CN107686840B - Pears transcription factor PyERF3 and its recombinant expression carrier and application - Google Patents

Abstract

The invention discloses pears transcription factor PyERF3 and its recombinant expression carrier and applications.One kind is isolated from that ' red eggplant ' pears have the transcription factor PyERF3 gene for promoting the operatic circle skin anthocyanin biological synthetic functional, and nucleotides sequence is classified as shown in SEQ ID No.1, and the amino acid sequence of coding is shown in sequence table SEQ ID No.2.By Agrobacterium tumefaciens-mediated Transformation method by transcription factor instantaneous conversion tobacco, strawberry and pear fruit, through biological function verification, show the function of the biosynthesis for the PyERF3 gene and others transcription factor PyMYB114 and PybHLH3 formation transcription control complex promotion the operatic circle skin anthocyanin that the present invention clones.The discovery of PyERF3 gene provides new genetic resources to promote the molecular breeding of the operatic circle skin anthocyanin synthesis to provide new genetic resources to implement green agriculture, the development and utilization of the genetic resources advantageously reduce agricultural cost and realize environmental-friendly.

Description

Pears transcription factor PyERF3 and its recombinant expression carrier and application

Technical field

The invention belongs to plant genetic engineering fields, are related to pears transcription factor PyERF3 and its recombinant expression carrier and answer With, and in particular to from ' separation, clone obtain the relevant AP2/ERF of a participation the operatic circle skin anthocyanin biosynthesis in red eggplant ' pears Family member PyERF3 gene and its application.

Background technique

Anthocyanin is the Plant Secondary Metabolites of biosynthesis in flavonoids metabolic pathway, it is a kind of water colo(u)r. It is widely present in plant kingdom, stem, leaf, flower and fruit is made to show chromatic colour.Anthocyanin in spending, which mainly attracts, awards Powder person, and the synthesis of the anthocyanin in seed and fruit attracts the feeding of birds and animal, is more conducive to seed dis persal (Holton et al.,1995).Anthocyanin also plays important role to disease resistance of plant, for example is resisting ultraviolet light, antioxidant activity Aspect effect (Bieza et al., 2001；Veeriah et al., 2006), there are also the works of various wholesome aspects With, such as provide nervous system protection and cardiovascular disease, cancer and diabetes etc. (Konczak et al., 2004； Butelli et al.,2008).Therefore, anthocyanin is important sanatory natural pigment (Camire et Al.2002), the significant contribution to fruit quality is made it.

The formation of anthocyanin is completed under a series of catalytic action of enzymes, it is flavonoids biosynthetic metabolism approach A branch, initial reaction be by phenylalanine lyases (PAL) catalysis cinnamic acid reaction, the main pass successively passed through Key catalyzing enzyme phenylalanine lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H), the successive reaction and during generating anthocyanin occurred, including dihydroflavonol 4-reductase (DFR), anthocyanin Synzyme (ANS) and UDP-glucose: flavonoids 3-O- glucosyltransferase (UFGT) ultimately forms anthocyanin (Takos et al., 2006).In addition, grinding in terms of the biosynthesis of the adjusted and controlled gene of transcription factor expressed and then cause anthocyanin Study carefully have in model plant relevant report (Zhang et al., 2003；Rowan et al.,2009).

In garden crop, the molecular mechanism that anthocyanin accumulates in fruit is also widely studied.R2R3-MYB transcription The factor has important regulating and controlling effect in anthocyanin biosynthetic process.Such as apple (Malus × domestica), MdMYB10 and MdMYB110a is cloned from red pulp apple ' Red Field ' and ' Sangrado ' respectively, they are demonstrate,proved Bright positive regulation type I/II apple pericarp color (Espley et al., 2007；Chagné et al.,2013).Also The transcription factor found in two apples, MdMYB1 and MdMYBA are to be promoted anthocyanin biosynthesis by photoinduction in apple Vital regulator (Takos et al., 2006；Ban et al.,2007；Li et al.,2012).Strawberry (Fragaria × ananassa), FaMYB10 and FaMYB1 (Fragaria × ananassa) be proved to it is positive respectively and The biosynthesis of negative regulation anthocyanin (Medina-Puche et al., 2014；Aharoni et al.,2001).Therefore, MYB Albumen is considered as the key compound for distributing expression of specific gene mode, and bHLH albumen, regulatory complex it is other at Member may have the adjusting target (Akagi et al., 2009) of overlapping.In fruit, the bHLH of flavonoids biosynthesis is participated in Albumen in grape, apple and strawberry it is identified (Hichri et al., 2010；Matus et al.,2008；Xie et al.,2012；Schaart et al.,2013).In grape, bHLH transcription factor VvMYC1 is shown and different MYB eggs White (Vv MYB5a, VvMYB5b, VvMYBA1/A2, VvMYBPA1) interaction participates in anthocyanin to induce in flavonoid path With the promoter of the gene of former anthocyanin biosynthesis (Hichri et al., 2010).WD40 albumen participates in anthocyanin biology Synthesis regulation complex is reported in 1 (TTG1) gene loci of arabidopsis TRANSPARENT TES-TA GLABRA first (Walker et al., 1999), its homologue be also accredited include apple (Brueggemann et al., 2010), grape (Kobayashi et al., 2004) pomegranate (Ben-Simhon et al., 2011) and strawberry (Schaart Et al., 2013) garden crops such as.In addition to this, anthocyanin biosynthesis can be activated there are also other transcription factors.In quasi- south In mustard, WRKY (Johnson et al., 2002), WIP (Sagasser et al., 2002), MADS domain (Nesi et al., 2002),PIF3(Shin et al.,2007),NAC(Morishita et al.,2009),CPC(Zhu et al., 2009),Ethylene (Jeong et al.,2010),phytochrome-interacting ankyrin repeat protein 2(PIA2)(Yoo et al.,2011),SPL9 (Gou et al.,2011),HY5(Shin et al., 2013), JASMONATE ZIM-domain (JAZ) (Qi et al., 2011), COP1 (Maier et al., 2013) and Report influences the biosynthesis of anthocyanin to MYB112 (Lotkowska et al., 2015) successively.These transcription factors can pass through Be integrated to MBW compound promote or inhibit anthocyanin biosynthesis (Li et al., 2013；Viola et al., 2016)；For example, TCP3 and TCP15 can be turned respectively by being integrated to miR156 targeting transcription factor SPL9 by interference MBW It records complex and forms negative regulation anthocyanin accumulation (Gou et al., 2011).Jia et al. (2015) report, miRNA858 Negative regulation anthocyanin biosynthesis passes through the expression of two transcription factor R2R3MYB transcription factors.Similarly, Sawano et Al. it is by regulating and controlling PAP1 that (2017), which report anthocyanin biosynthesis in dsRNA binding protein DRB3 negative regulation arabidopsis, Expression.In addition, it is by completely cutting off MYB that Xie et al. (2016) report DELLA albumen, which can promote the biosynthesis of anthocyanin, The expression of/bHLH/WD40 complex and inhibitor MYBL2 and JAZ.Equally, Wang et al. (2016) is reported, MiRNA858a and HY5 leads to the activation of anthocyanin biosynthesis pathway in arabidopsis to the inhibition expression of MYBL2.Therefore, cyanine Glycosides biosynthesis is formed the control of regulated and control network by MBW compound or other transcription factors.In fruit, it is related to fruit maturation tune Control some key genes be accredited, it is many be SEPALLATA- and SQUAMOSA-class MADS box or SBP box transcription factor (Seymour et al., 2012；Karppinen et al.,2013；Seymour et al., 2011).This regulated and control network seem be in different fruit trees it is conservative (Seymour et al., 2008；Seymour et al.,2011).Although nearest research has been discovered that effect of some transcription factors in the adjusting of flavonoids biosynthesis, But the connection between the developmental regulation factor and the downstream effector for participating in flavonoids biosynthesis is not yet unlocked.Mature related flower The SQUAMOSA-class MADS box transcription factor VmTDR4 of green glycosides biosynthesis is FRUITFULL in arabidopsis (FUL) base The connection between TDR4 in the homologous gene and tomato of cause is reported (Jaakola et al., 2010) in cowberry. Anthocyanin accumulation in the spatial and temporal expression and cranberry of VmTDR is consistent, and the silencing of VmTDR4 causes anthocyanin biology to close At significant reduction.The expression of SEPALLATA-class MADS box transcription factor PyMADS18 and the cyanine of red skin Pear varieties Glycosides accumulates related (Wu et al., 2013b).Zhou et al. (2015) report, the molecular genetic mechanism of red meat peach is to pass through The PpNAC1 transcriptional activation anthocyanin biosynthesis of QTL positioning identification.In pervious report, it is believed that AP2/ERFs is to participate in apple Fruit and plum fruit Mature Regulation (Whale et al., 2007；Manganaris et al.,2008).Then, Licausi et al. (2013) report, AP2/ERF transcription factor participate in nascent and secondary metabolites, growth and development Process.The research that AP2/ERF transcription factor participates in aspect in anthocyanin biosynthesis is also rarely reported.

