中國農業科學 ?? 2014, Vol. 47 ?? Issue (7): 1330-1340.doi: 10.3864/j.issn.0578-1752.2014.07.010

? 昆蟲幾丁質代謝與植物保護 ? 上一篇    下一篇

飛蝗幾丁質合成酶2基因的表達特性、功能及調控

 劉曉健, 崔淼, 李大琪, 張歡歡, 楊美玲, 張建珍   

  1. 山西大學應用生物學研究所,太原 030006
  • 收稿日期:2013-10-24 出版日期:2014-04-01 發布日期:2013-11-05
  • 通訊作者: 張建珍,Tel:0351-7018871;E-mail:[email protected] E-mail:[email protected]
  • 作者簡介:劉曉健,Tel:0351-7017846;E-mail:[email protected]
  • 基金資助:

    國家自然科學基金項目(31100925,31272380)、山西省高等學校留學回國人員科研資助項目(201163)、山西省科學技術發展計劃項目(20110311010)

Expression, Function and Regulation of Chitin Synthase 2 Gene in Locusta migratoria

 LIU  Xiao-Jian, CUI  Miao, LI  Da-Qi, ZHANG  Huan-Huan, YANG  Mei-Ling, ZHANG  Jian-Zhen   

  1. Research Institute of Applied Biology, Shanxi University, Taiyuan 030006
  • Received:2013-10-24 Online:2014-04-01 Published:2013-11-05

摘要: 【目的】幾丁質合成酶(chitin synthase,CHS)是昆蟲幾丁質合成過程中的關鍵酶之一,由于高等動物不存在該酶,而被認為是設計安全高效殺蟲劑的潛在靶標。論文在已克隆得到飛蝗幾丁質合成酶2基因cDNA序列(LmCHS2,GenBank登錄號:GU067731)的基礎上,進一步深入探討該基因在不同發育時期的表達特性、功能及調控,為基于RNA干擾技術的飛蝗有效控制提供科學依據。【方法】根據已知LmCHS2 基因核苷酸序列,設計特異性表達引物,運用RT-qPCR技術研究該基因在卵、若蟲及成蟲期的表達特性;體外合成LmCHS2的dsRNA后,分別注射至成蟲期第1天的雌蟲和雄蟲,收集第5天的中腸樣本提取RNA,反轉錄成cDNA后,采用RT-qPCR方法檢測LmCHS2沉默效果。解剖飛蝗整個腸道后,觀察中腸的形態變化及圍食膜的完整性,探討該基因在成蟲期的生物學功能;饑餓處理飛蝗不同時間后,再重新進食,觀察試蟲腸道的變化,進一步運用RT-qPCR技術檢測LmCHS2的表達。【結果】LmCHS2在飛蝗卵發育的前期和中期幾乎沒有可檢測到的表達,卵發育后期表達量急劇上升,在4齡、5齡若蟲和成蟲期穩定表達;分別對羽化后第1天的雌、雄成蟲注射dsCHS2,與對照組相比,處理組試蟲LmCHS2表達量均顯著下降,且取食量明顯減少,雌蟲和雄蟲死亡率分別達78%和85%;解剖消化道后發現,注射dsCHS2后飛蝗中腸幾乎不含有食物,中腸和胃盲囊長度顯著縮短;對中腸的組織學觀察結果表明對照組飛蝗圍食膜發育完整,而注射dsCHS2后圍食膜被嚴重破壞甚至缺失;饑餓處理飛蝗48 h后,與對照組相比,饑餓組中腸幾乎不含有食物,長度亦顯著縮短。H&E染色結果表明,饑餓組圍食膜被嚴重破壞,對照組圍食膜結構完整,與RNAi的結果非常相似;重新進食后圍食膜發育良好;饑餓處理24 h和48 h后LmCHS2的表達被顯著抑制,重新進食0.5 h后,其表達量快速上調,表明進食影響LmCHS2的表達。【結論】LmCHS2參與中腸圍食膜的形成,對飛蝗的生長發育至關重要,該基因的沉默影響中腸圍食膜的完整性,使飛蝗對食物的消化吸收困難,最終因饑餓而死亡;此外,該基因的表達受飛蝗進食的調控。

關鍵詞: 飛蝗 , 幾丁質合成酶2基因 , 表達特性 , RNA干擾 , 調控

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

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