【作者】 董占鹏；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2011， 博士

【摘要】 家蚕是一种重要的农业经济昆虫,蚕丝业是我国农业经济的重要组成部分,一直在改善民生和可持续发展中发挥着积极作用,深入研究家蚕生命活动规律和基因功能对蚕丝业的持续稳定发展具有重要意义。随着家蚕基因组框架图和精细图的完成,家蚕全基因组的遗传基因信息逐步得到诠释,为广泛和深入开展基因组及各种功能基因的研究提供了坚实的基础条件。清道夫受体(scavenger receptor, SR)是细胞表面的一种跨膜糖蛋白,能结合多种多聚阴离子并将其转移到细胞中降解,具有多种功能。B型清道夫受体(class B scavenger receptor, SR-B)是清道夫受体家族中具有CD36结构域的一个小超基因家族,它们能够结合多种配体,参与机体一系列的生命代谢活动,在动脉粥样硬化及其它心血管疾病的形成或抑制、机体免疫防御、凋亡细胞清除、类胡萝卜素代谢和转运、信息素感知与传导以及视觉传导等生理过程中起着重要作用。这种功能的多样性,引起了人们对家蚕SR-B基因家族研究的兴趣。家蚕作为鳞翅目的模式生物,对其SR-B基因家族的研究,既能够解析这些基因在家蚕中行使的功能,也可为其它昆虫特别是鳞翅目昆虫的相关研究提供重要参照和理论依据,这对揭示SR-B基因家族的功能多样性和作用机制有重要意义。本论文利用家蚕9×基因组及基因芯片数据,通过生物信息学方法对家蚕SR-B基因家族进行了鉴定与分析,并对家族中BmSCRBQ1、BmSCRBQ4基因的结构特征、表达模式和组织定位进行了研究,在体外细胞水平上初步探索了这两个基因的功能。主要研究结果如下：1.家蚕SR-B基因家族的生物信息学及表达模式分析基于家蚕9倍全基因组数据库,应用生物信息学方法,鉴定获得了14个家蚕B型清道夫受体基因,这些基因分布在至少5条染色体上,共包含120个内含子,平均每个基因拥有8.5个内含子。家蚕SR-B家族的基因数目接近果蝇、按蚊、赤拟谷盗等昆虫的SR-B基因数目,较人、小鼠、线虫、斑马鱼和原鸡等物种的SR-B基因数目多,说明昆虫SR-B基因在物种分化后形成了更多的拷贝。家蚕SR-B家族基因均具有CD36结构域的特征序列,不同基因的氨基酸序列相似性在20%～39%之间,保守位点分散,没有连续的氨基酸保守序列。家蚕SR-B基因与小鼠、人的SR-B基因的氨基酸序列相似性在20%-35%之间,与果蝇、按蚊、蜜蜂和赤拟谷盗的SR-B基因的氨基酸序列相似性在19～63%之间。系统发生分析的结果表明,昆虫的SR-B基因明显分成3个类群,每个类群又分别形成了若干亚群,家蚕与其它4个昆虫的SR-B基因构成了直向同源关系,没有一个物种形成独特的分群,说明该类基因的各个亚家族在物种分化之前就已经形成,表明SR-B基因家族在昆虫物种间具有相对保守的功能。EST数据、芯片数据和RT-PCR检测分析结果表明,家蚕SR-B基具有不同的表达模式,多数基因的表达存在组织特异性,在家蚕幼虫的主要组织中均有SR-B基因转录表达。2.家蚕BmSCRBQ1、BmSCRBQ4基因的克隆、序列分析与鉴定对BmSCRBQ1、BmSCRBQ4基因的全长CDS进行克隆测序,序列分析表明,BmSCRBQ4基因的核苷酸序列与家蚕数据库预测的序列完全一致,而BmSCRBQ1基因的核苷酸序列在家蚕品种间存在3种不同形式：一些品种的BmSCRBQ1核苷酸序列与数据库预测序列完全一致,即完整型；另一些品种的BmSCRBQ1核苷酸序列存在2种选择性剪切方式,即完整型和缺失了第8外显子的缺失型;还有一些品种的BmSCRBQ1核苷酸序列与数据库序列相比存在14个或15个碱基突变,即点突变型。BmSCRBQ1基因完整型或点突变型的ORF长1482 bp,编码493个氨基酸,由10个外显子和9个内含子组成；BmSCRBQ1基因缺失型的ORF长1377 bp,编码458个氨基酸,含9个外显子和8个内含子。BmSCRBQ4基因的ORF长1371bp,编码456个氨基酸,由9个外显子和8个内含子组成。利用Smart、TMHMM2.0和Motif scan软件进行结构域和跨膜结构分析表明,BmSCRBQ1基因编码的蛋白含有2个跨膜区,2个胞质区和1个胞外区,含有9个N-连接糖基化位点、5个酪蛋白激酶Ⅱ磷酸化位点、4个豆蔻酰化位点和1个蛋白激酶C磷酸化位点。BmSCRBQ1基因缺失型除了缺少靠近C-羧基端的2个豆蔻酰化位点外,其余结构域特征与BmSCRBQ1的一致。BmSCRBQ4基因编码的蛋白含有1个C-羧基端跨膜区,1个C-羧基端胞质区和1个含N-端区域的胞外区,在N-氨基端没有预测到跨膜区,序列中存在7个N-连接糖基化位点、7个酪蛋白激酶Ⅱ磷酸化位点、5个蛋白激酶C磷酸化位点、4个豆蔻酰化位点和1个酪氨酸激酶磷酸化位点。这些分析结果表明,BmSCRBQ1、BmSCRBQl两个基因均含有典型的CD36结构域,属于SR-B受体基因。BmSCRBQ1、BmSCRBQ4两个基因间氨基酸序列的相似性约为30%,其中蛋白激酶C共有序列、5个糖基化位点、3个酪蛋白激酶Ⅱ磷酸化位点和6个半胱氨酸高度保守。BmSCRBQ1、BmSCRBQ4基因氨基酸序列与人CD36、小鼠SR-BI和果蝇ninaD基因的氨基酸序列比较,它们中的蛋白激酶C共有序列和6个半胱氨酸以及几个甘氨酸、苯丙氨酸、脯氨酸等高度保守。3.家蚕BmSCRBQ1、BmSCRBQ4基因的表达模式RT-PCR检测结果表明,BmSCRBQ1、BmSCRBQ4基因在大造未受精卵和整个胚胎期、幼虫期的大多数时期以及家蚕BmE、BmN细胞系中都有表达。在大造5龄第3天的幼虫组织中,BmSCRBQl基因在中肠、血液、中部丝腺、卵巢、头部、体壁、精巢、脂肪体、后部丝腺和马氏管中均有表达；BmSCRBQ4基因在中肠、中部丝腺、头部、体壁、脂肪体、精巢、卵巢中均有表达,在血液、后部丝腺和马氏管中未检测到表达。BmSCRBQ1、BmSCRBQ4两个基因在大多数发育时期和多个组织均有表达,暗示着2个基因可能有持家基因的功能。Western blotting的结果表明,在大造5龄第3天幼虫的卵巢、血液、后部丝腺、中部丝腺、精巢、马氏管、脂肪体等组织中均能检测到BmSCRBQ1蛋白的阳性信号,在中肠中未检测到目的蛋白的阳性信号,BmSCRBQ1蛋白大小约为60kDa。仅在大造5龄第3天幼虫的中部丝腺、脂肪体和精巢中检测到BmSCRBQ4蛋白的阳性信号,其它组织未检测到目的蛋白的阳性信号,BmSCRBQ1蛋白大小在组织间存在差异,中部丝腺中约为50kDa,脂肪体中约为55kDa,精巢中约为60kDa。在N4 5龄第4天幼虫的精巢、卵巢中均检测到两个蛋白的阳性信号,BmSCRBQ1蛋白仅有一条阳性条带,大小约为60kDa,而BmSCRBQ4蛋白在两个组织中均有3条阳性条带,大小分别约为50kDa、57kDa和70kDa。在家蚕BmE、BmN细胞系中也能检测到两个蛋白的阳性信号,BmSCRBQ1蛋白在两个细胞中均出现一条明显的阳性条带,大小约为60kDa,而BmSCRBQ4蛋白在两个组织中均有2条阳性条带,大小分别约为50kDa和57kDa(或55kDa)。这些结果表明,BmSCRBQ1基因的表达蛋白形式单一,大小稳定；而BmSCRBQ4基因的表达蛋白大小不一,形式各样,推测可能与组织特异性、蛋白糖基化程度或与其结构和功能的多样性有关。4.家蚕BmSCRBQ1、BmSCRBQ4蛋白的组织定位基于BmSCRBQ1、BmSCRBQ4基因在转录水平和蛋白水平上的表达结果,利用石蜡切片技术和免疫组织化学的方法在家蚕品种大造5龄第3天幼虫的组织(精巢、脂肪体、中部丝腺、血细胞和卵巢)中对两个基因表达的蛋白进行组织定位。