›› 2018, Vol. 61 ›› Issue (8): 914-922.doi: 10.16380/j.kcxb.2018.08.004

• 研究论文 • 上一篇    下一篇

二化螟碱性神经酰胺酶和中性鞘磷脂酶基因RNA干扰技术体系的优化

孙娜1,2,#, 杨琼2,#, 韩阳春2, 张茹2, 王利华2, 方继朝1,2,*   

  1.  (1. 南京农业大学植物保护学院,南京 210095; 2. 江苏省农业科学院植物保护研究所,南京 210014)
  • 出版日期:2018-08-20 发布日期:2018-08-20

Optimization of the RNAi system of alkaline ceramidase and neutral sphingomyelinase genes in Chilo suppressalis (Lepidoptera: Pyralidae)

SUN Na1,2,#, YANG Qiong2,#, HAN Yang-Chun2, ZHANG Ru2, WANG Li-Hua2, FANG Ji-Chao1,2,*   

  1. (1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 2. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
  • Online:2018-08-20 Published:2018-08-20

摘要:  【目的】比较不同方法以及不同靶标基因的RNA干扰(RNA interference, RNAi)效率,建立并优化二化螟Chilo suppressalis碱性神经酰胺酶saCER和中性鞘磷脂酶snSMase基因的RNAi的技术体系。【方法】通过培养液浸泡法将果蝇Drosophila daCER基因的dsRNA导入果蝇S2细胞内;分别通过显微注射法和荧光纳米粒子介导喂食法将体外合成的二化螟saCER和snSMase基因的特异性双链RNA(dsRNA)导入二化螟3龄(注射法)和2龄(喂食法)幼虫体内,之后利用qPCR测定靶标基因的mRNA表达水平,比较不同方法对不同基因的RNA干扰效率。【结果】将果蝇S2细胞浸泡在浓度为15 ng/μL的含daCER dsRNA细胞培养液中培养72 h后,daCER基因的表达水平下降了约84%。dsRNA (5 000 ng/μL)注射二化螟3龄幼虫60 h后对saCER基因的干扰效率达到最高(41%),dsRNA (2 500 ng/μL)注射48 h后对snSMase基因的干扰效率最高(47%);给二化螟2龄幼虫喂食dsRNA(48 μg/d)后,分别在第7天和第8天对saCER(32%)和snSMase(52%)基因达到最大干扰效率。对于aCER基因,果蝇S2细胞浸泡法与二化螟注射法和喂食法相比,干扰效率差异极其显著;使用相同方法,对saCER基因和snSMase基因的干扰效率无显著差异;对于二化螟的同一基因(saCER或者snSMase),注射法与荧光纳米粒子喂食法之间干扰效率也无显著性差异。【结论】碱性神经酰胺酶基因和中性鞘磷脂酶基因对导入dsRNA进行RNAi的方法较敏感,本研究建立并优化的显微注射法和荧光纳米粒子介导喂食法RNAi技术体系在鞘脂质代谢酶基因功能的基础研究中具有可行性。注射法和喂食法对二化螟幼虫aCER基因的干扰效率远低于浸泡法对果蝇S2细胞中这一基因的干扰效率,进一步证实二化螟血淋巴中的RNA酶对dsRNA的快速降解以及中肠围食膜的阻隔很大程度上削减了dsRNA进入二化螟细胞内发挥干扰作用。

关键词: 二化螟, 碱性神经酰胺酶, 中性鞘磷脂酶, RNAi, 显微注射法, 荧光纳米粒子介导喂食法, 浸泡法

Abstract: 【Aim】 To establish and optimize the RNAi system for alkaline ceramide (saCER) and neutral sphingomyelin (snSMase) genes of Chilo suppressalis by comparing the RNA interference efficiencies based on different methods and different target genes.【Methods】 Double-stranded specific RNA (dsRNA) of Drosophila daCER gene was introduced into Drosophila S2 cells by soaking in dsRNA-containing medium. Specific dsRNA of saCER and snSMase genes of C. suppressalis were synthesized in vitro and introduced into the 3rd instar larvae (micro-injection) and the 2nd instar larvae (fluorescent nanoparticle-mediated feeding) of C. suppressalis, respectively. The expression levels of these target genes were measured by qPCR for investigating the RNAi efficiency among different methods as well as different genes. 【Results】 The expression level of daCER gene decreased by about 84% by soaking Drosophila S2 cells in dsRNA-containing medium at a concentration of 15 ng/μL for 72 h. The RNAi efficiency of saCER gene was the highest (41%) at 60 h post injection into the 3rd instar larvae at the dsRNA concentration of 5 000 ng/μL, and that of snSMase gene was the highest (47%) at 48 h post injection at the dsRNA concentration of 2 500 ng/μL. The highest RNAi efficiencies of saCER and snSMase genes were obtained on the 7th day (32%) and the 8th day (52%), respectively, after feeding the 2nd instar larvae with dsRNA (48 μg/d). For aCER gene in Drosophila S2 cells, the interference efficiency with soaking method was significantly higher than those with injection and feeding method of C. suppressalis. There was no significant difference in the RNAi efficiency of saCER and snSMase genes using the same introduction method, and between methods of micro-injection and fluorescent nanoparticle-mediated feeding for the same gene (saCER or snSMase) of C. suppressalis. 【Conclusion】 The aCER and nSMase genes are sensitive to RNAi by introducing dsRNA both in vivo and in vitro. The protocols of RNAi via micro-injection and fluorescent nanoparticle-mediated feeding developed and optimized in this study are feasible for the basic research of enzyme genes of the sphingolipid metabolism. The RNAi efficiencies of aCER gene in C. suppressalis larvae using injection and feeding methods were significantly lower than that in Drosophila S2 cells using soaking method, further confirming that the interference efficiency of dsRNA into the C. suppressalis cells is greatly reduced due to the rapid degradation of dsRNA by RNase in hemolymph and the barrier of midgut pericardial membrane.

Key words: Chilo suppressalis; alkaline ceramidase, neutral sphingomyelinase, RNAi, micro-injection method, fluorescent nanoparticle-mediated feeding method, soaking method