家蚕Apolipoprotein D基因克隆及功能分析

发布时间：2018-01-21 16:05

本文关键词： 家蚕载脂蛋白D 表达特征氧化应激细菌侵染　出处：《西南大学》2016年硕士论文　论文类型：学位论文

【摘要】：载脂蛋白D作为一种糖蛋白,主要存在于血液中的高密度脂蛋白(HDL),同时广泛分布于各组织中。载脂蛋白D主要作为脂类,固醇类物质的转运体,同时还参与中枢神经系统和周围神经系统的修复,免疫系统反应,抵抗外界压力信号,改变运动能力以及参与一些神经退行性疾病。载脂蛋白D广泛存在于不同物种中,通过由八个反向平行的β折叠形成的L-型桶状结构与一些疏水性的小分子结合,运输到靶组织,参与调控组织的发育等生理功能。载脂蛋白D在人类,小鼠,以及果蝇中有着深入的研究,然而在家蚕中研究却鲜有报道。作为一种重要的鳞翅目经济性昆虫的家蚕,一直以来受到广泛的关注。同时家蚕在发育过程中伴随着巨大形态学和功能学的变化。利用蛋白质组学技术,我们从家蚕蛹后期鉴定出了两个具有lipocalin结构域的载脂蛋白D(Apolipoprotein D)基因,在SilkDB编号为BGIBMGA002703,BGIBMGA002704。本研究主要通过生物信息学分析、基因克隆、原核表达、Western Blot技术、免疫组化、荧光定量PCR等方法鉴定到家蚕的两个载脂蛋白D蛋白,编号为BGIBMGA002703,BGIBMGA002704基因(ApoDⅠ和ApoDⅡ),并对其进行了克隆表达及抗体的制备研究,对其组织和时期表达特征进行探究及组织定位分析,同时对其功能进行了初步探究。主要研究结果如下:1、家蚕载脂蛋白D(ApoDⅠ、ApoDⅡ)的鉴定及生物信息学分析;通过对家蚕蛹后期的蜕皮液和血液的双向电泳分析,鉴定到了在SilkDB上编号为BGIBMGA002703,BGIBMGA002704的两个基因,基因定位于家蚕大造种的第五号染色体上,具体位置分别为nscaf2529:4191766..4194594(+strand)和nscaf2529:4202485..4205359(+strand)。芯片数据和RT-PCR显示,ApoDⅠ主要在家蚕的头部和精巢表达,ApoDⅡ主要在家蚕的头部和中部及后部丝腺表达。基因组结构显示它们都具有4个外显子和3个内含子,结构域预测结果显示apodⅠ具有一个lipocalin(pf00061)结构域,apodⅡ具有一个lipocalin_2(pf08212)结构域。两者氨基酸序列比对具有24.65%的相似性。对apodⅠ和apodⅡ基因进行序列分析发现,它们都具有信号肽序列。同时对两个基因的编码的蛋白的三级结构预测显示,都能形成一个由八个反向平行的β折叠形成的l-型桶状结构。进化关系显示家蚕的apodⅠ基因与美国白蛾的载脂蛋白d基因进化关系较近,apodⅡ基因与内华达白蚁载脂蛋白d基因的进化关系较近。利用silkdb进行多序列比对,鉴定出家蚕中9个lipocalin家族基因,共聚为三类,第一类主要在中肠特异表达,第二类主要在丝腺表达,第三类广泛的在生殖腺,马氏管,脂肪体,头和血淋巴中表达。2、家蚕载脂蛋白d基因的原核表达、纯化和抗体制备;通过对apodⅠ和apodⅡ基因的序列分析,设计不包含信号肽序列的表达引物。以蛹两天的cdna为模板进行pcr扩增,将获得的片段插入到p28(pet-28a改造)载体上,测序成功后转化到transetta(de3)表达菌株内,发现apodⅠ和apodⅡ都以包涵体的形式表达。纯化apodⅡ的包涵体,制备抗体。为获得可溶性的蛋白,又构建了pet-sumo-apodⅠ和pet-sumo-apodⅡ两个原核表达载体,测序正确后,转化入transetta(de3)表达菌株内,在上清中获得了sumo-apodⅠ和sumo-apodⅡ两个重组蛋白。3、家蚕载脂蛋白d的组织及时空表达分析及免疫组化;利用rt-pcr对五龄第3天家蚕各组织检测,发现apodⅠ基因主要在头部和精巢表达,apodⅡ基因主要在家蚕头部和丝腺表达。利用rt-pcr和q-pcr对五龄到蛹发育时期整蚕的核酸水平检测,发现apodⅠ基因主要在蛹期表达,其中在化蛹第3天表达量达到最高。apodⅡ基因在蛹前期及蛹后期、蛾期有高量表达。利用westernblot技术对apodⅡ蛋白水平的表达检测发现,apodⅡ蛋白主要存在家蚕五龄3天的头部,脂肪体,生殖腺,以及中部和后部丝腺中。对家蚕五龄到蛹期的时期血液检测发现,apodⅡ主要存在于上簇及蛹后期的血液中。对蛹期的头和卵巢的检测发现,在头中apodⅡ主要在蛹6天高量表达,卵巢中apodⅡ主要在蛹后期及蛾期有高表达。利用免疫荧光技术对载脂蛋白d(apodⅡ)进行组织定位,发现apodⅡ在家蚕五龄3天的精巢,卵巢,上簇时期的脑,化蛾当天的复眼中检测到了信号。4、家蚕载脂蛋白d基因对压力应激和细菌侵染的响应及调控初探选取uv和h2o2作为外界压力信号,大肠杆菌和沙雷氏菌作为细菌侵染的菌株,处理家蚕,通过q-pcr检测发现,apodⅠ和apodⅡ基因通过上调表达来响应外界压力信号,同时在细菌侵染实验中,ApoDⅠ和ApoDⅡ基因也以上调的方式来响应外界细菌侵染。为进一步解释可能调控方式,我们选取ApoDⅡ基因的5`上游1500bp的启动子序列,通过分析和结合文献,我们发现了与压力信号相关的参与DNA损伤修复的转录因子结合位点以及参与免疫活动相关的转录因子结合位点。通过在ApoDⅡ基因启动子区域发现大量BRC结合位点,通过蜕皮激素处理BmE细胞6小时后检测发现,ApoDⅡ基因的表达量显著上调,推测ApoDⅡ基因可能与蜕皮激素调控相关。
[Abstract]:Apolipoprotein D is a glycoprotein, high density lipoprotein is mainly found in the blood (HDL), and is widely distributed in various tissues. Apolipoprotein D mainly as lipids, steroid material transporter, also involved in the repair of central nervous system and peripheral nervous system, immune system response, resistance to outside the pressure signal, change the movement ability and participate in some neurodegenerative diseases. Apolipoprotein D widely exists in different species, through the combination of small molecule L- type barrel structure formed of eight antiparallel beta sheet and some hydrophobic, transported to the target tissue, the development of physiological functions involved in the regulation of tissue. Apolipoprotein