【作者】 高志宏；

【导师】 夏庆友；

【作者基本信息】 西南大学 ， 生化与分子生物学， 2010， 博士

【摘要】 家蚕(silkworm, Bombyx mori)属于昆虫鳞翅目蚕蛾科蚕蛾属家蚕种,是由野蚕驯化而来,家蚕咬食桑叶,吐丝,且容易饲养成本低廉,在我国广大农村被广泛饲养。摘桑养蚕在我国拥有几千年的历史。家蚕作为一种重要的经济昆虫,为人类社会的文化发展作出了重要贡献；同时它义是一种模式研究昆虫。家蚕具有超强的丝蛋白合成能力,一头家蚕经过不到一个月的生长生育,食下约22g的桑叶,合成并分泌0.5～1g的丝蛋白。家蚕丝蛋白由丝素和丝胶组成,丝蛋白的表达受到严格的时间和空间限制,具有在五龄中后期,在中部丝腺高量合成丝胶蛋白,在后部丝腺合成丝素蛋白的特点。在丝蛋白基因表达调控方面,一直以来倍受人们的关注,丝蛋白基因反复“开启”和“关闭”的表达模式,也是真核生物转录和翻译调控研究的典型模型。上世纪七八十年代众多科学家对家蚕进行了大量的研究,但直到今天,对丝腺高效的蛋白合成能力和基因表达的精密调控机制,仍知之甚少。家蚕是首个全基因组测序的鳞翅口昆虫,本研究基于家蚕基因组精细图以及全基因组组织芯片数据,对几个家蚕丝蛋白合成表达调控相关基因进行分析和研究,获得主要结果如下：1.家蚕丝腺表达的bHLH基因的克隆及表达谱分析根据家蚕基因组精细图数据和全基因组组织表达芯片数据,刘春等分析了家蚕高量表达的转录调控因子。我们发现其中存在一类转录调控因子——bHLH转录调控因子。家蚕丝腺bHLH基因有2个,对应的探针号为Sw15083和sw01142,基因编号为BGIBMGA005127和BGIBMGA007303,根据与果蝇的sage基因和dimm基因同源,分别命名为Bmsage和Bmdimm。家蚕中已经发现的转录调控因子大多数为homedomain类转录调控因子,bHLH类转录因子的发现对丝腺基因的表达调控有着重大的意义。Bmsage基因定位在第25号染色体,为单拷贝基因,大小为2.9Kb,全长mRNA为1,196 bp,包含一个702 bp的完整编码框,推导编码234个氨基酸,该基因由4个外显子和3个内含子组成。Bmsage基因的转录起始位点上游(-699～650)存在一个核心启动子,核心启动子上游包括基本转录元件-10域和CAAT框。在基因的3’端下游存在2个PolyA的终止信号。Bmdimm位于第17号染色体,为单拷贝基因,至少由4个外显子和3个内含子组成,该基因大小为4.6Kb,mRNA长度为1,593 bp,包含-个636 bp的开放阅读框,推导编码211个氨基酸,其中精氨酸、谷氨酸和丝氨酸含量较高,等电点为5.2。在Bmdimm基因上游-1,865～-1,914处存在一个可能的核心启动子,5’端上游存在多处可能的-10域和CAAT框,基因下游不含PolyA的终止信号。bHLH类转录因子在家蚕中的报道还比较少见。果蝇的sage基因对唾液腺特异表达基因具有调控作用,唾液腺被认为是丝腺最有可能的同源器官,与丝腺存在某些相似的特征。分析发现在FibH基因的内含子中存在多个bHLH的潜在结合位点,该结合位点与果蝇bHLH转录因子的结合基序非常相似,这些位点与丝腺转录因子FMBP-1的结合位点较近。组织原位杂交实验证实Bmsage基因在5龄三天的中部丝腺和后部丝腺细胞中有表达。Bmsage基因主要在丝腺中表达,且在食桑期上调表达,在眠期和变态期表达量下调。Bmdimm基因是后部丝腺特异表达基因,4龄眠期开始表达,5龄食桑期表达量较高,发育至第6天表达量下调,熟蚕期不表达。在FibH基因的内含子中存在多个bHLH基因的潜在结合位点,因此推测丝腺bHLH基因可能对FibH基因的表达起着重要的调控作用。2. AcMNPV介导下FibH启动子驱动报告基因在家蚕各组织中的表达研究丝素基因是家蚕丝腺的重要结构基因,具有明显的时间和空间特异性表达特征。FibH基因mRNA表达于后部丝腺,在食桑期积累,眠期表达量降低；FibH基因在眠期被关闭,前期积累的mRNA在眠期被降解。为了找寻新的FibH基因的顺式调控元件,分别克隆5’端截短的大小不同的FibH启动子,基因转移载体使用重组杆状病毒(rAcMNPV)。结果发现,3个FibH基因启动子在病毒介导下能在丝腺、脂肪体和血淋巴中高效驱动外源基因的表达,且可以直接通过体表检测到荧光。表明,FibH启动子组织特异性表达特性发生了改变。在对比实验中,Ser1启动子仍特异地在中部丝腺中驱动下游基因的表达。丝素重链启动子在家蚕组织中的异位表达,我们猜测，可能由于丝素重链5’端上游序列中包含多个转录增强元件,这些增强元件被其他细胞因子识别后增强了下游基因的转录；FibH被整合到病毒基因组,病毒本身的某些元件可能影响FibH启动子的活性,从而引起这种异位的表达：基因的表达或某种特性的维持是需要特定的环境,病毒感染后影响家蚕正常的生理生化反应,影响细胞的微环境,从而改变FibH基因原本的特性。病毒感染后环境条件的改变,使丝素重链启动子异位表达于脂肪体和血细胞。比较不同大小的FibH启动子的表达,在丝腺中,0.2kb和0.9kb的启动子在中部丝腺和后部丝腺均有启动活性,而2.1kb的启动子仅在后部丝腺中起始报告基因的表达。分析2.1kb的FibH启动子序列,推测2.1kb的FibH启动子可能使用不同的转录起始位点,从而转录出不同的mRNA。由于翻译的提前中止而不表达EGFP蛋白。3.AcMNPV病毒感染对丝素重链启动子的影响研究早期的研究表明,FibH基因在后部丝腺细胞中特异表达。是在家蚕幼虫盛食期(眠间期)特异地在后部丝腺中合成,在眠期包括在后部丝腺中其表达也被抑制。以AcMNPV作为基因转移载体,FibH启动子在家蚕中存在异位表达现象。首先推测AcMNPV病毒对FibH启动子可能产生影响。我们使用dornor AcMNPV对FibH转基因家蚕感染,结果发现EGFP的表达位置并没有发生变化,仍特异地在丝腺中表达。排除了由于杆状病毒的感染,激活家蚕基因表达发生变化,如基因的上调或下调表达,这些基因进一步激活FibH在其他组织中表达的可能性；同时也不是病毒特异表达的基因对启动子活性产生影响的结果。近年相继报道丝素基因在家蚕其他组织中存在渗漏转录的现象；P25基因不仅在后部丝腺高效转录,而且在家蚕幼虫期其他组织如卵巢组织中也有转录,在丝腺和卵巢组织中存在不同的转录起始位点；3种丝素蛋白基因和Ser1基因的启动子转染BmN细胞,都存在一定程度的表达。我们的实验及近几年丝素重链启动子研究的报道,共同说明,FibH基因在转录水平,在家蚕体内没有严格的组织特异性。尽管有报道认为FibH基因在中部丝腺以及其他组织存在渗漏表达现象,但至今未发现丝蛋白在丝腺以外的组织表达,包括转染实验丝蛋白启动子也仅在丝腺中表达。这说明丝腺基因在蛋白表达方面存在较严格的组织特异性。4.异位表达原因推测结合我们的研究,杆状病毒介导下,丝素基因启动子存在异位表达现象,我们推测,由于FibH启动子被整合到病毒基因组中,病毒本身的某些顺式元件影响FibH启动子的活性,从而改变FibH启动子原本的特性。FibH基因启动子在转录上并不严格,其他组织中的存在一定的转录,携带启动子的病毒在家蚕组织中的大量复制,使FibH启动子在家蚕细胞中的含量远大于正常情况下,也远大于转基因蚕中,最终产生可以检测到的蛋白表达,从而表现异位表达的现象。此推测还有待于实验的进一步证实。由此可见丝素重链基因的转录和翻译调控,比人们的想象要复杂很多,它的调控机理的阐明还需要进一步的研究。更多还原

