drunk purple bud tea tree breed in Institute of Zoology " Tea Germplasm garden, Guangdong Province ".The sample adopted uses liquid nitrogen flash freezer rapidly, takes back laboratory frozen in-80 DEG C of refrigerators, for determination of anthocyanin content, gene clone and gene expression analysis.
2. the isolation and determination of purple young tea leaves tree bud-leaf RNA
The extraction of purple young tea leaves tree bud-leaf RNA adopts RNA to extract test kit (Aidlab EASYspin Plus), and concrete grammar is with reference to specification sheets.With DNase I (QIAGEN RNeasy Mini kit) DNA digestion, concrete grammar is with reference to specification sheets.The concentration determination of RNA and purity check: get 1.0 μ l RNA solution dilution to 100.0 μ L, measure the absorbance value of 260,280 and 320nm, and calculate 260nm/280nm ratio with nucleic acid-protein detector.When the ratio of OD260/OD280 is between 1.80 ~ 2.00 time, RNA sample can be used for next step test.Total serum IgE sample concentration is by following formulae discovery: RNA concentration=OD260*40ng μ L-1*100.For evaluating the integrity of total serum IgE further, get the agarose gel electrophoresis detection that 2.0 μ L total serum IgE carry out 1.5%.
The synthesis of 3.cDNA first chain and original template homogenization
Utilize SMARTTM RACE cDNA Amplification Kit (Clontech) to synthesize cDNA to clone for RACE fragment.2.0 μ g total serum IgE all got by each sample for expressing, and utilize MLV-ReverseTranscriptase Kit (Invitrogen company) to synthesize cDNA first chain, concrete operations are all undertaken by test kit specification sheets.
4. design of primers
According to the fundamental principle of design of primers, design primer with DNAMAN, entrust BGI Technology Solutions Co., Ltd.'s synthesis.RACE fragment, total length and Rea-time pcr amplification the primer are in table 1.
Table 1 RACE fragment, total length and Real-time PCR primer
5.CsMYB1 the separation of gene and sequential analysis:
Based on est sequence according to the transcript profile of high-throughput Solexa order-checking acquisition, the est sequence that CsMYB2 gene pairs is answered is analyzed through ncbi database BLAST, turns out to be the homolgous molecule of myb gene, therefore called after CsMYB2.Then devise RCAE primer according to fragment, through amplification, obtaining 3 ＇ RACE fragments is about 490bp, and 5 ＇ RACE fragment 900bp, RACE step is see the SmartRACE specification sheets of Clontech company.Order-checking 5 ' RACE sequence of gained and 3 ' RACE sequence are carried out splicing to design primer and obtain total length and be respectively 1280bp, CDS encoding sequence 900bp, 292 amino acid of encoding.Nucleotide sequence and the aminoacid sequence of the ORF frame of described CsMYB2 are shown in as follows.Concrete cloning procedure electrophorogram is as Fig. 1.As shown in Figure 2, CsMYB2 full length nucleotide sequence is translated, containing R2 and R3 two incomplete tumor-necrosis factor glycoproteinss in the aminoacid sequence obtained; Structural domain [D/E] Lx2 [R/K] x3Lx6Lx3R of an allusion quotation, is called as bHLH Motif, it be can with the interactional region of bHLH mono-proteinoid (Zimmermann et al.2004); Above result shows that CsMYB2 is a member of R2R3-MYB gene family.
DNAman software building is utilized to comprise 16 regulation and control pycnogenolss of CsMYB2 and the systematic evolution tree of the biosynthetic R2R3-MYB transcription factor of anthocyanin, as shown in Figure 3.CsMYB2 with confirm that the biosynthetic transcription factor homolog of proanthocyanidin glycosides is higher, as persimmon (DkMYB4) (Akagi et al.2009), high clump cowberry (VcR2R3MYB transcription factor) (Zifkin etal.2011), cocoa (TcTT2like MYB transcription factor) (Liu et al.2009) etc.
The expression analysis of 6.CsMYB2 in purple young tea leaves tree
The expression analysis of CsMYB2 in purple young tea leaves tree adopts Real-Time PCR reaction, adopts
480II quantitative real time PCR Instrument (Roche) and
premixEx TaqTM (Perfect Real Time, Takara) test kit carries out.Concrete steps are with reference to the operational manual of instrument and the operation instruction of test kit.Prepare PCR reaction solution on ice, reaction volume is 20.0 μ L; Reaction conditions is as follows: 50 DEG C, 60s; 95 DEG C, 60s, 95 DEG C, 15s, 56 DEG C, 20s, 72 DEG C, 35s, 40 circulations; Carry out 55-95 DEG C of melting curve analysis afterwards.3 repetitions established by each sample.By the specificity of melting curve qualification product after reaction terminates.Reference gene selected by experiment is with reference to the ACTIN (HQ615689) reporting good stability.Gene relative expression component analysis uses 2
-△ △ CTmethod carries out data processing (Livak andSchmittgen2001), calculates the expression level of CsMYB2.All experiments all establish 3 repetitions, experiment ddH above
2o is negative control.
