CN106480063A - A kind of tea tree myb transcription factor CsAN1 and its application in regulation and control anthocyanidin metabolism - Google Patents

Abstract

The invention particularly discloses a kind of tea tree myb transcription factorCsAN1And its application in regulation and control anthocyanidin metabolism, the tea tree myb transcription factorCsAN1Nucleotide sequence such as SEQ ID NO：Shown in 1.CsAN1PBI121 carrier is connected to after being connected to 35S promoter, using Agrobacterium GV3101 mediated transformation tobacco, while can form the gene of compound by the gene and with whichCsGL3WithCsTTG1Transformation of tobacco, overexpresses plant tobacco leaf and reddens, detect anthocyanidin content by spectrophotometer and significantly improve, detect two kinds of primary pigments of cyanidin and delphinidin by HPLC respectively.

Description

A kind of tea tree myb transcription factorCsAN1And its application in regulation and control anthocyanidin metabolism

Technical field

A kind of the present invention relates to plant genetic engineering field, in particular it relates to tea tree myb transcription factorCsAN1And its Application in regulation and control anthocyanidin metabolism.

Background technology

Tealeaves is one of most important non-alcoholic drink in the world, and which is rich in Tea Polyphenols, theanine, caffeine and multiple types Flavonoid substances etc., these materials impart important effect of tealeaves health care.China is the big producing country of tealeaves, plants tea Leaf is the important sources of a lot of mountain area economies at present, and tealeaves is also the key farm products that China's export is earned foreign exchange.High anthocyanidin content Tealeaves more directly can not only bring multiple health-care efficacy for the person of drinking tea, it is high that its purpleization leaf characteristic possesses which Ornamental value, has large-scale application in sightseeing tea place.The molecular mechanism that tea tree purpleization bud-leaf is formed has important showing Sincere justice and extensive value.

Anthocyanidin is maximum water colo(u)r family in plant kingdom, and a member in flavonoid class family.Know Anthocyanin have kind more than 20, common are 6 kinds, i.e. cyanidin in plant（Cyanidin）, delphinidin （Delphidin）, pelargonidin（Pelargonidin）, paeonidin（Peonidin）, morning glory pigment（Petunidin） And malvidin（Malvidin）.Anthocyanidin is stored in the vacuole of plant, be root, stem, leaf, flower, fruit and seed etc. these Plant tissue shows purple, red and blue main cause（Koes et al., 2005；Tanaka et al., 2008）.Flower Blue or green element has very important effect to the growth metabolism of plant, such as attracts insect pollination and helps seed dispersal etc..In plant In vivo, anthocyanidin acts also as the protection mechanism for responding physiology and environmental change, is conducive to plant to tackle various biological and non-life Thing is coerced（Xu et al., 2015；Zhou et al., 2014）.

As anthocyanidin has multiple BAs, which also plays important angle in terms of human medical's health care Color.Anthocyanidin is a kind of natural antioxidant, human body can be protected from the injury of the harmful substances such as free radical, strengthening blood The aspects such as tubular elastic, prevention cardiovascular and cerebrovascular disease, protection liver play the role of important.Newest epidemiological study tables of data Bright：Anthocyanidin with lasting health-care efficacy and may can reduce the morbidity risk rate of chronic disease（Traka et al., 2011）.Espley（2014）Deng the corn with high anthocyanidin, tomato and apple carry out feeding trial to toy, as a result show： The food of these high anthocyanidin shows beneficial effect to the growth of toy.

Additionally, anthocyanidin is had a wide range of applications in food colour exploitation as the natural harmless pigment of one kind.Mesh Before, in food processing, pigment used mostly is synthetic dyestuff, has different degrees of toxicity, and long-term consumption meeting harmful to human is good for Health.With the raising of people's attention rate safe to food, present in this plant of anthocyanidin, natural colouring matter increasingly causes section Grind the concern in field.Therefore, the food seed selection of high anthocyanidin content becomes the topical subject of botanist's research.

