CN107686840A

CN107686840A - Pears transcription factor PyERF3 and its recombinant expression carrier and application

Info

Publication number: CN107686840A
Application number: CN201710768517.9A
Authority: CN
Inventors: 吴俊�; 姚改芳; 张绍铃; 明美玲; 谷超; 汪润泽; 杨广艳; 刘海楠
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2017-06-23
Filing date: 2017-08-31
Publication date: 2018-02-13
Anticipated expiration: 2037-08-31
Also published as: CN107686840B

Abstract

The invention discloses pears transcription factor PyERF3 and its recombinant expression carrier and application.One kind, which is isolated from ' red eggplant ' pears, has the transcription factor PyERF3 genes for promoting the operatic circle skin anthocyanin biological synthetic functional, and its nucleotides sequence is classified as shown in SEQ ID No.1, and its amino acid sequence encoded 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 that the PyERF3 genes of the invention cloned form the function of the biosynthesis of transcription control complex promotion the operatic circle skin anthocyanin with other transcription factor PyMYB114 and PybHLH3.The discovery of PyERF3 genes, to promote the molecular breeding of the operatic circle skin anthocyanin synthesis to provide new genetic resources, new genetic resources is provided to implement green agriculture, the utilization of the genetic resources advantageously reduce agricultural cost and realized environment-friendly.

Description

Pears transcription factor PyERF3 and its recombinant expression carrier and application

Technical field

The invention belongs to plant genetic engineering field, is related to pears transcription factor PyERF3 and its recombinant expression carrier and answers With, and in particular to separated from ' red eggplant ' pears, clone obtains one and participates in the related AP2/ of the operatic circle skin anthocyanin biosynthesis ERF family member PyERF3 genes and its application.

Background technology

Anthocyanin is the Plant Secondary Metabolites of biosynthesis in flavonoids metabolic pathway, and it is a kind of water colo(u)r. It is widely present in plant kingdom, stem, leaf, flower and fruit is showed chromatic colour.Anthocyanin in spending is mainly that attraction is awarded 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 effect to disease resistance of plant, for example is resisting ultraviolet, Antioxidation activity aspect effect (Bieza et al., 2001；Veeriah et al., 2006), it is also various good for health Aspect effect, the protection and angiocardiopathy of nervous system, cancer and diabetes etc. (Konczak et are such as provided al.,2004；Butelli et al.,2008).Therefore, anthocyanin is important sanatory natural pigment (Camire Et al.2002), make it the significant contribution to fruit quality.

