【作者】 栗凤鹏；

【导师】 吴金美；

【作者基本信息】 江苏科技大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 三磷酸腺苷结合盒转运蛋白(ATP-binding cassette, ABC)广泛分布于从细菌到人类等各种生物体中,是最大的膜蛋白家族之一,它主要利用ATP水解释放能量实现多种底物的跨膜转运。ABC转运子在生物体内以全分子或半分子转运子的形式存在,全分子转运子由2个核苷酸结合域(nucleotide binding domain,NBD)和2个跨膜结构域(transmembrane domain,TMD)构成,每个半分子转运子包括1个NBD和1个TMD,每个TMD一般由6个α螺旋构成,半分子转运子依赖形成同源或异源二聚体发挥作用,它们通过形成一个跨膜通道以实现底物分子的跨膜运输。利用生物信息学知识,本文初步鉴别了家蚕基因组数据库中潜在的47个ABC转运蛋白,这些蛋白成员涵盖了ABC转运蛋白的8个亚族(ABCA-ABCH)。家蚕ABCC亚族是含有成员最多的亚族,有15个成员,其中有5个同源于果蝇的的多药耐药糖蛋白(multidrug resistance,MDR); ABCB亚族有9个成员,有4个与果蝇的多药耐药相关蛋白(multidrug resistance-associated proteins,MRPs)同源。目前,家蚕ABC转运蛋白研究相对较少,通过对家蚕ABC转运蛋白的初步鉴定,为进一步研究家蚕ABC转运蛋白的功能提供了条件。三磷酸腺苷结合盒转运子B亚族成员ABCB6 (ATP-binding cassette transporter isoform B6, ABCB6)是一个非常重要的半分子转运子,在哺乳动物中主要参与细胞内卟啉类化合物的转运和铁离子平衡的调节。利用人的ABCB6基因编码氨基酸序列在家蚕EST数据库中进行tblastn检索,把检索到的EST序列进行电子拼接,根据拼接结果设计引物进行RT-PCR扩增、克隆测序验证。通过反转录聚合酶链式反应(RT-PCR)成功克隆了家蚕Abcb亚族基因第1个成员BmAbcb6的完整开放阅读框。生物信息学方法分析发现,BmAbcb6有16个外显子和15个内含子,编码850个氨基酸残基,分子质量为96.35 kD,等电点8.23;BmAbcb6含有1个NBD和有10个α螺旋构成的TMD,是家蚕Abcb亚族成员。半定量RT-PCR方法分析BmAbcb6在家蚕5龄第3天幼虫不同组织中均有表达,其中以精巢中的表达量相对最高。研究结果为进一步研究该基因的功能奠定了基础。通过反转录聚合酶链式反应(RT-PCR)扩增、克隆测序了家蚕ABC转运子Bmwh2完整的开放阅读框。生物信息学方法分析发现,Bmwh2有14个外显子和13个内含子,编码689个氨基酸残基,分子质量为77.38 kD,等电点8.42。Bmwh2含有1个NBD和6个α螺旋构成的TMD,为半转运子,属于家蚕ABC转运子超家族G亚族成员。半定量RT-PCR方法分析Bmwh2在家蚕5龄第3天幼虫不同组织的表达水平,以精巢中的表达量相对最高。通过显微注射仪,用合成的siRNA注射胚胎发育时期的蚕卵,对Bmwh2进行干涉研究,获得了白卵和嵌合体的干涉表型。根据实验结果,推测Bmwh2参与家蚕浆液膜色素前体的转运。更多还原

【Abstract】 ATP-binding cassette (ABC) transporter proteins constitute one of the largest protein superfamilies and are present in all organisms from bacteria to human. These proteins utilize the energy derived from ATP binding hydrolysis to drive substrate translocation across the membrane. ABC transporters have been subdivided into either full or half transporters in all organisms. Full transporters contain two nucleotide binding domains (NBD, also called ATP-binding cassettes) and two six-transmembrane helices, referred to as the“transmembrane domain”(TMD), on a single polypeptide. As their name suggests, half transporters contain one ABC domain and one TMD on a single polypeptide. Half transporters are dependent upon the formation of hetero- or homo-dimers. The silkworm genome sequence has been analyzed to find ATP-binding cassette transport protein genes based on the knowledge of bioinformatics. We identified 47 ABC transport proteins in the silkworm genome sequences, which possesses members of all current ABC subfamilies A to H. The largest silkworm is the ABCC genes which have 15 members, and the second largest silkworm group is the ABCB subgroup with 9 members. Silkworm showed 5 proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and 4 proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCCsubfamily). At present, ABC transport proteins were studied very limited in silkworm. The present study provides a useful foundation for studying the function of ABC transport proteins in silkworm.ATP-binding cassette transporter isoform B6 (ABCB6) has been considered to be a very important half-transporter. In mammals, ABCB6 was shown to be functionally active in cellular e?ux of certain porphyrins from cells and to be involved in regulation of iron homeostasis. The nucleic acid sequence of reported human ABCB6 was used to TBLASTN search against the silkworm EST database. The ESTs with high score were clustered and assembled into a consensus sequence. Based on the consensus sequence, the open reading frame (ORF) encoding BmAbcb6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced, which is the first member of Abcb subfamily in silkworm, termed as BmAbcb6. Further bioinformatic analysis shows that the BmAbcb6 cDNA has 16 exons and 15 introns, encoding 850 amino acid residues with pI 8.23 and a predicted molecular mass of 96.35 kD, respectively. The BmAbcb6 protein contains a cytosolic nucleotide binding domain and one transmembrance domain that consists of 10α-helices, it belongs to the Abcb subfamily. By Semi-quantitative RT-PCR analysis, BmAbcb6 gene expression was analyzed in different tissues on the third day of the fifth instar larvae, and the result shows that BmAbcb6 is expressed in every tissue and is detected the highest expression level in testis. This result provides a foundation for studying the function of BmAbcb6 gene in silkworm.The open reading frame (ORF) encoding Bmwh2 gene of Bombyx mori was amplified by reverse transcription-polymerase chain reaction and sequenced. Further bioinformation analysis showed that the Bmwh2 cDNA has 14 exons and 13 introns, which codes for 689 amino acid residues with pI 8.42 and a predicted molecular mass of 77.38 kD. Bmwh2 protein is a half-transporter with one cytosolic nucleotide binding domain and one transmembrance domain that consists of 6α-helices. It belongs to subfamily G of the Bombyx mori ABC transporter superfamily. Semi-quantitative RT-PCR analysis to the expression of Bmwh2 gene in different tissues of the 3rd day larvae of the 5th instar indicated that the highest expression level was in testis. A number of white eggs and chimeric RNAi (RNA interference) phenotypes were obtained by microinjecting synthesized siRNA corresponding to the Bmwh2 gene into Bombyx mori eggs at embryonic development stage. Base on the above results, it is suggested that Bmwh2 may be involved in transportation of the precursors of the serosal pigments in silkworm egg.更多还原