Tea tree is initiated by a kind of important xylophyta of China, and China's major part mountain area Main Cultivation at present One of industrial crops.In production cultivation, purple is one of relatively conventional proterties of tea leaf, except some special tea trees Resource young sprout bud-leaf is throughout the year in outside aubergine（As " purple beautiful "）, some other tea tree breed grown shape in season in spring and autumn The impact of the factors such as state, illumination, temperature, its young tender shoots leaf also occur different degrees of purpleization phenomenon, and further physics and chemistry divides Analysis shows Purple tea shoots, and often anthocyanidin content is higher（Ji Pengzhang etc., 2010；Li Shuanling etc., 2009；Xiao Lizheng etc., 2009）. It is considered that, the height of the anthocyanidin content in tealeaves and tea leaf quality, should not be making containing purpuric bud-leaf in negative correlation Green tea.But the molecular composition, biochemical function, the discovery of medical value with anthocyanidin, the anthocyanidin in tealeaves become one One of individual study hotspot.

Lv etc.（2015）Anthocyanins to " purple beautiful " are measured finding：" purple beautiful " mainly contains 8 kinds of anthocyanidin, its Middle corn flower -3-O- galactolipin（cyanidin-3-O-galactoside）Content highest.The accumulation of high anthocyanidin makes " purple Beautiful " possess strong anti-oxidative activity.Horse spring thunder etc.（2012）Purple bud and green bud are analyzed using biochip technology, screening To 43 difference expression genes, these genes are primarily involved in energetic supersession, secondary metabolism and transcriptional control etc.；And clone 2 Individual tea tree transcription factor gene CsMYB1（Accession number：HQ660373）And CsMYB2（Accession number：HQ660374）Full length gene, Found with quantitative fluorescent PCR detection：CsMYB2 is more than 100 times of root in the expression of blade, and shading treatment significantly improves tea The content of anthocyanidin is set, at this moment the expression of CsMYB1 also increases.Chen Linbo etc.（2012）Using cDNA-AFLP technology to " purple Beautiful " gene expression of young leaflet tablet and mature leaf is analyzed, and screens 59 difference expression genes, in young tender purple portion Position up-regulated expression have 26 fragments, 33 are lowered expression, and these genes mainly have metabolism GAP-associated protein GAP, transcription factor, letter Number albumen and some assume albumen.Recently, Zhou Qiongqiong etc.（2015）Research finds：In tea tree, 9 anthocyanidin route of synthesis are crucial Enzyme gene PAL, C4H, CHS, CHI, F3H, F3'H, F3'5'H, DFR and ANS are in all up-regulated expression in the tender purple leaf of children.Ultraviolet The environmental condition of light, low temperature and nitrogen stress is conducive to the accumulation of tea tree anthocyanidin, and this is mainly closed by inducing anthocyanidin synthesis The expression of bond structure gene, so as to affect the accumulation of anthocyanidin（Li Zhi, 2014）.At present, to " purple beautiful " tea tree germ, the second leaf, Open face leaf, the sequencing of the transcript profile in four periods of climax leaves also to have completed, this is by for being the heredity of research tea tree purple gene Breeding lays the foundation（Chen Linbo etc., 2015）.

Although the studies above has carried out Primary Study, tea tree anthocyanidin metabolism way to the metabolism research of tea tree anthocyanidin The mechanism of action of the regulatory mechanism of footpath transcriptional level and anthocyanidin synthesis associated transcription factor is still not clear, and awaits grinding further Study carefully.

Content of the invention

In order to overcome the disadvantages mentioned above of prior art and deficiency, the primary and foremost purpose of the present invention is to provide a kind of tea tree MYB Transcription factorCsAN1Gene.

Further object is that providing above-mentioned tea tree myb transcription factorCsAN1Albumen.

Further object is that provide a pair to be used for expanding tea tree myb transcription factorCsAN1Primer.

Further object is that providing above-mentioned tea tree myb transcription factorCsAN1Close in regulation and control Anthocyanin Application during becoming.

Further object is that providing a kind of containing above-mentioned tea tree myb transcription factorCsAN1Recombinant expressed Carrier.

Further object is that providing a kind of containing above-mentioned tea tree myb transcription factorCsAN1Transgenosis plant Strain.

Above-mentioned purpose of the present invention is achieved through the following technical solutions.

A kind of tea tree myb transcription factorCsAN1, its nucleotide sequence such as SEQ ID NO：Shown in 1, maximum ORFs （Code area）For 765bp.