The formation of anthocyanin is completed under a series of catalytic action of enzymes, and it is flavonoids biosynthetic metabolism approach A branch, initial reaction be by phenylalanine lyases (PAL) catalysis cinnamic acid reaction, that passes through successively is main Crucial catalyzing enzyme phenylalanine lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavanones 3- hydroxyls Change enzyme (F3H), and the successive reaction occurred during anthocyanin is generated, including dihydroflavonol 4-reductase (DFR), Anthocyanin synzyme (ANS) and UDP-glucose:Flavonoids 3-O- glucosyltransferases (UFGT), ultimately form anthocyanin (Takos et al., 2006).In addition, the expression of the adjusted and controlled gene of transcription factor and then causing the biosynthesis of anthocyanin The research of aspect in model plant existing 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 is transcribed The factor has important regulating and controlling effect in anthocyanin biosynthetic process.Such as apple (Malus × domestica), MdMYB10 and MdMYB110a ' is cloned, they are demonstrate,proved from red pulp apple in Red Field ' and ' Sangrado ' respectively Bright positive regulation type I/II apple pericarps color and luster (Espley et al., 2007；Chagné et al.,2013).Also The transcription factor found in two apples, MdMYB1 and MdMYBA, it is 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 pattern, and bHLH albumen, regulatory complex it is other into Member may have overlapping regulation target (Akagi et al., 2009).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 factors VvMYC1 is shown and different MYB eggs (Vv MYB5a, VvMYB5b, VvMYBA1/A2, VvMYBPA1) interaction participates in anthocyanin to induce in vain 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 (TTG1) gene locis of arabidopsis TRANSPARENT TES-TA GLABRA 1 first (Walker et al., 1999), its homologue be also accredited including apple (Brueggemann et al., 2010), grape (Kobayashi et al., 2004) pomegranate (Ben-Simhon et al., 2011) and strawberry (Schaart Et al., 2013) garden crop such as.In addition, also other transcription factors can activate anthocyanin biosynthesis.Intending In southern 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 attached to MBW compounds promote or suppress anthocyanin biosynthesis (Li et al., 2013；Viola et al., 2016)；For example, TCP3 and TCP15 respectively can be by disturbing MBW to turn by being attached to miR156 targeting transcription factors SPL9 Record complex and form negative regulation anthocyanin accumulation (Gou et al., 2011).Jia et al. (2015) reports, miRNA858 The expression that the biosynthesis of negative regulation anthocyanin passes through 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 associated proteins 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 complexs and inhibitor MYBL2 and JAZ.Equally, Wang et al. (2016) are reported, Suppression expression of the miRNA858a and HY5 to MYBL2 causes the activation of anthocyanin biosynthesis pathway in arabidopsis.Therefore, flower Blue or green glycosides biosynthesis is formed regulated and control network by MBW compounds or other transcription factors and controlled.In fruit, be related to fruit into Some key genes of ripe regulation and control have been accredited, and many of which is SEPALLATA- and SQUAMOSA-class MADS box Or SBP box transcription factors (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 work of some transcription factors in the regulation of flavonoids biosynthesis With, but the contact between the developmental regulation factor and the downstream effector of participation flavonoids biosynthesis is not yet untied.Ripe phase The SQUAMOSA-class MADS box transcription factors VmTDR4 for closing anthocyanin biosynthesis is FRUITFULL in arabidopsis (FUL) contact between the TDR4 in the homologous gene and tomato of gene be reported in cowberry (Jaakola et al., 2010).Anthocyanin accumulation in VmTDR spatial and temporal expression and cranberry is consistent, and VmTDR4 silence causes anthocyanin The significant reduction of biosynthesis.SEPALLATA-class MADS box transcription factors PyMADS18 expression and red skin Pear varieties Anthocyanin accumulation it is relevant (Wu et al., 2013b).Zhou et al. (2015) reports, the molecular genetic mechanism of red meat peach It is the PpNAC1 transcriptional activation anthocyanin biosynthesis by QTL positioning identifications.In former report, it is believed that AP2/ERFs Be participate in apple and plum fruit Mature Regulation (Whale et al., 2007；Manganaris et al.,2008).With Afterwards, Licausi et al. (2013) are reported, AP2/ERF transcription factors participate in that regulation and control are nascent and secondary metabolites, growth and Growth course.The research that AP2/ERF transcription factors participate 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.Comparatively, it is Europe to have more red skin Pear varieties Pears (P. communis).For most of east pears, the product of anthocyanin can be just only shown connecing maturescent period Tired (Yang et al, 2014)；And the coloring of European pears, early stage fruit development, the fruit of coloring can continue until the maturity period. In European pears, Dondini et al. (2008) reports, ' Max Red Bartlett ' Red color traits navigate to linkage group 4 (LG4).Then, Pierantoni et al. (2010) are reported, although PyMYB10 that Identification arrives and apple MdMYB10 very high homologies, and MdMYB10 control apple pericarp color and luster, but ' Max Red Bartlett ' and In ' Williams ' Pear varieties, it is not the red skin of direct transcriptional control and the gene of yellow rind character, positions pears genome Different regions.Wu et al. (2013b) also report, European pear ' early red Kao Misi ' and the red green of other green bud mutations The mutant of pericarp color, anthocyanin synthesis in the operatic circle skin may be take part in by identifying MADS transcription factors.However, MYB10 Have been reported that regulation and control anthocyanin biosynthesis some Pear varieties (Feng et al., 2010；Zhang et al.,2011；Yu et al.,2012).' formation of Max Red Bartlett ' green bud mutation is relevant for the methylation level of MYB10 promoter regions (Wang et al.,2012).Anthocyanin biosynthesis gene MYB10 and bHLH expression do not show related to WD40 Property, propose to regulate and control a kind of regulatory mechanism (Yang et al., 2014) of anthocyanin biosynthesis in pears.

The content of the invention

It is an object of the invention 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 application of the gene.

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

One kind, which is isolated from ' red eggplant ' pears, has the transcription factor PyERF3 genes for promoting anthocyanin biological synthetic functional, belongs to AP2/ERF family members, its nucleotides sequence are classified as shown in SEQ ID No.1, the ORFs comprising 1200bp；Coding 399 Individual amino acid, its amino acid sequence encoded is shown in sequence table SEQ ID No.2, isoelectric point 6.60, molecular weight 43.18 kDa。

Recombinant expression carrier containing PyERF3 genes of the present invention.