Pears (Pyrus) have plantation in the world, are broadly divided into two types: Asian pear and European pears.Asian pear has The red skin pears main cause of seldom high quality is that its coloring is unstable.In contrast, more red skin Pear varieties are Europe Pears (P. communis).For most of east pears, the product of anthocyanin only can be shown connecing maturescent period Tired (Yang et al, 2014)；And the coloring of European pears is early stage fruit development, the fruit of coloring can continue until the maturity period. In European pears, Dondini et al. (2008) report, ' Max Red Bartlett ' Red color trait navigates to linkage group 4 (LG4).Then, Pierantoni et al. (2010) is reported, although PyMYB10 that Identification arrives and apple MdMYB10 very high homology, and MdMYB10 control apple pericarp color, but ' Max Red Bartlett ' and In ' Williams ' Pear varieties, it is not the gene of direct transcriptional control red skin and yellow rind character, positioning pears genome Different regions.Wu et al. (2013b) also reports, the red green fruit of European pear ' early red Kao Misi ' and other green bud mutations The mutant in color of the leather pool, anthocyanin synthesis in the operatic circle skin may be taken part in by identifying MADS transcription factor.However, MYB10 also has Report regulation anthocyanin biosynthesis some Pear varieties (Feng et al., 2010；Zhang et al.,2011；Yu et al.,2012).' formation of the green bud mutation of Max Red Bartlett ' is related for the methylation level of MYB10 promoter region (Wang et al.,2012).Anthocyanin biosynthesis gene MYB10 does not show related to the expression of bHLH to WD40 Property, propose a kind of regulatory mechanism (Yang et al., 2014) for regulating and controlling anthocyanin biosynthesis in pears.

Summary of the invention

The object of the present invention is to provide a kind of transcription factor PyERF3 genes for promoting anthocyanin biosynthesis.

It is a further object of the present invention to provide the applications of the gene.

The purpose of the present invention can be achieved through the following technical solutions:

One kind be isolated from ' red eggplant ' pears have promote anthocyanin biological synthetic functional transcription factor PyERF3 gene, belong to AP2/ERF family member, nucleotides sequence are classified as shown in SEQ ID No.1, the open reading frame comprising 1200bp；Coding 399 A amino acid, the amino acid sequence of coding are isoelectric point 6.60 shown in sequence table SEQ ID No.2, molecular weight 43.18 kDa。

Recombinant expression carrier containing PyERF3 gene of the present invention.

The recombinant expression carrier is preferably the carrier that sets out with pSAK277, and the insertion point of the PyERF3 gene is Between EcoR I and Xhol I.

Host strain containing PyERF3 gene of the present invention.

Clone the primer pair of PyERF3 gene cDNA sequence of the present invention, upstream primer PyERF3-F1 sequence such as SEQ Shown in ID No.3, downstream primer PyERF3-R1 sequence is as shown in SEQ ID No.4.

PyERF3 gene of the present invention is in the application for promoting the anthocyanin biosynthesis of the operatic circle skin.

Recombinant expression carrier of the present invention promotes the application of the operatic circle skin anthocyanin biosynthesis.

Beneficial effect

Compared with prior art, the present invention has the following advantages and effects:

The discovery of 1.PyERF3 gene, to promote the molecular breeding of biosynthesis of the operatic circle skin anthocyanin to provide new gene Resource provides new genetic resources to implement green agriculture, the development and utilization of the genetic resources advantageously reduce agricultural cost and It realizes environmental-friendly.

2. passing through mediated by agriculture bacillus heredity transient transformation methods for transcription factor PyERF3 and other transcription factor cotransformations Tobacco, strawberry and pear fruit, and through biological function verification, show that the PyERF3 gene that the present invention clones has promotion the operatic circle skin The function of anthocyanin biosynthesis.

Detailed description of the invention

Fig. 1 is expression analysis thermal map of the PyERF3 gene of the present invention in the red skin Pear varieties of 6 different color modes.

Fig. 2 is PyERF3 of the present invention and other transcription factors form regulation complex cotransformation tobacco and promote anthocyanin biology Synthesis.