Above-mentioned tea tree myb transcription factorCsAN1, its amino acid sequence such as SEQ ID NO：Shown in 2.The sequence has 254 Amino acid, the typical structural characteristics with R2R3MYB transcription factor, and it is distinctive to possess regulation and control anthocyanidin synthesis transcription factor KPRPR [S/T] F domain（See Fig. 1）.

It is used for for a pair expanding tea tree myb transcription factorCsAN1Primer, including upstream and downstream primer, its nucleotide sequence divides Not as SEQ ID NO：3 and SEQ ID NO：Shown in 4.

This three albuminoid of bHLH, MYB, WD40, is the major transcription regulatory factor for participating in anthocyanin synthesis.They pass through The specific bond of DNA sequence dna and the interaction of protein-protein, reach activation or suppression target gene transcriptional expression, so as to adjust The synthesis of control plant anthocyanin.In general, transcription factor can regulate and control multiple anthocyanidin route of synthesis expression of structural gene, from And largely affect the synthesis of anthocyanidin.The related key transcription factor of overexpression anthocyanidin synthesis（Generally MYB family）, Such as：ArabidopsisAtMYB75/PAP1, tomatoSlANT1, appleMdMYB10WithMdMYB110a, cauliflowerBoMYB2 And beetBvMYB1Etc. the content that can significantly improve anthocyanidin.In contrast, if myb gene afunction or Expression is suppressed, it will cause anthocyanidin normally can not accumulate in plant body.

Above-mentioned tea tree MYB transcription becauseCsAN1Application in regulation and control plant anthocyanin building-up process.

A kind of recombinant expression carrier, by SEQ ID NO：Tea tree myb transcription factor shown in 1CsAN1Connect with 35S promoter After connecing, it is connected on expression vector.

A kind of preparation method of transfer-gen plant, by SEQ ID NO：Tea tree myb transcription factor shown in 1CsAN1With 35S After promoter connection, it is connected on expression vector and is containedCsAN1Recombinant vector；By recombinant vector by agriculture bacillus mediated Conversion plant, you can obtain transfer-gen plant.

Preferably, the plant is tobacco.

It is highly preferred that will containCsAN1Recombinant vector by agrobacterium mediation converted plant while, also willCsGL3 WithCsTTG1Gene withCsAN1Plant is converted together.

A kind of preparation method of transgene tobacco, comprises the steps：

（1）By SEQ ID NO：Tea tree myb transcription factor shown in 1CsAN1After being connected with 35S promoter, it is connected to expression and carries Contained on bodyCsAN1Recombinant vector, by recombinant vector convert Agrobacterium competent cell, obtain recombinational agrobacterium, to Isopyknic acetosyringone containing 0.2mmol/L, the MgCl of 10mM is added in recombinational agrobacterium₂Slow with the MES infiltration of 10mM Liquid is rushed, is preparedCsAN1Infect liquid；

（2）According to step（1）Method prepare respectivelyCsGL3WithCsTTG1Infect liquid；

（3）WillCsAN1、CsGL3WithCsTTG1Infect liquid equal-volume mixing after, squeeze into the back side of tobacco leaf, incubated Obtain final product transgene tobacco within 10 days.

Compared with prior art, the present invention has the advantages that：

The present invention obtains a kind of tea tree myb transcription factorCsAN1Nucleotide sequence, and its amino acid sequence is analyzed, It was found thatCsAN1Conserved domain with R2R3-MYB transcription factor, and the binding site with bHLH and regulation and control anthocyanidin conjunction KPRPR [S/T] the F domain for becoming.CsAN1PBI121 carrier is connected to after being connected to 35S promoter, using Agrobacterium GV3101 Mediated transformation tobacco, while can form the gene of compound by the gene and with whichCsGL3WithCsTTG1Difference transformation of tobacco, It is observed that blade starts anthocyanidin occur after 4 days, tobacco leaf reddens, and reaches maximum to the 10th day anthocyanin accumulation content Value.After the anthocyanidin extraction of tobacco leaf, anthocyanidin content is detected by spectrophotometer and is significantly improved, examined by HPLC Measure cyanidin,（cyanidin-3-O-galactoside）And delphinidin（delphinidin-3-O- galactoside）Two kinds of primary pigments.