Described recombinant expression carrier, preferably using pSAK277 as the carrier that sets out, the insertion point of the PyERF3 genes is Between EcoR I and Xhol I.

Host Strains containing PyERF3 genes of the present invention.

Clone the primer pair of PyERF3 gene cDNA sequences of the present invention, sense primer PyERF3-F1 sequences such as SEQ Shown in ID No.3, anti-sense primer PyERF3-R1 sequences are as shown in SEQ ID No.4.

PyERF3 genes of the present invention are promoting the application of the operatic circle skin anthocyanin biosynthesis.

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 advantages below and effect：

The discovery of 1.PyERF3 genes, to promote the molecular breeding of the biosynthesis of the operatic circle skin anthocyanin to provide new gene Resource, new genetic resources is provided to implement green agriculture, the utilization of the genetic resources advantageously reduce agricultural cost with Realize environment-friendly.

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

Brief description of the drawings

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

Fig. 2 is that PyERF3 of the present invention forms regulation and control complex cotransformation tobacco promotion anthocyanin biology with other transcription factors Synthesis.

Wherein：A. after instantaneous conversion 7 days tobacco leaf anthocyanin accumulation phenotypic map.a：(zero load is as negative by pSAK277 Control)； b.PyMYB114+PyMYB10,c.PyMYB10+PybHLH3；d.PyMYB114+PybHLH3；e.PyMYB10 + PybHLH3+PyERF3；d.PyMYB114+PybHLH3+PyERF3；B. tobacco leaf anthocyanin is measured by color difference meter to colour The pigment accumulation in region, a*/b* ratio switch to be changed into red from green on the occasion of the color for representing strawberry from negative value.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 represents that difference shows The horizontal P of the property<0.01, small letter represents the significant horizontal P of property of difference<0.05.

Fig. 3 is that PyERF3 of the present invention forms regulation and control complex cotransformation strawberry promotion anthocyanin biology with other transcription factors Synthesis.

Wherein：The phenotypic map of the coloring of the strawberry fruit of ' YW5AF7 ' that is A. converted by injection.A and b.pSAK277 and Vertical section；C and d.PyMYB10 and vertical section；E and f.PyMYB114 and vertical section；G and h.PyMYB114+PyMYB10 and vertical Section；I and j.PyMYB10+PybHLH3 and vertical section；K and l.PyMYB114+PybHLH3 and vertical section；M and N.PyMYB114+PybHLH3+PyERF3 and vertical section.B. pigment is detected by painted areas of the color difference meter in strawberry fruit Change, a*/b* ratio switch to be changed into red from green on the occasion of the color for representing tobacco leaf from negative value.Error line is measurement 6 The average value of individual painted areas ratio.C. to the measure of strawberry fruit Anthocyanin content.D.UPLC is to strawberry fruit anthocyanin Component and content.Corn flower 3- Arabinosides, peonidin 3- galactosides.Capitalization shows the significant horizontal P of property of difference< 0.01, lowercase shows the significant horizontal P of property of difference<0.05.

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

Wherein：A. it is the phenotypic map that PyERF3 promotes anthocyanin coloring with other transcription factor cotransformation pear fruits.I： PSAK277 (zero load is used as negative control) ' is early crisp ' pears；II.PyMYB114+PybHLH3；III.PyMYB114+PybHLH3+ PyERF3.B. switched to using the change of color difference meter measure the operatic circle skin painted areas pigment, a*/b* ratio from negative value on the occasion of table Show that the color of tobacco leaf is changed into red from green.Error line is to measure the average value of 6 painted areas ratio.Lowercase Show that difference has the significant horizontal P of property<0.05.C. instantaneous conversion the operatic circle skin coloration station determines pigment content by UPLC.D. spend The relative expression quantity analysis of blue or green glycosides biosynthesis related genes.The table of the biosynthesis of E.RNA AF panel the operatic circle skin anthocyanins Type 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.Using the change of color difference meter measure the operatic circle skin painted areas pigment；G. determined by UPLC RNA AF panels the operatic circle skin Anthocyanin Content and component.Cy 3-gal:Corn flower 3- galactosides, Cy 3-gly:Corn flower 3- glucosides, Cy 3-ara:Corn flower 3- Arabinosides, Pe 3-gal:Peonidin 3- galactosides, Pe 3-gly:Chinese herbaceous peony Medicine 3-glucoside of element, Pe 3-ara:Peonidin 3- Arabinosides.H. using anthocyanin phase in RT-qPCR analysis the operatic circle skins The expression of correlation gene.