Wherein: the anthocyanin of tobacco leaf accumulates phenotypic map after A. instantaneous conversion 7 days.(zero load is as negative by a:pSAK277 Control)； b.PyMYB114+PyMYB10,c.PyMYB10+PybHLH3；d.PyMYB114+PybHLH3；e.PyMYB10 + PybHLH3+PyERF3；f .PyMYB114+PybHLH3+PyERF3；B. the coloring of tobacco leaf anthocyanin is measured by color difference meter The pigment accumulation in region, the ratio of a*/b*, which switchs to positive value from negative value, indicates that the color of strawberry becomes red from green.Error line is Measure the average value of 6 painted areas ratio.C. the content of the total anthocyanin of spectrophotometric determination is utilized.Capitalization indicates that difference is aobvious Horizontal P < 0.01 of property, small letter indicate significant horizontal P < 0.05 of property of difference.

Fig. 3 is PyERF3 of the present invention and other transcription factors form regulation complex cotransformation strawberry and promote anthocyanin biology Synthesis.

Wherein: the phenotypic map that A. passes through the coloring of the strawberry fruit of ' YW5AF7 ' of injection conversion.A and b.pSAK277 and Longitudal section；C and d.PyMYB10 and longitudal section；E and f.PyMYB114 and longitudal section；G and h.PyMYB114+PyMYB10 and vertical Section；I and j.PyMYB10+PybHLH3 and longitudal section；K and l.PyMYB114+PybHLH3 and longitudal section；M and N.PyMYB114+PybHLH3+PyERF3 and longitudal section.B. the painted areas by color difference meter in strawberry fruit detects pigment Variation, the ratio of a*/b*, which switchs to positive value from negative value, indicates that the color of tobacco leaf becomes red from green.Error line is measurement 6 The average value of a painted areas ratio.C. to the measurement of strawberry fruit Anthocyanin content.D.UPLC is to strawberry fruit anthocyanin Component and content.Corn flower 3- Arabinoside, peonidin 3- galactoside.Capitalization show the significant horizontal P of property of difference < 0.01, lowercase shows significant horizontal P < 0.05 of property of difference.

Fig. 4 is that PyERF3 of the present invention overexpression and RNAi carrier convert pear fruit and influence anthocyanin biosynthesis.

Wherein: A. is the phenotypic map that PyERF3 and other transcription factor cotransformation pear fruits promote anthocyanin coloring.I: PSAK277 (zero load is used as negative control) ' is early crisp ' pears；II.PyMYB114+PybHLH3；III.PyMYB114+PybHLH3+ PyERF3.B. using the variation of color difference meter measurement the operatic circle skin painted areas pigment, the ratio of a*/b* switchs to positive value table from negative value Show that the color of tobacco leaf becomes red from green.Error line is the average value for measuring 6 painted areas ratio.Lowercase alphabet Bright difference has significant horizontal P < 0.05 of property.C. instantaneous conversion the operatic circle skin coloration station measures pigment content by UPLC.D. cyanine The relative expression quantity analysis of glycosides biosynthesis related genes.The phenotype of the biosynthesis of E.RNA AF panel the operatic circle skin anthocyanin Figure.A.pSAK277 (zero load is used as negative control) ' is red early crisp ' pears；b.PyMYB114-RNAi c.PybHLH3- RNAi.d.PyERF3-RNAi..F measures the variation of the operatic circle skin painted areas pigment using color difference meter；G. it is measured by UPLC RNA AF panel the operatic circle skin Anthocyanin Content and component.Cy 3-gal: corn flower 3- galactoside, Cy 3-gly: corn flower 3- Glucoside, Cy 3-ara: corn flower 3- Arabinoside, Pe 3-gal: peonidin 3- galactoside, Pe 3-gly: Chinese herbaceous peony Plain 3-glucosides, Pe 3-ara: peonidin 3- Arabinoside.H. related using anthocyanin in RT-qPCR analysis the operatic circle skin The expression of gene.

Fig. 5 be Dual-luciferase reportor systerm measure PyERF3 of the present invention and others transcription factor PyMYB114 and Activity analysis of the PybHLH3 cotransformation to promoter.

Wherein: firefly luciferase (LUC) and renilla luciferase (REN) active ratio LUC/REN indicate to transcribe For transcription complex to the transcriptional activation activity of the promoter of anthocyanin metabolic pathway structural gene, ratio is higher, and transcriptional activation is living Property it is also higher, error line indicates that 3 biology repeat the average value of ratio.PDFR, pANS, pUFGT indicate structural gene The promoter sequence of PyDFR, PyANS, PyUFGT.

Fig. 6 is gene PyERF3 of the present invention and the Yeast two hybrid assay of other transcription factor interactions is analyzed.

Wherein: A.I-V respectively represents the length of the amino acid residue at the gene end PyMYB114 C- or the end N-；B. pass through Yeast two-hybrid verifies the site to interact between gene PyMYB114 and PybHLH3 in vitro；C. pass through yeast two hybrid The site of the interaction of outer verifying gene PyMYB114 and PyERF3；D. by yeast two-hybrid verify in vitro PyMYB10 and The interaction of PybHLH3.E. yeast two-hybrid verifies the interaction of PyERF3 and PybHLH3 in vitro；Wherein, I-V distinguishes Represent the length of the amino acid residue at the gene end PyERF3 C- or the end N-.

The working model of Fig. 7 transcription factor PyMYB114, PybHLH3 and PyERF3 transcriptional control anthocyanin biosynthesis.

Wherein: in red skin pears formed transcription control complex MYB114-bHLH3-ERF3, MYB10-bHLH3-ERF3 and MYB114-MYB10 can be formed complex be individually integrated to anthocyanin biosynthesis pathway key gene (such as DFR, ANS, UFGT) promoter region, activate the expression of downstream gene, promote the biosynthesis of the operatic circle skin anthocyanin.But it transcribes In the absence of regulating and controlling complex (MYB114-bHLH3-ERF3), transcription factor cannot be individually bonded to promoter region, therefore, The gene in downstream cannot be activated, and the biosynthesis of anthocyanin is suppressed.Therefore, red skin pears have reformed into shagreen pears.

Specific embodiment

The present invention is described in detail below in conjunction with specific embodiment.According to it is below description and these embodiments, Those skilled in the art can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, Various changes and modifications can be made to the present invention, so that it is applicable in various uses and condition.