结果表明,BmSCRBQ1蛋白主要存在于精巢的生精囊膜和内膜中、脂肪细胞的细胞膜上、中部丝腺丝腺细胞的内缘与外缘细胞膜上、血液中原白血球和颗粒细胞的细胞膜上以及卵巢中卵泡细胞的细胞膜区域中。而BmSCRBQ4蛋白则存在于在精巢的精原细胞的细胞膜和外膜中、脂肪细胞的细胞膜上和中部丝腺丝腺细胞的外缘细胞膜上,在血细胞和卵巢中未检测到BmSCRBQ4蛋白的阳性信号。在不同组织中的定位结果表明,两个蛋白主要分布在组织的组成膜和细胞的细胞膜上,推测其功能可能与各组织中脂类物质的流动和代谢、代谢产物和外源物的结合与吞噬、转运以及信号传递等活动有关。5.家蚕BmSCRBQ1、BmSCRBQ4基因真核表达蛋白结合细菌的初步研究将BmSCRBQ1、BmSCRBQ4基因ORF框全长序列克隆进哺乳动物细胞表达载体pcDNA3.1,构建重组载体,在HEK293细胞中进行表达,利用真核细胞表达系统在体外细胞水平上研究BmSCRBQ1、BmSCRBQ4基因的功能。转染结果表明,两个基因的重组载体都能在HEK293细胞中表达目的蛋白,表达的目的蛋白明显位于细胞的细胞膜区域。通过优化转染条件,重组载体的转染效率可保持在20%-30%。将FITC标记的大肠杆菌和金黄色葡萄球菌分别与转染后的细胞孵育,免疫荧光处理后在荧光显微镜下观察,发现两种细菌的部分菌体附于表达BmSCRBQ1、BmSCRBQ4蛋白的细胞边缘,推测两种蛋白可能均具有结合细菌的能力,对它们之间的结合关系尚需进一步研究。更多还原

【Abstract】 Silkworm is an important insect of agricultural economy, the silk industry is an important part in China’s agricultural economy, and has been playing an active role in improving people’s livelihood and sustainable development constantly. Researching in the law of silkworm vital movement and gene function in-depth is a significance for maintaining sustained and steady development of silk industry. As the accomplishing of silkworm genome framework map and detailed map, the genie informations of silkworm complete genome have been being annotated gradually, and providing a solid foundation conditions for studying on genomes and functions of various genes widespreadly and thoroughly. Scavenger receptors (SR) is a transmembrane glycoprotein in the cell surface, they can bind varied polyanions and transfer these polyanions to cells on degradation, performing many kinds of functions. The family of class B scavenger receptor (SR-B) is a small super-gene family of scavenger receptors family, these genes with CD36 domain can bind a variety of ligands, and participate in a series of the life metabolic activities, and plays an important role in physiological processes, such as in the formation and inhibition of cardiovascular disease including atherosclerosis, immune defense, clearing apoptotic cells, carotenoid metabolism and transport, pheromone sensory conduction and visual transduction and so on. This functional diversity evokes people’s interest to study silkworm SR-B genes. Bombyx mori is a Lepidoptera model insect, researching the silkworm SR-B genes not only can resolve the functions of the silkworm SR-B genes, but also provides an important reference and theoretical study basis for relevant studies of other insects, specially for Lepidoptera insects, it is an importantance of revealing the functional diversity and mechanism of action of SR-B genes family. In this thesis, utilizing 9×genome data and microarray database. SR-B genes family of silkworm was identified and analyzed through bioinformatics. The structural features, expression patterns and tissues localizations of BmSCRBQ1 and BmSCRBQ4 genes from this family have been researched and analyzed, and the functions of these two genes were initially explored in cell level in vitro. The main results are as follows:1 Analysis in bioinformatics and expression patterns of silkworm SR-B genes familyBased on silkworm 9×whole genome database, utilizing bioinformatics methods,14 class B cavenger receptor genes of silkworm are identified, these genes distributed in at least five