本文關(guān)鍵詞： 家蠶 載脂蛋白D 表達(dá)特征 氧化應(yīng)激 細(xì)菌侵染　出處：《西南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：載脂蛋白D作為一種糖蛋白,主要存在于血液中的高密度脂蛋白(HDL),同時(shí)廣泛分布于各組織中。載脂蛋白D主要作為脂類,固醇類物質(zhì)的轉(zhuǎn)運(yùn)體,同時(shí)還參與中樞神經(jīng)系統(tǒng)和周?chē)窠?jīng)系統(tǒng)的修復(fù),免疫系統(tǒng)反應(yīng),抵抗外界壓力信號(hào),改變運(yùn)動(dòng)能力以及參與一些神經(jīng)退行性疾病。載脂蛋白D廣泛存在于不同物種中,通過(guò)由八個(gè)反向平行的β折疊形成的L-型桶狀結(jié)構(gòu)與一些疏水性的小分子結(jié)合,運(yùn)輸?shù)桨薪M織,參與調(diào)控組織的發(fā)育等生理功能。載脂蛋白D在人類,小鼠,以及果蠅中有著深入的研究,然而在家蠶中研究卻鮮有報(bào)道。作為一種重要的鱗翅目經(jīng)濟(jì)性昆蟲(chóng)的家蠶,一直以來(lái)受到廣泛的關(guān)注。同時(shí)家蠶在發(fā)育過(guò)程中伴隨著巨大形態(tài)學(xué)和功能學(xué)的變化。利用蛋白質(zhì)組學(xué)技術(shù),我們從家蠶蛹后期鑒定出了兩個(gè)具有l(wèi)ipocalin結(jié)構(gòu)域的載脂蛋白D(Apolipoprotein D)基因,在SilkDB編號(hào)為BGIBMGA002703,BGIBMGA002704。本研究主要通過(guò)生物信息學(xué)分析、基因克隆、原核表達(dá)、Western Blot技術(shù)、免疫組化、熒光定量PCR等方法鑒定到家蠶的兩個(gè)載脂蛋白D蛋白,編號(hào)為BGIBMGA002703,BGIBMGA002704基因(ApoDⅠ和ApoDⅡ),并對(duì)其進(jìn)行了克隆表達(dá)及抗體的制備研究,對(duì)其組織和時(shí)期表達(dá)特征進(jìn)行探究及組織定位分析,同時(shí)對(duì)其功能進(jìn)行了初步探究。主要研究結(jié)果如下:1、家蠶載脂蛋白D(ApoDⅠ、ApoDⅡ)的鑒定及生物信息學(xué)分析;通過(guò)對(duì)家蠶蛹后期的蛻皮液和血液的雙向電泳分析,鑒定到了在SilkDB上編號(hào)為BGIBMGA002703,BGIBMGA002704的兩個(gè)基因,基因定位于家蠶大造種的第五號(hào)染色體上,具體位置分別為nscaf2529:4191766..4194594(+strand)和nscaf2529:4202485..4205359(+strand)。芯片數(shù)據(jù)和RT-PCR顯示,ApoDⅠ主要在家蠶的頭部和精巢表達(dá),ApoDⅡ主要在家蠶的頭部和中部及后部絲腺表達(dá)�；蚪M結(jié)構(gòu)顯示它們都具有4個(gè)外顯子和3個(gè)內(nèi)含子,結(jié)構(gòu)域預(yù)測(cè)結(jié)果顯示apodⅠ具有一個(gè)lipocalin(pf00061)結(jié)構(gòu)域,apodⅡ具有一個(gè)lipocalin_2(pf08212)結(jié)構(gòu)域。兩者氨基酸序列比對(duì)具有24.65%的相似性。對(duì)apodⅠ和apodⅡ基因進(jìn)行序列分析發(fā)現(xiàn),它們都具有信號(hào)肽序列。同時(shí)對(duì)兩個(gè)基因的編碼的蛋白的三級(jí)結(jié)構(gòu)預(yù)測(cè)顯示,都能形成一個(gè)由八個(gè)反向平行的β折疊形成的l-型桶狀結(jié)構(gòu)。進(jìn)化關(guān)系顯示家蠶的apodⅠ基因與美國(guó)白蛾的載脂蛋白d基因進(jìn)化關(guān)系較近,apodⅡ基因與內(nèi)華達(dá)白蟻載脂蛋白d基因的進(jìn)化關(guān)系較近。利用silkdb進(jìn)行多序列比對(duì),鑒定出家蠶中9個(gè)lipocalin家族基因,共聚為三類,第一類主要在中腸特異表達(dá),第二類主要在絲腺表達(dá),第三類廣泛的在生殖腺,馬氏管,脂肪體,頭和血淋巴中表達(dá)。