Expression analysis of the PyERF3 gene of the present invention of embodiment 1 in the red skin Pear varieties of different color modes

Show that anthocyanin biosynthesis is by transcription control complex regulating and expressing in pervious report.We select The transcription factor and candidate gene screened in the red green pericarp RNA-seq data (Yang et al., 2015) studied before taking PyMYB114 does correlation analysis.Ironically find 5 APETALA2/ERFs (AP2/ERFs) transcription factors in red skin pears The pericarp up-regulated expression of ' red eggplant ', and consistent expression pattern is shown with PyMYB114.The naming method of AP2/ERFs gene Be according to the unnamed gene of the highest sequence of similarity in ncbi database comparison result for reference.In our current research, Wo Menxuan The red skin Pear varieties of 6 different developing stage verify candidate gene PyMYB114 and AP2/ by RT-qPCR analysis Whether the expression pattern of ERFs is consistent.The forward primer of PyMYB114 RT-qPCR analysis is 5 '- GCCACATCCGTCATAAGACCTC-3 ' (SEQ ID NO.19), reverse primer is 5 '-GCCACTCATGTGTAACCCTTC-3 ' (SEQ ID NO.20).The forward primer of PybHLH3 RT-qPCR analysis is 5 '-TTGTGGAGGGAAGTGGCGGT-3 ' (SEQ ID NO.23), reverse primer is 5 '-AGCTCCCTAAGTGTTTGCATCAC-3 ' (SEQ ID NO.24).PyMYB10RT- The forward primer of qPCR analysis is 5 '-GACCAATGTGATAAGACCTCAGCC-3 ' (SEQ ID NO.27), and reverse primer is 5'-CCGTTCTTTGTTGACGACGAC-3'(SEQ ID NO.28).The forward primer of PyERF3RT-qPCR analysis is 5 '- CTTCCACAACCACCACAACAGC-3 ' (SEQ ID NO.7), reverse primer is 5 '-GATGGAGTGGTTGACGATGCAG- 3'(SEQ ID NO.8).The forward primer of PyERF73RT-qPCR analysis is 5 '-TCCTCAGGAGAGGGAGAGCG-3 ' (SEQ ID NO.9), reverse primer is 5 '-TCCTCAGGAGAGGGAGAGCG-3 ' (SEQ ID NO.10).PyERF2RT-qPCR points The forward primer of analysis is 5 '-CGATCCTCGTGCCAATGAAAATTC-3 ' (SEQ ID NO.11), and reverse primer is 5 '- CTTCTCATCCCTGTAAGGAGCCT-3'(SEQ ID NO.12).The forward primer of PyERF27RT-qPCR analysis is 5 '- TGCGGTGGTGCTATAATCTCCG-3 ' (SEQ ID NO.13), reverse primer is 5 '-CGAGTCGGTGTGAGTAGTTGGG- 3'(SEQ ID NO.14).The forward primer of PyERF113RT-qPCR analysis is 5 '-GCGAGCCCAGGATTAAAGGTG-3 ' (SEQ ID NO.15), reverse primer are 5 '-CAGCTTCCGTCCAGGATGATTC-3 ' (SEQ ID NO.16).As a result table Bright, only PyERF3 and PyMYB114 show significantly to be positively correlated (Fig. 1 and table 1) in 5 Pear varieties, in addition to ' red bar The coloring of pears ' fruit development mid-term is obvious.PyMYB10 and 5 AP2/ERFs gene is shown significantly in different Pear varieties Positive correlation in ' red eggplant pearss ' kind.It is worth noting that PyMYB10 and PyMYB114 show significantly to be positively correlated at 5 In Pear varieties, in addition to ' red early crisp ' shows red green alternate striped coloring.This shows that PyERF3 may be with candidate gene PyMYB114 regulates and controls anthocyanin biosynthesis jointly.

The red skin pears of 16, table different color modes are between fruit development different phase Anthocyanin Content and transcription factor Correlation analysis

The clone of the PyERF3 gene of the present invention of embodiment 2 and construction of recombinant vector

RNA is extracted from ' red eggplant ' the operatic circle skin, the first chain cDNA obtained through reverse transcription is complete for expanding PyERF3 gene It is long.RNA, which is extracted, uses Plant Total RNA Isolation Kit Plus (Foregene, RE-05022), according to the examination The operational manual operation that agent box provides.The synthesis of first chain cDNA First Script Strand cDNA Synthesis SuperMix (Transgene, AE301-02) reverse transcription reagent box (operates) according to the specification that the kit provides.Amplification Gene primer is to for PyERF3-F1:5 '-ACTAGTGGATCCAAAgaattcATGTTTTTGGGGTACAGTCGGG-3’(SEQ ID No.3)；PyERF3-R1:

5’-CAGGACTCTAGAAGTACTctcgagTCAACTGGATGAGGATGGATTGTTGC-3’(SEQ ID No.4).Super fidelity dna polymeraseSuper-Fidelity DNA Polymerase (P505-d1) is only praised purchased from promise Biotechnology company.The reaction system of amplification be 50 μ L in include 200ng cDNA, 2 × Phanta Max Buffer, 25 μ L, On 10 mM dNTP, 1 μ L, Phanta Max Super-Fidelity DNA Polymerase (1U/ μ l) 1 μ L, 10 μM of 2 μ L Primer is stated, ddH is added₂O to 50 μ L.PCR reaction is completed on eppendorf amplification instrument by following procedure: 95 DEG C, initial denaturation 3 is divided Clock, 95 DEG C are denaturalized 15 seconds, and 60 DEG C are annealed 15 seconds, and 72 DEG C extend 90 seconds, 35 thermal cycles, and 72 DEG C extend 5 minutes, 4 DEG C of preservations.It produces A raw single PCR band product.

PCR product (is thought after 1% agarose gel electrophoresis detection with a small amount of plastic recovery kits of AxyGEN purchased from love Into biotechnology Hangzhou Co., Ltd, China) DNA fragmentation is recycled, step is referred to explanation.The DNA solution of recovery purifying with The linear pSAK277 carrier of double digestion (EcoRI/XhoI) is attached reaction, recombinaseII One Step Cloning Kit (article No.: C112-01) is purchased from Nuo Weizan biotechnology company, and by specification step operation.Connect reactant Be total volume it is 10 μ L, linearizes cloning vector, 50~200ng including 5 × CE II Buffer, 50~200ng of 2 μ L Insert Fragment amplified production, 1 μ LII.37 DEG C of connection 30min.To after the reaction was completed, reaction is placed in ice immediately Cooling 5min, reaction product can be converted directly in water-bath.Conversion is using thermal shock method (referring to " molecular cloning experiment handbook " the Three editions, Science Press, 2002) conversion bacillus coli DH 5 alpha, is screened in the LB solid plate containing 50mg/L spectinomycin Positive colony, 5 positive colony sequencings of picking (are completed) by Shanghai Ying Jun Bioisystech Co., Ltd.Sequencing result shows PyERF3 full length gene is 1200bp, and nucleotides sequence is classified as shown in SEQ ID NO.1, and the interpretation of result of BLAST is proved from pears In the gene that newly obtains be an AP2/ERF gene family member, this unnamed gene is PyERF3 by applicant.Recombinant vector It is named as pSAK277-PyERF3.