chromosomes, containing a total of 120 introns, one gene has 8.5 introns in average.. The number of silkworm SR-B genes is close to Drosophila melanogaster’s. Anopheles gambiae’s and Tribolium castaneum’s, and more than the numbers of SR-B genes from Homo sapiens, Mus musculus, Caenorhabditis elegans, Danio rerio and Gallus gallus, indicating insect SR-B genes developed more copies after species differentiation.All SR-B genes of silkworm have characteristic domain of CD36, the similarities of amino acid sequences between silkworm SR-B genes are 20% to 39%, and conservative sites scattered, no continuous conserved sequences of amino acids in silkworm SR-B genes. The similarities amino acid sequences between silkworm SR-B genes and D.melanogaster’s, A.gambiae’s, A.mellifera’s and T.castaneum’s are 19% to 63%, between silkworm SR-B genes and mice’s and human’s are 20% to 35%.The results of phylogenetic analysis showed that insect SR-B genes divided into three groups, each group formed several subgroups respectively, SR-B genes from silkworm and other five insects formed orthologous relationship, no a insect species formed a unique grouping, indicating that each sub-group of such genes had been formed before species differentiato, and that the SR-B gene family in insect species could have relatively conservative functions.The results from silkworm EST and microarray database,analysis and RT-PCR testing showed that the SR-B genes of silkworm have different expression patterns, the exepression of most genes presented tissue specificity, there are the transcriptional expression of SR-B genes in main larval tissues of silkworm.2 Clone, sequences analysis and identification of silkworm BmSCRBQl and BmSCRBQ4 genesFull CDS length of BmSCRBQ1 and BmSCRBQ4 genes were cloned and sequenced. Sequence analysis showed that nucleotide sequences of BmSCRBQ4 is concordant with predicted sequences of silkworm databases, but there are three differenct types for the nucleotide sequences of BmSCRBQ1 in the silkworm strains and tissues:the nucleotide sequences of BmSCRBQ1 in some strains are consistent with database sequence predicted, called integrated type; the nucleotide sequences of BmSCRBQ1 in some strains show two alternative splicing, this is the integrity type and the deletion type which deleted exon 8; and the nucleotide sequences of BmSCRBQ1 in other strains exist 14or 15 bases mutation comparing with database sequences, called the point mutant type.The ORF lengths of BmSCRBQl integrated type or point mutant type is 1482bp, encoding 493 amino acids, with 10 exons and 9 introns; the ORF length of BmSCRBQ1 deletion type is 1377bp, encoding 458 amino acids, with nine exons and eight introns. The ORF length of BmSCRBQ4 gene is1371bp, encoding 456 amino acids, with 9 exons and 8 introns.Analyzing the structural domain and transmembrane domain by Smart, TMHMM2.0 and Motif Scan software revealed that the protein encoded by BmSCRBQ1 gene has two transmembrane domains, two terminal cytoplasmic region and an extracellular domain. There are nine N-linked glycosylation site, five casein kinase II phosphorylation sites,4 myristoylation site and a protein kinase C phosphorylation site in the protein encoded by BmSCRBQ1 