Description of the drawings

Fig. 1 isCsAN1Amino acid sequence analysis figure.

Fig. 2 isCsAN1The structure schematic diagram of overexpression vector.

Fig. 3 is for converting respectivelyCsAN1、CsAN1+ CsGL3 andCsAN1The tobacco leaf of+CsGL3+CsTTG1 micro- Structure chart.

Fig. 4 is control tobacco leaf, conversionCsAN1、CsAN1+ CsGL3 and conversionCsAN1The super table of+CsGL3+CsTTG1 Reach the anthocyanidin extract figure of tobacco leaf.

Fig. 5 is control tobacco leaf, conversionCsAN1And conversionCsAN1+ CsGL3+CsTTG1 overexpression tobacco leaf Anthocyanidin content figure.

Fig. 6 is control tobacco leaf, conversionCsAN1Overexpression plant tobacco leaf,CsAN1+ CsGL3 and conversionCsAN1The anthocyanidin HPLC figure of+CsGL3+CsTTG1 tobacco leaf.

Fig. 7 is that anthocyanidin route of synthesis key structure gene is compareing and overexpressing the expression situation map in tobacco.

Specific embodiment

The present invention is made with reference to Figure of description and specific embodiment and further elaborating, the embodiment It is served only for the present invention is explained, is not intended to limit the scope of the present invention.Test method used in following embodiments is for example nothing special Different explanation, is conventional method；The material that used, reagent etc., if no special instructions, are the reagent for commercially obtaining And material.

1 tea tree myb transcription factor of embodimentCsAN1Separate and clone

1st, design of primers：Early stage of the present invention carries out transcript profile sequencing to the leaves at the different development stages of tea tree " purple beautiful "（Peace promise is excellent Reach Gene science（Beijing）Co., Ltd）, obtain in transcript profile databaseCsAN1Total length, complete according to known sequence Long design amplificationCsAN1Special primer：

F：5’-TACTTATTCAGACAAACGCACAC-3’；

R：5’-ATTTCAACTCTAATTTCAACCCA-3’；

2nd, the extraction of RNA：Using Hipure Plant RNA Mini Kit（Mei Ji, Guangzhou）Tea tree " purple beautiful " terminal bud is extracted, the Two leaves, the total serum IgE of third and fourth leaf aggregate sample and old leaf, OD260/OD280 and OD260/ is determined with nucleic acid-protein instrument OD230 value, judges quality and the yield of RNA, detects the integrality of RNA using 1.0% agarose gel electrophoresis.

3rd, the synthesis of the first chain of cDNA

The synthesis of cDNA uses PrimeScript 1st Strand cDNA Synthesis Kit（TaKaRa, Dalian）Enter OK, comprise the following steps that：

（1）Following system is prepared in aseptic PCR pipe：

RNA	2 μg
		cDNA synthesis Primer	1 μL
SMARTⅡ Oligonucleotide	1 μL
		Rnase-free H2O	Up to 5 μL

（2）Biased sample, 72 DEG C of 2 min of insulation.

（3）The of short duration sample that is collected by centrifugation keeps test tube in room temperature in ttom of pipe.

（4）Following reagent is added in reaction tube：

5×First-Strand Buff	2 μL
		DTT	1 μL
50×dNTP Mix	1 μL
		Script Reverse Transcriptase	1 μL

（5）Mix, with 42 DEG C of 30 min of insulation of PCR instrument

（6）Add the TE buffer solution of 30 μ L

（7）85 DEG C of test tube of heating, 7 min

（8）It is stored in -20 DEG C of refrigerators standby.

4th, PCR amplification

Reaction condition：95 DEG C of denaturations 5min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 34 circulations；72 DEG C of extensions 5min.The PCR primer that amplification is obtained is connected to PMD19-T carrier（Purchase to TaKaRa）, screening positive clone is simultaneously sequenced, and obtains institute Full length gene is needed, is named asCsAN1.

Sequence analysis finds：CsAN1Maximum ORFs be 765 bases, such as SEQ ID NO:Shown in 1, coding 254 amino acid（As SEQ ID NO:Shown in 2）, conserved domain with R2R3-MYB transcription factor, and with bHLH's Binding site and KPRPR [S/T] the F domain of regulation and control anthocyanidin synthesis（See Fig. 1）.