The template of confactor PyMYB114 clone is ' August is red ' the operatic circle skin cDNA, the forward primer sequence of PCR amplification It is: 5 '-ACTAGTGGATCCAAAgaattcATGAGGAAGGGTGCCTGG-3 ' (SEQ ID NO.17)；Reverse primer sequences It is: 5 '-CAGGACTCTAGAAGTACTctcgagCTAAATCTTAGTTATCTCTTCTTCTAGATTCCA-3’ (SEQ ID NO.18).The template of confactor PybHLH3 clone is ' August is red ' the operatic circle skin cDNA, and the forward primer sequence of PCR amplification is: 5’-ACTAGTGGATCCAAAgaattcATGGCTGCACCGCCGCCAAG-3'(SEQ ID NO. 19)； 5'-CAGGACTCT AGAAGTACTctcgagTTAAGAGTCAGATTGGGGTATAATTTGATTTATC-3' (SEQ ID NO.20).PyMYB10 grams Grand template is ' August is red ' the operatic circle skin cDNA, the forward primer sequence of PCR amplification: 5 '-cgcggtggcggccgcggatccA TGGAGGGATATAACGTTAACTTG-3 ' (SEQ ID NO.21), reverse primer sequences: 5 '-gggccccccctcgagaagc ttCTATTCTTCTTTTGAATGATTCCAA-3'(SEQ ID NO.22).The product of PCR amplification passes through 1% Ago-Gel After electrophoresis detection, (pursued progress biotechnology Hangzhou Co., Ltd purchased from love, China) recycling with a small amount of plastic recovery kits of AxyGEN DNA fragmentation, the DNA solution of recovery purifying is attached with the linear pSAK277 carrier of double digestion (EcoRI/XhoI) to react, RecombinaseII One Step Cloning Kit (article No.: C112-01) only praises biotechnology public affairs purchased from promise Department, and by specification step operation.Coupled reaction system total volume is 10 μ L, including 5 × CE II Buffer of 2 μ L, 50~200ng linearizes cloning vector, 50~200ng Insert Fragment amplified production, 1 μ LII.37 DEG C of connections 30min.To after the reaction was completed, reaction is placed in ice-water bath cooling 5min immediately, reaction product can be converted directly.Turn Change using thermal shock method (referring to " molecular cloning experiment handbook " third edition, Science Press, 2002) conversion bacillus coli DH 5 alpha, The screening positive clone in the LB solid plate containing 50mg/L spectinomycin, 5 positive colony sequencings of picking are (by Shanghai English fine horse Bioisystech Co., Ltd completes).Sequencing result shows that PyMYB114 full length gene is 687bp, and nucleotides sequence is classified as SEQ Shown in ID NO.5, building recombinant vector is named as pSAK277-PyMYB114；PybHLH3 full length gene is 2130bp, core Nucleotide sequence is shown in SEQ ID NO.6, and building recombinant vector is named as pSAK277-PybHLH3.PyMYB10 full length gene For 735bp, nucleotides sequence is classified as shown in SEQ ID NO.23, and building recombinant vector is named as pSAK277-PyMYB10；Using Above-mentioned recombinant vector is imported into Agrobacterium GV3101 by freeze-thaw method.

Acting factor PyMYB114 and PybHLH3 cotransformation causes the PyERF3 gene of the present invention of embodiment 3 altogether with others The synthesis of tobacco anthocyanin

The recombinant vector of building passes through mediated by agriculture bacillus instantaneous conversion tobacco leaf.Turn the result shows that individually converting one The factor is recorded not it is observed that the accumulation of Anthocyanin Content, and need two transcription factor PyMYB114 of cotransformation and PybHLH3 is just it is observed that accumulation of the anthocyanin in tobacco leaf on a small quantity.And PyERF3 and PyMYB114 and PybHLH3 3 three Transcription factor cotransformation can accelerate anthocyanin biosynthesis, show deeper color and more strong reaction (Fig. 2A).This Outside, PyMYB114 and PyMYB10 cotransformation can also have faint pigment deposition.The tobacco leaf of anthocyanin accumulation passes through color difference Instrument and spectrophotometer detection pigment content (Fig. 2 B, C), it is as a result consistent with phenotypic map.Therefore, PyERF3 and other auxiliary turn The record factor PybHLH3 and PyMYB114 cotransformation can promote a large amount of synthesis of tobacco leaf anthocyanin.

The PyERF3 gene of the present invention of embodiment 4 and other transcription factor PyMYB114 and PybHLH3 cotransformation lead to strawberry Anthocyanin synthesis

' Yellow wonder ' 5AF7 (YW5AF7) fruit is (2 after spending for the recombinant vector instantaneous conversion yellow strawberry of building Week).The result shows that cotransformation PyMYB114 and PybHLH3 it is observed that a small amount of anthocyanin accumulation, when PyERF3 of the present invention with Two above transcription factor PyMYB114, PybHLH3 and when cotransformation, cause the coloring of strawberry holder injection areas to significantly increase. Pigment accumulation is not observed after unloaded pSAK277 conversion.Similar conversion has also been made in PyMYB10 as control, discovery The similar result of the color of PyERF3 and PyMYB114 and PybHLH3 and cotransformation accumulation.In addition, our also cotransformations PyMYB114 and PyMYB10 has found a large amount of pigment depositions (Fig. 3 A) inside holder.Further use color difference meter, spectrophotometer It is as a result consistent with phenotypic map with the content of UPLC measurement anthocyanin.PyERF3 and PyMYB114 and PybHLH3 cotransformation anthocyanin Content highest (Fig. 3 B, C, D).In addition, anthocyanin component and content in Ultra Performance Liquid Chromatography instrument analysis coloring strawberry.It increases The total Anthocyanin Content of the cotransformation strawberry fruit of PyERF3 of the invention is up to 68 μ g/g FW, this, which is apparently higher than, only turns Change the content of PyMYB114 and PybHLH3.The highest corn flower 3- galactoside of Anthocyanin Content, Zhan are total in inducing strawberry fruit 68% or more (Fig. 3 D) of Anthocyanin Content.

