利用RNAi抑制褐飞虱重要功能基因提高水稻抗虫性的研究

The brown planthopper (BPH) (Nilaparvata lugens Hemiptera: Delphacidae) is one of the most destructive pests of rice (Oryza sativa L.). In China, BPH has caused rice damage amounting to millions of tons per year. Cultivation and exploitation of insect-resistant rice is one of the effective control strategies. To strengthen resistance level of rice, NADH-quinone oxidoreductase 51kDa subunit (NQO) gene and the ribosomal small subunit protein 25 (RPS25) gene of BPH (biotype 3) will be selected as targets to design RNAi expression cassettes, driven by phloem-specific promoter RSs1, respectively. The endosperm-specific promoter Glub-4 will be used to control gene-scavenge enzyme gene. Expression vectors will be constructed which contains the two expression cassettes. Firstly, transgenic rice will be generated by transforming the vectors in BPH-susceptible rice MH63 with Agrobectium-mediated method. Secondly, RNAi expression level will be monitered in the rice phloem. Thirdly, temporal and spatial expression levels of NQO and RPS25 will be investigated in BPH fed with the novel rice or not. Lastly, relative growth rate, the mount of honeydew and mortality of BPH, the tolerance index, resistance index, the starch content and the mass of 1000 grains will be tested. The deletion efficiency of T-DNA in the rice endosperm will be evaluated as well. The data obtained in this work will supply a novel way in molecular breeding of highly resistant rice and attempting in solving the food safety of transgenic rice.

褐飞虱是水稻的主要害虫之一，其为害严重影响了我国稻米产量。培育抗性水稻新品种被认为是控制该害虫为害的有效途径之一。为提高水稻抗褐飞虱水平，本项目拟以生物型3褐飞虱的NADH辅酶Q氧化还原酶(NQO)基因和核糖体小亚基25蛋白(RPS25)基因为靶标，构建RNAi表达载体，使用韧皮部特异性启动子RSs1驱动RNAi表达框，胚乳特异性启动子Glub-4控制删除酶基因，转化水稻MH63以获得抗性水稻新种质。检测转化植株中RNAi分子在韧皮部表达特异性。进行褐飞虱饲喂，检测虫体内NQO、RPS25基因的时空表达变化，及RNAi分子在虫体内的组织分布特点。测定褐飞虱的相对生长率、蜜露量和死亡率；测定转基因水稻的耐虫指数和抗生指数、稻米的淀粉含量及千粒重；检测胚乳细胞中T-DNA的删除效果。本研究将为提高水稻抗褐飞虱的分子育种和转基因水稻的食品安全性探讨一条新途径。

褐飞虱是水稻的主要害虫之一，其为害严重影响了我国稻米产量。培育抗性水稻新品种被认为是控制该害虫为害的有效途径之一。为提高水稻抗褐飞虱水平，本项目拟以褐飞虱的NADH辅酶Q氧化还原酶(NQO)基因为靶标，构建RNAi表达载体，使用韧皮部特异性启动子RSs1驱动RNAi表达框，胚乳特异性启动子Glub-4控制删除酶基因，转化水稻MH63以获得抗性水稻新种质。获得了RNAi分子在韧皮部特异性表达的转化植株，结果表明RNAi分子主要在韧皮部细胞中表达，在导管博比细胞中也有低水平表达。进行褐飞虱饲喂，检测虫体内NQO基因的时空表达变化，结果表明在6、12、24、36、48、60小时的时间段内，虫体内NQO表达水平持续下降。测定褐飞虱的相对生长率、蜜露量和死亡率，结果表明取食转基因MH63后，若虫存活率显著下降，只有19.80%，即死亡率达到80.2%；4龄褐飞虱若虫在水稻HM63和转基因水稻MH63上的蜜露量分别为21和8.5 mg/d；若虫体重为负增长，相对生长率RGR几乎为0 mg.mg-1d-1。测定了遗传修饰水稻的耐虫指数和抗生指数，结果显示，被1-2龄若虫取食后，遗传修饰的苗期MH63的抗生性指数和耐虫性指数分别为0.7745和0.3392；而被3-4龄若虫取食后，该水稻苗期的抗生性指数和耐虫性指数分别为0.6833和0.4518，可见抗生性指数略有下降而耐虫性指数有所上升；但到成株期，该水稻的的抗生性指数和耐虫性指数分别为0.8052和0.7349，两个指标均有明显升高。这表明转NQO-RNAi表达框的MH63具有非常高的抗虫水平。此外检测胚乳细胞中T-DNA的删除效果初步显示胚乳细胞中无T-DNA存在的信号，删除效果良好。本研究将为提高水稻抗褐飞虱的分子育种和尝试解决遗传修饰水稻的食品安全性探讨一条新途径。