CsMYB2 sets the result of expression between same plant different development degree blade (red-violet colour leaf/greenery) as Fig. 4 at purple young tea leaves.Result shows, the expression level of CsMYB2 in the heliotrope new shoot of anthocyanin accumulation is significantly higher than greenery.This illustrates that the expression of CsMYB2 has specificity.
The Function Identification of 7.CsMYB2 gene
In order to verify the function of CsMYB2 gene, the Overexpression vector of CsMYB2 through Gateway technique construction.By BP and LR two-step reaction, goal gene can be cloned into object expression vector.First, according to the full length cDNA sequence of gene C sMYB2, design Auele Specific Primer CsMYB2 (attB) N and CsMYB2 (attB) C adding applicable recombination site (attB) for a pair, with the cDNA of purple young tea leaves tree red-violet colour leaf for template, amplify the ORF fragment with recombination site (attB), provide according to Invitrogen.
bP Clonase
tMiI Enzyme Mix with
lR Clonase
tMiI Enzyme Mix test kit carries out BP and LR reaction, refers to specification sheets.
BP reaction utilizes between a DNA fragmentation with two attB sites and a donor vehicle pDONOR221 with two attP sites to carry out recombining reaction, create an entry clones, concrete grammar is: with the 30%PEG 8000/30mM MgCl in test kit
2solution obtains carrying out purifying with the PCR primer of recombination site (attB) goal gene to amplification, recombining reaction (BP reacts) is at room temperature carried out again with the donor vehicle pDONR221 with attP site, the connection product obtained is the entry clones of this gene, produces two attL sites.Then this is connected product and proceed to bacillus coli DH 5 alpha, the resistance culture base of kantlex screens, picking transforms mono-clonal bacterial strain, carry out bacterium colony PCR qualification, bacterium amplification is shaken to being accredited as positive bacterium colony, extract plasmid and deliver to order-checking, upgrading grain, LR recombining reaction 4h is carried out with the object expression vector pB2WG7 with two attR sites under room temperature, CsMYB2 is connected on pB2WG7 carrier, obtain the plant expression vector of the CsMYB2 gene of the 35S promoter driving built, called after pB2WG7-CsMYB2, by freeze-thaw method by its transformation Agrobacterium EHA105 bacterial strain competent cell.Infect through Agrobacterium, Dual culture, screening and culturing and take root screening after, obtain turning CsMYB2 gene resistance Arabidopsis plant.The accumulation of the anthocyanidin in transgenic arabidopsis is shown in Fig. 5 and Fig. 6.The anthocyanidin content of result display transgenic arabidopsis is obviously high than contrast.The structure primer of plant expression vector is as shown in table 2.
Table 2 plant expression vector construction primer
MYB is the important transcription factor family of regulating plant secondary metabolism, wherein relevant to cyanidin(e) metabolism mainly R2R3-MYB mono-class.By the method that RT-PCR and RACE combines, we are separated to a R2R3-MYB gene from purple bud tea leaf, called after CsMYB2.The R2R3MYB aminoacid sequence of CsMYB2 and other species has very high homology, and all has R2R3-DNA binding domains at N end, therefore infers that CsMYB2 gene may participate in the synthesis of purple young tea leaves tree anthocyanidin.The expression that CsMYB2 sets in different development degree blade at purple young tea leaves all has obvious difference.CsMYB2 does not almost express not accumulating the mature leaf of anthocyanin (greenery), and the level expressed in purpureal bud-leaf higher (see Fig. 4).In Arabidopis thaliana, overexpression CsMYB2 can impel Arabidopsis leaf to accumulate anthocyanin, specify that CsMYB2 regulates and controls the function of purple young tea leaves tree bud-leaf anthocyanin further.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. a purple young tea leaves tree R2R3-MYB gene C sMYB2, is characterized in that: concrete nucleotide sequence is as shown in SED NO ID:1.
2. purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 1, is characterized in that: the aminoacid sequence of CsMYB2 is as shown in SED NO ID:2.
3. the application of the purple young tea leaves tree R2R3-MYB gene C sMYB2 described in claim 1 or 2, is characterized in that: described purple young tea leaves tree R2R3-MYB gene C sMYB2 application in the anthocyanin accumulation improving plant.
