Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida)

Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17–77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control.双斑叶蝉（Amrasca biguttula biguttula (Ishida)，俗称棉叶蝉）是棉花与秋葵的重大害虫。由于缺乏基因组与转录组数据，目前学界对该昆虫的分子层面研究仍十分有限。为开展该昆虫的功能基因组学研究，我们针对15种常见持家基因（housekeeping genes）——包括Tub、B-Tub、EF alpha、GADPH、UbiCF、RP13、Ubiq、G3PD、VATPase、Actin、18s、28s、TATA、ETF、SOD及溶细胞型肌动蛋白（Cytolytic actin）——在不同发育阶段及饥饿胁迫下的表达情况进行了评估。我们选取早期（1、2龄）、晚期（3、4龄）若虫以及成虫作为实验材料，借助geNorm、NormFinder、BestKeeper及RefFinder四款软件筛选稳定表达的内参基因（reference genes）。基于上述不同算法，RP13与VATPase被确定为最适用于逆转录实时定量PCR（RT-qPCR）进行基因表达定量的内参基因。综合三款算法结果的RefFinder分析显示，成虫体内的RP13、晚期若虫体内的微管蛋白（Tubulin, Tub）、早期若虫体内的28S以及饥饿胁迫下的UbiCF为稳定性最优的内参基因。我们还建立了将双链RNA（dsRNA）掺入饲料进行饲喂的实验方法。饲喂靶向Snf7、IAP、AQP1及VATPase的dsRNA后，相较于对照组，靶标基因的沉默效率可达56.17%~77.12%，处理组昆虫的死亡率较对照组提升16%~48%。