Fig. 5 be Dual-luciferase reportor systerm determine PyERF3 of the present invention and other transcription factor PyMYB114 and Activity analysis of the PybHLH3 cotransformations to promoter.

Wherein：Firefly luciferase (LUC) and the ratio LUC/REN of renilla luciferase (REN) activity represent to transcribe Transcription complex is to the transcriptional activation activity of the promoter of anthocyanin metabolic pathway structural gene, and ratio is higher, and transcriptional activation is lived Property it is also higher, error line represents that 3 biology repeat the average value of ratio.PDFR, pANS, pUFGT represent structural gene PyDFR, PyANS, PyUFGT promoter sequence.

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

Wherein：A.I-V represents gene PyMYB114 C- ends, or the length of the amino acid residue at N- ends respectively；B. pass through Yeast two-hybrid verifies the site of gene PyMYB114 and PybHLH3 interphase interaction in vitro；C. yeast two hybrid is passed through The site of outer checking gene PyMYB114 and PyERF3 interaction；D. verified in vitro by yeast two-hybrid PyMYB10 and PybHLH3 interaction.E. yeast two-hybrid verifies PyERF3 and PybHLH3 interaction in vitro；Wherein, I-V distinguishes Represent gene PyERF3 C- ends, or the length of the amino acid residue at N- ends.

The working model of Fig. 7 transcription factors 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 attached 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 turn When record regulation and control complex (MYB114-bHLH3-ERF3) is not present, transcription factor can not be individually bonded to promoter region, because This, the gene in downstream can not be activated, and the biosynthesis of anthocyanin is suppressed.Therefore, red skin pears have reformed into shagreen pears.

Embodiment

The present invention is described in detail below in conjunction with specific embodiment.According to following description and these embodiments, Those skilled in the art can determine the essential characteristic of the present 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 various uses and condition.

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

Showed in former report, anthocyanin biosynthesis is by transcription control complex regulating and expressing.We select Take the transcription factor and candidate gene of screening in the red green pericarp RNA-seq data (Yang et al., 2015) studied before PyMYB114 does correlation analysis.Ironically find 5 APETALA2/ERFs (AP2/ERFs) transcription factors in red skin The pericarp up-regulated expression of pears ' red eggplant ', and show consistent expression pattern with PyMYB114.The name side of AP2/ERFs genes Formula be according to the unnamed gene of similarity highest sequence in ncbi database comparison result for reference.In our current research, we The red skin Pear varieties of 6 different developing stage have been selected to verify candidate gene PyMYB114 and AP2/ by RT-qPCR analyses Whether ERFs expression pattern is consistent.The forward primer of PyMYB114 RT-qPCR analyses 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 analyses is 5 '-TTGTGGAGGGAAGTGGCGGT-3 ' (SEQ ID NO.23), reverse primer are 5 '-AGCTCCCTAAGTGTTTGCATCAC-3 ' (SEQ ID NO.24). The forward primer of PyMYB10RT-qPCR analyses is 5 '-GACCAATGTGATAAGACCTCAGCC-3 ' (SEQ ID NO.27), Reverse primer is 5 '-CCGTTCTTTGTTGACGACGAC-3 ' (SEQ ID NO.28).The forward direction of PyERF3RT-qPCR analyses Primer is 5 '-CTTCCACAACCACCACAACAGC-3 ' (SEQ ID NO.7), reverse primer is 5 '- GATGGAGTGGTTGACGATGCAG-3’(SEQ ID NO.8).The forward primer of PyERF73RT-qPCR analyses is 5 '- TCCTCAGGAGAGGGAGAGCG-3 ' (SEQ ID NO.9), reverse primer is 5 '-TCCTCAGGAGAGGGAGAGCG-3 ' (SEQ ID NO.10).The forward primer of PyERF2RT-qPCR analyses is 5 '-CGATCCTCGTGCCAATGAAAATTC-3 ' (SEQ ID NO.11), reverse primer are 5 '-CTTCTCATCCCTGTAAGGAGCCT-3 ' (SEQ ID NO.12). The forward primer of PyERF27RT-qPCR analyses is 5 '-TGCGGTGGTGCTATAATCTCCG-3 ' (SEQ ID NO.13), instead It is 5 '-CGAGTCGGTGTGAGTAGTTGGG-3 ' (SEQ ID NO.14) to primer.The forward direction of PyERF113RT-qPCR analyses Primer is 5 '-GCGAGCCCAGGATTAAAGGTG-3 ' (SEQ ID NO.15), reverse primer is 5 '- CAGCTTCCGTCCAGGATGATTC-3’(SEQ ID NO.16).As a result show, only PyERF3 and PyMYB114 is in 5 pears Notable positive correlation (Fig. 1 and table 1) is all shown in kind, except the coloring of ' red bar of pears ' fruit development mid-term is obvious.Not Significant positive correlation is shown in ' red eggplant pearss ' kind with PyMYB10 and 5 AP2/ERFs gene in Pear varieties.It is worth note Meaning is that PyMYB10 and PyMYB114 shows significant positive correlation in 5 Pear varieties, except ' red early crisp ' show it is red Green alternate striped coloring.This shows that PyERF3 may regulate and control anthocyanin biosynthesis jointly with candidate gene PyMYB114.