4. the application of purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 3, is characterized in that: described purple young tea leaves tree R2R3-MYB gene C sMYB2 application in the anthocyanin accumulation improving transgenic plant.
5. the application of purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 4, is characterized in that: described transgenic plant are the plant proceeding to CsMYB2 gene.
6. the application of purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 4, is characterized in that: described transgenic plant are the Arabidopis thaliana proceeding to CsMYB2 gene.
7. the application of purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 6, it is characterized in that: described proceeds to CsMYB2 gene for be connected on pDONR221 plasmid and pB2GW7 carrier by replacement(metathesis)reaction by CsMYB2, obtain recombinant plasmid, by recombinant plasmid transformed Agrobacterium EHA105 bacterial strain competent cell; Complete and proceed to CsMYB2 gene.
8. the application of purple young tea leaves tree R2R3-MYB gene C sMYB2 according to claim 7, is characterized in that: described is converted into freeze-thaw method transform mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510419264.5A CN104962565B (en) | 2015-07-16 | 2015-07-16 | A kind of purple young tea leaves tree R2R3 MYB gene Cs sMYB2 and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510419264.5A CN104962565B (en) | 2015-07-16 | 2015-07-16 | A kind of purple young tea leaves tree R2R3 MYB gene Cs sMYB2 and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104962565A true CN104962565A (en) | 2015-10-07 |
| CN104962565B CN104962565B (en) | 2017-10-17 |
Family
ID=54216655
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510419264.5A Active CN104962565B (en) | 2015-07-16 | 2015-07-16 | A kind of purple young tea leaves tree R2R3 MYB gene Cs sMYB2 and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962565B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105936898A (en) * | 2016-07-20 | 2016-09-14 | 中国农业科学院茶叶研究所 | Tea tree violet bud related protein CsGST and encoding gene and application thereof |
| CN106480063A (en) * | 2016-08-22 | 2017-03-08 | 华南农业大学 | A kind of tea tree myb transcription factor CsAN1 and its application in regulation and control anthocyanidin metabolism |
| CN109362548A (en) * | 2018-11-05 | 2019-02-22 | 无锡市茶叶品种研究所有限公司 | The method of comprehensive induction tea tree bud-leaf purple |
| CN110106275A (en) * | 2019-05-09 | 2019-08-09 | 中国农业科学院茶叶研究所 | A kind of InDel molecular labeling of tealeaves purple bud close linkage and its application |
| CN110669866A (en) * | 2019-11-14 | 2020-01-10 | 安徽农业大学 | InDel marker for identifying purple tea tree varieties and combination and application thereof |
| CN117604028A (en) * | 2023-08-09 | 2024-02-27 | 贵州大学 | Tea tree MYB transcription factor gene CsMYB4 and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009061214A1 (en) * | 2007-11-05 | 2009-05-14 | The New Zealand Institute For Plant And Food Research Limited | Compositions and methods for modulating pigment production in plants |
| CN101580543A (en) * | 2008-11-06 | 2009-11-18 | 北京农学院 | Transcription factor, namely MYB encoding genes synthesized by plant regulatory anthocyanin |
| CN103131715A (en) * | 2013-02-27 | 2013-06-05 | 中国科学院武汉植物园 | Plant flavonoid synthesis regulation gene and its application |
| CN103725693A (en) * | 2014-01-09 | 2014-04-16 | 华南农业大学 | Litchi R2R3-MYB gene LcMYB1 and application thereof |
-
2015
- 2015-07-16 CN CN201510419264.5A patent/CN104962565B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009061214A1 (en) * | 2007-11-05 | 2009-05-14 | The New Zealand Institute For Plant And Food Research Limited | Compositions and methods for modulating pigment production in plants |
| CN101580543A (en) * | 2008-11-06 | 2009-11-18 | 北京农学院 | Transcription factor, namely MYB encoding genes synthesized by plant regulatory anthocyanin |
| CN103131715A (en) * | 2013-02-27 | 2013-06-05 | 中国科学院武汉植物园 | Plant flavonoid synthesis regulation gene and its application |
| CN103725693A (en) * | 2014-01-09 | 2014-04-16 | 华南农业大学 | Litchi R2R3-MYB gene LcMYB1 and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| NCBI: "GenBank Accession No.AB503701.1", 《NCBI GENBANK》 * |
| NCBI: "GenBank Accession No.GU324346.1", 《NCBI GENBANK》 * |
| NCBI: "GenBank Accession No.JQ085966.1", 《NCBI GENBANK》 * |
| 王弘雪: "与茶树类黄酮合成相关的MYB转录因子的克隆及表达研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 贡年娣等: "茶树2个MYB转录因子基因的克隆及功能验证", 《茶叶科学》 * |
