飞蝗肠道核酸酶的特性及其对饲喂dsRNA干扰效率的影响

RNA interference (RNAi) is an emerging new strategy for insect pest management. Silencing a target gene effectively by feeding dsRNA is crucial for successful application of this technology. Locusts are extremely sensitive to RNAi by injection, but insensitive by feeding. It is hypothesized that the dsRNA degrading enzymes in the insect gut could play an important role determining the efficiency of oral RNAi. This project intends to molecularly characterize gut specific dsRNases and examine the effect of these enzymes on RNAi efficiency in migratory locust. First, dsRNase genes from L. migratoria will be identified based on EST and genome database of locust. The full length cDNA sequences of dsRNase genes will be sequenced by RACE-PCR. The gut specific dsRNase genes of locust will be selected using tissue-dependent expression analysis by RT-qPCR. Second, mRNA expression of gut specific dsRNase genes will be silenced by using RNAi to evaluate its effect on the degradation of dsRNA in midgut digestive juice. Further, the gut-specific dsRNase will be heterogeneously expressed in sf9 cells, and the purified enzyme will be used to evaluate its ability of degrading dsRNA. Third, chintinase 10 from L. migratoria (LmCht10) will be used as a target gene to investigate how dsRNase affect RNAi efficiency induced by feeding dsRNA. The silencing efficiency of LmCht10 and locust phenotype will be analyzed after feeding dsLmCht10 to locust nymphs and the expression of the dsRNase gene is suppressed by injection of its dsRNA. To further clarify the biological function of gut-specific dsRNase, transgenic locust with gut specific dsRNase gene knockout will be constructed by using CRISPR/Cas9 technology. After transgenic locusts at nymphal and adult stage are fed with dsLmCht10, the development and molting process of the insects will be observed. These results will reveal if poor RNAi efficiency by feeding dsRNA due to rapid degradation of dsRNA by dsRNases in the locust. This research is expected to facilitate the development of RNAi technology for insect pest management.

RNA干扰技术已成为植保领域害虫防治的新型策略，饲喂dsRNA能否有效沉默靶基因是该技术成功应用的关键。飞蝗体腔注射dsRNA具有明显的干扰效果，但饲喂dsRNA无效，推测昆虫肠道核酸酶是影响饲喂RNAi效率的关键因素。据此本项目拟开展飞蝗肠道核酸酶分子特性及其对饲喂dsRNA干扰效率的影响机制研究。主要内容如下：1）基于飞蝗转录组和基因组数据库，采用RACE技术克隆核酸酶基因全长cDNA；研究其组织分布，筛选肠道高表达的核酸酶基因；2）利用RNAi抑制dsRNase表达，分析其对中肠消化液降解dsRNA影响；异源表达dsRNase蛋白，体外检测其降解dsRNA的能力；3）以几丁质酶10为靶基因，研究核酸酶对饲喂靶基因dsRNA的沉默效率及表型影响；4）采用CRISPR/Cas9技术获得敲除dsRNase转基因飞蝗，深入探索该基因的生物学功能。研究结果将促进RNAi技术在害虫防治中的应用。

RNA干扰技术已成为植保领域害虫防治的新型策略，饲喂dsRNA能否有效沉默靶基因是该技术成功应用的关键。飞蝗体腔注射dsRNA具有明显的干扰效果，但饲喂dsRNA无效，推测昆虫肠道核酸酶是影响饲喂RNAi效率的关键因素。据此本项目拟开展飞蝗肠道核酸酶分子特性及其对饲喂dsRNA干扰效率的影响机制研究。本项目的主要研究内容和结果如下：1）克隆获得4条飞蝗dsRNase酶基因，分别命名为LmdsRNase1 、LmdsRNase2 、LmdsRNase3 和LmdsRNase4。组织特异表达结果显示，LmdsRNase2 和LmdsRNase3基因在飞蝗中肠高表达。2）免疫组化结果显示LmdsRNase2和LmdsRNase3蛋白定位于中肠细胞质。3）RNAi after RNAi实验结果显示：沉默LmdsRNase2 后分别饲喂两个靶基因dsRNA 的处理组虫体均出现蜕皮困难而死亡的表型，进一步采用qPCR和几丁质染色实验证实，飞蝗出现此表型的原因是靶基因表达量下降导致飞蝗表皮几丁质合成和降解受阻而产生。4）采用Bac-to-Bac昆虫细胞杆状病毒表达系统成功获得LmdsRNase2 和LmdsRNase3 融合蛋白，发现LmdsRNase2 可以在pH 6-10 的环境条件下快速降解dsRNA。5）本研究采用CRISPR/Cas9技术构建了敲除LmdsRNase2基因的转基因飞蝗，通过饲喂靶标基因dsLmCht10后，发现表皮中LmCht10表达下降90%，旧表皮降解受阻引起蜕皮困难而死亡。综上所述，昆虫dsRNases 受其生理pH条件的影响，最终影响着昆虫RNAi 的效率。本文的研究结果为昆虫RNAi 效率差异机制提供了理论基础，为利用RNAi 技术防治害虫提供了新思路和新方法，促进了RNAi 技术在害虫防治中的应用。