【作者】 唐顺明；

【导师】 李奕仁； 张志芳；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2004， 博士

【摘要】 从家蚕、野桑蚕基因组DNA克隆了LSP和HSC70-4基因启动子调控区，以luc为报告基因，构建了系列报告质粒，经脂质体转染昆虫细胞或蚕体内瞬时表达，对BmLSP和Bm／Bmand HSC70-4基因启动子的功能特性进行了分析研究，为进一步阐明LSP、HSC70-4基因的表达调控分子机理奠定了基础，同时为HSC70-4基因启动子实际应用进行了探索研究。本论文的主要研究内容如下： 一、Bm／Bmand LSP5’FR克隆与BmLSP启动子功能特性分析 克隆的Bm和Bmand LSP5’FR片段经测序分析，涵盖了LSP的第一内含子，第一外显子、核心启动子区及其5′上游区四部分；在启动子区域内有经典的TATA盒，脂肪体内组织特异性表达基因的共有序列和推定的激素响应元件，5′上游区发现了与家蚕丝素轻链基因第一内含子区域的高度同源序列和失活的部分水手转座子元件(MLE)。采用PCR法和限制性内切酶法获得了系列缺失BmLSP基因启动子，在BmN细胞瞬时表达系统考察了第一内含子(Ⅰ)、含丝素轻链基因第一内含子区域同源序列(S)以及MLE对BmLSP基因启动子的转录活性的影响，结果表明Ⅰ能显著增加启动子的转录活性达5.4-5.8倍，S增加启动子转录活性4.42倍，暗示在I、S中可能存在类似增强子的元件。而MLE则抑制启动子的转录活性。昆虫激素JHA对BmLSP启动子转录活性影响呈现剂量依赖效应，1μg／ml处理增加2.97倍，2-6μg／ml处理不影响，8μg／ml处理显著降低转录活性2.85倍；而MH处理对其活性没有显著影响。 二、Bm／Bmand HSC70-4 5’FR克隆及启动子功能特性分析 克隆的BmandHSC70-4、BmHSC70-45’FR经测序分析涵盖了部分第一外显子和启动子区：推测了可能存在的顺式作用元件有6个GATA盒、2个CAAT盒和2个热激响应因子(HSF)结合元件，但没有发现经典的TATA盒与GC盒。在常态下BmHSC70-4启动子、BmandHSC70-4启动子在BmN细胞、sf21细胞和五龄蚕体内都呈现高转录活性；37℃热激处理BmN、Sf21细胞和四龄蚕体能增加启动子的转录活性，且在sf21细胞中热激效果更为明显，而在五龄家蚕体内热激处理却抑制其转录活性；在五龄家蚕体内，JHA不影响BmHSC70-4启动子转录活性，低剂量MH处理显著提高转录活性，而高剂量(5μg MH／头)处理没有显著影响：在家蚕五龄蚕体内不同发育阶段BmHSC70-4启动子的转录活性有所波动，到熟蚕期达到最高。以上结果表明Bm、BmandHSC70-4启动子既具有组成型特征，又有一定的诱导型特征。 三、BmNPVhr3对Bm／Bmand HSC70-4启动子活性的增强功能 hr3是来源于BmNPV同源序列，具有BmNPV复制起始位点和增强子的双重功能。结果表明hr3能增强Bm／Bmand HSC70-4启动子活性，在BmN细胞内增强16-18倍，且与hr3接入方向无关：在蚕体内分别增强190.57倍，242.19倍：在五龄家蚕体内，JHA不影响BmHSCTO-4／hr3组合转录活性，不同剂量MH处理显著提高转录活性；在家蚕五龄蚕体内不同发育阶段BmHSC70-4／hr3启动子组合的转录活性有所波动，到熟蚕期达到最高，这与BmHSC70-4启动子一致，说明hr3只是增强转录活性，并不改变特性。以上结果显示HSC70-4／hr3启动子组合更适合在细胞稳定表达系统或转基因家蚕中驱动目的蛋白基因的转录表达。更多还原

【Abstract】 The regulation promoter region of LSP and HSC70-4 are cloned from genomic DNA from B. mori and B. mandarina. A series of luciferase reporter plasmids, driven by LSP and HSC70-4 promoters, are constructed, respectively. Via the transient expression system in BmN cells or silkworm transfected by lipofectin, the functional and characterestic analysis of the promoter are investigated. This will be helpful to further elucidate the regulation mechanism of LSP and HSC70-4 expression and to apply of the promoters in stable transformation cell system or transgenic silkworm.1. The Cloned BmandLSP and BmLSP 5’FR and Functional analysis of BmLSP promoterThe Cloned BmandLSP and BmLSP 5’FR, consisting of the first intron, the first exon, the core promoter region and 5’-upstream region, harbor the classic TATA box, sequences commonly found in insect genes that were specifically transcribed in fat body and the deduced ERE. The 5’-upstream region contains the homologous sequence with the first intron of Fib-L (S) and the inactive mariner like element (MLE). Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are constructed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hormones on the BmLSP promoter activity are investigated. The results show that the BmLSP promoter activity is enhanced by the intron with 5.4-5.8 folds, and by S with 4.42 folds, suggesting that the intron and S harbor the enhancer-like element. However, MLE in 5’-upstream region presents a negative effect on promoter activity. The effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity appear the typical dose-dependent manner, that is, low concentration treatments increase the BmLSP promoter activity and high concentration treatments decrease it. Meanwhile, insect ecdysone (MH) treatment present no significant effect2. The Cloned BmandHSC70-4 and BmHSC70-4 5’FR, and Characterestic and functional analysis of BmandHSC70-4 and BmHSC70-4 promotersThe Cloned BmandHSC70-4 and BmHSC70-4 5’FR, consisting of the partial first exon, the central promoter region and 5’-upstream region, harbor six GATAs, two CAATs and two HSFs, but no TATA and GC box. Under normal condition, transcriptional activities of BmandHSC70-4 or BmHSC70-4 promoter are high in BmN, Sf21 cells and the 5lh instar larvae. By heat-shock treatment at 37癈 for 2hr, the enhancements of BmandHSC70-4 or BmHSC70-4 promoter activity are achieved in BmN cells, sf21 cells and 4lh instar silkworm larvae, respectively. But heat-shock treatment for 5th instar silkworm larvae lead to a drastically decrease of the promoters’ activity. In 5th instar silkworm larvae, JHA treatments have no significant effect, and injection of low level of MH (1-3 V g per os) can increase BmHSC70-4 promoter activity, while high level of MH (5 M g per os) presents no effect. Transcriptional activity of promoter is changed with the different developmental stages and reaches the highest level at the wandering stage in 5" instar. From the above results obtained from in vitro and in vivo, we can deduceIIIthat BmandHSC70-4 or BmHSC70-4 promoter is both constitutive and inducible promoter, suggesting that its potential application in cell stable expression system or transgenic Bombyx mori.3. BmNPV hr3 enhancing BmHSC70-4 and BmandHSC70-4 promoter transcriptional activityBmNPV hr3, derived from B. mori Nucleopolyhedrovirus, functions as replication initiation site in viral DNA and enhancer for several promoters’ transcription. Here, hr3 also can increase the transcriptional activiy of the BmHSC70-4 and BmandHSC70-4 promoter by 16-18 folds in BmN cells and 190.57-242.19 folds in 5th instar larvae. The ArJ-mediated enhancement is orientation-independent.In the 5th instar larvae, MH treatments enhance the transcriptional activity of the promoter combination. The maximum stimulating effect, by 5ug MH per os treatment, is achieved about 17.54 times, compared with the cont更多还原