5 PyMYB114 of embodiment promotes pear flower blueness glycosides to synthesize with the homologous expression of its total acting factor

Choosing ' precocious strains ' for spending latter 30 days or so is test material, with reference to Clough et al. (1998) vacuum infiltration The carrier of the overexpression of building is transferred to the operatic circle skin by method.The result shows that infect jointly conversion PyERF3 and PyMYB114 and The effect of PybHLH3 is more stronger than the effect of cotransformation PyMYB114 and PybHLH3 at the region that the operatic circle skin infects (Fig. 4 A).This Outside, the coloring of color difference meter (Minolta chromameter, Japan) measurement cotransformation PyERF3 and PyMYB114 and PybHLH3 The ratio of the a*/b* in region is up to up to 0.38 (Fig. 4 B), the addition enhancing cyanine of further explanation PyERF3 of the present invention The coloring of glycosides.The content of UPLC measurement anthocyanin shows consistent with phenotypic data trend, its main component is corn flower 3- half Lactoside, and the increase of Anthocyanin Content is mainly the raising (Fig. 4 C) of corn flower 3- galactoside content.Anthocyanin biology The expression pattern of synthesis related gene also further analyzes (Fig. 4 D), the results showed that the addition of PyERF3 of the present invention makes anthocyanin The different degrees of raising of key structure gene PyDFR, PyANS and the PyUFGT expression quantity of biosynthesis promotes the operatic circle ginned cotton green Glycosides biosynthesis.Therefore, PyMYB114 plays an important role during transcriptional control anthocyanin biosynthesis, and PyERF3 plays the effect of enhancing.

The function of PyERF3 gene further interferes gene expression system to be verified by RNA.In order to avoid silencing its Its homologous gene, the amino acid fragment for choosing transcription factor close to the specificity at 3 ' ends are cloned, and are inserted into pGreenII 62- SK transient expression vector, constructs recombinant vector: PyERF3-RNAi, PyMYB114-RNAi and PybHLH3-RNAi.Agrobacterium Mediated transformation pGreenII 62-SK (zero load), PyMYB114-RNAi and PybHLH3-RNAi and PyERF3-RNAi are injected respectively To ' red early crisp ' the operatic circle skin close to the maturity period.Conversion 7 days after observe PyMYB114-RNAi and PybHLH3-RNAi and It colours and reduces around the position that the operatic circle skin of PyERF3-RNAi infects, there is no send out for pGreenII 62-SK (zero load) the operatic circle skin The variation (Fig. 4 E) of existing fruit colour.The variation for infecting the fruit colour around position is measured (Fig. 4 F) with color difference meter, unloaded The ratio of pGreenII 62-SK a*/b* reaches 0.6, and it is negative that the ratio of the a*/b* of the operatic circle skin of gene inhibition expression, which is, Value illustrates that they are all significantly lower than unloaded (P < 0.05).The measurement of total Anthocyanin Content shows similar trend, UPLC measurement The main component of anthocyanin is corn flower 3- galactoside, and the reduction of Anthocyanin Content is also mainly corn flower 3- galactolipin The reduction (Fig. 4 G) of glycosides content.In addition, the transcriptional level of the key gene of RT-qPCR analysis anthocyanin synthesis, the results showed that, with Zero load is compared, PyMYB114-RNAi, and the expression quantity of PybHLH3-RNAi and PyERF3-RNAi have different degrees of decline (figure 4H).These are the result shows that PyERF3 gene of the present invention participates in the regulation of the operatic circle skin anthocyanin biosynthesis.

6 Dual-luciferase reportor systerm of embodiment verifies the interaction between PyERF3 and PyMYB114

In order to verify whether total PyERF3 of the present invention and other transcription factor cotransformations can activate anthocyanin biosynthesis generation The expression for thanking to the promoter of the structural gene PyDFR, PyANS and PyUFGT of access passes through instantaneous conversion protoplasts of Arabidopsis thaliana broken by ultrasonic, And it is detected with Dual-luciferase reportor systerm.The result shows that PyERF3 of the invention and PyMYB114 and PybHLH3 corotation Change the transcriptional activation activity ability ratio PyMYB114 and PybHLH3 cotransformation activity stronger (Fig. 5) to promoter.Of the invention PyERF3 arrives similar result with PyMYB10 and PybHLH3 cotransformation.In addition, PyMYB114 and PyMYB10 cotransformation also can Activate the activity of promoter.These results also indicate that PyERF3 and PyMYB114 and PybHLH3 form transcription control complex, It is integrated to the promoter region of anthocyanin metabolic pathway structural gene, activates the expression of downstream gene, promotes anthocyanin in the operatic circle Synthesis in skin.

7 yeast two-hybrid of embodiment verifies the interaction between PyERF3 and PyMYB114

Yeast two-hybrid is whether there is between the transcription factor of gene PyERF3 and cotransformation of the invention to verify Interaction, PyMYB114 CDS full length sequence and the end C- or N- residue amino acid sequence are cloned respectively, and are inserted into pGBKT7 load Body, verifying self-excitation are activity.The purpose of having cloned PybHLH3 and PyERF3 CDS full length sequence insertion pGADT7 carrier simultaneously is inspection It surveys and whether there is interactions between protein with PyMYB114.Training of the cotransformation PybHLH3 and PyMYB114 first in two scarce SD-Trp-Leu It supports and is grown on base, then transformant is transferred on the culture medium of four scarce SD-Trp-Leu-His-Ade and is screened, the results showed that, The amino acid sequence (V) of PyMYB114 CDS overall length and 160, the end C- amino acid residue (169-229) (IV) show very strong Transcriptional activation activity, N- Amino End Group end residue MYB114^1-93(I) and MYB114^1-160(II) and C-terminal amino acid residue MYB114^136-229(III) transcriptional activation activity is not present.When cotransformation yeast, N- Amino End Group end residue MYB114^1-160(II) with PybHLH3 can not only be grown on the culture medium in two scarce SD-Trp-Leu, and can be in four scarce SD-Trp-Leu-His-Ade It is grown on culture medium, this illustrates the site of interaction between PyMYB114 and PybHLH3 in N- Amino End Group end residue MYB114^1-160 (II) site (Fig. 6 A, B).Similar cotransformation yeast (Fig. 6 C) between PyMYB114 and PyERF3, the results showed that, N- and 93, the end C- amino acid residue (I, III) can not only grow on two scarce SD-Trp-Leu culture mediums, and can be in four scarce SD- It is grown on Trp-Leu-His-Ade culture medium.So between PyMYB114 and PyERF3 interaction site at least one, it Be respectively present in the regions of 93 amino acid residues in the end N- and C-.In addition, in order to verify between PyERF3 and PybHLH3 whether There are interaction, PyERF3 CDS full length sequence and the end C- or N- residue amino acid sequence are cloned respectively, then cotransformation respectively PybHLH3 and PyERF3 CDS full length sequence and the end C- or N- residue amino acid sequence.The result shows that PyERF3 amino acid residue Sequence IV and V self-excitation is activity most strong, remaining end C- or N- residue amino acid sequence (I, II, III) and PybHLH3 are two It lacks and is grown on the culture medium of two scarce SD-Trp-Leu, but cannot be grown on four scarce SD-Trp-Leu-His-Ade culture mediums, this Show the site (Fig. 6 E) that interaction is not present between PybHLH3 and PyERF3.In addition, the yeast of PyMYB114 and PyMYB10 is total Conversion shows interaction site between the two in the area (Fig. 6 D) of 93 amino acid residues (III) in the end PyMYB114 C-.To sum up institute It stating, PyERF3 and transcription factor PybHLH3 and PyMYB114 interaction form transcription control complex ERF3-MYB114-bHLH3, It is integrated to the promoter region of PyDFR, PyANS and PyUFGT, collectively promotes anthocyanin biosynthesis in the operatic circle skin.