gene. The protein domain characteristics of BmSCRBQl deletion type is consistent with BmSCRBQl’s except the lack of two myristoylation site close to C-carboxyl terminal. The protein encoded by BmSCRBQ4 gene has a transmembrane domain in the C- carboxyl terminal with a C-carboxyl terminal cytoplasmic region and an extracellular domain including N-terminal area, no the N-amino terminal transmembrane domain was predicted. There are 7 N-linked glycosylation sites, seven casein kinaseⅡphosphorylation sites, five protein kinase C phosphorylation sites,4 myristoylation site and one tyrosine kinase phosphorylation site in the protein encoded by BmSCRBQ4 gene. These analyzing results showed that two genes do have CD36 domain, belonging to SR-Bgene.Similarity of amino acid sequences between BmSCRBQ1 and BmSCRBQ4 is about 30%, there are high conserved protein kinase C consensus sequence,5 conserved glycosylation sites, three conserved casein kinaseⅡphosphorylation sites and six conserved cysteine residues. Comparing with human CD36’s and mouse SR-BI’s and Drosophila ninaD’s reveals high conserved protein kinase C consensus sequence, six conserved cysteine residues, and several conserved glycine, phenylalanine, and proline residues.3 The expression patterns of BmSCRBQl and BmSCRBQ4 genesRT-PCR results showed that BmSCRBQ1 and BmSCRBQ4 genes expressed in in unfertilized eggs and whole embryonic period, in majority of larvae period of Dazao strain and in cell lines BmE and BmN of silkworm. In silkworm strain Dazao tissues of 3-days-old 5th instar larvae, BmSCRBQ1 gene expressed in midgut, hemolymph, middle silk gland, ovary, head, integumentum, testis, fat body, posterior silk gland and in malpighian tubules; BmSCRBQ4 gene expressed in midgut, silk gland, head, integumentum, fat body, testis and in ovary, no expressions were detected in hemolymph, posterior silk gland and in malpighian tubules. The expression patterns of BmSCRBQ1 and BmSCRBQ4 genes expressing in mostly growth periods and in majority of larvae tissues hinted that two genes may act as housekeeping genes.Western blotting results showed that the positive signals of BmSCRBQ1 protein can be detected in ovary, hemolymph, posterior silk gland, middle silk gland, testis, malpighian tubules, fat body, head and in integumentum of 3-days-old 5th instar larvae of Dazao, no positive signal of target protein was detected in midgut. The size of BmSCRBQ1 protein is approximately 60kDa in each tissue (about 65kDa in). But. in tissues of 3-days-old 5th instar larvae of Dazao, the positive signals of BmSCRBQ4 protein were be detected only in the middle silk gland, fat body and in testes, no positive signals of the target proteins were not detected in other tissues. There are different in protein size between tissues, about 50kDa in the middle silk gland, approximately 55kDa in fat body, and approximately 60kDa in testis. In testis and ovary of 4 days 5th instar larvae of N4 stains, the positive signals of two proteins were detected, and BmSCRBQ1 protein has only one positive band with the size of about 60kDa; BmSCRBQ4 protein has three positive bands in each tissue, with the size of approximately 50kDa,57kDa and 70kDa respectively. In silkworm two cell