飞蝗几丁质合成酶2基因的表达特性、功能及调控

doi:10.3864/j.issn.0578-1752.2014.07.010

摘要/Abstract

摘要： 【目的】几丁质合成酶（chitin synthase，CHS）是昆虫几丁质合成过程中的关键酶之一，由于高等动物不存在该酶，而被认为是设计安全高效杀虫剂的潜在靶标。论文在已克隆得到飞蝗几丁质合成酶2基因cDNA序列（LmCHS2，GenBank登录号：GU067731）的基础上，进一步深入探讨该基因在不同发育时期的表达特性、功能及调控，为基于RNA干扰技术的飞蝗有效控制提供科学依据。【方法】根据已知LmCHS2 基因核苷酸序列，设计特异性表达引物，运用RT-qPCR技术研究该基因在卵、若虫及成虫期的表达特性；体外合成LmCHS2的dsRNA后，分别注射至成虫期第1天的雌虫和雄虫，收集第5天的中肠样本提取RNA，反转录成cDNA后，采用RT-qPCR方法检测LmCHS2沉默效果。解剖飞蝗整个肠道后，观察中肠的形态变化及围食膜的完整性，探讨该基因在成虫期的生物学功能；饥饿处理飞蝗不同时间后，再重新进食，观察试虫肠道的变化，进一步运用RT-qPCR技术检测LmCHS2的表达。【结果】LmCHS2在飞蝗卵发育的前期和中期几乎没有可检测到的表达，卵发育后期表达量急剧上升，在4龄、5龄若虫和成虫期稳定表达；分别对羽化后第1天的雌、雄成虫注射dsCHS2，与对照组相比，处理组试虫LmCHS2表达量均显著下降，且取食量明显减少，雌虫和雄虫死亡率分别达78%和85%；解剖消化道后发现，注射dsCHS2后飞蝗中肠几乎不含有食物，中肠和胃盲囊长度显著缩短；对中肠的组织学观察结果表明对照组飞蝗围食膜发育完整，而注射dsCHS2后围食膜被严重破坏甚至缺失；饥饿处理飞蝗48 h后，与对照组相比，饥饿组中肠几乎不含有食物，长度亦显著缩短。H&E染色结果表明，饥饿组围食膜被严重破坏，对照组围食膜结构完整，与RNAi的结果非常相似；重新进食后围食膜发育良好；饥饿处理24 h和48 h后LmCHS2的表达被显著抑制，重新进食0.5 h后，其表达量快速上调，表明进食影响LmCHS2的表达。【结论】LmCHS2参与中肠围食膜的形成，对飞蝗的生长发育至关重要，该基因的沉默影响中肠围食膜的完整性，使飞蝗对食物的消化吸收困难，最终因饥饿而死亡；此外，该基因的表达受飞蝗进食的调控。

关键词: 飞蝗 , 几丁质合成酶2基因 , 表达特性 , RNA干扰 , 调控

Abstract: 【Objective】Chitin synthase is one of the key enzymes responsible for chitin synthesis in insects. As this enzyme is absent in higher animals, it could be served as a potential target for developing safe and effective insecticides. In our earlier research, the cDNA of chitin synthase 2 gene (LmCHS2, GenBank accession number: GU067731) in Locusta migratoria was cloned. The objectives of this paper are to further study the expression, function and regulation of LmCHS2, and to provide a scientific basis for effective pest control using RNAi methods.【Method】Based on the nucleotide sequence of LmCHS2, a pair of specific expression primers was designed, the expression patterns of LmCHS2 were studied in eggs, nymphs and adults by RT-qPCR. The dsRNA of LmCHS2 was synthesized in vitro, and then injected into the female or male adults on day 1, respectively. The midguts dissected from the injected insects on day 5 were pooled for each RNA extraction. cDNA synthesis and RT-qPCR were performed to determine the down-regulation of LmCHS2. After dissected the whole gut, the midgut changes and integrity of peritrophic matrix (PM) were observed to explore the biological functions of this gene in L. migratoria adults. Locusts were maintained with no food in different times, and feeding again, to observe the changes of guts. Then the transcript levels of LmCHS2 were detected by RT-qPCR. 【Result】 LmCHS2 was almost undetectable during the early and middle embryogenesis, but dramatically up-regulated in late eggs. It was consistently expressed throughout the nymphal and adult stages. After dsCHS2 was injected into the female or male adults on day 1, significantly reduced transcript of LmCHS2 was observed as compared with that of the controls, and resulted in a decreased feeding and a high mortality of insects (78% for female and 85% for male adults). After dissection, it was found that there was virtually no food contained in dsCHS2-injected insects and the average length of midguts and gastric caeca was shorter than that of the control. Furthermore, histological observation of midguts showed that the control locusts contained a fully developed PM, however, locusts injected with dsCHS2 exhibited a disrupted PM or even absence of the PM. Locusts were treated under starvation for 48 h, the midguts hardly contained food and the average length of midguts was significantly shorter than that of the control midguts. From the H & E stained results, it was found that the PM was almost absent in non-fed midguts while the PM of control midguts was well-structured, which was very similar with the RNAi. But after fed again, the insects contained a fully developed PM. When locusts were maintained with no food for 24 h and 48 h, the transcript levels of LmCHS2 were suppressed significantly. When locusts were fed for another 0.5 h period, the transcript levels increased to the control level rapidly, which suggested that feeding affected the expression of LmCHS2. 【Conclusion】LmCHS2 is responsible for chitin biosynthesis of peritrophic matrix of the midgut and plays a key role for the development of L. migratoria. The decreased expression of this gene affected the integrity of the PM, thus hindered the food absorption and led to the mortality of the locusts. In addition, feeding regulated the expression of LmCHS2.

Key words: Locusta migratoria , chitin synthase gene , expression characteristics , RNA interference , regulation

刘晓健, 崔淼, 李大琪, 张欢欢, 杨美玲, 张建珍. 飞蝗几丁质合成酶2基因的表达特性、功能及调控[J]. 中国农业科学, 2014, 47(7): 1330-1340.

LIU Xiao-Jian, CUI Miao, LI Da-Qi, ZHANG Huan-Huan, YANG Mei-Ling, ZHANG Jian-Zhen. Expression, Function and Regulation of Chitin Synthase 2 Gene in Locusta migratoria[J]. Scientia Agricultura Sinica, 2014, 47(7): 1330-1340.

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