转录因子ABI4调控草莓果实成熟的分子机制

[目的]探讨转录因子ABI4在草莓果实发育过程中的作用.[方法]以八倍体草莓品种’甜查理’为试材,利用基因同源克隆技术获得了1个草莓转录因子FaABI4编码基因,运用ClustalX软件和MEGA 5软件分析了草莓中FaABI4与其他物种间的序列同源性和亲缘关系,并利用荧光定量PCR技术分析了FaABI4基因随着草莓果实发育的表达模式.通过植物双元表达载体pBI121构建的基因瞬时表达系统对草莓果实中的FaABI4基因表达水平进行了调控,FaABI4基因表达量瞬时调控出现表型后,分别采取空载体pBI121和干扰载体pBI121-FaABI4侵染过的草莓果实并且去掉种子,分析其果实色素含量、脱落酸含量、果实硬度和蔗糖含量等生理指标,以及FaCHS和FaUFGT基因的表达水平变化.[结果]获得了1个编码330个氨基酸的FaABI4转录因子,与AP2家族的特征一样,含有1个57个氨基酸残基的AP2结构区域,与其他物种如梅花、葡萄和苹果的亲缘关系很近.FaABI4基因表达量在草莓果实发育前期很低,在果实发育中期和后期表达量剧烈上升.草莓果实瞬时干扰FaABI4基因14 d后发现,与注射空载体pBI121相比,注射pBI121-FaABI4的草莓果实表面出现了部分不能正常上色现象.切取不能上色部分的果肉进行生理和分子指标分析发现,与对照果实pBI121相比,pBI121-FaABI4降低了果实花色苷和蔗糖含量,促使果实硬度变大,但对ABA含量未有影响,同时降低了花色苷合成路径中的2个关键酶基因FaCHS和FaUFGT的表达水平.[结论]草莓中的转录因子FaABI4跟其他物种一样具有典型的AP2结构域,属于AP2家族.FaABI4正向调控草莓果实发育,可通过影响FaCHS和FaUFGT基因表达水平影响花色苷积累,同时可影响果实硬度及蔗糖积累,最终调控草莓果实的发育成熟进程.
[Objectives]In order to elucidate the putative function of the ABI4 transcription factor during the strawberry fruit development,RNA interfere of ABI4 gene was performed. [Methods]Octaploid strawberry cultivar(Fragaria × ananassa Duch’Sweet Charlie’)was used as test material,and one ABI4 transcription factor encoding gene FaABI4 was isolated from strawberry based on a gene homologous cloning strategy. Sequence homology of strawberry FaABI4 with other species and genetic relationship were analyzed using software ClustalX and MEGA 5. Expression patterns of FaABI4 gene during strawberry fruit development was analyzed by fluorescent quantitative PCR technology. We constructed the gene transient expression system based on the plant binary expression vector pBI121,and successfully regulated the expression level of in strawberry fruit. After the phenotype appeared in the FaABI4 gene silencing fruit,the control fruit of pBI121,and pBI121-FaABI4 infected fruits were collected,and seeds were peeled,respectively. The physiological indexes,such as fruit pigment and abscisic acid ABA content,fruit hardness,sucrose content,molecular levels changes of FaCHS and FaUFGT gene expression level,were analyzed. [Results]An ABI4 transcription factor,with 330 amino acids and similar with AP2 family characteristics,which contains a 57 amino acid residues of AP2 domain area,was isolated from strawberry fruit. It had close genetic relationship with plum,grapes and apples. The expression level of FaABI4 gene was low during the early development stage of strawberry fruit,but increased sharply during the middle and later development stages. RNA instantaneous interference of FaABI4 mRNA transcript levels was performed in strawberry fruit,and 14 days later,the pBI121-FaABI4 infected fruit