【Abstract】 Silkworm (Bombyx mori) is an insect Lepidoptera Saturniidae moth silkworm, domesticated by the wild silkworm. Bombyx mori feeds on mulberry, spinning silk and breeding widely in China. Sericulture in China has been thousands of years of history. Silkworm is a key economic insect, which made important contributions to cultural development in human society. And silkworm is also a model insect in research. Silkworm has super ability to synthesize silk protein. After less than a month, a silkworm feeds on mulberries about 22g, synthesizes and secretes 0.5～1g of silk protein. Silk protein is made of fibroin and sericin. The expression of silk protein gene is strictly subject to time and space constraints, which sericin protein is largely synthesized in middle silk gland and fibroin protein is highly expressed during the middle and late fifth instar. In the expression and regulation of silk protein gene, it is concerned that the "open" and "close"expression pattern of silk protein gene is a typical research model in eukaryotic transcription and translation.70s and 80s last century, many scientists made a huge number of research on silkworm. Until now, it is seldom known about the ability to be effectively synthesized in silk gland and the precise regulation mechanism which a gene is expressed and synthesized. Silkworm (Bombyx mori) is the first Lepidoptera insect that is whole genome sequencing. This research analyzes and studies the specifically expressed genes on Ch.25 of silkworm, based on silkworm precise mapping and tissue chip data of whole genome. The main findings follow:1. The cloning and expression profile of bHLH gene in silkwormAccording to the data of precise mapping and the tissue chip data of whole genome, it is found that 2 bHLH genes expressed in silk gland by Liu C. The expressed bHLH genes in silk gland, the probe are sw15083 and sw01142, the gene ID are BGIBMGA005127 and BGIBMGA007303. According to the homologous to sage and dimm gene in fruit fly, named Bmsage and Bmdimm. Most of the known transcriptional and regulation factors belong to homedomain families. The discovery of bHLH gene in silk gland is great significant to regulation of silk gene expression. Bmsage gene is on CH.25th, the gene length is 2.9kb, the total length of mRNA is 1199bp which includes a completed 720bp of coding frame that predicts to code 234 amino acids. This gene is single copy gene, made of 4 exons and 3 introns; there is a core promoter in the upstream(-699-650) of transcription initiation site. The upstream of the promoter contains basic elements:-10 region and CAAT frame; there are two polyA termination signals in the downstream of 3’ terminal.Bmdimm gene is on CH.17th single copy gene, made of 4 exons and 3 introns. the gene length is 4.6kb, the total length of mRNA is 1,593bp which includes a completed 636bp of coding frame that predicts to code 211 amino acids. There is a core promoter in the upstream(-1865～-1914) of transcription initiation site. The upstream of the promoter contains servral basic elements:-10 region and CAAT frame; there are not polyA termination signals in the downstream of 3’terminal.It is seldom reported about the bHLH-class transcriptional factors. Sage gene of fruit fly regulates salivary gland-specific expression. Salivary is recognized as the most possible homologous tissue, which is similar characteristic of silk gland. In the in situ hybridization experiment, it is demonstrated that Bmsage gene is expressed in silk gland nuclei during the day 5 of fifth in star. Bmsage gene is up-regulated in the intermolt period, and down-regulated in the molt period and metamorphosis. Bmdimm gene is expressed specifically in silk gland, Bmsage gene is up-regulated in the 4th instar molt period and previous period of 5th instar, and down-rcgulated in the later period and metamorphosis. It is found that there are many bIILH binding sites in the FibH gene introns, which are adjacent to the binding site of FMBP-1, a transcriptional factor. It is speculated that Bmsage gene is important to regulate the expression of FibH gene.2. Study of the FibH promoter expression mediated by AcMNPV in the silkworm tissuesFibroin gene is a important structural gene in the silk gland of silkworm. The mRNA of FibH gene is expressed in posterior silk gland. The expression of FibH gene is accumulated in the inter-molt period, decreased in the molt period. FibH gene is closed in the molt period, in which the early accumulated mRNAs are degraded. In order to analysis the new cis-regulatory domain of FibH gene in silkworm, three 5’ terminal-truncated FibH promoters that are different from sizes has been cloned. The recombinant Autographa california multiple nuclear polyhedrosis virus (rAcMNPV) are used as gene transfer vectors. It is found that three FibH gene promoters effectively expressed, mediated by virus, the exogenous genes in silk gland, fat body and hemolymph, which the fluorescence is detected on the surface. It is suggested that the tissue-specifically expressed feature of FibH promoter changed. In contrast experiment, Serl promoter still specifically promotes to downstream gene expression in the posterior silk gland. Compared the expression of different FibH promoters in size,0.2K and 0.9K of promoters express genes in the middle and posterior silk glands, but 2. 1K of promoter is express genes only in the posterior silk gland. The ectopic expression of FibH promoter in the silkworm tissues, we speculate, is probably because that there are many transcriptional enhancement elements in 5’upstream of FibH, which are enhanced the downstream gene transcription after recognized by other cell factors. FibH is integrated into the virus genome. Some of the elements in virus itself probably affect the activity of FibH promoter. Hence, it