Leading reference

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Sequence table

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Gly His Ile Gly Ser Gly Gly Val Thr Ser Ser Phe Gly Gln Leu Tyr

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Glu Ser Gly Asn Arg Ala Phe Arg Gly Gly Tyr Ser Asp Tyr Arg Gly

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Glu Ser Thr Asn Phe Ser Thr Ala Pro Thr Thr Ala Thr Val Ser Val

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Thr Thr Ala Val Pro Thr Thr Pro Ser Ser Thr Glu Ser Val Ser Phe

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Glu Glu Thr Gly Glu Arg Lys Arg Arg Tyr Arg Gly Val Arg Gln Arg

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Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro His Lys Ala Ala

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Arg Val Trp Leu Gly Thr Phe Asp Thr Ala Glu Ala Ala Ala Arg Ala

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Tyr Asp Glu Ala Ala Leu Arg Phe Arg Gly Asn Arg Ala Lys Leu Asn

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Phe Pro Glu Asn Val Arg Leu Val Gln Pro Pro Pro Pro Pro Pro Pro

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Thr Leu Gln Thr Phe Asn Ser Asn Ser Arg Pro Thr Gln Tyr Ala Gln

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Pro Leu Gln Pro Pro Pro Pro Leu Pro Gln Pro Pro Gln Gln Gln Leu

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Tyr His Ser Gln Gln Ala Phe Gln Pro Ser Ser Asp Leu Leu Arg Asp

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Tyr Phe Asp Tyr Ser Gln Leu Leu Gln Ser Ser Ala Asp Phe His Pro

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Gln Gln Gln Gln Gln Gln Gln Pro Ser Ser Leu Leu Gln Gln Met Tyr

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Tyr Asn Ser Gln Leu Ala Ser Leu Gln Ser Ser Ile Leu Gln Pro Ala

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Ser Ser Thr Thr Pro Ser Ser Ala Leu Pro Ser Ser Ala Ser Ser Ser

340 345 350

Ala Ser Phe Pro Leu Phe Phe Ser Glu Gln Asn Gln Gln Leu Gly Phe

355 360 365

Phe Pro Gln Pro Gln Asn Pro Asn Gln Gly Gly Ser Ser Asp Phe Gln

370 375 380

Ala Pro Ser Trp Ser His Ser Gly Asn Asn Pro Ser Ser Ser Ser

385 390 395

<210> 3

<211> 50

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

caggactcta gaagtactct cgagtcaact ggatgaggat ggattgttgc 50

<210> 4

<211> 50

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

caggactcta gaagtactct cgagtcaact ggatgaggat ggattgttgc 50

<210> 5

<211> 687

<212> DNA

<213>' August is red ' pears (pear)

<400> 5

atgaggaagg gtgcctggac tcaacaggaa gatgatattc tgaggcagta cgttgaaaag 60

catggagatg gaaagtggca ccaggttcct cgcgaaacag gtctaaacag atgcaggaaa 120

agctgcagac agaggtggtt gaactatttg aagccgaatc tcaagagcgg agatttcaca 180

gaggatgaaa tagatctaat ccatagactt cagaaacttt tgggaaacag gtggtcaata 240

attgctggaa gactcccagg aagaacagca ggcaaggtaa aaaattattg gaatagcaag 300

caacgaaagg agttggaata tatgaaggat aaatccaaag aaagaacaaa agccacatcc 360

gtcataagac ctcaaccacg gagagctaga gttgcaattt ttcaatctga agagaactgt 420

agcaggttat tacagacatc atcaccacct acagaaaacg ctattgattc atggaaggcc 480

atgttgcatg atacagacaa tgttgatgga acaccatttt ctagtttagg gttaggggaa 540

gacctcttca caaacttttg ggttgaagat attgcacagt cgacaatggt aggcatgaat 600

tctgctgatg aagggttaca catgagtggc aacttttcct ttagagagaa cttttggaat 660

ctagaagaag agataactaa gatttag 687

<210> 6

<211> 2130

<212> DNA

<213>' August is red ' pears (pear)

