【作者】 刘岩；

【导师】 于涟； 陆长德；

【作者基本信息】 浙江大学 ， 生物医学工程， 2006， 博士

【摘要】 家蚕(Silkworm)是最早人工饲育的经济昆虫之一。几千年来,家蚕因为其强大的合成和分泌丝蛋白(丝素和丝胶)能力而备受人类关注。丝腺是合成和分泌丝蛋白的特殊器官,从形态和功能上可以分为三部分:前部丝腺(ASG)、中部丝腺(MSG)和后部丝腺(PSG)。丝蛋白的大量合成和分泌是在五龄起第3日开始的,后部丝腺特异性的合成和分泌丝素蛋白,而中部丝腺特异性地合成和分泌丝胶蛋白。两种丝蛋白基因的时间特异性和组织特异性表达现象,引起了研究者们广泛的兴趣。基因表达受多种调控因子的多级调节,其中启动子的调控占有十分重要的地位。因此,研究丝蛋白基因启动子的功能序列对于深入了解基因的表达调控机制具有十分重要的意义。 目前已有5种丝胶基因被克隆和分离,分别被称为Ser1、Ser2、MSGS3、MSGS4和MSGS5,其中Ser1基因是丝胶蛋白的主要表达基因。Guo XY等将EGFP报告基因连接在1.6 Kbp长的Ser1基因启动子序列及信号肽序列下游,构建含上述表达框的重组杆状病毒(Acserpegfp △EGT)。五龄起蚕时,将Acserpegfp △EGT注射入家蚕体腔,通过荧光解剖显微镜观察发现:报告基因EGFP能够丝腺特异性地分泌表达。然而,Ser1基因这种组织特异性表达现象的内在机理还很不清楚。 本研究利用AcMNPV介导的体内瞬时表达方法,对Ser1基因的启动子序列进行部分删除,通过分析EGFP报告基因的表达,研究Ser1启动子序列中与丝腺组织专一性表达相关的元件。首先,对Ser1基因全长启动子进行了5’端分段顺次缺失,用PCR方法构建得到6种不同长度的Ser1启动子序列。将这6种Ser1启动子序列分别与报告基因EGFP连接构建表达盒,分别装入转移载体pFFa2中,成功获得6种重组的转移载体:pFFa2-1.2serEGFP,pFFa2-0.92serEGFP,pFFa2-0.64serEGFP,pFFa2-0.43serEGFP,pFFa2-0.27serEGFP和pFFa2-0.18serEGFP。通过Bac-to-Bac系统制备6种重组杆状病毒Ac1.2serEGFP、Ac0.92serEGFP、Ac0.64serEGFP、Ac0.43serEGFP、Ac0.27serEGFP和Ac0.18serEGFP。这些重组病毒分别经注射感染五龄起家蚕,通过荧光显微镜观测蚕组织中EGFP基因的表更多还原

【Abstract】 Bombyx mori is an economically important organism and has been used extensively in sericulture industry for the commercial production of silk. The silk glands are functionally divided into three distinct compartments, the anterior (ASG), middle (MSG) and posterior (PSG) silk glands. Fibroins, the fibroin L and H chains and fibrohexamerin (formerly known as P25), are expressed exclusively in posterior silk gland (PSG), whereas the sericins are produced specifically in MSG. In this regard, the silk gland of Bombyx mori constitutes an attractive model for studying mechanism governing spatially and temporally programmed transcription.Sericin is a group of glue proteins which ensure the cohesion of the cocoon by aticking the silk threads together. Six kinds of sericin protein molecules are coded by two genes, ser1 and ser2, through alternative splicing. The Ser1 gene is the major constituents of sericins. Within the upstream sequence of the Ser1 gene, three specific protein binding sites are well known now. SA (-103 to -85), SB (-149 to -135) and SC (-204 to -183) have been characterized by in vitro transcription and DNase I foot printing. Silk gland factor I (SGF-1) that binds to the SA site exists specifically in the silk gland extract while SGF-3/POU-M1 was found to interact with the SC site. Current knowledge concerning the Ser1 promoter and factors associated with transcription regulation has been either partially or totally derived from in vitro assays. Recently reports have shown that there are some silkworm strains susceptible to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which could serve as a novel in vivo gene delivery tool for the silkworm. Therefore developments in recombinant DNA technology and the advent of in vivo baculovirus expression system offer an approach to the study of the molecular mechanisms of transcriptional controls in silk gene in vivo.Owning to the poor understanding of mechanism responsible for thetissue-specific expression of the Ser\ gene, the present studies are carried to analyze the related region in the sericin-1 gene promoter. In the present study, we address to explore the possible cis-acting region(s) that takes part in determining the tissue-specific expression of the sericin-1 gene in vivo.To identify element involved in this transcription-dependent spatial restriction, EGFP reporter gene with SerJ promoter truncated in 5’ terminal is introduced into the larvae with recombinant AcMNPV vector. A 209 bp DNA sequence upstream from the transcriptional start site (-586~-378) is found necessary to suppress the ectopic expresson. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500~-476) suppresses the ectopic expression of the Serl promoter. Moreover, electrophoretic mobility shift assays (EMSA) were performed for a preliminarily analysis of the protein binding to this inhibitory region. Results showed that an unknown factor that binds to this 25 bp fragment is abundant in fat body. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the sericin-1 promoter.Together with what have been reported, we supposed that there have been two kinds of mechanism involved in the control the expression of silk genes, they are activating in silk gland and repressing in other tissues. The specific expression of the sericin-1 gene could be the result of the co-ordination of multiple factors and cis-acting elements. This still needs further investigate.In general, the present studies may not only contribute to understanding the mechanisms of tissue-specific gene expression and benefit to improving the future production of silk-manufacture industry, but also do good to develop silk gland bioreactor in the future.更多还原