飞蝗表皮Tweedle家族基因的功能及几丁质代谢的调控机制

发布时间：2018-06-25 21:40

本文选题：飞蝗 + 蜕皮　；参考：《山西大学》2016年硕士论文

【摘要】：昆虫表皮是抵御外界复杂环境的第一道屏障。为适应环境的变化、满足个体的成长需求,在昆虫不同的发育阶段,必须定期蜕去旧表皮并合成新表皮。昆虫表皮主要由蛋白质和几丁质构成,并通过一系列复杂的生化反应形成精细结构。本研究以我国重要农业害虫飞蝗为研究对象,通过对表皮蛋白中的Tweedle蛋白和参与几丁质代谢途径中重要的miRNA进行研究,探讨Tweedle蛋白在表皮形成和代谢中的作用以及miRNA参与几丁质代谢途径的转录后调控功能的机理。1)Tweedle蛋白是表皮蛋白家族重要成员之一,其特点是具有4个特异性保守区,能与几丁质结合。在果蝇中,当Tweedle D发生突变时,幼虫表皮合成受损,无法维持正常体型,表明Tweedle家族在表皮的合成途径中起重要作用。本研究通过搜索飞蝗转录组数据库,获得表皮蛋白基因LmTwdl1及LmTwdl2的序列,并证实这两个基因属于Tweedle家族。通过Real-Time PCR分析了 Tweedle基因表达谱,结果表明,飞蝗Tweedle基因在表皮与前肠中表达量非常高,其他组织中基本不表达;同时,表皮不同发育阶段表达变化显示,Tweedle基因在5龄前期及中期低表达,后期表达量迅速升高。原位杂交实验表明,LmTWl1转录物定位于体壁上皮细胞胞质中。为研究飞蝗Tweedle基因的生物学功能,我们进行了 RNA干扰实验。结果发现RNA干扰LmTwdl1后,飞蝗出现蜕皮异常并大量死亡。HE染色与几丁质染色结果显示,飞蝗表皮与对照相比无显著差异,而电镜结果显示,RNAi后飞蝗新表皮中的上表皮厚度变薄,原表皮几丁质排布出现紊乱。以上结果表明LmTwdl1在飞蝗的蜕皮发育中起关键作用。研究结果将有助于进一步加深对Tweedle基因功能的理解。2)几丁质代谢是表皮合成的关键步骤。几丁质合成酶(chitin synthase,CHS)与几丁质酶(chitinase,CHT)是几丁质代谢途径中的两种关键酶;miR-71与miR-263通过抑制靶基因LmCHS1及LmCHT10的表达,参与几丁质的合成与降解,并调节蜕皮发育。通过注射miRNA激动剂,分析miRNA过表达对靶基因表达量的影响,并通过原位杂交实验证明miRNA与其对应靶基因在表皮细胞中共定位,同时发现miRNA过表达或RNA干扰靶基因后,飞蝗均出现表皮蜕皮异常现象。结果表明,miRNA参与几丁质代谢途径的转录后调控。以上研究结果有助于我们进一步探索依赖LmCHS1及LmCHT10的几丁质代谢途径的机制,阐明了蜕皮发育在飞蝗生活史中的重要意义,同时也为蝗灾控制提供了一种新的思路。
[Abstract]:Insect epidermis is the first barrier against complex environment. In order to adapt to the change of environment and meet the needs of individual growth, it is necessary to shed the old epidermis and synthesize new epidermis regularly at different stages of insect development. Insect epidermis is mainly composed of protein and chitin and forms fine structure through a series of complex biochemical reactions. In this study, the Tweedle protein in epidermal protein and miRNA involved in chitin metabolism pathway were studied with locust migratory locust, an important agricultural pest in China. To explore the role of Tweedle protein in epidermal formation and metabolism and the mechanism of miRNA involved in posttranscriptional regulation of chitin metabolism pathway. 1. Tweedle protein is one of the important members of epidermal protein family, which is characterized by four specific conserved regions. Can combine with chitin. In Drosophila, when Tweedle D mutated, the biosynthesis of larval epidermis was damaged, which indicated that Tweedle family played an important role in the synthesis of epidermis. In this study, the epidermal protein genes LmTwdl1 and LmTwdl2 were obtained by searching the transcriptome database of locust migratory locust, and the two genes were confirmed to belong to the Tweedle family. The expression profile of Tweedle gene was analyzed by Real-Time PCR. The results showed that the expression of Tweedle gene was very high in epidermis and foregut, but not in other tissues. The expression changes of Tweedle gene in epidermis at different developmental stages showed that the expression of Tweedle gene was low in the early and middle stages of the 5th instar, and the expression level of Tweedle increased rapidly In situ hybridization showed that LmTWl1 transcripts were located in the cytoplasm of human wall epithelial cells. In order to study the biological function of Tweedle gene, RNA interference experiment was carried out. The results showed that after RNA interference LmTwdl1, the migration locust had abnormal ecdysis and a large number of death. The results of HE staining and chitin staining showed that there was no significant difference in the epidermis between the migratory locust epidermis and the control, but the electron microscope showed that the upper epidermis of the new locust epidermis became thinner after RNAi. The arrangement of chitin in the original epidermis was disordered. These results suggest that LmTwdl1 plays a key role in the development of decidua. The results will be helpful to further understand the function of Tweedle gene (.2) chitin metabolism is a key step in epidermal synthesis. Chitin synthase (CHS) and chitinase (chitinase) are two key enzymes in chitin metabolism pathway: miR-71 and miR-263 participate in the synthesis and degradation of chitin by inhibiting the expression of target genes LmCHS1 and LmCHT10, and regulating the development of decidua. The effect of over expression of miRNA on the expression of target gene was analyzed by injection of miRNA agonist. The results of in situ hybridization showed that miRNA and its corresponding target gene were co-located in epidermal cells, and that miRNA overexpression or RNA interference target gene was found at the same time. The abnormal epidermal molting was found in all locust migratory locust. The results showed that miRNA was involved in the posttranscriptional regulation of chitin metabolism pathway. The above results are helpful for us to further explore the mechanism of chitin metabolic pathway dependent on LmCHS1 and LmCHT10, clarify the importance of molting development in the life history of migratory locusts, and provide a new idea for locust disaster control.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q963

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