2、家蠶載脂蛋白d基因的原核表達(dá)、純化和抗體制備;通過(guò)對(duì)apodⅠ和apodⅡ基因的序列分析,設(shè)計(jì)不包含信號(hào)肽序列的表達(dá)引物。以蛹兩天的cdna為模板進(jìn)行pcr擴(kuò)增,將獲得的片段插入到p28(pet-28a改造)載體上,測(cè)序成功后轉(zhuǎn)化到transetta(de3)表達(dá)菌株內(nèi),發(fā)現(xiàn)apodⅠ和apodⅡ都以包涵體的形式表達(dá)。純化apodⅡ的包涵體,制備抗體。為獲得可溶性的蛋白,又構(gòu)建了pet-sumo-apodⅠ和pet-sumo-apodⅡ兩個(gè)原核表達(dá)載體,測(cè)序正確后,轉(zhuǎn)化入transetta(de3)表達(dá)菌株內(nèi),在上清中獲得了sumo-apodⅠ和sumo-apodⅡ兩個(gè)重組蛋白。3、家蠶載脂蛋白d的組織及時(shí)空表達(dá)分析及免疫組化;利用rt-pcr對(duì)五齡第3天家蠶各組織檢測(cè),發(fā)現(xiàn)apodⅠ基因主要在頭部和精巢表達(dá),apodⅡ基因主要在家蠶頭部和絲腺表達(dá)。利用rt-pcr和q-pcr對(duì)五齡到蛹發(fā)育時(shí)期整蠶的核酸水平檢測(cè),發(fā)現(xiàn)apodⅠ基因主要在蛹期表達(dá),其中在化蛹第3天表達(dá)量達(dá)到最高。apodⅡ基因在蛹前期及蛹后期、蛾期有高量表達(dá)。利用westernblot技術(shù)對(duì)apodⅡ蛋白水平的表達(dá)檢測(cè)發(fā)現(xiàn),apodⅡ蛋白主要存在家蠶五齡3天的頭部,脂肪體,生殖腺,以及中部和后部絲腺中。對(duì)家蠶五齡到蛹期的時(shí)期血液檢測(cè)發(fā)現(xiàn),apodⅡ主要存在于上簇及蛹后期的血液中。對(duì)蛹期的頭和卵巢的檢測(cè)發(fā)現(xiàn),在頭中apodⅡ主要在蛹6天高量表達(dá),卵巢中apodⅡ主要在蛹后期及蛾期有高表達(dá)。利用免疫熒光技術(shù)對(duì)載脂蛋白d(apodⅡ)進(jìn)行組織定位,發(fā)現(xiàn)apodⅡ在家蠶五齡3天的精巢,卵巢,上簇時(shí)期的腦,化蛾當(dāng)天的復(fù)眼中檢測(cè)到了信號(hào)。4、家蠶載脂蛋白d基因?qū)毫?yīng)激和細(xì)菌侵染的響應(yīng)及調(diào)控初探選取uv和h2o2作為外界壓力信號(hào),大腸桿菌和沙雷氏菌作為細(xì)菌侵染的菌株,處理家蠶,通過(guò)q-pcr檢測(cè)發(fā)現(xiàn),apodⅠ和apodⅡ基因通過(guò)上調(diào)表達(dá)來(lái)響應(yīng)外界壓力信號(hào),同時(shí)在細(xì)菌侵染實(shí)驗(yàn)中,ApoDⅠ和ApoDⅡ基因也以上調(diào)的方式來(lái)響應(yīng)外界細(xì)菌侵染。為進(jìn)一步解釋可能調(diào)控方式,我們選取ApoDⅡ基因的5`上游1500bp的啟動(dòng)子序列,通過(guò)分析和結(jié)合文獻(xiàn),我們發(fā)現(xiàn)了與壓力信號(hào)相關(guān)的參與DNA損傷修復(fù)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)以及參與免疫活動(dòng)相關(guān)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。通過(guò)在ApoDⅡ基因啟動(dòng)子區(qū)域發(fā)現(xiàn)大量BRC結(jié)合位點(diǎn),通過(guò)蛻皮激素處理BmE細(xì)胞6小時(shí)后檢測(cè)發(fā)現(xiàn),ApoDⅡ基因的表達(dá)量顯著上調(diào),推測(cè)ApoDⅡ基因可能與蛻皮激素調(diào)控相關(guān)。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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