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

The clone of the PyERF3 genes 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 genes It is long.RNA extractions use Plant Total RNA Isolation Kit Plus (Foregene, RE-05022), according to the examination The operational manual operation that agent box provides.First chain cDNA synthesis First Script Strand cDNA Synthesis SuperMix (Transgene, AE301-02) reverse transcription reagent box (specification provided according to the kit Operation).Amplification gene primer pair is 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) only praise purchased from promise Biotechnology company.The reaction system of amplification is that 50 μ L include 200ng cDNA, the μ L of 2 × Phanta Max Buffer 25, 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, adds ddH₂O to 50 μ L.PCR reactions are completed on eppendorf amplification instruments by following procedure：95 DEG C, pre-degeneration 3 is divided Clock, 95 DEG C are denatured 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. Produce a single PCR bands product.

PCR primer (is thought after 1% agarose gel electrophoresis detection with a small amount of glue reclaim kits of AxyGEN purchased from love Enter biotechnology Hangzhou Co., Ltd, China) DNA fragmentation is reclaimed, step consults and uses explanation.The DNA solution of recovery purifying Reaction, recombinase are attached with the linear pSAK277 carriers of double digestion (EcoRI/XhoI) II One Step Cloning Kit (article No.s：C112-01 Nuo Weizan biotechnologies company, and by specification step operation) are purchased from.Connection Reaction system cumulative volume is 10 μ L, including 2 μ L 5 × CE II Buffer, 50~200ng linearisation cloning vectors, and 50 ~200ng Insert Fragment amplified productions, 1 μ LII.37 DEG C of connection 30min., immediately will be anti-after the completion of question response It should be positioned in ice-water bath and cool down 5min, reaction product can be converted directly.Conversion uses thermal shock method (reference《Molecular cloning Laboratory manual》The third edition, Science Press, 2002) conversion bacillus coli DH 5 alpha, consolidate in the LB containing 50mg/L spectinomycins Screening positive clone in body flat board, 5 positive colony sequencings of picking (are completed) by Shanghai Ying Jun Bioisystech Co., Ltd.Survey Sequence result shows that PyERF3 full length genes are 1200bp, and its nucleotides sequence is classified as shown in SEQ ID NO.1, BLAST result The gene that analytical proof newly obtains from pears is an AP2/ERF gene family member, and this unnamed gene is by applicant PyERF3.Recombinant vector is named as pSAK277-PyERF3.

