中國農業科學 ?? 2014, Vol. 47 ?? Issue (7): 1313-1320.doi: 10.3864/j.issn.0578-1752.2014.07.008

? 昆蟲幾丁質代謝與植物保護 ? 上一篇    下一篇

中華稻蝗幾丁質酶基因10(OcCht10)的分子特性及功能

 李大琪1, 王燕1, 張建琴1, 李濤1, 孫毅2, 張建珍1   

  1. 1、山西大學應用生物學研究所,太原 030006;
    2、山西省農業科學院生物技術研究中心,太原030031
  • 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:0351-7017098;E-mail:[email protected] E-mail:[email protected]
  • 作者簡介:李大琪,Tel:0351-7016102;E-mail:[email protected]
  • 基金資助:

    國家自然科學基金項目(31272380,31201710)、教育部博士點專項基金(博導類)(20121401110008)、教育部留學回國人員科研啟動基金(20111139)、山西省高等學校優秀青年學術帶頭人項目(2011)

Molecular Characterization and Function of Chitinase 10 Gene (OcCht10) from Oxya chinensis

 LI  Da-Qi-1, WANG  Yan-1, ZHANG  Jian-Qin-1, LI  Tao-1, SUN  Yi-2, ZHANG  Jian-Zhen-1   

  1. 1、Research Institute of Applied Biology, Shanxi University, Taiyuan 030006;
    2、Biotechnology Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031
  • Received:2013-10-24 Online:2014-04-01 Published:2013-11-05

摘要: 【目的】獲得中華稻蝗(Oxya chinensis)幾丁質酶基因10(OcCht10)的cDNA序列,預測其功能域,并做聚類分析明確該基因的系統發育關系。繪制OcCht10在5齡若蟲不同組織部位和發育時間的表達圖譜,研究其在中華稻蝗蛻皮過程中的生物學功能,為害蟲防治提供高效安全的候選基因。【方法】從中華稻蝗轉錄組數據庫搜索OcCht10 cDNA片段,經過blast分析、比對、拼接后,翻譯為氨基酸序列,預測其功能域,并與其他昆蟲幾丁質酶家族基因進行聚類分析,進一步確認該基因的系統發育關系并進行準確命名;選取中華稻蝗5齡第6天不同組織部位及5齡若蟲不同天數表皮樣本,總RNA提取后反轉錄為cDNA模板,采用Real-time quantitative PCR(qPCR)方法獲得OcCht10在5齡稻蝗若蟲不同組織部位和不同發育階段的表達譜;設計OcCht10 dsRNA引物,用試劑盒體外合成dsOcCht10,采用注射法進行RNA干擾;取注射24 h稻蝗表皮樣本,用qPCR方法檢測OcCht10基因沉默效率;并仔細觀察dsRNA注射后稻蝗表型,統計其死亡率。【結果】經對中華稻蝗轉錄組數據庫的搜索,獲得OcCht10 cDNA片段長度為9 318 bp,開放閱讀框為8 613 bp,編碼2 870個氨基酸;其3′非編碼區為705 bp,推測OcCht10 5'端有500多堿基尚未獲得;預測其氨基酸序列具有多個結構域,包含有5個幾丁質酶催化域和6個幾丁質結合域;與其他昆蟲幾丁質酶基因聚類分析結果顯示OcCht10屬于昆蟲幾丁質酶家族基因Ⅱ組,該組基因與昆蟲蛻皮相關;5齡若蟲組織特異性分析表明該基因主要在表皮、前腸和后腸等外胚層發育而成的組織器官中表達,提示OcCht10可能參與表皮幾丁質代謝;發育時間表達圖譜表明該基因在5齡第6—7天,即蛻皮前的表皮中高表達,表明OcCht10可能負責蛻皮時表皮幾丁質降解;5齡若蟲第2天注射該基因的dsRNA,24 h后取表皮檢測其干擾效率,結果顯示該基因被沉默70%;與注射dsGFP的對照組相比,注射dsOcCht10 5齡若蟲表現為齡期延長,舊表皮無法開裂,蛻皮受阻,昆蟲無法正常活動導致死亡,死亡率達到100%。【結論】獲得OcCht10的部分cDNA序列,該基因在昆蟲蛻皮前表皮中高表達;OcCht10參與中華稻蝗的蛻皮發育,注射dsOcCht10可有效沉默靶基因,并導致試蟲無法完成正常蛻皮而死亡。

關鍵詞: 中華稻蝗 , 幾丁質酶基因 , 實時定量PCR , RNA干擾

Abstract: 【Objective】 The objectives of this study are to obtain cDNA sequence of chitinase 10 gene (OcCht10) from Oxya chinensis, analyze its functional domain and phylogenetic relationship with chitinases from other known insect species, investigate its expression patterns and biological function during molting process, and to provide a new candidate gene for pest control.【Method】 cDNA fragments of OcCht10 were searched from O. chinensis’ transcriptome database. After blast analysis, the cDNA sequence of OcCht10 was assembled and translated, the functional domains of OcCht10 were predicted by bioinformatics methods. Phylogenetic analysis was performed with other insect chitinase 10 amino acid sequences. The first-stranded cDNAs were synthesized by using RNA isolated from integument of each day of 5th instar nymphs and various tissues of the 6th day in 5th instar nymphs. Reverse transcription quantitative PCR (qPCR) was carried out to analyze the gene expression patterns. Biological function of OcCht10 was studied by RNA interference method. The dsRNA primers were designed for dsOcCht10 synthesis in vitro. The dsRNAs were injected into the 2nd day of 5th instar nymphs for RNA interference, integument was dissected for silencing efficiency detection at 24 h after injection by using qPCR method. The phenotype was carefully observed and mortality was calculated till control insects molted to adults.【Result】 The obtained cDNA (9 318 bp) of OcCht10 contained an open reading frame of 8 613 bp, encoding 2 870 amino acid residues and a non-coding region of 705 bp at 3′ end. There were about 500 bp lost in 5′ end. The deduced amino acid sequence included five chitinase catalytic domains and six chitin binding domains. Phylogenetic analysis showed that OcCht10 belonged to chitinase group Ⅱ, the genes from this group were crucial for insect molting based on references. Tissue specific expression analysis of OcCht10 showed that it was predominately expressed in the integument, foregut and hindgut, which developed from ectoderm. The results suggested that OcCht10 may be involved in chitin metabolism of insect integument. Developmental expression patterns showed that OcCht10 was highly expressed before and after molting stages, lower in middle stages of 5th instar nymphs, which implied that OcCht10 could digest chitin of integument during molting process. RNA interference results indicated that the corresponding transcript level was silenced by 70% after OcCht10 dsRNA injection. Compared with the dsGFP injected control group, the nymphs injected with OcCht10 dsRNA displayed slow development and failed to detach old cuticle during molting, the mortality reached 100%.【Conclusion】 The partial cDNA sequence of OcCht10 was obtained from O. chinensis, the mRNA expression of OcCht10 was higher in the integument before molting; OcCht10 is involved in O. chinensis molting process, and dsOcCht10 injection can effectively silence mRNA expression of this gene and result in the block of ecdysis and even death of O. chinensis.

Key words: Oxya chinensis, chitinase gene, reverse transcription quantitative PCR, RNA interference

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