【作者】 刘晓健；

【导师】 张建珍； 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2013， 博士

【摘要】 几丁质是昆虫体壁和中肠围食膜的主要成分,几丁质合成是一个高度复杂的生化生理过程,受激素等信号途径的调控。由于高等动物不存在几丁质,因此昆虫几丁质合成途径是设计环境友好杀虫剂的理想靶标,具有重要的研究价值。本文以飞蝗(Locusta migratoria)为研究材料,对几丁质合成关键基因谷氨酸盐：果糖-6-磷酸转氨酶(LmGfat)、 UDP-N-乙酰葡糖胺焦磷酸化酶(LmUAPs)和几丁质合成酶(LmCHSs)分子特性及转录调控进行了较为系统和深入的研究,主要内容如下：一、几丁质合成关键基因的mRNA表达特性分析利用qPCR技术分析了几丁质合成关键基因在飞蝗不同组织和发育阶段的mRNA表达特性,结果表明：LmGfat、LmUAP1和LmCHSl在若虫体壁高表达,卵发育前期和后期及若虫蜕皮后表达量较高,龄期中间表达量较低；成虫期体壁低表达,LmGfat和LmUAP1在卵巢中恒定高表达,LmCHS1随着卵巢的发育表达量逐渐降低；LmUAP2和LmCHS2分别在脂肪体、中肠和胃盲囊中高表达,且在飞蝗整个发育期稳定表达；进一步的研究发现LmCHS2在中肠前、中、后段的表达量逐渐降低,原位杂交实验显示其转录本主要在胃盲囊柱状细胞形成的顶端区域中。几丁质合成关键基因mRNA表达特性不同,表明其生理功能的差异。二、几丁质合成关键基因的功能研究利用RNA干扰技术进一步研究了几丁质合成关键基因的功能,结果表明：LmGfat、 LmUAP1和LmCHS1负责体壁几丁质的合成；注射各个基因的dsRNA后,体壁切片的组织学观察表明,与对照组相比,新表皮几丁质合成量显著减少,导致飞蝗因蜕皮困难死亡；LmCHS2基因对中肠围食膜几丁质的合成起关键作用,注射dsCHS2组中肠围食膜不完整或完全缺失,影响食物的消化吸收从而使飞蝗因饥饿死亡。三、LmBR-C和LmE74基因的分子特性及功能分析Broad-Complex和E74是蜕皮激素信号传导的早期应答基因,通过搜索飞蝗转录组数据库,获得6个BR-C和2个E74的同型异构体。以共同区域设计引物进行qPCR,结果表明：LmBR-C和LmE74具有不同的组织分布和发育阶段表达特性,在肌肉和脂肪体中表达量最高,体壁中表达量也较高；这2个基因均在卵-若虫-成虫期表达,但呈现不同的表达特性。在四龄或五龄若虫期注射LmBR-C的dsRNA后,LmBR-C基因没有明显下调,而LmE74基因的表达则被显著沉默,但E74表达下调后并未影响飞蝗的生长发育,注射dsE74的飞蝗亦能成功羽化为成虫。四、LmEcR和LmRXR基因的分子特性及功能分析LmEcR和LmRXR在脂肪体、肌肉、体壁和前肠中高表达,卵发育后期及若虫蜕皮后表达量均较高。注射dsEcR或dsRXR均导致飞蝗高致死率,五龄第2天注射dsEcR组飞蝗发育时间延迟,当对照组已完成正常蜕皮进入成虫时,处理组仍处于五龄若虫期,直至死亡。而注射dsRXR组飞蝗发育时间与对照组无明显差异,均因蜕皮困难死亡；切片的组织学观察显示,注射dsEcR组飞蝗旧表皮与皮细胞层没有分离,而注射dsRXR组飞蝗表皮与对照组相比无明显变化。五、几丁质合成关键基因的转录调控利用RNAi技术将LmEcR和LmRXR基因和早期转录因子LmE74的表达沉默后,并不影响几丁质合成关键基因(LmGfat、LmUAPl、LmCHSl和LmCHS2)的mRNA表达；解剖飞蝗体壁进行体外组织培养,加入20E后,几丁质合成关键基因的表达亦没有显著变化,表明几丁质合成关键基因不受20E的调控。研究发现进食影响LmCHS2基因的表达,饥饿处理后LmCHS2基因在中肠中的表达被显著抑制,重新进食后,该基因的表达快速上调。基于上述结果推测几丁质合成关键基因的调控模式存在差异。六、氟虫脲处理飞蝗后差异表达基因的DGE分析氟虫脲浸渍法处理飞蝗后,试虫均在蜕皮时死亡,进一步利用数字基因表达谱(Digital Gene Expression)分析氟虫脲处理前后差异表达的基因,结果显示：氟虫脲处理后差异表达基因主要集中在生物代谢,涉及蛋白、脂类、核酸、糖类等；部分几丁质酶、表皮蛋白及解毒酶系(羧酸酯酶、谷胱甘肽S-转移酶和P450)基因在氟虫脲处理后也有显著变化。分析认为氟虫脲可能通过影响多个代谢通路的基因而使飞蝗致死,这些变化基因的生物学功能和氟虫脲的潜在作用机理还有待进一步研究和探讨。更多还原