lines BmE and BmN, the positive signals of two proteins were detected also, BmSCRBQl protein has one apparently positive band with the size of about 60kDa, BmSCRBQ4 protein has two positive bands in each cell, with the size of approximately 50kDa,57kDa(or 55 kDa) espectively. These results indicated that the proteins expressed by BmSCRBQ1 gene have single pattern and and stable size, but the proteins expressed by BmSCRBQ4 gene showed no uniform size and variform patterns, guessing that this may be related to the tissue specificity, degree of protein glycosylation or to the diversity of its structure and function.4 Tissue positioning of BmSCRBQl and BmSCRBQ4 proteinsBased on the expression results of BmSCRBQ1 and BmSCRBQ4 genes in the transcription level and protein level, using paraffin section and immunohistochemistry methods, tissues of 3-days-old 5th instar larvae of Dazao strain(such as testis, fat body, middle silk gland, haemocyte and ovaries)were selected to locate two protein in tissues. The results showed that BmSCRBQ1 protein were detected mainly in the spermatotheca membrane and intima of testis. in the plasma membrane of adipocyte, in both inner and outer membrane of silk gland cells of the middle silk gland, in the plasma membrane of original leukocyte and granulosa cells in haemocyte, and in the membrane area of follicle cells of ovary. However, BmSCRBQ4 protein were detected in the spermatogonia membrane and the outer membrane of testis, in the plasma membrane of adipocyte and in the outer membrane of silk gland cells of the middle silk gland, but no BmSCRBQ4 protein can be detected in haemocyte and ovary. The location results in different tissues showed that all two proteins exist in the constitutive tunicas of tissues and (or) in the plasmalemma area of cells, guessing that the functions of two proteins may be involved in lipid flow and metabolism, recognition and phagocytosis and transportation of metabolic waste and xenobiotics, and signaling transduction and so on.5 Initiatory study on the proteins of eukaryotic expression of BmSCRBQ1 and BmSCRBQ4 genes binding bacteriaFull ORF sequences of BmSCRBQl and BmSCRBQ4 genes were amplified and subcloned into the mammalian expression vector pcDNA3.1 after enzyme to construct the recombinant vector for expression in HEK293 cells, in order to study the functions of BmSCRBQ1 and BmSCRBQ4 genes in the cellular level in vitro using eukaryotic cell expression system. Transfection results have shown that the two recombinant vectors can express the target proteins in HEK293 cells and the target proteins obviously located in the cell membrane area of HEK293 cells.Optimizing conditions of transfection showed that the expression efficiency of the recombine vectors can be maintained in 20% to 30%. The HEK293 cells after transfection incubated with the FITC labeled E.coli and Staphylococcus aureus respectively, the samples were observed under a fluorescence microscope after immunofluorescence treatment, found that a small number of two bacteria were adhere to the edge of cell expressing BmSCRBQ1 and BmSCRBQ4 proteins, guessing that two proteins may be able to bind bacteria, further study on the binding action between them would be needed.更多还原