【作者】 甘丽萍；

【导师】 徐世清； 司马杨虎；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2011， 博士

【摘要】 家蚕(Bombyx mori)作为生产用经济昆虫,雄性在体质、丝蛋白质合成效率和蚕丝品质等方面都优于雌性,其调控机理目前还了解很少。限性遗传是家蚕的一种应用广泛的遗传方式,其中茧色限性遗传方式,与斑纹限性一样广泛用于蚕种生产过程的雌雄分离制种。家蚕天然有色茧是具有重大应用前景的遗传资源,有色茧的茧丝颜色种类、色度、色牢度、色匀度等茧色相关遗传控制,不仅仅受数十个(推测)色素结合与转运主基因控制,还受到更加复杂的基因网络影响,分子遗传研究表明相关基因未完全确认。家蚕黄茧限性品种(雌为黄茧,雄为白茧),为研究黄、白茧之间差异表达基因提供了非常好的材料。本文使用该系统Ysh蚕品种,构建了不同性别的LongSAGE表达谱文库,筛选了差异表达基因。通过构建的apEST电子表达谱、在线芯片表达谱平台(BmMDB)的查询以及RT-PCR、Real-Time PCR、免疫组化和Western blotting等实验技术,调查了雌雄差异表达基因在Ysh及其它几个品种中的时空表达谱、激素处理和限食处理后的基因调控变化,测定了相关品种组织中的总蛋白酶和胰蛋白酶活性,并对丝氨酸蛋白酶前体(SPP)和丝氨酸蛋白酶抑制剂1(SPI1)进行了蛋白水平上的调查,获得的主要结果如下。1 apEST提供了电子表达谱的查询与分析平台构建了基于家蚕dbEST为数据源(截止2011年3月20有316,223条EST)的基因电子表达谱分析平台(apEST)(http://jysw.suda.edu.cn/bombyx/Download.html)。通过和其它基因表达谱分析平台的比较,明确了其可靠性和可行性。该平台能为挖掘特异性基因提供有利参考,在本实验室已进行了广泛的应用。2构建了黄茧限性系统雌雄的Long SAGE文库根据家蚕幼虫色素快速积累时期和组织特异性,构建了以整个5龄幼虫期中肠和丝腺为材料的的Ysh蚕品种雌雄Long SAGE文库,总共获得了32,099个标签,筛选出202个差异标签(Tag),其中108个Tag在雌性中上调表达,94个Tag在雄性中上调表达。借助家蚕全基因组公共数据库,通过参考文库和改进的GLGI方法,对其中76个Tag进行了注释,为茧色相关基因发掘,特别是色素结合与转运主基因以外的新基因发掘创造了平台。SAGE文库提供的大量性别差异标签,得到其全长并进行功能研究对于阐明限性黄茧品种雌雄差异性状机理是有价值的。3丝氨酸蛋白酶(SPs)和丝氨酸蛋白酶抑制剂(Serpins)是互作关系的酶类在家蚕Ysh品种5龄前中期,幼虫中肠特异表达的SPs基因的mRNA表达量,以及胰蛋白酶活性都高于5龄末期,也高于眠期,同时眠期检测不到SPP蛋白的表达。Spp基因受饥饿处理的瞬时调控,推测食物进入中肠后诱导了参与中肠食物蛋白质消化与吸收的SPs分泌,后者又启动了SPs基因的表达,而停止喂食,蛋白酶分泌也停止。SPs的表达特征提供了SPs参与中肠食物蛋白的分子证据。SPs基因在5龄后期雌蚕的中肠表达量高于雄性,推测与雌性造卵需要更多的蛋白质营养有关。5龄中后期出现的丝腺特异表达的Serpins基因表达活性,在分子水平上印证了家蚕吐丝前需要Serpins保护丝腺中大量积累的丝物质不被蛋白酶降解的生理现象,不同时期和组织间(MSG和PSG)所需Serpins种类差异,体现了家蚕复杂的丝物质合成与储藏调控网络。而Spi1基因在雄性丝腺中表达和蛋白质翻译的持续时间比雌性中长,推测与雄性丝物质质量和数量比雌性有优势有关。外源性的蜕皮激素(MH)处理5龄3日家蚕幼虫,SPs和Serpins基因呈现不同的调控机制,保幼激素类似物(JHA)对这2个基因的表达具有类似的正调控作用。激素对基因的调控主要与发育时期及激素剂量有关。Spi1基因在幼虫饥饿处理后出现下调比Spp晚,暗示Serpins受取食的直接影响比SPs慢,也说明了食物供需对丝腺的反应迟于对中肠的影响。SPs和Serpins基因的这种组织表达特异性,为它们参与相应组织中的特异性转录调控提供了佐证,而发育时期表达差异和激素处理后调控变化体现了基因执行功能上的时序性和特殊性。Serpins中的许多成员通过抑制SPs的同功酶来参与SPs的活性调控,保持体内环境的平衡。4推测表皮蛋白基因CPH45克隆与功能研究根据SAGE文库中标签GCTTCCGCCGTGCCTGC(Tag.1161),克隆到了一个长度为676 bp的全长cDNA序列,序列分析只有1个外显子,没有内含子,推导的编码蛋白序列长度为165AA,位于家蚕第19号染色体的nscaf 2 767上。该基因在中肠特异表达,与家蚕CPH家族的4条蛋白序列同源性在42%以上,根据已有的家蚕CPH基因名称顺序,新基因命名为中肠特异表达的CPH基因(CPH45)。CPH45表达量在Ysh品种雄性显著高于雌性,受到5龄中期外源性MH的负调控和JHA的正调控,雄性对外源昆虫激素的感受更敏感。食物的供缺对CPH45基因的表达水平没有显著影响,推测其不直接参与食物的消化。功能研究表明CPH45在维持家蚕中肠形态中发挥作用。CPH45基因在蚕品种之间的表达差异显示,它在不同的品种中参与路径的位点不同,作用可能存在差异。5保幼激素甲基转移酶基因JHAMT2克隆与分析根据SAGE文库中标签序列GAAACAGCACGCACGCT(Tag.1221),克隆到了一个长度为1 007 bp的全长cDNA序列,序列分析有4个外显子,定位于家蚕第12号染色体的nscaf 2 993上。推导的编码蛋白序列长度为266AA,有一个甲基转移酶超家族功能位点,与烟草天蛾(Manduca sexta)JHAMT同源性达到38%,因此克隆基因命名为家蚕保幼激素甲基转移酶基因(JHAMT2)。JHAMT2基因在丝腺、特别是中部丝腺组织特异表达,发育时期表现为4龄期有比较高的表达量,5龄期只在5龄第3日有表达,性别差异表现为雄性体内持续表达时间比雌性中长。JHAMT2基因时期表达特征在分子水平上印证了家蚕JH的合成在4龄期比较旺盛,在5龄第3日后受到抑制,对位于其合成上游的JHAMT需要量也相应的降低。更多还原

【Abstract】 The domesticated silkworm, Bombyx mori, serves as an importantly economic insect. As is known to all, male silkworms are better than females in respect of physical qualities, synthetic efficiency of silk proteins and silk qualities. At present, the regulatory mechanisms are not thoroughly clear. The sex-limited heredity is widely obtained in Bombyx mori, in which, the cocoon and color sex-limited inherited method, like the stripe sex-limited, is applied to separate males and females in silkworm breeding. Natural colored-cocoons are genetic resources with a promising and significant application future. Genetic controls related to silk color kinds, chroma, color fastness, color well degrees are influenced not only by tens main genes (predicted) of pigment combination and transportation, but more complicated gene nets. Molecular genetics suggests that it hasn’t been confirmed of the related genes yet. The yellow cocoon sex-limited strain (female cocoons are yellow while male cocoons are white) provides excellent materials to research differently expressed genes between yellow and white cocoons.This study constructed LongSAGE libraries of Ysh strain and