中华稻蝗几丁质酶基因10（OcCht10）的分子特性及功能

doi:10.3864/j.issn.0578-1752.2014.07.008

摘要/Abstract

摘要： 【目的】获得中华稻蝗（Oxya chinensis）几丁质酶基因10（OcCht10）的cDNA序列，预测其功能域，并做聚类分析明确该基因的系统发育关系。绘制OcCht10在5龄若虫不同组织部位和发育时间的表达图谱，研究其在中华稻蝗蜕皮过程中的生物学功能，为害虫防治提供高效安全的候选基因。【方法】从中华稻蝗转录组数据库搜索OcCht10 cDNA片段，经过blast分析、比对、拼接后，翻译为氨基酸序列，预测其功能域，并与其他昆虫几丁质酶家族基因进行聚类分析，进一步确认该基因的系统发育关系并进行准确命名；选取中华稻蝗5龄第6天不同组织部位及5龄若虫不同天数表皮样本，总RNA提取后反转录为cDNA模板，采用Real-time quantitative PCR（qPCR）方法获得OcCht10在5龄稻蝗若虫不同组织部位和不同发育阶段的表达谱；设计OcCht10 dsRNA引物，用试剂盒体外合成dsOcCht10，采用注射法进行RNA干扰；取注射24 h稻蝗表皮样本，用qPCR方法检测OcCht10基因沉默效率；并仔细观察dsRNA注射后稻蝗表型，统计其死亡率。【结果】经对中华稻蝗转录组数据库的搜索，获得OcCht10 cDNA片段长度为9 318 bp，开放阅读框为8 613 bp，编码2 870个氨基酸；其3′非编码区为705 bp，推测OcCht10 5'端有500多碱基尚未获得；预测其氨基酸序列具有多个结构域，包含有5个几丁质酶催化域和6个几丁质结合域；与其他昆虫几丁质酶基因聚类分析结果显示OcCht10属于昆虫几丁质酶家族基因Ⅱ组，该组基因与昆虫蜕皮相关；5龄若虫组织特异性分析表明该基因主要在表皮、前肠和后肠等外胚层发育而成的组织器官中表达，提示OcCht10可能参与表皮几丁质代谢；发育时间表达图谱表明该基因在5龄第6—7天，即蜕皮前的表皮中高表达，表明OcCht10可能负责蜕皮时表皮几丁质降解；5龄若虫第2天注射该基因的dsRNA，24 h后取表皮检测其干扰效率，结果显示该基因被沉默70%；与注射dsGFP的对照组相比，注射dsOcCht10 5龄若虫表现为龄期延长，旧表皮无法开裂，蜕皮受阻，昆虫无法正常活动导致死亡，死亡率达到100%。【结论】获得OcCht10的部分cDNA序列，该基因在昆虫蜕皮前表皮中高表达；OcCht10参与中华稻蝗的蜕皮发育，注射dsOcCht10可有效沉默靶基因，并导致试虫无法完成正常蜕皮而死亡。

关键词: 中华稻蝗 , 几丁质酶基因 , 实时定量PCR , RNA干扰

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

李大琪1, 王燕1, 张建琴1, 李涛1, 孙毅2, 张建珍1. 中华稻蝗几丁质酶基因10（OcCht10）的分子特性及功能[J]. 中国农业科学, 2014, 47(7): 1313-1320.

LI Da-Qi-1, WANG Yan-1, ZHANG Jian-Qin-1, LI Tao-1, SUN Yi-2, ZHANG Jian-Zhen-1. Molecular Characterization and Function of Chitinase 10 Gene (OcCht10) from Oxya chinensis[J]. Scientia Agricultura Sinica, 2014, 47(7): 1313-1320.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.07.008

https://www.chinaagrisci.com/CN/Y2014/V47/I7/1313

参考文献

[1]Merzendorfer H. Insect chitin synthases: a review. Journal of Comparative Physiology B, 2006, 176: 1-15.

[2]Vega H, Specht C A, Liu Y, Robbins P W. Chitinases are a multi-gene family in Aedes, Anopheles and Drosophila. Insect Molecular Biology, 1998, 7(3): 233-239.

[3]Zhu Q, Deng Y, Vanka P, Brown S J, Muthukrishnan S, Kramer K J. Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics, 2004, 20(2): 161-169.

[4]Zhu Q, Arakane Y, Banerjee D, Beeman R W, Kramer K J, Muthukrishnan S. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochemistry and Molecular Biology, 2008, 38: 452-466.

[5]Zhang J Z, Zhang X, Arakane Y, Muthukrishnan S, Kramer K J, Ma E B, Zhu K Y. Comparative genomic analysis of chitinase and chitinase-like genes in the African Malaria mosquito (Anopheles gambiae). PLoS ONE, 2011, 6(5): e19899.