| 马春雷等: "茶树2个MYB转录因子基因的克隆及表达分析", 《林业科学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105936898A (en) * | 2016-07-20 | 2016-09-14 | 中国农业科学院茶叶研究所 | Tea tree violet bud related protein CsGST and encoding gene and application thereof |
| CN105936898B (en) * | 2016-07-20 | 2019-06-14 | 中国农业科学院茶叶研究所 | A kind of tea tree purple bud GAP-associated protein GAP CsGST and its encoding gene and application |
| CN106480063A (en) * | 2016-08-22 | 2017-03-08 | 华南农业大学 | A kind of tea tree myb transcription factor CsAN1 and its application in regulation and control anthocyanidin metabolism |
| CN109362548A (en) * | 2018-11-05 | 2019-02-22 | 无锡市茶叶品种研究所有限公司 | The method of comprehensive induction tea tree bud-leaf purple |
| CN110106275A (en) * | 2019-05-09 | 2019-08-09 | 中国农业科学院茶叶研究所 | A kind of InDel molecular labeling of tealeaves purple bud close linkage and its application |
| CN110106275B (en) * | 2019-05-09 | 2023-05-09 | 中国农业科学院茶叶研究所 | InDel molecular marker closely linked with tea purple buds and application thereof |
| CN110669866A (en) * | 2019-11-14 | 2020-01-10 | 安徽农业大学 | InDel marker for identifying purple tea tree varieties and combination and application thereof |
| CN110669866B (en) * | 2019-11-14 | 2022-12-27 | 安徽农业大学 | InDel marker for identifying purple tea tree varieties and combination and application thereof |
| CN117604028A (en) * | 2023-08-09 | 2024-02-27 | 贵州大学 | Tea tree MYB transcription factor gene CsMYB4 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104962565B (en) | 2017-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| XIE et al. | The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples | |
| CN104962565A (en) | Purple bud tea tree R2R3-MYB gene CsMYB2 and application thereof | |
| Li et al. | A poplar B‐box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light | |
| Zhang et al. | A newly isolated Na+/H+ antiporter gene, DmNHX1, confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana | |
| CN109161550A (en) | A kind of the SlbHLH59 gene and application method of regulation tamato fruit ascorbic acid content | |
| CN103725693A (en) | Litchi R2R3-MYB gene LcMYB1 and application thereof | |
| Xin et al. | Overexpression of the Ginkgo biloba WD40 gene GbLWD1-like improves salt tolerance in transgenic Populus | |
| Li et al. | Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus | |
| Wu et al. | The endo‐beta mannase MAN7 contributes to cadmium tolerance by modulating root cell wall binding capacity in Arabidopsis thaliana | |
| CN105132433A (en) | cDNA sequence of MYB transcription factors for positively regulating anthocyanin synthesis | |
| CN110964740B (en) | Preparation method and application of tobacco with high flavonol content | |
| Zhang et al. | Heterologous expression of GmSIP1; 3 from soybean in tobacco showed growth retardation and tolerance to hydrogen peroxide | |
| Li et al. | The MdCBF1/2‐MdTST1/2 module regulates sugar accumulation in response to low temperature in apple | |
| CN110452917A (en) | The application of bryony VyGOLS gene and its coding albumen in drought stress | |
| CN110295193A (en) | Application method of the tomato miR6027 gene in control fruit color | |
| Liu et al. | Regulation of Anthocyanin Accumulation by a Transcription Factor LcTT8 From Lonicera caerulea L. | |
| CN115725646A (en) | Application of tomato transcription factor SlMYB1 in regulation and control of tomato fruit quality, gray mold resistance and fruit shape | |
| CN115976039A (en) | Actinidia arguta photoresponse gene AaHY5like9 and application thereof | |
| CN115197951A (en) | Tea tree flavonol synthesis candidate gene CsNAC086 and application thereof | |
| Yue et al. | Physiological and transcriptome analyses reveal that mid-fruit load improves the strength of source and sink in grapevine (Vitis Vinifera L.) | |
| CN107904238A (en) | Thick boisiana high salt, drought-inducible promoter IpLEA PRO and its application | |
| CN110272906B (en) | Tamarix chinensis salt stress response gene TcSBP1, miRNA resistance target rTcSBP1 thereof and application | |
| Li et al. | AcMYB1 interacts with AcbHLH1 to regulate anthocyanin biosynthesis in Aglaonema commutatum | |
| CN103917087A (en) | Method for obtaining a transformed ipomoea batatas plant body including highly accumulated carotenoid and anthocyanin, and plant body obtained by same | |
| KR101711848B1 (en) | Method for producing functional stay-green transgenic plant with improved tolerance to abiotic stress using ONAC106 gene and the plant thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |