转录因子FvWRKY46通过果胶裂解酶FvPLC调控草莓果实软化的分子机理

It is an important to prevent strawberry fruit ripening and softening and extend its shelf life.Recently, we isolated a transcription factor, FvWRKY46 from diaploid strawberry fruit.It is very interested that the fruit extend its softening stage in FvWRKY46-RNAi lines and Northern blot results show that the gene expression of pectate lyase C (FvPLC) is blocked in transgenic fruit. FvPLC is screened to be one of target genes of FvWRKY46 by pull-down assay,suggesting that FvWRKY46 may be involved in the regulation in fruit softening by modulating FvPLC in strawberry. To verify this hypothesis, firstly, Electrophoretic mobility shift assay (EMSA) is to be performed to confirm the results of the pull-down assay in vitro. Subsequently, yeast one-hybrid, ChIP-PCR assays and LUC/REN will be carried out to.make sure if FvWRKY46 impacts on the promoters of FvPLC. Finally, transgenic strawberry to over-express FvWRKY46 is used to exam the gene and protein expression and pectate lyase activity and characterize the biological function of FvWRKY41 in fruit softening in strawberry. This study aims to elucidate the mechanism and pathway by which FvWRKY46 influences fruit ripening and softening in strawberry, maybe by the regulation of FvWRKY46 in the function of FvPLC protein.

防止草莓软化，延长果实货架期，是草莓生产中亟待解决的问题。在前期研究中，申请人从草莓中克隆到一个FvWRKY46转录因子，发现其基因沉默能够使果胶裂解酶FvPLC基因表达降低且阻止果实软化。用pull-down方法鉴定FvPLC是FvWRKY46的直接靶基因之一，表明FvWRKY46可能通FvPLC调控草莓果实软化。为验证该设想，本研究拟先采用电泳迁移变动分析（EMSA）验证pull-down结果；而后利用酵母单杂交、ChIP(染色质免疫沉淀)-PCR及LUC/REN双荧光瞬时表达分析从体外、体内实验确证FvWRKY46对FvPLC启动子的转录调控方式；最后，检测FvWRKY46转基因草莓果实中FvPLC基因、蛋白表达及果胶裂解酶的活性，确定FvWRKY46在果实软化中的生物学功能，解释FvWRKY46通过FvPLC调控果实软化的分子机理。

防止草莓软化，延长果实货架期，是草莓生产中亟待解决的问题。本项目通过对草莓的一个转录因子FvWRKY48的转基因研究，发现其基因沉默能够使果胶裂解酶基因表达降低且阻止果实软化。通过进一步研究酵母单杂交（Y1H），发现FvWRKY48可以与FvPLA和FvPLB基因的启动子物理结合。进而通过电泳迁移变动分析（EMSA）验证Y1H的结果；利用ChIP(染色质免疫沉淀)-PCR及LUC/REN双荧光瞬时表达分析从体外、体内实验确证FvWRKY48对FvPLA启动子的转录调控方式；最后，检测FvWRKY48转基因草莓果实中FvPLA基因、蛋白表达及果胶裂解酶的活性，确定FvWRKY48在果实软化中的生物学功能，解释FvWRKY48通过FvPLA调控果实软化的分子机理。