Functional Analysis Of Strawberry(Fragaria Vesca) Transcription Factor FvTCP9 In Fruit Ripening

The strawberry,which has high commercial and nutritive value,is an important model plant for scientific research.TCP(Teosinte branched1 Cincinnata Proliferating cell factor)transcription factor is a plant-specific gene family which plays vital roles in regulating plant growth and development.It has been reported that TCP may be related to fruit development.However,the biological functions of TCP genes were little known in strawberry.Our laboratory has identified TCP gene family in strawberry(Fragaria vesca)and 19 FvTCP genes were obtained.Based on this,FvTCP9,a gene related to the ripening of strawberry fruits,was cloned from diploid woodland strawberry accession’Heilongjiang-3’.Transient transformation technology was used to verify the function of different expression levels of FvTCP9 in fruit ripening.The molecular mechanism of FvTCP9 was studied by real-time fluorescence quantitative PCR(RT-q PCR)and protein interaction.Finally,the result showed FvTCP9 promoted fruit ripening by regulating ABA and anthocyanin signaling pathways,and this result was verified by FvTCP5 gene.The main research results are as follows:1.FvTCP9 and FvTCP5,two genes involved in fruit ripening,were cloned from diploid woodland strawberry accession ’Heilongjiang-3’.FvTCP9 and FvTCP5 respectively had a total length of 1356 bp and1320 bp,and encoded 451 and 439 amino acids.Amino acid sequence analysis revealed that they all had an atypical b HLH domain(basic Helix-Loop-Helix).2.Transient transformation technology was used to overexpress and silence FvTCP9 in strawberry fruits.Results revealed that overexpression of FvTCP9 made fruit turn red in advance and promoted fruit ripening.Silencing FvTCP9 in fruits kept the injection site white and inhibited fruit ripening.3.The physiological indexes of instant fruits were detected.Results showed that the content of ABA and anthocyanin in the over-expressed fruits was significantly higher than silent fruits.Transcriptional pattern analysis showed that FvTCP9 regulated the expression of ABA biosynthesis gene Fa NCED1 and promoted the biosynthesis of ABA in fruits.And FvTCP9 altered the transcript of three ABA related genes Fa PYR1,Fa Sn RK2 and Fa ABI5.This meant FvTCP9 was involved in regulating ABA signaling pathway.The expression of7 anthocyanin structural genes in transient fruits showed significantly difference,which was up-regulated in FvTCP9 overexpression fruit and down-regulated in FvTCP9 silenced fruit.What’s more,a yeast two-hybrid result showed that FvTCP9 protein can interact with Fa MYC1 to regulate anthocyanin signaling pathway.4.Similar results were obtained by verifying in FvTCP5,a member of strawberry TCP family Class II subfamily the same as FvTCP9.The interaction between FvTCP5 protein and Fa MYB1,Fa TTG and Fa PYR1 was detected by yeast two-hybrid screening,and the results were verified by Bi FC.FvTCP5 promoted fruit ripening by regulating ABA and anthocyanin signaling pathways.