D in humans, mice and Drosophila have in-depth study, but there were few studies in the silkworm Bombyx mori. As an important economic insect of Lepidoptera, has received extensive attention. At the same time in the process of the development of silkworm with great morphology and function. By using proteomics technology, we have produced two lipocalin domains of apolipoprotein D from Silkworm (Apolipoprotein D) later identified in the SilkDB gene, named BGIBMGA002703, BGIBMGA002704., this research mainly through bioinformatics analysis, gene cloning and prokaryotic expression of Western, Blot, immunohistochemistry, fluorescence quantitative PCR methods to identify two apolipoprotein D protein of Bombyx mori, numbered BGIBMGA002703, BGIBMGA002704 gene (ApoD I and ApoD II), and has carried on the study on the preparation of cloning and expression of antibodies, expression characteristics and exploration the organization location analysis on its structure and period, and the function has carried on the preliminary exploration. The main results are as follows: 1, silkworm apolipoprotein D (ApoD I and ApoD II) identification and analysis of biological information; Through two-dimensional electrophoresis of the late silkworm molting fluid and blood analysis were identified in the SilkDB number BGIBMGA002703, two of BGIBMGA002704 gene, gene mapping in the silkworm created a specific location on chromosome fifth, respectively nscaf2529:4191766..4194594 (+strand) and nscaf2529:4202485..4205359 (+strand). The chip data and RT-PCR display ApoD I mainly expressed in silkworm head and testis, mainly in the silkworm ApoD II and the head of the middle and posterior silk gland. The expression of the genome structure show they have 4 exons and 3 introns, domain prediction results show that the APOD I have a lipocalin (pf00061) domain, APOD II has a lipocalin_2 (pf08212) domain. The two amino acid sequence has 24.65% similarity. Sequence analysis showed the APOD I and APOD II gene, it has signal peptide sequence As predicted at the same time. The three stage structure of two genes encoding proteins that can form a consists of eight antiparallel beta folding type l- barrel structure. The evolutionary relationship shows close evolutionary relationship of the apolipoprotein D gene APOD gene of silkworm and American white moth, more recent evolution the relationship between APOD gene and apolipoprotein D gene in Nevada termites. Multiple sequence alignment was performed using silkdb, a identification 9 lipocalin family genes in the silkworm, copolymerization into three categories, the first category is mainly in the midgut specific expression, second class is mainly expressed in silk gland, third kinds of widely in gonad, Malpighian tubules, fat body, head and blood with Bazhong.2 expression, prokaryotic expression of silkworm apolipoprotein D gene, purification and antibody preparation; the sequence of APOD I and APOD II gene expression analysis, primer design does not contain a signal peptide sequence. With the pupa two days of cDNA as the template for PC R was inserted into the