2 tea tree myb transcription factor of embodimentCsAN1The structure of overexpression vector and conversion

1st, the structure schematic diagram of overexpression vector is as shown in Fig. 2 concretely comprise the following steps：To implement the positive colony that row 1 are obtained PMD19T-CsAN1Carrier is template, designs special primer：pBI121-CsAN1-F:5’- GGACTCTAGAGGATCCATGGACATTGTTTGTTGTGT-3 ', pBI121-CsAN1-R:5’- GACCACCCGGGGATCCTCATCATTCATCACCTAACA-3 ', enters performing PCR amplification.While usingBamHI（TaKaRa）Digestion After pBI121 over-express vector linearizes which, using ClonExpress II One Step Cloning Kit（Nanjing promise Wei Zan bio tech ltd）It is attached.Digestion system：BamH1 μ L+Buffer of I, 1 μ L+pBI121 plasmid, 3 μ L+ H₂O polishing is to 10 μ L.37 DEG C of water-bath digestion 4h are put, goes to inactivation 20min in 85 DEG C of water-baths afterwards.

The linked system of 1 ClonExpress II One Step Cloning Kit of table

ddH2O	Up to 20 μl
		5×CE II Buffer	4 μl
Linearisation cloning vector	50～200 ng
		Insert Fragment amplified production	20～200 ng
Exnase® II	2 μl

After the completion of system is prepared, gently blown and beaten with pipettor up and down and mix each component several times, it is to avoid produce bubble and (acutely please don't shake Swing or be vortexed mixing).It is placed in 37 DEG C of 30 min of reaction.After the completion of question response, reaction tube is placed in ice-water bath cools down immediately 5 min.Afterwards, product directly can be converted；Also -20 DEG C can be stored in, conversion of thawing when needed.

2nd, the competent preparation of Agrobacterium

（1）Picking Agrobacterium GV3101（Agrobacterium tumefaciens）Single bacterium colony, is inoculated in 10 mL YEP culture In base, 28 DEG C of shaken cultivation are overnight；

（2）Take 400 μ L bacterium solution to be placed in 50 mL YEP nutrient solutions, 28 DEG C of 5～6 h to OD600 of shaken cultivation are 0.5；

（3）Bacterium solution is poured in 50 mL centrifuge tubes, after 6000 rpm are centrifuged 5 min, abandons supernatant；

（4）Thalline is resuspended in the NaCl of 10 mL, 0.15 M, 6000 rpm are centrifuged 5 min afterwards, abandon supernatant；

（5）Thalline 20 mM CaCl of l mL precooling₂Solution gently suspends, and adds 15% glycerine of precooling, and 100 μ L divide Dress, -80 DEG C of Refrigerator stores are standby.

3rd, recombinant plasmid transformed Agrobacterium competent cell

（1）The 100 μ L Agrobacterium competent cells for taking -80 DEG C of preservations are put on ice, gently cell are suspended after thawing completely；

（2）5 μ L plasmids are added to place 30 min on ice after mixing；

（3）Centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, 1 min of heat shock in 37 DEG C of water-baths, will go to rapidly on ice, ice bath 2 min；

（4）Add 1 mL YEP fluid nutrient medium, 28 DEG C of 1 h of shaken cultivation；

（5）4000 rpm are centrifuged 5 min, plus 100 μ L YEP nutrient solution suspension cells；

（6）Bacterium solution is coated on the YEP flat board containing corresponding antibiotic, 28 DEG C of 32～48 h of culture；

（7）The Agrobacterium bacterium colony that picking grows shakes 24 h of bacterium for 28 DEG C in YEP fluid nutrient medium, then bacterium colony is detected with PCR, As a result it is positive explanation expression vector establishment success.

4th, prepare and infect liquid

（1）Picking positive monoclonal bacterium colony is in 5 mL YEP nutrient solutions（Containing Kan⁺）, 28^o180 rpm activation culture of C shaking table 18h～24h, until bacterium solution OD value reaches 0.6 or so；

（2）Take step（1）In 1mL bacterium solution be inoculated in the YEP nutrient solution of 50 mL（Containing Kan⁺）, identical condition is in shaking table Expand further and shake, treat that bacterium solution OD value reaches 0.6 or so；

（3）3000 rpm in the centrifuge, are centrifuged 10 min, and collects thalline adds isopyknic permeabilization buffer（Containing 0.2 The acetosyringone of mmol/L, the MgCl of 10 mM₂With 10 mM MES）Resuspension thalline.

