家蚕Sox家族基因BmDichaete的克隆与表达分析

发布时间：2018-08-31 08:06

【摘要】：【目的】Sox家族是一类转录调控因子,在多细胞动物的发育过程中起到了极其重要的作用。本研究旨在获得家蚕Bombyx mori Sox家族基因,并分析其在幼虫不同组织和发育阶段的表达模式及功能。【方法】采用cDNA末端快速扩增方法(rapid amplification of cDNA ends,RACE)克隆家蚕Sox家族基因,并对其序列进行生物信息学分析。构建原核表达载体,诱导原核表达并得到其蛋白的多克隆抗体,并以Western blot验证其蛋白在家蚕胚胎发育时期(产卵后24,48,72,96,120,144,168,192和216 h)和蚁蚕的表达特征。同时,使用半定量RT-PCR技术分析这一基因在家蚕整个发育过程以及5龄第3天幼虫不同组织(精巢、卵巢、神经节、头部、丝腺、脂肪体、马氏管、中肠、后肠、表皮、血液、气管)中的表达谱。【结果】克隆获得一个家蚕Sox家族基因,将其命名为BmDichaete(Gen Bank登录号:KY914554),其cDNA序列全长为1 541 bp,开放阅读框长741 bp,编码246个氨基酸,预测分子量大小为27.3 kD,等电点为9.89。同源序列比对和系统进化分析显示,BmDichaete具有Sox家族的典型HMG结构域,并与其他鳞翅目昆虫的Dichaete蛋白具有较高的同源性。对其表达模式分析发现,BmDichaete在家蚕整个发育时期都有表达,从胚胎发育后期到蛹前期持续高表达,在蛹后期及成虫期表达量有所下降,并且该基因在家蚕5龄第3天幼虫的卵巢、头部、丝腺及后肠组织中显著高表达。Western blot分析结果显示,BmDichaete在家蚕胚胎发育过程中持续而稳定地表达。【结论】家蚕BmDichaete具有典型HMG结构域,属于Sox家族成员。BmDichaete在家蚕胚胎发育的各个时期都发挥作用。本研究为进一步探索该基因在家蚕中的功能奠定了基础。
[Abstract]:Objective: Sox family is a class of transcriptional regulators, which plays an important role in the development of multicellular animals. The purpose of this study was to obtain Bombyx mori Sox family genes of Bombyx mori, and to analyze their expression patterns and functions in different tissues and developmental stages of larvae. [methods] the Sox family genes of Bombyx mori were cloned by cDNA terminal rapid amplification method (rapid amplification of cDNA ends,RACE). The sequence was analyzed by bioinformatics. The prokaryotic expression vector was constructed to induce the prokaryotic expression and the polyclonal antibody of the protein was obtained. The Western blot was used to verify the expression characteristics of the protein during the embryonic development of Bombyx mori (24: 48 / 721 / 96120144168192 and 216 h after oviposition) and in ant silkworm (Bombyx mori). At the same time, semi-quantitative RT-PCR technique was used to analyze the different tissues (spermary, ovary, ganglion, head, silk gland, fat body, Markov duct, midgut, hindgut, epidermis, blood) of silkworm (Bombyx mori) during the whole development process and the 3rd day of the 5th instar (spermary, ovary, ganglion, head, silk gland, fat body). [results] A silkworm Sox family gene was cloned and named as BmDichaete (Gen Bank accession number: KY914554). Its cDNA sequence was 1 541 bp, open reading frame 741 bp, encoding 246 amino acids. The predicted molecular weight of 27.3 kD, isoelectric point is 9.89. Homologous sequence alignment and phylogenetic analysis showed that BmDichaete had a typical HMG domain of the Sox family and had high homology with the Dichaete proteins of other Lepidoptera insects. The expression pattern of BmDichaete was found to be highly expressed in silkworm during the whole development period, from the later stage of embryonic development to the early stage of pupae, and the expression level of BmDichaete was decreased in the later stage of pupae and adult stage, and the expression of BmDichaete gene was found in the ovary of the 5th instar larvae of Bombyx mori on the 3rd day of silkworm. The results of Western blot analysis showed that BmDichaete was continuously and stably expressed in the embryonic development of Bombyx mori. [conclusion] Silkworm BmDichaete has a typical HMG domain. BmDichaete, a member of the Sox family, plays an important role in the development of silkworm embryos. This study laid a foundation for further exploring the function of this gene in silkworm.
【作者单位】：西南大学家蚕基因组生物学国家重点实验室;西南大学农业部蚕桑生物学与遗传育种重点实验室;
【基金】：国家自然科学基金项目(31472152,31572466)
【分类号】：Q78;S881.2

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