摘要
【目的】研究东亚飞蝗几丁质酶家族基因的时空表达特性及其在蜕皮发育中的生物学功能,筛选在飞蝗发育过程中致死性的几丁质酶基因,为实现基于RNAi的飞蝗有效控制提供重要的基础数据。【方法】在东亚飞蝗EST数据库搜索几丁质酶基因片段,用生物信息学方法分析所获得的基因片段序列;以飞蝗不同龄期第4天若虫和5龄东亚飞蝗不同组织部位为材料提取RNA并反转录为cDNA,应用RT-PCR方法研究时空表达特性;选取在表皮高表达的几丁质酶基因LmCht6,采用RNA干扰方法研究其生物学功能。【结果】基于东亚飞蝗EST库搜索和进一步的序列分析,共获得7条几丁质酶基因片段;不同组织部位与不同龄期RT-PCR结果表明这7条几丁质酶基因的时空表达特性存在明显差异,其中LmCht6主要在表皮表达;LmCht6的RNA干扰试验表明:2龄飞蝗注射dsLmCht6后,其几丁质酶基因LmCht6表达量明显降低;飞蝗从2龄到3龄的龄期转化过程中,表现为发育延迟,新表皮与旧表皮无法完全分离,导致死亡率达到72.2%。【结论】东亚飞蝗拥有多个几丁质酶基因,它们的时空表达特性存在差异;LmCht6在飞蝗蜕皮过程中具有十分重要的生物学功能,该基因被沉默后飞蝗无法完成蜕皮而导致死亡。
【Objective】In order to provide important basis for effective pests control based on RNAi,the temporal and spatial expression characterizations and the biological functions of chitinase family genes from L.migratoria manilensis were investigated and chitinase genes causing locusts death during development were screened.【Method】Chitinase-like gene fragments were obtained from locustDB and the sequences of these genes were analyzed by bioinformatics methods.The first-strand cDNAs were synthesized using RNA isolated from the fourth day of different locust stages and the various tissues of the 5th nymphs and RT-PCR was carried out to analyze the gene expression patterns.Biological functions of LmCht6 that was highly expressed in the integument were studied by RNA interference.【Result】Seven chitinase-like gene fragments were gained from locustDB.The RT-PCR results showed that the temporal and spatial expression patterns among seven chitinase genes were significantly different and LmCht6 was highly expressed in the integument.RNA interference of LmCht6 showed that the corresponding transcript level was reduced in the nymphs after being injected with LmCht6 dsRNA compared with the control insects.The nymphs injected with LmCht6 dsRNA displayed slow development and failed to detach old cuticle during molting.Consequently,72.2% of mortalities were observed.【Conclusion】More than one chitinase gene was present in locust and the temporal and spatial expression profiles of these genes were different.The RNAi results indicated that LmCht6 play an important role in the molting process of L.migratoria manilensis,and gene silencing can result in its ecdysis block and even death.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第3期485-492,共8页
Scientia Agricultura Sinica
基金
国家自然科学基金(30970410)
山西省回国留学人员科研资助项目(2009)
国家转基因生物新品种培育重大科技专项(2008ZX08003-001)
国家公益性行业科技项目(200903021)
