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通过RNAi策略转化小麦,以降低小麦种子中直链淀粉的含量。小麦中直链淀粉合成的关键酶是颗粒结合型淀粉合成酶(GranuleboundstarchsynthaseI,GBSSI,即WAXY蛋白),通过RTPCR方法从小麦种子中分离出Waxy基因。Southern杂交分析表明,在基因组中存在3个Waxy基因。Northern杂交分析显示出在授粉后的小麦种子中检测到WaxymRNA。利用RNA沉默策略,将Waxy编码区683bp的正向和反向片段以及150bp内含子,连接于表达载体pCAMBIA3300中玉米ubi1启动子下游。以扬麦10号授粉后15d的幼胚为外植体,利用农杆菌介导的方法进行转化。通过PCR、RTPCR和叶片离体褪绿实验鉴定出4株转基因植株。小麦胚乳I2KI染色和直链淀粉含量测定表明这4株转基因植株直链淀粉含量明显下降。研究结果表明Waxy基因的RNA沉默使转基因小麦种子直链淀粉的含量下降。
Wheat is transformed by RNAi strategy to reduce amylose content in wheat seeds. The key enzyme in the synthesis of amylose in wheat is the granule-bound starch synthase (GBSSI, WAXY protein). The Waxy gene was isolated from wheat seeds by RTPCR. Southern blot analysis showed that there are three Waxy genes in the genome. Northern blot analysis showed that WaxymRNA was detected in pollinated wheat seeds. The 683bp forward and reverse fragments of Waxy coding region and 150bp intron were ligated into the expression vector pCAMBIA3300 downstream of the ubi1 promoter by RNA silencing strategy. The young embryos 15 days after pollination of Yangmai 10 were used as explants and transformed by Agrobacterium-mediated method. Four transgenic plants were identified by PCR, RTPCR and leaf chloroplast in vitro. I2KI staining and amylose content determination of wheat endosperm showed that the amylase content of the four transgenic plants was significantly decreased. The results show that RNA silencing of Waxy gene reduces the amylose content of transgenic wheat seeds.