leads to ectopic expression. The gene expression or maintenance of some specificity needs specific circumstances. The normal physiological and biochemical reactions of silkworm are influenced by virus infection. The micro-environment of cells is also affected. Thus, it changed the original feature of FibH gene. After virus infection, the change of environmental condition makes FibH promoter ectopically express genes in fat body and hemolymph.3. The influence of FibH promoter by AcMNPV infectionThe early research suggests that FibH gene is specifically expressed in the posterior silk gland. It is specifically synthesized in the posterior silk gland during the inter-molt period, but the expression is inhibited during the molt period.FibH promoter has the phenomenon of ectopic expression, if AcMNPV is as the gene transfer vector. In order to detect that whether AcMNPV affects FibH promoter or not, we infected FibH-transgenic silkworm, using donor AcMNPV. It is found that the expressed position of EGFP did not change, is still expressed in the silk gland. After eliminating virus infection, the up-regulation of some of unexpressed genes activated, and these genes further activated the expressed probability of FibH in other tissues. Meantime, it is not the results that virus-specific expressed genes influence the activity of promoter. In recent years, it is reported that fibroin gene has the phenomenon of leakage transcription in other silkworm tissues. p25 gene not only transcribes effectively in the posterior silk gland, but also transcribes in other tissues, such as ovarian tissue, during the larva period. There are different transcriptional initiation sites in silk gland and ovary. Three types of fibroin protein genes infected BmN cells, which is expressed, to certain extent.4. Speculation the reason of protein ectopic expressionCombining our research, it is speculated that the FibH promoter is integrated into the virus genome, some cis-elements of virus itself affect the activity of FibH promoter, which change the original features. The transcription of FibH gene promoter is not strict, the transcription in other tissues also exists. Due to the virus large reproduction, the contents of FibH promoter in silkworm tissues is much more than that in the normal condition. The accumulation of a little transcription ultimately produces the phenomenon of protein ectopic expression, which is able to be detected. The speculation needs to be the experimental support of FibH gene expression and regulation research. Although, it is reported that FibH gene exists leakage phenomenon in the middle silk gland and other tissues, it is not found that the silk protein is expressed in tissues except from silk gland. The genes in the silk gland have strict tissue specificity. This shows the transcriptional and translational regulation of FibH gene is more complicated than that humans think. It is necessary to do further research in order to illustrate the regulation mechanism.更多还原