【作者】 李文学； 刘刚； 柴春利； 鲁成；

【Author】 LI Wen xue1 LIU Gang1 CHAI Chun li2 LU Cheng2 (1Sericultural Research Institute;Sichuan Academy of Agricultural Sciences;Nanchong 637000;Sichuan;2The Key Sericultural Laboratory of Agricultural Ministry;College of Sericulture and Biote hnolog y,Southwest University,Chongqing 400716,China)

【机构】 四川省农业科学院蚕业研究所； 西南大学蚕学与生物技术学院农业部蚕桑学重点开放实验室；

【摘要】 为阐明家蚕生殖发育的调控机制,在家蚕基因组、EST数据以及芯片数据分析的基础上,本文对果蝇中已报道的生殖质决定相关基因在家蚕中的同源基因进行了鉴定,从FlyBase下载果蝇生殖质形成相关基因18个,通过与家蚕基因组数据进行BLAST比对分析,发现13个基因在家蚕中均有同源基因,并对其表达模式进行了分析。然后对高度保守的mago nashi基因进行了克隆和原核表达。克隆的mago nashi基因完整的编码区序列为441bp,编码146个氨基酸残基。该基因位于nscaf2655上,只有一个完整的拷贝,在基因组上有1611bp,共有两个外显子,外显子/内含子边界处均符合GT/AG规则。RT-PCR检测了该基因在发育各时期各组织器官的表达情况,发现该基因在所检测的所有发育时期、组织器官均有表达,是一个非特异表达基因。将家蚕mago nashi基因的编码区序列克隆到原核表达载体pET28-a中,并将构建成的pET-mago nashi重组质粒转化表达宿主菌Escherichia coli BL21(DE3)细胞,IPTG诱导后进行SDS-PAGE检测分析。结果表明,同对照相比,重组质粒在23.5KD处有一条十分明显的新增蛋白带,与预测的蛋白质大小一致,说明该基因体外重组表达成功。更多还原

【Abstract】 To further clarify the Silkworm reproductive and developmental mechanisms,based on the analysis of the silkworm genome,the large scale EST datas and microarray data,this paper analyzed the homology and Spatio-temporal expression of the key gene which had been reported to determine the formation of germ plasm;Downloaded 18 reproductive genes playing essential roles in germ plasm formation in the Drosophila from FlyBase,Used BLAST to compare them with silkworm genome data,13 genes in Silkworm have homologous gene.According to the gene array and the gene expression microarray data to analyz the expression patterns of the predicted genes which may have influence on the formation of germ cells. For the expression analysis of Bmmago gene,we cloned the coding sequence of the Bmmago gene by RT-PCR,then the clone sequenced. The Bmmago gene,a single copy locating on the genome,spans 1611bp and is comprised of 2 exons and 1 intron. The gene comprised an open reading frame of 441bp,and the encoded protein was 146 amino acids. The expression of Bmmago gene was foundin all kinds of tissues,organs and all stages,thus the gene is a non-specific expression gene. In order to further research of the characteristics and function of the encoding protein,a prokaryote express vector,pET-Bmmago,containing the complete ORF of Bmmago,had been constructed.The recombinant plasmid was induced by IPTG.,then performed the SDS-PAGE,we found the recombinant protein expressed. Comparing with the reference,between the marker 31KD-20.1KD,recombinant protein shown a clearly new band. This molecular weight correspond the predicted protein size.更多还原