【Abstract】 Chitin is the main component of the integument and peri trophic matrix (PM) of the midgut in insects. Chitin biosynthesis is a complicated chemical and physiological process and regulated by hormone. As chitin is absent in higher animals, the pathway of insect chitin biosynthesis has attracted much research interest in serving as a potential target for developing safe and effective insecticides. In this thesis, the molecular characteristics and transcriptional regulation of key genes involved in chitin biosynthesis from Locusta migratoria were analyzed comprehensively. The genes include glutamine: fructose-6-phosphate aminotransferase (LmGfat), UDP-N-acetylglucosamine pyrophosphorylase (LmUAP) and chitin synthase (LmCHS). The main contents are as follows:1. Analysis of the mRNA expression characteristics of key genes involved in chitin biosynthesisqPCR was used to analyze the mRNA expression of key genes involved in chitin biosynthesis in different tissues and developmental stages of L. migratoria. The results showed that LmGfat, LmUAP1and LmCHS1were highly expressed in the nymphal integument. High expression was detected during early embryogenesis after eggs laid, then decreased greatly, and slowly increased before eggs hatch. During nymphal development, the highest expression of these genes appeared after molting but declined in each intermolt period and then increased before molting to the next stage. During the development of adults, they were lowly expressed in the integument but consistently expressed in the ovary of adults expect for LmCHS1. LmUAP2and LmCHS2were highly expressed in fat body, midgut and gastric caeca during the developmental stages, respectively. For LmCHS2, the decreased expression was drastic from the anterior to the posterior regions of the midgut. In situ hybridization indicated LmCHS2transcripts were mainly detected in the apical regions of brush border forming columnar cells in gastric caeca. The different expression patterns of these genes suggested they have different physiological functions. 2. Functional study on key genes involved in chitin biosynthesisThe functions of key genes involved in chitin biosynthesis were further explored by using RNAi. The results showed that LmGfat, LmUAPl and LmCHSl were responsible for chitin synthesis of the integument. Injection of dsRNA of each gene lead to the reduction of chitin synthesis in new cuticle and insects died during the molting process. LmCHS2played key roles in chitin synthesis of peritrophic matrix of the midgut. Nymphs injected with dsLmCHS2showed a significantly reduced or even loss peritrophic matrix, which hindered the food absorption and caused locusts died by the starvation.3. Molecular characteristics and functional study on LmBR-C and LmE74Broad-Complex and E74are primary response genes involved in the ecdysone signalling pathway. By searching the transcriptome database of locust, six BR-C and two E74isoforms were obtained. The common region was used to design primers. qPCR analysis showed that LmBR-C and LmE74have different tissue and developmental expression patterns. LmBR-C and LmE74were highest expressed in muscle and fat body, respectively, and also highly expressed in the integument. The mRNA expression of two genes was detectable during the developmental stages from eggs to adults, but exhibited different characteristics. When dsRNA of LmBR-C was injected into the4th and5th instar nymphs, the mRNA expression of LmBR-C did not be repressed. The mRNA expression of LmE74could be silenced after dsE74injection with30μg, however, down-regulated expression of LmE74had no effect on the development of locusts. Locusts injected with dsE74molted to adults successfully.4. Molecular characteristics and functional analysis of LmEcR and LmRXRLmEcR and LmRXR were highly expressed in the fat body, muscle, integument and foregut in late eggs and after nymphal moult. Injection of dsEcR or dsRXR resulted in high mortality. Compared to the control, the development of nymphs injected with dsEcR delayed and insects died still in the5th instar. However, there is no difference of the developmental time between nymphs injected with dsRXR and control, all insects injected with dsRXR died during the molting process. The cuticle did not separate from epidermis in dsEcR injected nymphs by histological observation. There is no remarkably difference in the histological structure of cuticle between dsRXR injected nymphs and control.5. The transcriptional regulation of key genes involved in chitin biosynthesisThe down-regulated expression of LmEcR or LmRXR or primary transcription factor LmE74did not affect the expression of LmGfat, LmUAP1, LmCHS1and LmCHS2. No significant change of the mRNA expression of these genes was detected after adding20E to the incubated integument in vitro. These results suggested that20E did not regulate the expression of key genes involved in chitin biosynthesis. However, feeding changed mRNA expression of LmCHS2. When locusts were maintained with no food, the transcript levels of LmCHS2in midguts were lower than those of control. The expression increased to the control level rapidly after feeding again. Based on these results, it is deduced that the regulation mechanism of key genes involved in chitin biosynthesis may be different.6. DGE analysis of differentially expressed genes after flufenoxuron treatmentWhen insects were treated with flufenoxuron by immersion, the insects died during the molting process, suggesting flufenoxuron is a good pesticide to control locusts. We further analyzed differently expressed genes by using Digital Gene Expression (DGE). The results showed that the differently expressed genes mainly focus on the metabolism, including proteins, lipids, nuclear acid and hydrocarbons. Some genes encoding chitinase and detoxification enzymes like carboxylesterases, gluthathione S-transferases and P450s were also significantly affected after flufenoxuron treatment. The biological meaning of the differentially expressed genes and the potential mechanism of flufenoxuron need to be further studied.更多还原