5th, transformation of tobacco blade

（1）The program request of Ben's tobacco on the Nutrition Soil of moistening, sprout by overlay film moisturizing to seed, and the Ben's tobacco seedling of sprouting is 20 DEG C illumination box（14h illumination/10h is dark）Can be used to infect after 2 week of middle culture；

（2）Above-mentioned is infected the back side that liquid squeezes into Ben's tobacco leaf with the needleless injector of 1mL；Similarly, willCsEGL3WithCsTTG1Infect liquid respectively withCsAN1Infect the mixing of liquid equal-volume, configurationCsAN1+CsEGL3WithCsAN1+CsEGL3+CsTTG1Liquid is infected, infects Ben's tobacco leaf in the same way, plant is placed in 24 DEG C of insulating boxs trains afterwards Foster 48h；

（3）Start to observe the color change of tobacco leaf after 4 days, the Ben's tobacco leaf after 10 days to have accumulated anthocyanidin takes Sample, is analyzed further to which.

3 tea tree myb transcription factor of embodimentCsAN1The anthocyanidin measurement of overexpression plant

1st, conversion is taken respectivelyCsAN1、CsAN1+ CsGL3 andCsAN1The blade of the tobacco of+CsGL3+CsTTG1, using micro- The microstructure of the various transgenic tobacco leaf of sem observation, is as a result shown in Fig. 3.

2nd, anthocyanidin extraction：Fresh for the blade of 3 g sample is added liquid nitrogen to grind in mortar, is transferred into test tube afterwards In, add the extraction buffer solution of 3 mL（By 18% normal propyl alcohol, 1% hydrochloric acid：81% water is formulated）, 3 min of boiling water bath, room temperature are quiet Put overnight.Leaching results are shown in Fig. 4.

3rd, the measurement of anthocyanidin：Leaching liquor is determined in A with ultraviolet specrophotometer₅₃₅And A₆₅₀Light absorption value, final flower Blue or green cellulose content (A₅₃₅～A₆₅₀) g -1 (FW) expression.Measurement result is shown in Fig. 5.

4th, Anthocyanins are determined：Take 10 μ L（1）Leaching liquor be expelled to XSelect HSS C-18 SB pipe（4.6× 250 mm, 5 μm, water generation Science and Technology Ltd.）, use 5% formic acid（A）With 100% methyl alcohol（B）As mobile phase, in 520 nm Detection anthocyanidin, concrete operations are with reference to the document that delivers（Shan et al. 2009）Testing result such as Fig. 6.Result from Fig. 6 Understand, in transgene tobacco, cyanidin detected,（cyanidin-3-O-galactoside）And delphinidin （delphinidin-3-O-galactoside）Two kinds of primary pigments.

Embodiment 4

In order to further appreciate that tea tree myb transcription factorCsAN1The mechanism of action in anthocyanidin synthesis, the present embodiment have studied Anthocyanidin route of synthesis key structure gene is compareing and is overexpressing the expression situation in tobacco.Concretely comprise the following steps, according to The sequences Design special primer of genes of interest and reference geneActinPrimer（It is shown in Table 2）, using Hipure Plant RNA Mini Kit（Guangzhou U.S. base）Respective sample total serum IgE is extracted, and cDNA is inverted to, and it is anti-qRT-PCR to be carried out with the product as template Should, each reaction sets 3 repetitions, and qRT-PCR reaction system is as follows：0.25 5 μ of μ L, 2 × SYBRGreenMIX of cDNA template L, 10 μm of olL^-10.5 μ L of forward primer, 10 μm of ol L^-10.5 μ L of reverse primer, adds water to cumulative volume and reaches 10 μ L. QRT-PCR program：94℃,10 min；94 DEG C, 10 s, 55 DEG C, 15 s, 72 DEG C, 30 s, 40 circulations.Product is with melting Tracing analysis, and verified with agarose gel electrophoresis.Data are collected using 480 software of LightCylcer of Roche company, no Relative expression quantity with 3 kinds of functional genes of transgene tobacco sample room is with the 2 of CT value^-△△CTMethod is processed, and as a result sees Fig. 7.

