家蚕丝腺因子SGF-1的基因克隆及序列结构和表达特征与亚细胞定位  

Gene Cloning,Sequence Structrue,Expression Pattern and Subcelluar Localization of Silk Gland Factor SGF-1 of Bombyx mori

作　　者：王叶菁[1,2] 刘莉娜 高春雁 李珍珍[2] 常怀普[1,2] 赵萍[2] 何华伟[1,2] 

机构地区：[1]西南大学生物技术学院,重庆市蚕丝纤维新材料工程技术研究中心,重庆400715 [2]家蚕基因组生物学国家重点实验室,西南大学,重庆400715

出　　处：《蚕业科学》2016年第2期219-227,共9页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划“973”项目(No.2012CB1146-02);国家自然科学基金项目(No.31402139);中央高校基本科研业务费项目(No.XDJK2013C049;XDJK2013A019);西南大学博士基金项目(No.SWU112086;SWU112111)

摘　　要：Fox类转录因子在细胞生长、增殖、分化和胚胎发育等过程中发挥重要功能,SGF-1是家蚕丝腺细胞转录因子,属于Fox类转录因子家族。家蚕SGF-1（BGIBMGA005101-TA）基因位于第25号染色体nscaf2823：112743～113792（＋strand）,含有1个外显子,CDS全长1 050 bp,编码349个氨基酸。家蚕品种大造5龄第3天幼虫的基因芯片表达谱显示,SGF-1基因在大多数组织中有表达,在丝腺中高量表达,其中在前部和中部丝腺的表达量略高于后部丝腺,在雄蚕中的表达量略高于雌蚕。氨基酸组成分析显示,SGF-1蛋白富含脯氨酸、丝氨酸、丙氨酸和亮氨酸,且多数为亲水性氨基酸。序列结构分析表明,SGF-1包含典型的Forkhead结构域,其两端分别为Forkhead_N末端结构域和HNF3_C末端结构域,其中大多数氨基酸残基形成了无规则卷曲结构,α-螺旋与β-折叠结构分别约占18.0%和0.6%。从家蚕后部丝腺克隆了SGF-1基因,并构建麦芽糖结合蛋白标签融合表达载体,表达并纯化了重组SGF-1蛋白。通过RT-PCR与Western blotting检测分别从基因转录水平和蛋白质表达水平上证实,SGF-1在家蚕5龄第3天幼虫丝腺中高量表达。亚细胞定位实验显示SGF-1定位于细胞核中。这些结果为深入研究SGF-1的结构和功能提供了有益的基础信息。Fox family transcription factors play important roles in cell growth,proliferation,differentiation and embryonic development. SGF-1 is a silk gland cell-specific transcription factor,which belongs to Fox transcription factor family. SGF-1gene（ BGIBMGA005101-TA） of silkworm（ Bombyx mori）is positioned at nscaf2823： 112743- 113792（ ＋ strand） of the 25 th chromosome and contains an exon. The CDS full length of SGF-1 is 1 050 bp,encoding 349 amino acids.Microarray expression profiles of Bombyx mori variety Dazao show that SGF-1 gene was widely expressed in most tissues and highly expressed in silk gland on day 3of the 5th instar silkworm larvae. The expression level of SGF-1 in anterior and middle silk gland was higher thanthat in posterior silk gland. The expression level of SGF-1 in male silkworm was higher than that in female silkworm. Amino acid composition analysis showed that proline,serine,alanine and leucine were enriched in SGF-1 protein. Most amino acid residues of SGF-1 protein were hydrophilic. Structural analysis showed that SGF-1 contained a typical Forkhead domain of which two ends were termed as Forkhead N-terminal domain and HNF3 C-terminal domain,respectively. Most residues in these domains were in random coil conformation. The content of α-helix and β-sheet structures in SGF-1 was about 18. 0% and 0. 6%,respectively. In this study,we cloned the SGF-1 gene from posterior silk gland of silkworm,constructed the expression vector of SGF-1 fused with maltose binding protein tag,and expressed and purified the recombinant SGF-1 protein. RT-PCR and Western blotting assay confirmed that SGF-1 was highly expressed in the silk gland of day 3 B. mori larvae of the 5th instar at the levels of mRNA transcription and protein expression. Subcellular localization analysis showed that SGF-1 was located in the nucleus of cells. These results provide a beneficial basic information for further studies on the structure and function of SGF-1.

关 键 词：家蚕 丝腺细胞转录因子SGF-1 基因克隆 序列结构 表达谱 亚细胞定位 

分 类 号：S881.24[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]