| 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. |
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