fragment to p28 vector (pET-28a transformation), sequencing was transformed into transetta (DE3) expression strain, APOD I and APOD II are expressed in the form of inclusion bodies. The purification of inclusion body of APOD, the preparation of antibody. In order to obtain soluble protein, and construct the pet-sumo-apod I and pet-sumo-apod II two prokaryotic expression vector after sequencing and transformed into transetta (DE3) expression strain, obtained the sumo-apod I and sumo-apod II two.3 recombinant protein in the supernatant, and the spatial and temporal expression analysis and immunohistochemistry of silkworm apolipoprotein D; detection of five at the age of third days the silkworm tissues by RT-PCR, found APOD gene mainly expressed in head and testis, APOD II gene mainly expressed in silkworm silk gland and head. Detected by RT-PCR and Q-PCR to the five instar developmental period of pupae on the nucleic acid levels of whole silkworm, APOD I gene The expression in the pupal stage, of which the highest expression of.Apod II gene in pupa and pupa stage in the late pupation for third days, the moth has high quantity expression. Found on the expression of APOD II protein level detected by Westernblot technology, APOD II protein mainly exists five silkworm 3 days old head, fat body and gonads, and the middle and posterior silk gland of Bombyx mori. Five instar to the pupal period blood test found that APOD II mainly exists in the cluster and later on pupal blood. Detection of the pupal stage head and ovary, head in APOD II is mainly expressed in pupae 6 days, mainly in the ovary of APOD II late pupa and moth stages had high expression by immunofluorescence on apolipoprotein D (APOD II) were found in the tissue localization, APOD II five 3 day old silkworm testis, ovary, brain on the cluster period, the eye of the moth was detected in the.4 signal, silkworm apolipoprotein D gene on pressure Study on regulation of stress and stress response and bacterial infection using UV and H2O2 as the external pressure signal, Escherichia coli and Serratia strains as bacterial infection, treatment of silkworm, detected by Q-PCR, APOD I and APOD II gene in response to external pressure signal through upregulation of expression, at the same time in the bacterial infection in the experiment, ApoD I and the ApoD II gene also above a way to respond to external bacterial infection. In order to further explain the possible regulation, we selected 5` 1500bp upstream of the promoter sequence of ApoD gene, through analysis and combined with the literature, we found that the transcription factor involved in DNA damage repair related pressure signal binding sites and transcription factors involved in immune the activities related to the binding sites in the promoter regions. Through the ApoD II gene found a large number of BRC binding sites by ecdysone treatment of BmE cells after 6 hours of detection, ApoD The expression of gene was upregulated and presumably ApoD gene with ecdysone.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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