screened differently expressed genes. In this study, the spatio-temporal expression patterns and gene regulation changes after the treatment with hormone and food limitation were investigated, meanwhile, the activities of total proteinase and trypsin were measured with two representative genes researched at the level of proteins by constructing apEST electronic expression profile, querying online BmMDB and some experimental technologies, such as RT-PCR, Real-Time PCR, immunohistochemistry and Western blotting. The main results are as follows:1 apEST provides the platform to obtain and analyze electronic expression profilesGene electronic expression profile analysis platform(apEST)was constructed based on the data resources of Bombyx mori dbEST( http://jysw.suda.edu.cn/bombyx/Download.html). (316,223 ESTs by Mar. 20, 2011). Compared with other platforms providing gene electronic expression profile analysis, the reliability and feasibility of ours have been ensured. This platform can provide advantageous references to unearth special genes, which has been widely used in our laboratory.2 The construction of Long SAGE libraries between the males and females of the yellow cocoon sex-limited strain, YshAccording to the tissue specificity and the stage when pigment is rapidly accumulated in larvae, the Long SAGE libraries between the males and females were constructed based on the materials of the midgut and silkgland of the fifth instar larvae. A total number of 32,099 tags were obtained, of which 202 differently expressed tags were singled out. Among these tags, 108 were up-regulated in females while 94 were up-regulated in males. And 76 of these tags were annotated with the help of reference libraries and the improved GLGI methods, which created a platform for cocoon color-related genes exploration, especially for new ones besides main genes involved in the pigment combination and transportation. Since a large quantity of sexual different tags are provided by SAGE libraries, it is valuable to clone the full length of these tags and research the functions of them which will definitely contribute to characterize the mechanisms of the character difference between males and females of the yellow cocoon sex-limited strain, Ysh.3 Serine protease (SPs) and Serine protease inhibitor (Serpins) belong to interactional enzymesDuring early and medium stages of 5th instar lavae of Ysh,the especially expressed mRNA levels of SPs precursor gene (Spp) in the midgut was higher than the last stages of the fifth instar larvae as well as the molting stages which was the same of the activity of trypsin. Meanwhile, proteins could not be detected in the molting stages. Spp is transiently regulated by the hunger treatment suggesting that the food induced the secretion of SPs, which participated in protein digestion and absorption, thereby started the expression of SPs genes. When feeding stopped, the secretion of proteinases stopped, either. Thus, the expression character of SPs provided molecular evidence that SPs participated in food protein digestion. The SPs gene expression level in female is higher than that of male in the midgut of the last stages of the fifth instar larvae, suggesting that the females need more protein nutrition during oviposition.The specifically expressed activity of Serpins showed in the silkgland of the middle and late stage of the fifth instar, confirming the physiological phenomenon that a large number of Serpins was needed to protect mass of accumulated silk materials in the silkgland from the degradation by proteinase bofore spinning. The differences between Serpins types needed in different periods and tissues (MSG and PSG) reflected the complicated synthesizing and storing regulation nets of silk materials in Bombyx mori. However, the expression and