芸薹属原花青素跨膜转运蛋白TT12、AHA10基因家族RNA干扰载体的构建

Construction of RNAi Vectors of Brassica TT12 and AHA10 Gene Families which Encode Transmembrane Transporters of Proanthocyanidins

拟南芥TT12和AHA10基因分别编码MATE类型和H+泵类型的跨膜转运蛋白,介导原花青素单体向液泡的转运,对种皮色素的积累起重要作用。甘蓝型油菜是重要的油料作物,但缺乏黄籽基因型,现有黄籽材料表型不稳定,黄籽性状的分子机理不清,缺乏相关的分子育种。克隆了芸薹属TT12和AHA10基因家族的RNA干扰载体片段BTT12I和BA-HA10I,以基于pFGC5941而改造的植物RNA干扰基础载体pFGC5941M为骨架,构建了相应的RNAi载体pFGC5941M-BTT12I(简称pBTT12I)和pFGC5941M-BAHA10I(简称pBAHA10I),通过分子鉴定后转化农杆菌,获得工程菌株,有利于通过基因沉默技术研究TT12和AHA10基因在芸薹属种皮色素跨膜转运中的作用和机理,探索黄籽性状的分子育种。

Arabidopsis thaliana TT12 and AHA10 gene encode a MATE type and a H＋-ATPase type transmembrane transporters respectively,mediating the transporting of proanthocyanidin monomers into the vacuoles and affecting the deposition of seed coat pigments.Brassica napus is an important oil crop,but it lacks yellow-seed genotypes.Current yellow-seeded stocks have unstable phenotypes,the molecular mechanism of yellow seed trait is unclear,and little molecular breeding on this trait has been reported.In this study,fragments BTT12I and BAHA10I targeted to RNAi of Brassica TT12 and AHA10 gene families,respectively,were cloned,and the corresponding RNAi vectors pFGC5941M-BTT12I（simplified as pBTT12I）and pFGC5941M-BAHA10I（simplified as pBAHA10I）were constructed using pFGC5941M as the plant RNAi basic vector which is an improved version of pFGC5941.After molecular identifications,they were transformed into Agrobacterium to generate engineering strains,which could promote the investigation of the actions and mechanisms of TT12 and AHA10 gene in transmembrane transporting of seed coat pigments and the molecular breeding of Brassica yellow seed trait by gene silencing technology.