The template of confactor PyMYB114 clones is ' August is red ' the operatic circle skin cDNA, the forward primer sequence of PCR amplifications 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 clones is ' August is red ' the operatic circle skin cDNA, the forward primer sequence of PCR amplifications It is：5’-ACTAGTGGATCCAAAgaattcATGGCTGCACCGCCGCCAAG-3’(SEQ ID NO. 19)； 5’- CAGGACTCTAGAAGTACTctcgagTTAAGAGTCAGATTGGGGTATAATTTGATTTATC-3’ (SEQ ID NO.20)。 The template of PyMYB10 clones is ' August is red ' the operatic circle skin cDNA, the forward primer sequence of PCR amplifications：5‘- cgcggtggcggccgcggatccATGGAGGGATATAACGTTAACTTG-3 ' (SEQ ID NO.21), reverse primer sequences： 5‘-gggccccccctcgagaagcttCTATTCTTCTTTTGAATGATTCCAA-3’(SEQ ID NO.22).PCR amplifications After product is detected by 1% agarose gel electrophoresis, (pursued progress biotechnology purchased from love with a small amount of glue reclaim kits of AxyGEN Hangzhou Co., Ltd, China) recovery DNA fragmentation, the DNA solution of recovery purifying is linear with double digestion (EcoRI/XhoI) PSAK277 carriers are attached reaction, recombinaseII One Step Cloning Kit (article No.s： C112-01 Nuo Weizan biotechnologies company, and by specification step operation) are purchased from.Coupled reaction system cumulative volume is 10 μ L, Including 2 μ L 5 × CE II Buffer, 50~200ng linearisation cloning vectors, the amplification production of 50~200ng Insert Fragments Thing, 1 μ LII.37 DEG C of connection 30min.After the completion of question response, reaction is positioned in ice-water bath cooled down immediately 5min, reaction product can be converted directly.Conversion uses thermal shock method (reference《Molecular cloning experiment handbook》The third edition, science Publishing house, 2002) conversion bacillus coli DH 5 alpha, the screening positive clone in the LB solid plates containing 50mg/L spectinomycins, 5 positive colony sequencings of picking (are completed) by Shanghai Ying Jun Bioisystech Co., Ltd.Sequencing result shows, PyMYB114 genes Total length is 687bp, and its nucleotides sequence is classified as shown in SEQ ID NO.5, and structure recombinant vector is named as pSAK277- PyMYB114；PybHLH3 full length genes are 2130bp, and its nucleotides sequence is classified as shown in SEQ ID NO.6, build recombinant vector It is named as pSAK277-PybHLH3.PyMYB10 full length genes are 735bp, and its nucleotides sequence is classified as shown in SEQ ID NO.23, Structure recombinant vector is named as pSAK277-PyMYB10；Above-mentioned recombinant vector is imported into Agrobacterium GV3101 using freeze-thaw method In.

Acting factor PyMYB114 and PybHLH3 cotransformation causes the PyERF3 genes of the present invention of embodiment 3 altogether with others Tobacco anthocyanin synthesizes

The recombinant vector of structure passes through agriculture bacillus mediated instantaneous conversion tobacco leaf.As a result show, individually conversion one turns 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 Individual transcription factor cotransformation, can accelerate anthocyanin biosynthesis, show deeper color and more strong reaction (Fig. 2A). In addition, PyMYB114 can also have faint pigment deposition with PyMYB10 cotransformations.The tobacco leaf of anthocyanin accumulation passes through color Poor instrument and spectrophotometer detection pigment content (Fig. 2 B, C) is as a result consistent with phenotypic map.Therefore, PyERF3 and others are auxiliary Transcription factor PybHLH3 and PyMYB114 cotransformation is helped to promote a large amount of synthesis of tobacco leaf anthocyanin.

The PyERF3 genes of the present invention of embodiment 4 cause strawberry with other transcription factor PyMYB114 and PybHLH3 cotransformations Anthocyanin synthesizes

' Yellow wonder ' 5AF7 (YW5AF7) fruit is (2 after spending for the recombinant vector instantaneous conversion yellow strawberry of structure Week).As a result show, cotransformation PyMYB114 and PybHLH3 is it is observed that the accumulation of a small amount of anthocyanin, as PyERF3 of the present invention During with two above transcription factor PyMYB114, PybHLH3 and cotransformation, the coloring of strawberry holder injection areas is caused significantly to increase By force.Pigment accumulation is not observed after unloaded pSAK277 conversions.PyMYB10 as control have also been made similar conversion, hair The similar result that existing PyERF3 accumulates to the color and luster of PyMYB114 and PybHLH3 and cotransformation.In addition, our also cotransformations PyMYB114 and PyMYB10, find 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 measure anthocyanins.PyERF3 and PyMYB114 and PybHLH3 cotransformation cyanine Glycosides content highest (Fig. 3 B, C, D).In addition, anthocyanin component and content in Ultra Performance Liquid Chromatography instrument analysis coloring strawberry.Increase Added the PyERF3 of the present invention total Anthocyanin Content of cotransformation strawberry fruit to be up to 68 μ g/g FW, this apparently higher than Only convert PyMYB114 and PybHLH3 content.Anthocyanin Content highest corn flower 3- galactolipins in inducing strawberry fruit Glycosides, account for more than 68% (Fig. 3 D) of total Anthocyanin Content.

