中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能

doi:10.3864/j.issn.0578-1752.2014.07.009

摘要/Abstract

摘要： 【目的】研究中华稻蝗（Oxya chinensis）几丁质脱乙酰基酶（chitin deacetylase，CDA）基因的分子特性和生物学功能，为新型农药靶标筛选提供科学依据。【方法】采用生物信息学方法在稻蝗转录组数据库中搜索获得几丁质脱乙酰基酶基因cDNA序列，对其进行保守区域分析，选取赤拟谷盗（Tribolium castaneum）、果蝇（Drosophila melanogaster）、冈比亚按蚊（Anopheles gambiae）、家蚕（Bombyx mori）和云杉卷叶蛾（Choristoneura fumiferana）等昆虫物种的同源序列与中华稻蝗几丁质脱乙酰基酶氨基酸序列进行聚类分析，进一步构建系统发育树；运用real-time PCR方法检测几丁质脱乙酰基酶基因在中华稻蝗5龄若虫不同组织部位和不同发育天数表皮中的表达特性；进一步采用RNA干扰（RNAi）技术研究该基因对中华稻蝗蜕皮发育的影响。【结果】在中华稻蝗转录组数据库中搜索获得2条几丁质脱乙酰基酶基因的cDNA全长序列，生物学软件分析其氨基酸，发现2条序列均具有信号肽，开放阅读框包含3个几丁质脱乙酰基酶保守区域：几丁质结合区（ChBD）、低密度脂蛋白受体区（LDLa）和几丁质脱乙酰基催化结合域（CDA）。聚类分析表明2条序列分别与所选用的5种昆虫CDA2聚为一支。剪切子聚类分析发现2条序列分别与5种昆虫CDA2a和CDA2b相聚为一支，综合序列分析和聚类结果，认为所获得的序列属于几丁质脱乙酰基酶2基因的2个不同剪切子，分别命名为OcCDA2a和OcCDA2b。定量PCR分析表明OcCDA2a和OcCDA2b均在中华稻蝗表皮、前肠和后肠中高表达；在5龄第1天的表皮中表达量较高，之后逐步下降，蜕皮前又略有上升；RNAi研究发现5龄第2天的若虫注射dsOcCDA2a或dsOcCDA2b 24 h后，与注射dsGFP的对照相比，目的基因表达量显著降低，沉默效率分别为96.72%和80.43%；进一步观察试虫的表型，与对照组相比，注射dsOcCDA2和dsOcCDA2a的大多数虫体出现龄期延长，脊线开裂，旧表皮不能顺利剥离而导致虫体扭曲，最终死亡。少数若虫在蜕皮前死亡，未出现异常表型，部分若虫虽蜕至成虫，但四肢无力，行动缓慢。死亡率分别达到87.5%和94.4%；注射dsOcCDA2b的试虫无可见表型，可正常蜕皮发育至成虫。【结论】中华稻蝗几丁质脱乙酰基酶2基因具有2个剪切子，分别为OcCDA2a和OcCDA2b。OcCDA2a参与虫体蜕皮，对其生长发育起着重要的作用，该基因的沉默导致中华稻蝗因蜕皮困难而死亡，而OcCDA2b对中华稻蝗的生长发育没有影响。

关键词: 中华稻蝗 , 几丁质脱乙酰基酶 , mRNA表达 , RNA干扰 , 表型

Abstract: 【Objective】 In order to provide a theoretical basis for selecting novel target for pest control, molecular characteristics and biological function of chitin deacetylase 2 gene (OcCDA2) from Oxya chinensis were studied.【Method】The cDNA sequences of OcCDA2 were searched from transcriptome of O. chinensis by bioinformatics method, and the conserved domain was anaylzed, the homologous sequences from Tribolium castaneum, Drosophila melanogaster; Anopheles gambiae, Bombyx mori and Choristoneura fumiferana were selected to construct phylogenetic tree. The real-time PCR was applied to detect the relative expression of OcCDA2 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference (RNAi) was performed to study the biological function of the OcCDA2 during molting and development of O. chinensis. 【Result】The full length cDNA sequences of two chitin deacetylase genes were identified. The amino acid analysis showed that they possessed the signal peptide, the open reading frame contained three conserved domains: chitin binding domain (ChBD), low-density lipoprotein receptor chass A domain (LDLa) and chitin deacetylase catalytic domain (CDA). The phylogenetic analysis showed that two variants of OcCDA2 clustered with the CDA2s of five insect species, and the two variants differed only in one exon consisting of about 120 nucleotides, the alternatively spliced regions gathered with CDA2a or CDA2b, respectively. Based on sequence alignment and phylogenetic analysis, two variants of OcCDA2 were named as OcCDA2a and OcCDA2b. Tissue-specific expression analysis indicated that both OcCDA2a and OcCDA2b were mainly expressed in the integument, foregut and hindgut. Developmental expression patterns showed that the relative expressions of OcCDA2a and OcCDA2b were higher in the 1st day of 5th instar nymphs, then decreased gradually, and began to ascend before molting. RNAi results showed that the expression of target genes was significantly reduced in 24 h after dsOcCDA2a or dsOcCDA2b was injected into 1st day of 5th instar nymphs, the silence efficiency of genes was 96.72% and 80.43%, respectively. Compared with dsGFP injected controls, the majority of insects injected with dsOcCDA2 and dsOcCDA2a were unable to shed the old cuticle and led to body warping, finally to death. A minority of nymphs died before molting without obvious phenotypes. A few nymphs could develop to the adults, however, the legs appeared strengthless and the movement became slowly. The mortality rates were 87.5% and 94.4%, respectively. While insects injected with dsOcCDA2b alone could successfully molt to adults, and no phenotype was observed.【Conclusion】The OcCDA2 has two alternatively spliced variants, namely OcCDA2a and OcCDA2b. OcCDA2a plays a key role in the development of O. chinensis. The silence of OcCDA2a leads to the ecdysis failure and death of O. chinensis. However, OcCDA2b is not essential to molting process of O. chinensis.

Key words: Oxya chinensis , chitin deacetylase , mRNA expression , RNA interference , phenotype

于荣荣, 丁国伟, 郭亚平, 马恩波, 张建珍. 中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能[J]. 中国农业科学, 2014, 47(7): 1321-1329.

YU Rong-Rong, DING Guo-Wei, GUO Ya-Ping, MA 恩Bo, ZHANG Jian-Zhen. Molecular Characterization and Functional Analysis of Chitin Deacetylase 2 Gene in Oxya chinensis[J]. Scientia Agricultura Sinica, 2014, 47(7): 1321-1329.

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