干扰( RNA interference, RNAi)是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。本研究以稻纵卷叶螟可溶型海藻糖酶基因( CmTre1)为靶标,设计RNAi靶位点并将其命名为CmTre1∗,在其两端分别添加BamH I与Spe I酶切位点并进行人工合成,然后经双酶切后与p1301植物表达载体连接,构建重组表达载体p1301-CmTre1∗。 PCR、双酶切和测序鉴定结果证明重组表达载体p1301-CmTre1∗构建成功,并将其成功转入农杆菌LBA4404,构建基因工程菌。用含有p1301-CmTre1∗质粒的农杆菌LBA4404浸染中花11号水稻的愈伤组织,经过愈伤组织的抗性筛选、分化和植株再生,获得26株阳性转基因植株。用取食非转基因水稻的稻纵卷叶螟作为对照,用实时荧光定量 PCR 方法分株测定取食转基因水稻稻纵卷叶螟体内CmTre1基因的mRNA表达水平,并检测其体内海藻糖酶活性变化。结果显示,相较于对照组,取食转基因水稻稻纵卷叶螟体内CmTre1基因表达量下降了44．79%,海藻糖酶活力平均下降了14．94%。研究结果表明转基因水稻的RNA干扰效应相当明显,能对取食其叶片的稻纵卷叶螟CmTre1基因起到明显的沉默作用。%RNA interference ( RNAi) refers to a biological phenomenon in which RNA molecules inhibit gene expression, typically by causing the degradation of homologous mRNA molecules. RNAi is a gene silencing process induced by double-stranded RNA molecules and is a highly evolutionarily conserved mechanism of gene regulation. In this paper, target sequence specific to soluble trehalase gene (CmTre1) from Cnaphalocr-ocis medinalis was designed and synthesized with BamH I and Spe I restriction enzyme cutting sites at both sides, and was named CmTre1∗. After double enzyme digestion, this fragment was inserted into p1301 to construct recombinant expression vector p1301-CmTre1∗. The results of PCR, enzyme digestion, and se-quencing showed that the recombinant vector had been successfully constructed. Then this vector p1301 -CmTre1∗was transformed into Agrobacterium tumefaciens LBA4404 to construct genetic engineering bacteria. The calli of rice Zhonghua 11 were infected by A. tumefaciens LBA4404 that contained p1301-CmTre1∗. Then the calli were screened by higromycin. After differentiation of the resistant calli and and plant regenera-tion, 26 positive transgenic rice plants were obtained. CmTre1 mRNA expression levels in C. medinalis that fed on these transgenic rice were detected with real time quantitative PCR ( RT-qPCR) and trehalase activi-ties in vivo were tested by the Trehalase Kit, using C. medinalis that fed on non-transgenic rice as controls. The results showed that the average expression level of CmTre1 in C. medinalis feeding on transgenic rice de-creased by 44. 79% and the average trehalase activity in vivo fell by 14. 94%, compared to the control groups. The findings indicated that the transgenic rice plants containing the interference sequence CmTre1∗could strongly silence CmTre1 in C. medinalis that fed on these transgenic rice.
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