The PyMYB114 of embodiment 5 promotes pear flower green grass or young crops glycosides to synthesize with the homologous expression of its common acting factor

It is test material to choose ' precocious strains ' for spending latter 30 days or so, with reference to Clough et al. (1998) vacuum infiltration The carrier of the overexpression of structure is transferred to the operatic circle skin by method.As a result show, infect jointly conversion PyERF3 and PyMYB114 and PybHLH3 effect is more stronger than cotransformation PyMYB114 and PybHLH3 effect at the region that the operatic circle skin infects (Fig. 4 A).This Outside, color difference meter (Minolta chromameter, Japan) measure cotransformation PyERF3 and PyMYB114 and PybHLH3 The a*/b* in color region ratio is up to up to 0.38 (Fig. 4 B), further illustrating PyERF3 of the present invention addition strengthens flower The coloring of blue or green glycosides.The content of UPLC measure anthocyanins shows consistent with phenotypic data trend, and its main component is corn flower 3- Galactoside, and the increase of Anthocyanin Content is mainly the rise (Fig. 4 C) of corn flower 3- galactoside contents.Anthocyanin gives birth to The expression pattern of thing synthesis related gene also further analyzes (Fig. 4 D), the results showed that PyERF3 of the present invention addition, makes cyanine The different degrees of raising of key structure gene PyDFR, PyANS and the PyUFGT expression quantity of glycosides biosynthesis, promotes the operatic circle ginned cotton Blue or 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 genes further disturbs gene expression system to be verified by RNA.In order to avoid silence its Its homologous gene, choose transcription factor and be cloned close to the specific amino acid fragment at 3 ' ends, be inserted into pGreenII 62- SK transient expression vectors, construct 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 noted respectively It is mapped to ' red early crisp ' the operatic circle skin close to the maturity period.Conversion 7 days after observe PyMYB114-RNAi and PybHLH3-RNAi and Colour and reduce around the position that PyERF3-RNAi the operatic circle skin infects, pGreenII 62-SK (zero load) the operatic circles skin is not sent out The change (Fig. 4 E) of existing fruit colour.The change for infecting the fruit colour around position is determined (Fig. 4 F) with color difference meter, unloaded PGreenII 62-SK a*/b* ratio reaches 0.6, and it is negative that the a*/b* for the operatic circle skin that gene suppresses to express ratio, which is, Value, illustrates that they are all significantly lower than zero load (P<0.05).The measure of total Anthocyanin Content shows similar trend, and UPLC is surveyed The main component for determining anthocyanin is corn flower 3- galactosides, and the reduction of Anthocyanin Content is also mainly corn flower 3- half The reduction (Fig. 4 G) of lactoside content.In addition, the transcriptional level of the key gene of RT-qPCR analysis anthocyanin synthesis, as a result table Bright, compared with zero load, PyMYB114-RNAi, PybHLH3-RNAi and PyERF3-RNAi expression quantity has different degrees of Decline (Fig. 4 H).These results show that PyERF3 genes of the present invention participate in the regulation and control of the operatic circle skin anthocyanin biosynthesis.

Interaction between the Dual-luciferase reportor systerm of embodiment 6 checking PyERF3 and PyMYB114

In order to verify whether common PyERF3 of the present invention and other transcription factor cotransformations can activate anthocyanin biosynthesis generation Thank to the expression of the structural gene PyDFR, PyANS and PyUFGT of path promoter by instantaneous conversion protoplasts of Arabidopsis thaliana broken by ultrasonic, And detected with Dual-luciferase reportor systerm.As a result show, PyERF3 of the invention and PyMYB114 and PybHLH3 are total to Convert more stronger than PyMYB114 and PybHLH3 cotransformation activity (Fig. 5) to the transcriptional activation activity ability of promoter.The present invention PyERF3 and PyMYB10 and PybHLH3 cotransformations arrive similar result.In addition, PyMYB114 and PyMYB10 cotransformations Also the activity of promoter can be activated.These results also indicate that PyERF3 and PyMYB114 and PybHLH3 form transcriptional control and answered Zoarium, the promoter region of anthocyanin metabolic pathway structural gene is attached to, activates the expression of downstream gene, promote anthocyanin Synthesis in the operatic circle skin.

Interaction between the yeast two-hybrid of embodiment 7 checking PyERF3 and PyMYB114

