RNAi介导的GdHsp60和GdHsp70基因沉默对沙葱萤叶甲幼虫抗寒性的影响

【目的】本研究旨在比较不同RNAi方法对沙葱萤叶甲Galeruca daurica幼虫热激蛋白基因(GdHsp60和GdHsp70)的沉默效率，以选择一种可高效降低靶基因表达水平的研究方法，明确这两个热激蛋白在沙葱萤叶甲幼虫抗寒性中的作用。【方法】分别通过饲喂法和显微注射法进行RNAi沉默沙葱萤叶甲1和2龄幼虫的GdHsp60和GdHsp70，采用qPCR检测GdHsp60和GdHsp70的沉默效率；通过显微注射法分别 对GdHsp60和GdHsp70进行RNAi后24 h，用热电偶法测定沙葱萤叶甲2龄幼虫过冷却点和结冰点，生物测定沙葱萤叶甲2龄幼虫暴露于不同低温条件下(-6~-14℃) 2 h的半致死温度(Ltemp₅₀)以及在-5℃低温条件下处理不同时间后的半致死时间(Ltime₅₀)。【结果】用饲喂法和显微注射法进行RNAi均可以降低GdHsp60和GdHsp70的表达水平，但显微注射法的沉默效率更高。与对照组(显微注射dsGFP)相比，沙葱萤叶甲2龄幼虫分别显微注射dsGdHsp60和dsGdHsp70 24 h后，GdHsp60和GdHsp70的表达水平均降至最低，分别降低了84.15%和92.38%。在沙葱萤叶甲2龄幼虫中，显微注射dsGdHsp60 24 h后其过冷却点、结冰点、Ltemp₅₀及Ltime₅₀值分别-10.56±0.42℃，-7.66±0.56℃，-8.33℃和49.25 h，显微注射dsGdHsp70 24 h后其过冷却点、结冰点、Ltemp₅₀及Ltime₅₀值分别为-9.08±0.23℃，-6.09±0.28℃, -8.20℃和52.21 h，而对照组的分别为-14.71±0.11℃，-13.94±0.09℃，-10.63℃和87.13 h。与对 照组(显微注射dsGFP)相比，在沙葱萤叶甲2龄幼虫分别显微注射dsGdHsp60和dsGdHsp70 24 h后过冷却点 、结冰点和Ltemp₅₀显著上升，而Ltime50值显著缩短。【结论】显微注射法可作为沙葱萤叶甲Hsp相关基 因RNAi的主要干扰方法；沉默GdHsp60和GdHsp70基因均会显著降低了沙葱萤叶甲幼虫的抗寒能力。

Effects of RNAi-mediated gene silencing ofGdHsp60 and GdHsp70 on the cold hardiness ofGaleruca daurica(Coleoptera: Chrysomelidae) larvae

【Aim】 This study aims to compare the efficiency of different RNAi methods forsilencing the heat shock protein genes (GdHsp60 and GdHsp70) of Galeruca daurica, so as toselect a method for efficiently reducing the expression level of target gene expression forthe functional study to clarify the function of these two genes in the cold hardiness of G.daurica larvae. 【Methods】 GdHsp60 and GdHsp70 in the 1st and 2nd instar larvae of G.daurica were silenced by RNAi via feeding and microinjection, respectively, and theirsilencing efficiencies were detected by qPCR. After RNAi of GdHsp60 and GdHsp70 bymicroinjection for 24 h, the super-cooling point and freezing point of the 2nd instarlarvae of G. daurica were determined with thermocouple method, and the median lethaltemperature (Ltemp₅₀) for the 2nd instar larvae exposed to different low temperatures (-6--14℃) for 2 h and the median lethal time (Ltime₅₀) for exposed to -5℃ for different periods were determined by bioassay. 【Results】 GdHsp60 and GdHsp70 could be silenced byRNAi via both feeding and microinjection, but microinjection-based RNAi had highersilencing efficiency. After microinjection of dsGdHsp60 and dsGdHsp70 into the 2nd instarlarvae of G. daurica for 24 h, the expression levels of GdHsp60 and GdHsp70 decreased tothe lowest point, being decreased by 84.15% and 92.38%, respectively, as compared to thatin the control (microinjected with dsGFP). In the 2nd instar larvae of G. daurica, thesuper-cooling point, freezing point, and Ltemp₅₀ and Ltime₅₀ values after microinjectionof dsGdHsp60 for 24 h were -10.56±0.42℃, -7.66±0.56℃, -8.33℃ and 49.25 h,respectively, while those after microinjection of dsGdHsp70 for 24 h were -9.08±0.23℃,-6.09±0.28℃, -8.20℃, and 52.21 h, respectively, and those in the control were 14.71±0.11℃, 13.94±0.09℃, -10.63℃ and 87.13 h, respectively. Compared with the control(microinjected with dsGFP), the super-cooling point, freezing point and Ltemp50 in the 2ndinstar larvae of G. daurica microinjected with dsGdHsp60 and dsGdHsp70 for 24 hsignificantly increased, while the Ltime₅₀value significantly shortened. 【Conclusion】Microinjection method can be used to efficiently silence the Hsp-related genes in G.daurica. Silencing GdHsp60 and GdHsp70 can significantly decrease the cold hardiness of G.daurica larvae.