translation time of the serine protease inhibitor 1 gene (Spi1) lasted longer in the male silkgland than that in female, suggesting that this phenomenon might to be related to the advantage of males on both quality and quantity of silk materials compared with females.The fifth larvae on day 3 were treated by the exogenous molting hormone (MH), and SPs and Serpins presented different regulation mechanisms. Juvenile hormone analogue (JHA) had a similar positive regulation effect on expression of these two genes.The effect of hormones on gene expression is mainly related to the development stages and hormone doses.The down-regulation of Spi1 gene occurred later than Spp gene after hunger treatment on the larvae, suggesting that the direct influence by food on Serpins was slower than SPs, and also showing that the reaction of food supply and demand to the silkgland was later than the influence on the midgut. The tissue expression specificity of SPs and Serpins genes provided a proof that they were involved in transcription regulation of the corresponding tissues. Moreover, both of the expression differences during the developmental period and regulation changes after the hormone treatment reflected the time sequence and particularity of these gene functions. Many members in Serpins participated in the activity regulation of SPs via inhibiting SPs isoenzymes to keep balance of homeostasis.4 Cloning and functional research on putative cuticle protein gene, CPH45A cDNA sequence of 676 bp was cloned according to the tag‘GCTTCCGCCGTGCCTGC’(Tag.1161)in SAGE libraries containing only one exon with no intron. The length of deduced protein was 165aa, located on the nscaf 2767 of the 19th chromosome of Bombyx mori. This gene specifically expressed in the midgut and owning more than 42% homology with 4 protein sequences of CPH family of Bombyx mori. According to the existing gene name order of CPH gene in Bombyx mori, the novel gene was named putative cuticle protein gene 45, CPH45. The expression level of CPH45 in males was apparently higher than that in females of Ysh, which was negatively regulated by exogenous MH and positively regulated by JHA during the middle stage of the 5th instar. Males were more sensitive to exogenous insect hormones. The expression level of CPH45 appear to be an obvious effect on suggesting that it did not directly participate in food digestion. Functional research showed that CPH45 had an influence on maintaining shape of the midgut of Bombyx mori. The expression differences of CPH45 in different silkworm strains showed that this gene might be implicated in different pathways.5 Cloning and characterization of juvenile hormone methyltransgerase gene, JHAMT2A cDNA sequence, 1007 bp, was cloned according to the tag‘GAAACAGCACGCACGCT’(Tag.1221)in SAGE libraries. This gene contains four exons, located on the nscaf 2 993 of the 12th chromosome of Bombyx mori. The deduced protein sequence is 266aa with a functional locus of methyltransgerase super family. It owns 38% homology with JHAMT in Manduca sexta and therefore the cloning gene was named as juvenile hormone methyltransgerase gene JHAMT of Bombyx mori, JHAMT2. The JHAMT2 specially expressed in the midgut, especially in the middle midgut. During different developmental stages, it had a relatively higher expression level in the 4th instar larvas, while it only expressed on day 3 during the whole fifth larvae. The sexual differences were reflected by a longer continuous expression time in males than that in females. In molecular level, the period expression features of JHAMT2 verified that the synthesis of JH was relatively exuberant in the 4th instar larvae while inhibited after day 3 of the fifth instar larvae. Meanwhile, the requirement of JHAMT, catalyzing the synthesis of JH, reduced somewhat.更多还原