[6]Nakabachi A, Shigenobu S, Miyagishima S. Chitinase-like proteins encoded in the genome of the pea aphid, Acyrthosiphon pisum. Insect Molecular Biology, 2010, 19(Suppl.2): 175-185.

[7]Pan Y, Lü P, Wang Y, Yin L J, Ma H X, Ma G H, Chen K P, He Y Q. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome. Journal of Insect Science, 2012, 12: Article 150.

[8]李大琪, 杜建中, 张建琴, 郝耀山, 刘晓健, 王亦学, 马恩波, 张建珍, 孙毅. 东亚飞蝗几丁质酶家族基因的表达特性与功能研究. 中国农业科学, 2011, 44(3): 485-492.

Li D Q, Du J Z, Zhang J Q, Hao Y S, Liu X J, Wang Y X, Ma E B, Zhang J Z, Sun Y. Study on expression characteristics and functions of chitinase family genes from Locusta migratoria manilensis (Meyen). Scientia Agricultura Sinica, 2011, 44(3): 485-492. (in Chinese)

[9]Zhu Q, Arakane Y, Beeman R W, Kramer K J, Muthukrishnan S. Functional specialization among insect chitinase family genes revealed by RNA interference. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(18): 6650-6655.

[10]Khajuria C, Buschman L L, Chen M S , Muthukrishnan S, Zhu K Y. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochemistry and Molecular Biology, 2010, 40: 621-629.

[11]Shi L, Paskewitz S M. Identification and molecular characterization of two immune-responsive chitinase-like proteins from Anopheles gambiae. Insect Molecular Biology, 2004, 13(4): 387-398.

[12]Kawamura K, Shibata T, Saget O, Peel D, Bryant P J. A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells. Development, 1999, 126: 211-219.

[13]Schultz J, Milpetz F, Bork P, Ponting C P. SMART, a simple modular architecture research tool: Identification of signaling domains. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95: 5857-5864.

[14]Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 2001, 29(9): 2002-2007.

[15]Arziman Z, Horn T, Boutros M. E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Research, 2005, 33: 582-588.

[16]Arakane Y, Muthukrishnan S. Insect chitinase and chitinase-like proteins. Cellular and Molecular Life Sciences, 2010, 67: 201-216.

[17]Yan J, Cheng Q, Narashimhan S, Li C B, Aksoy S. Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochemistry and Molecular Biology, 2002, 32: 979-989.

[18]Krishnan A, Nair P N, Jones D. Isolation, cloning, and characterization of new chitinase stored in active form in chitin-lined venom reservoir. The Journal of Biological Chemistry, 1994, 269(33): 20971-20976.

[19]Royer V, Fraichard S, Bouhin H. A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis. Biochemical Journal, 2002, 366: 921-928.

[20]Tomoyasu Y, Miller S C, Tomita S, Schoppmeier M, Grossmann D, Buche G. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium. Genome Biology, 2008, 9: R10.

[21]Dong Y, Friedrich M. Nymphal RNAi: Systemic RNAi mediated gene knockdown in juvenile grasshopper. BMC Biotechnology, 2005, 5: 25-32.

[22]Zhang J Z, Zhang J Q, Yang M L, Jia Q D, Guo Y P, Ma E B, Zhu K Y. Genomics-based approaches to screening carboxylesterase-like genes potentially involved in malathion resistance in oriental migratory locust (Locusta migratoria manilensis). Pest Management Science, 2011, 67: 183-190.

[23]Price D R G, Gatehouse J A. RNAi-mediated crop protection against insects. Cell, 2008, 26(7): 393-400.

[24]Mao Y B, Cai W J, Wang J W, Hong G J, Tao X Y, Wang L J, Huang Y P, Chen X Y. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology, 2007, 25(11): 1307-1313.

[25]Zhu J Q, Liu S M, Ma Y, Zhang J Q, Qi H S, Wei Z J, Yao Q, Zhang W Q, Li S. Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR. PLoS ONE, 2012, 7(6): e38572.

[26]Baum J A, Bogaert T, Clinton W, Heck G R, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J. Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 2007, 25(11): 1322-1326.

[27]Mao Y B, Tao X Y, Xue X Y, Wang L J, Chen X Y. Cotton plants expressing CYP6AE14 double-stranded RNA show enhanced resistance to bollworms. Transgenic Research, 2011, 20: 665-673.

[28]He B C, Chu Y, Yin M Z, Müllen K, An C J, Shen J. Fluorescent nanoparticle delivered dsRNA toward genetic control of insect pests. Advanced Materials, 2013, 25: 4580-4584.

[29]Khan A M, Ashfaq M, Kiss Z, Khan A A, Mansoor S, Falk B W. Use of recombinant Tobacco mosaic virus to achieve RNA interference in plants against the citrus mealybug Planococcus citri (Hemiptera: Pseudococcidae). PLoS ONE, 2013, 8(9): e73657.