家蚕Ssu72蛋白基因的克隆及序列与表达分析

Cloning,Sequence and Expression Analyses of BmSsu72 Gene of Silkworm,Bombyx mori

Ssu72(suppressor of sua7-1 clone 2)蛋白的命名源于对酵母sua7-1基因突变的抑制作用,推测其在真核生物RNA聚合酶Ⅱ作用的转录过程中有重要功能。从家蚕蛹cDNA文库中筛选到家蚕Ssu72蛋白基因(BmSsu72)的cDNA序列,利用生物信息学方法分析该基因ORF为579 bp,编码蛋白质含有192个氨基酸残基,具有完整的Ssu72保守结构域,三级结构包含9个α螺旋和4个β折叠,推测α螺旋和β折叠之间可能形成锌指结构。以家蚕蛹cDNA为模板PCR克隆到BmSsu72基因的完整ORF序列,通过构建载体pET-28a(+)-BmSsu72在大肠杆菌中表达并纯化了重组BmSsu72蛋白,进而利用纯化蛋白免疫新西兰大白兔获得多克隆抗体。采用荧光定量PCR检测到BmSsu72基因随着家蚕的生长发育转录水平呈逐渐下降趋势,在卵期的转录水平最高,在蛾期的转录水平最低;该基因在5龄幼虫各组织中的转录水平以马氏管最高,卵巢和丝腺次之。Western blotting检测BmSsu72在家蚕5龄幼虫各个组织中均有表达,其中在丝腺中的表达水平最高。推测BmSsu72可能与家蚕丝腺的分泌相关。

Ssu72 （suppressor of sua7-1 clone 2） protein was so named based on its inhibitory function to mutation of sua7-1 gene in yeast, suggesting that it plays a very important role during transcription carried out by eukaryotic RNA polymerase II. We screened and obtained a cDNA sequence of the silkworm （ Bornbyx mori） Ssu72 （BmSsu72） gene from silkworm pupa cDNA library. Bioinformatic analysis indicated that ORF of this gene is 579 bp long and encodes 192 amino acid residues with the complete Ssu72 conserved domain. Tertiary structure of the protein contains 9 e-helices and 4 B-strands. It is predicted that zinc finger structure may exist between some of the e-helices and 16-strands. The complete ORF sequence of BmSsu72 gene was cloned by PCR using silkworm pupa cDNA as template. Through constructing expression vector pET-28a（＋）-BmSsu72, the recombinant protein BmSsu72 was expressed in E. coil and further pu-rifled. Then, the purified recombinant protein was used to immunize New Zealand rabbit to prepare polyclonal antibody. By using fluorescence quan-titative PCR, it was detected that the transcriptional level of BmSsu72 decreased progressively with the growth and development of Bombyx mori. The transcriptional level in egg stage was the highest while in adult stage it was the lowest. The transcriptional level of this gene in different tissues of the 5th instar larvae was found to be the highest in Malpighian tubule followed by ovary and silk gland. Western blot analysis revealed that expression of BmSsu72 was detectable in all tissues of the 5th instar larvae, with the highest expression level in silk gland. It is thus presumed that BmSsu72 is related to the secretion in silk gland of Bombyx mori