利用二化螟内源microRNA培育抗二化螟转基因水稻的机理及应用研究

Transgenic plant-mediated RNAi has become a promising strategy of developing insect resistant crops, in which dsRNAs or artificial microRNAs (amiRNAs) aiming on pest target genes are overexpressed. In this project, a novel RNAi method of developing striped stemborer (SSB) resistant rice is studied. miRNAs play an important regulatory role in the growth and development of insects. It is hypothesized that the corresponding miRNA amount in SSB would rise abnormally when SSB feeds on transgenic rice overexpressing SSB endogenous miRNAs. Then the regulatory functions of miRNAs would be interfered, causing developmental abnormalities or mortality in SSB. In the previous study, 20 SSB miRNA sequences were selected and assessed, and among them 5 miRNAs caused obvious RNAi effects of the feeding SSB larvae when overexpressed in transgenic rice, including growth inhibition, abnormal eclosion, or higher mortality. The further research would be conducted including identifying the target genes of 5 selected miRNAs and studying their biological functions, developing dual amiRNA rice and evaluating their SSB resistance, and conducting SSB resistant evaluation under field conditions.

基于转基因植物表达针对害虫靶标基因的双链RNA（dsRNAs）或人工microRNAs（amiRNAs）诱导取食害虫的RNAi效应，已经成为一种重要的抗虫转基因作物培育策略。我们拟对一种新的利用RNAi培育抗二化螟水稻的方法进行研究。miRNA在昆虫的生长发育过程中扮演重要的调控角色。我们假设在水稻中超表达二化螟的miRNA，则取食该水稻的二化螟吸收水稻组织中过表达的二化螟miRNA后，体内miRNA水平异常上升，扰乱其调控功能，从而出现发育异常或致死。在前期工作中，20个二化螟miRNA序列被挑选和评价，其中5个miRNA在水稻中过表达后导致取食二化螟幼虫出现生长抑制、羽化异常、死亡率上升等RNAi表型。我们拟进一步对5个选择的miRNA开展靶标基因研究及生物学功能分析，双价amiRNA水稻培育及抗性评估，以及相应的田间抗性试验。

RNA干扰（RANi）已成为一种具有广阔应用前景的抗虫植物培育策略。利用该策略，一般在植物中过表达的靶向昆虫目标基因的双链 RNA (dsRNA)，然后基于小干扰 RNA (siRNA) 途径介导的靶基因沉默，导致取食害虫的发育异常或致死。然而，除鞘翅目外，其他大多数昆虫如鳞翅目、双翅目、膜翅目和半翅目等对dsRNA诱导的RNAi的敏感性难以预测。这成为了利用RNAi策略培育抗虫植物的主要障碍。少量的报道表明microRNA (miRNA)介导的RNAi在开发抗虫作物方面具有潜力。我们之前的研究表明，水稻主要害虫二化螟(SSB)对dsRNA诱导的RNAi具有耐受性，但对人工miRNA (amiRNA)介导的RNAi相对敏感。本研究利用amiRNA表达技术在水稻中过表达SSB内源性的miRNA获得5种表达不同二化螟内源miRNA的抗虫转基因水稻csu15, csu53, csu9, csu19, csu260。基于室内二化螟抗性评价，最终确定表达二化螟内源miRNA csu-novel-260（靶向二化螟蜕皮激素合成途径disembodied基因）的转基因水稻csu260抗虫表现最佳。通过一系列分子检测，我们挑选了两个代表性csu260品系csu260-16和csu260-18进行深入研究。小RNA测序表明，csu260-16和csu260-18中的amiRNA主要被加工理成两种20 bp和21 bp的序列。大田抗虫性测试表明，csu260-16和csu260-18的枯心率相对于非转基因亲本对照分别下降62.9%和50.4%；白穗率相对于非转基因亲本对照分别下降74.6%和54.0%。农艺性状考察显示，虽然csu260-16和csu260-18的某些农艺性状相对对照有所改变，但产量潜力无显著差异。上述结果表明本研究获得的csu260水稻具有良好商业化应用潜力。