芸薹属ANR(BAN)基因家族RNA干扰载体的构建

Construction of RNAi Vector of Brassica ANR (BAN) Gene Family

花青素还原酶(ANR)是原花青素生物合成的关键酶,对种皮色素的积累起重要作用。黄子是油菜的重要优质性状,但甘蓝型油菜中其基因型缺乏,表型不稳定,分子机理不清。该实验克隆了芸薹属ANR基因家族的RNA干扰片段BANRI,将其反义片段、正义片段采用NcoI+AatII、BamHI+XbaI分别插入到改进型植物RNA干扰基础载体pFGC5941M的启动子与间隔区、间隔区与终止子之间,形成重组载体pFGC5941M-BANRI(简称为pBANRI),复合PCR鉴定表明载体构建成功,并转化根癌农杆菌菌株LBA4404,获得工程菌株。pBANRI的构建有助于揭示芸薹属物种种皮色素合成的机理,探索对油菜等植物种皮色素进行分子育种的可能性。

Anthocyanidin reductase （ANR/ BAN） was a key enzyme in proanthocyanidin biosynthesis, playing an important role in the deposition of seed coat pigments. Yellow seed was an important good-quality trait, but in Brassica napus its genotypes were rare, the phenotypes were unstable, the molecular mechanisms were unclear, and no molecular breeding was reported. In this study, an RNAi fragment targeted to Brassica ANR gene family was cloned. Its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites of an improved plant RNAi basic vector pFGC5941M using NcoI ＋AatII and BamHI ＋XbaI double digestions respectively. The resultshowed recombinant vector was pFGC5941M-BANRI （simplified as pBANRI）, which was verified by multiple PCR checking and successfully transformed into Agrobacterium tumefaciens strain LBA4404 to form engineering strain. Construction of pBANRI will promote the clarifying of the mechanism and molecular breeding of seed coat pigment biosynthesis in Brassica.