<400> 6

atggctgcac cgccgccaag cagcagccgc ctccgtggta tgttgcaggc ctcagtccaa 60

tatgtccaat ggacttacag tctcttctgg caaatctgtc cccaacaagg gatcttagta 120

tggtcagatg ggtactataa tggagccatc aagacgagga agacggtgca accaatggaa 180

gtgagtgccg aggaggcatc tctccagagg agccagcaac tcagagaact ctacgactct 240

ttgtccgctg gagagacaaa ccagccccca gcacgccgcc cttgcgcttc cttgtccccg 300

gaggacttaa ccgaatccga atggttctac ttgatgtgtg tctcattctc ctttcccccc 360

ggcgtcgggt tgccagggaa agcatacgca aggaggcagc atgtatggct caccggtgca 420

aacgaggtcg atagcaaaac cttttccaga gctattttgg caaagagtgc tcgtatacag 480

accgtagtgt gcattcctct tctagatggc gtcgtagaat ttggcaccac agagagggtt 540

ccagaagacc acgccttagt cgaacacgtc aaaaccttct tcgttgacca ccaccaccct 600

ccgccaccaa aacccgccct ctccgagcac tccacatcca accccgccac ctcatccgat 660

cacccacatt tccactctcc gcaccttctc cagaccatgt gcaccaaccc tcctctcaac 720

gccgcccaag aagacgaaga ggacgaagaa gaagatgata atcaggagga ggacgacgga 780

ggagacgagt ccgactccga agccgaaacg ggtcgcaatg gtggagccgt tgttcccgcc 840

gcaaaccctc ctcaggtttt ggccgcggta gccgagccaa gcgagctcat gcaactcgag 900

atgtccgaag acatccggct gggctccccg gacgatgcct caaataactt ggactctgat 960

ttccacttgt tagctgtgag tcagtctagg aacccagcgg atcagcagag acaagctgac 1020

tcgtatcgag ccgagtcgac caggcggtgt ccgtcagtac aagagccgct gagcagtggc 1080

cttcaaccgc cgcacacagg acccttagct ttagaggagt tgacacatga tgacgacaca 1140

cattactcgg agacggtctc caccatactg cagggacaag cgactcggtg gacggattca 1200

tcgtccacca actacacagc ttgcttgact cagtcggctt tcgccaagtg gtcgagccgg 1260

attgatcacc acttcctcat cccggttgag ggcacgtccc aatggctttt gaaatatatt 1320

ttatttagtg taccattcct ccactcaaaa tatcgcgacg aaaactcgcc aaaatctcaa 1380

gagggcgaag gctcgacgcg tttgaggaaa gggaccccac aagacgagct cagtgccaat 1440

catgtgttag cggaacgacg tcgtagagag aagcttaatg agaggtttat tatactaagg 1500

tccctagtgc cttttgtgac aaaaatggac aaggcttcga tattagggga cacaatcgag 1560

tatgtgaagc aactgcgtaa caaaattcag gatctcgagg cacgtaacat gctgatggag 1620

gaagatcaac gatcgagatc atccggggaa atgcaaaggt ccagtagttg taaagagttg 1680

cgaagtgggc tcacggtagt ggagcggacc caaggaggtc caccggggtc cgataaaagg 1740

aagttgagga ttgtggaggg aagcggcggt gtcgccattg gtaaggctaa agtaatggag 1800

gactcaccgc ctccaccgcc cccgccacca cctcagccag aaccttcacc gacacctatg 1860

gtgacgggga cttctctaga ggtgtcgata atcgagagtg atgggctgtt ggagctccaa 1920

tgcccgtata gagaagggtt attgcttgat gtgatgcaaa cacttaggga gctaagaatt 1980

gagaccacgg tggtccagtc ctcattgaat aacggattct tcgtagctga actaagagcc 2040

aaggtgaagg ataacgtgag tggcaagaaa gtaagtatta cggaagtgaa gagggtgata 2100

aatcaaatta taccccaatc tgactcttaa 2130

<210> 7

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cttccacaac caccacaaca gc 22

<210> 8

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gatggagtgg ttgacgatgc ag 22

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tcctcaggag agggagagcg 20

<210> 10

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

tcctcaggag agggagagcg 20

<210> 11

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

cgatcctcgt gccaatgaaa attc 24

<210> 12

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

cttctcatcc ctgtaaggag cct 23

<210> 13

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

tgcggtggtg ctataatctc cg 22

<210> 14

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cgagtcggtg tgagtagttg gg 22

<210> 15

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gcgagcccag gattaaaggt g 21

<210> 16

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cagcttccgt ccaggatgat tc 22

<210> 17

<211> 39

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

actagtggat ccaaagaatt catgaggaag ggtgcctgg 39

<210> 18

<211> 57

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

caggactcta gaagtactct cgagctaaat cttagttatc tcttcttcta gattcca 57

<210> 19

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

actagtggat ccaaagaatt catggctgca ccgccgccaa g 41

<210> 20

<211> 58

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

caggactcta gaagtactct cgagttaaga gtcagattgg ggtataattt gatttatc 58

<210> 21

<211> 45

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

cgcggtggcg gccgcggatc catggaggga tataacgtta acttg 45

<210> 22

<211> 46

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

gggccccccc tcgagaagct tctattcttc ttttgaatga ttccaa 46

<210> 23

<211> 735

<212> DNA

<213>' August is red ' pears (pear)

<400> 23

atggagggat ataacgttaa cttgagtgtg agaaaaggtg cctggactcg agaggaagac 60

aatcttctca ggcagtgcat tgagattcat ggagagggaa agtggaacca agtttcatac 120

aaagcaggct taaacaggtg caggaagagc tgcagacaaa gatggttaaa ctatctgaag 180

ccaaatatca agagaggaga ctttaaagag gatgaagtag atcttatact tagacttcac 240

aggcttttgg gaaacaggtg gtcattgatt gctagaagac ttccaggaag aacagcgaat 300

gatgtgaaaa attattggaa cactcgattg cggatcgatt ctcgcatgaa aacgttgaaa 360

aataaatctc aagaaacgag aaagaccaat gtgataagac ctcagcccca aaaattcatc 420

aaaagttcat attacttaag cagtaaagaa ccaattctag aacatattca atcagcagaa 480

gatttaagta cgccatcaca aacgtcgtcg tcaacaaaga acggaaatga ttggtgggag 540

accttgttcg aaggcgagga tacttttgaa agggctgcat gtcccagcat tgagttagag 600

gaagaactct tcacaacttt ttggtttgat gatcgactgt cggcaagatc atgtgccaat 660

tttcctgaag aaggacaaag tagaagtgaa ttctccttta gcatggacct ttggaatcat 720

tcaaaagaag aatag 735

Claims (2)

1.PyERF3Gene associationPyMYB114Gene andPybHLH3Gene is promoting answering in the anthocyanin biosynthesis of the operatic circle skin With；DescribedPyERF3Gene nucleotide series are as shown in SEQ ID No.1；DescribedPyMYB114Gene nucleotide series As shown in SEQ ID No.5；DescribedPybHLH3Gene nucleotide series are as shown in SEQ ID No.6.

2. containingPyERF3The recombinant expression carrier of gene, which is combined, to be containedPyMYB114The recombinant vector of gene and containPybHLH3Gene Recombinant vector promote the anthocyanin biosynthesis of the operatic circle skin in application；DescribedPyERF3Gene nucleotide series such as SEQ Shown in ID No.1；DescribedPyMYB114Gene nucleotide series are as shown in SEQ ID No.5；DescribedPybHLH3Gene Nucleotide sequence is as shown in SEQ ID No.6.