纳米粒子载体对灰飞虱RNAi效率的影响

【目的】筛选针对灰飞虱Laodelphax striatellus RNAi的dsRNA高效纳米递送载体。【方法】使用壳聚糖、碳量子点(carbon quantum dot, CQD)和lipofectamine 2000作为代表性纳米粒子，分别与dsRNA进行混合，形成稳定的3种不同复合颗粒，利用光谱法检测其dsRNA装载率。以灰飞虱膜结合型海藻糖酶基因LsStre为靶标基因来测试3种纳米粒子的RNAi效率。用不同纳米粒子包裹的dsLsStre喂食后，通过荧光实时定量PCR(qPCR)检测喂食后2 d灰飞虱2龄若虫中LsStre mRNA表达水平，并检测和计算灰飞虱2龄若虫在6 d内的校正死亡率，以裸dsLsStre引起的2龄若虫死亡率为对照，评价3种纳米载体对LsStre的RNAi增效作用。【结果】3种纳米载体均能负载dsEgfp，且这3种纳米载体复合dsEgfp的效率均在95%以上。3种纳米材料对灰飞虱2龄若虫的毒性均较弱。同未用纳米粒子包裹的裸dsLsStre喂食组(对LsStre表达量的抑制率为46%)相比，壳聚糖和CQD能够显著提高LsStre的RNAi效率(对LsStre表达量的抑制率分别为78%和84%)，而lipofectamine 2000不能显著提高LsStre的RNAi效率(抑制率为52%)。壳聚糖和CQD可以有效提高喂食dsLsStre对灰飞虱2龄若虫的致死效应，在连续喂食6 d后，灰飞虱2龄若虫的校正死亡率分别达到76%和82%，与直接用裸dsLsStre处理的对照组校正死亡率(35%)相比，增效系数分别达到2.17和2.34，lipofectamine 2000的增效能力最弱，其与dsLsStre的复合颗粒处理引起灰飞虱2龄若虫死亡率为38%，增效系数为1.09。【结论】壳聚糖和CQD纳米载体能够显著提高灰飞虱对于喂食dsRNA的敏感性，而lipofectamine 2000的RNAi增效作用较弱。研究结果有助于评价纳米载体在灰飞虱RNAi中的增效作用，为进一步开发和筛选有效的RNAi纳米载体，实现害虫绿色防控，提供了理论依据和应用策略。

Effects of nanoparticle carriers on the RNAi efficiency in the small brown plant hopper,Laodelphax striatellus(Hemiptera: Delphacidae)

【Aim】 To screen the high efficient delivery nanocarrier of dsRNA used for RNAi in Laodelphax striatellus. 【Methods】 Chitosan, carbon quantum dot (CQD), and lipofectamine 2000 were used as representative nanoparticles (NPs). The dsRNA loading efficacy of each NP was measured by spectrophotometry. The membrane-bound trehalase gene of L. striatellus, LsStre, was selected as the target gene to test the RNAi efficiency mediated by the three NPs. The mRNA expression levels of LsStre in the 2nd instar nymphs of L. striatellus at 2 d after being fed with dsLsStre mediated by different NPs were quantified by using real-time quantitative PCR (qPCR) strategy, and the corrected mortality rates induced by dsRNAs in 6 d were also detected and calculated. The synergic effects of the three NPs to RNAi of LsStre were evaluated based on the corrected mortality rates of the 2nd instar nymphs after RNAi with dsLsStre mediated by different NPs compared with that caused by naked dsLsStre (the control). 【Results】 All the three NPs could load dsEgfp, with the loading efficiencies of the three NPs coupled with dsEgfp all over 95%. All the three NPs showed low toxicity to the 2nd instar nymphs of L. striatellus. The qPCR results demonstrated that compared to the control group fed with naked dsLsStre not encapsulated with nanoparticles (causing 46% inhibition rate on the expression level of LsStre), chitosan and CQD significantly increased the RNAi efficiency of LsStre via feeding delivery, causing 78% and 84% inhibition rate, respectively, on the expression level of