Table 2 detects the primer sequence of expression related gene for qRT-PCR

NbF3’H-F	TAAGGCTTCATCCATCCACC
		NbF3’H-R	CAAAGTCATTTCCTCGCACA
NbDFR-F	TTGTTGGTCCATTCCTCACG
		NbDFR-R	CCACGGGCAAGTCCTTATCG
NbLDOX-F	AGTATGCTAATGACCAACCCTC
		NbLDOX-R	AGTCCCAGCCCAATAGAAAG
NbActin-F	AGTCCTCTTCCAGCCATCCA
		NbActin-R	TAGGAGCCAAAGCCGTGATT

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>A kind of tea tree myb transcription factor CsAN1 and its application in regulation and control anthocyanidin metabolism

<130>

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 765

<212> DNA

<213>The ORF of CsAN1

<400> 1

atggacattg tttgttgtgt tccattagga gtgagaaaag gtgcatggac tggagaagaa 60

gatagtttgc taaagaactg cattgagcaa tatggagagg gaatgtggca ccaaattcct 120

tccagagcag gattgaatag atgcagaaaa agttgtagac taagatggct gaattatctg 180

agaccaaaca taaaaagagg caattttacg atggatgaag ttgatctcat tatcaagctt 240

cagaagctgc taggcaatag atggtcgttg attgcgggta gacttccagg aagaacagca 300

aatgatgtaa aaaactattg gaataccaac ttacagaaga aactgatcac ccaaagagaa 360

aaggtgaaag ccaagactca agagaagatg gaaaccataa ttatacgacc tcggcctcga 420

atcttctcca aaaatcaacc acggttaatg gacaaaactg ccattataga aaatattcga 480

acaagagaca accttagcga gccatttcca ctaccgctac cgctaccacc atcgcgggat 540

gatggaatat catgggataa cattgttgtt aactccaaaa ttaacaatgg aatcacatgg 600

tccgcaaatg gcttgattga ggaggccaat atagggaatt gggatgggaa aataagacca 660

ggtacacata catcttgtgg caatttcatt gatgaagacc agagtgattg gagtgacatt 720

ttctttaata atgtgaacct ttgggatctg ttaggtgatg aatga 765

<210> 2

<211> 254

<212> PRT

<213>CsAN1 amino acid sequence

<400> 2

Met Asp Ile Val Cys Cys Val Pro Leu Gly Val Arg Lys Gly Ala Trp

1 5 10 15

Thr Gly Glu Glu Asp Ser Leu Leu Lys Asn Cys Ile Glu Gln Tyr Gly

20 25 30

Glu Gly Met Trp His Gln Ile Pro Ser Arg Ala Gly Leu Asn Arg Cys

35 40 45

Arg Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn Ile

50 55 60

Lys Arg Gly Asn Phe Thr Met Asp Glu Val Asp Leu Ile Ile Lys Leu

65 70 75 80

Gln Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro

85 90 95

Gly Arg Thr Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr Asn Leu Gln

100 105 110

Lys Lys Leu Ile Thr Gln Arg Glu Lys Val Lys Ala Lys Thr Gln Glu

115 120 125

Lys Met Glu Thr Ile Ile Ile Arg Pro Arg Pro Arg Ile Phe Ser Lys

130 135 140

Asn Gln Pro Arg Leu Met Asp Lys Thr Ala Ile Ile Glu Asn Ile Arg

145 150 155 160

Thr Arg Asp Asn Leu Ser Glu Pro Phe Pro Leu Pro Leu Pro Leu Pro

165 170 175

Pro Ser Arg Asp Asp Gly Ile Ser Trp Asp Asn Ile Val Val Asn Ser

180 185 190

Lys Ile Asn Asn Gly Ile Thr Trp Ser Ala Asn Gly Leu Ile Glu Glu

195 200 205

Ala Asn Ile Gly Asn Trp Asp Gly Lys Ile Arg Pro Gly Thr His Thr

210 215 220

Ser Cys Gly Asn Phe Ile Asp Glu Asp Gln Ser Asp Trp Ser Asp Ile

225 230 235 240

Phe Phe Asn Asn Val Asn Leu Trp Asp Leu Leu Gly Asp Glu

245 250

<210> 3

<211> 23

<212> DNA

<213>Forward primer

<400> 3

tacttattca gacaaacgca cac 23

<210> 4

<211> 23

<212> DNA

<213>Reverse primer

<400> 4

atttcaactc taatttcaac cca 23

<210> 5

<211> 36

<212> DNA

<213> pBI121-CsAN1-F

<400> 5

ggactctaga ggatccatgg acattgtttg ttgtgt 36

<210> 6

<211> 36

<212> DNA

<213> pBI121-CsAN1-R

<400> 6