Yeast two-hybrid is to whether there is to verify between the gene PyERF3 of the present invention and the transcription factor of cotransformation Interaction, PyMYB114 CDS full length sequences and C- or N- ends residue amino acid sequence are cloned respectively, and are inserted into pGBKT7 loads Body, checking self-excitation are activity.Having cloned the purpose of PybHLH3 and PyERF3 CDS full length sequences insert pGADT7 carriers simultaneously is Detection whether there is interactions between protein with PyMYB114.Cotransformation PybHLH3 first and PyMYB114 lacks SD-Trp-Leu's two Grown on culture medium, then transformant be transferred on four culture mediums for lacking SD-Trp-Leu-His-Ade and screened, the results showed that, The amino acid sequence (V) of PyMYB114 CDS total lengths and 160, C- ends amino acid residue (169-229) (IV) show very strong Transcriptional activation activity, N- Amino End Groups end residue MYB114^1-93And MYB114 (I)^1-160And C-terminal amino acid residue MYB114 (II)^136-229(III) transcriptional activation activity is not present.During cotransformation yeast, N- Amino End Groups end residue MYB114^1-160(II) with PybHLH3 can not only grow on the culture medium for lacking SD-Trp-Leu two, and can lack SD-Trp-Leu-His-Ade four Grown on culture medium, the site of interaction is in N- Amino End Groups end residue MYB114 between this explanation PyMYB114 and PybHLH3^1-160 (II) site (Fig. 6 A, B).Similar cotransformation yeast (Fig. 6 C) between PyMYB114 and PyERF3, the results showed that, N- and 93, C- ends amino acid residue (I, III) can not only lack two to be grown on SD-Trp-Leu culture mediums, and can lack SD- four Grown on Trp-Leu-His-Ade culture mediums.So between PyMYB114 and PyERF3 interaction site at least one, it Be respectively present in the regions of 93 amino acid residues in N- and C- ends.In addition, it is between PyERF3 and PybHLH3 to verify No interaction to be present, PyERF3 CDS full length sequences and C- or N- ends residue amino acid sequence are cloned respectively, then cotransformation respectively PybHLH3 and PyERF3 CDS full length sequences and C- or N- ends residue amino acid sequence.As a result show, PyERF3 amino acid is residual Basic sequence IV and V self-excitation is activity most strong, and remaining C- or N- ends residue amino acid sequence (I, II, III) exists with PybHLH3 Grown on two scarce two scarce SD-Trp-Leu culture mediums, but can not lack four and be grown on SD-Trp-Leu-His-Ade culture mediums, This shows the site (Fig. 6 E) that interaction is not present between PybHLH3 and PyERF3.In addition, PyMYB114 and PyMYB10 yeast Cotransformation shows interaction site between the two at the area of 93 amino acid residues (III) in PyMYB114 C- ends (Fig. 6 D).It is comprehensive Upper described, PyERF3 forms transcription control complex ERF3-MYB114- with transcription factor PybHLH3 and PyMYB114 interaction BHLH3, PyDFR, PyANS and PyUFGT promoter region are attached to, collectively promote anthocyanin biosynthesis in the operatic circle skin.

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Sequence table

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Ser Ser Ser Phe Pro Ser Ala Ser Pro Leu Ser Ala Ala Phe Ser Ser

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Thr Ala Ser Pro Ala Ser Asn Asn Trp Val Gly Gln Lys Arg Gly Arg

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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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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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

1. one kind be isolated from ' red eggplant ' pears promoted in the form of complex the operatic circle skin anthocyanin biological synthetic functional transcription because Sub- PyERF3 genes, its nucleotide sequence is as shown in SEQ ID No.1.

2. the albumen of the transcription factor PyERF3 gene codes described in claim 1, its amino acid sequence such as SEQ ID No.2 institutes Show.

3. the recombinant expression carrier containing gene described in claim 1.

4. recombinant expression carrier according to claim 3, it is characterised in that, will by right using pSAK277 as the carrier that sets out Ask obtained by between insertion EcoR I and the Xhol I sites of 1 gene.

5. the Host Strains containing gene described in claim 1.

6. clone the primer pair of gene cDNA sequence described in claim 1, it is characterised in that sense primer PyERF3-F1 sequences are such as Shown in SEQ ID No.3, anti-sense primer PyERF3-R1 sequences are as shown in SEQ ID No.4.

7. application of the PyERF3 genes in the operatic circle skin anthocyanin biosynthesis is promoted described in claim 1.

8. application according to claim 7, it is characterised in that the PyERF3 gene associations PyMYB114 described in claim 1 Application of one or two gene in the operatic circle skin anthocyanin biosynthesis is promoted in gene and PybHLH3 genes；Described PyMYB114 gene nucleotide series are as shown in SEQ ID No.5, described PybHLH3 gene nucleotide series such as SEQ ID Shown in No.6.

9. application of the recombinant expression carrier in the operatic circle skin anthocyanin biosynthesis is promoted described in claim 3.

10. application according to claim 9, it is characterised in that the recombinant expression carrier described in claim 3, which is combined, to be contained One or two carrier in the recombinant vector of PyMYB114 genes and the recombinant vector of the gene containing PybHLH3 is promoting the operatic circle skin Application in anthocyanin biosynthesis.