gaccacccgg ggatcctcat cattcatcac ctaaca 36

<210> 7

<211> 20

<212> DNA

<213> NbF3'H-F

<400> 7

taaggcttca tccatccacc 20

<210> 8

<211> 20

<212> DNA

<213> NbF3'H-R

<400> 8

caaagtcatt tcctcgcaca 20

<210> 9

<211> 20

<212> DNA

<213> NbDFR-F

<400> 9

ttgttggtcc attcctcacg 20

<210> 10

<211> 20

<212> DNA

<213> NbDFR-R

<400> 10

ccacgggcaa gtccttatcg 20

<210> 11

<211> 22

<212> DNA

<213> NbLDOX-F

<400> 11

agtatgctaa tgaccaaccc tc 22

<210> 12

<211> 20

<212> DNA

<213> NbLDOX-R

<400> 12

agtcccagcc caatagaaag 20

<210> 13

<211> 20

<212> DNA

<213> NbActin-F

<400> 13

agtcctcttc cagccatcca 20

<210> 14

<211> 20

<212> DNA

<213> NbActin-R

<400> 14

taggagccaa agccgtgatt 20

Claims (9)

1. a kind of tea tree myb transcription factorCsAN1Gene, it is characterised in that its nucleotide sequence such as SEQ ID NO：Shown in 1.

2. a kind of tea tree myb transcription factorCsAN1Albumen, it is characterised in that its amino acid sequence such as SEQ ID NO：Shown in 2.

3. it is used for for a pair expanding tea tree myb transcription factorCsAN1Primer, it is characterised in that including upstream and downstream primer, its nucleosides Acid sequence is respectively as SEQ ID NO：3 and SEQ ID NO：Shown in 4.

4. tea tree myb transcription factor described in claim 1CsAN1Application in regulation and control Anthocyanin synthesis.

5. a kind of recombinant expression carrier, it is characterised in that by the tea tree myb transcription factor described in claim 1CsAN1With 35S After promoter connection, it is connected on expression vector.

6. a kind of preparation method of transfer-gen plant, it is characterised in that by the tea tree myb transcription factor described in claim 1CsAN1After being connected with 35S promoter, it is connected on expression vector and is containedCsAN1Recombinant vector；Recombinant vector is passed through Agrobacterium mediation converted plant, you can obtain transfer-gen plant.

7. the preparation method of transfer-gen plant according to claim 6, it is characterised in that the plant is tobacco.

8. the preparation method of the transfer-gen plant according to claim 6 or 7, it is characterised in that will containCsAN1Restructuring While carrier is by agrobacterium mediation converted plant, also willCsGL3WithCsTTG1Gene withCsAN1Plant is converted together.

9. a kind of preparation method of transgene tobacco, it is characterised in that comprise the steps：

（1）By the tea tree myb transcription factor described in claim 1CsAN1After being connected with 35S promoter, expression vector is connected to On containedCsAN1Recombinant vector, by recombinant vector convert Agrobacterium competent cell, obtain recombinational agrobacterium, Xiang Chong Isopyknic acetosyringone containing 0.2mmol/L, the MgCl of 10mM are added in group Agrobacterium₂MES infiltration buffering with 10mM Liquid, preparesCsAN1Infect liquid；

（2）According to step（1）Method prepare respectivelyCsGL3WithCsTTG1Infect liquid；

（3）WillCsAN1、CsGL3WithCsTTG1Infect liquid equal-volume mixing after, squeeze into the back side of tobacco leaf, incubated Obtain final product transgene tobacco within 10 days.