家蚕生物钟基因Bmcry1与Bmcry2的克隆及生物信息学分析

Molecular cloning and bioinformatic analysis of biological clock genes Bmcry1 and Bmcry2 in Bombyx mori

隐花色素基因(cryptochrome gene,Cry)是已确认的主要生物钟基因之一,它广泛分布于细菌和真核生物中。昆虫Cry基因分为Cry1和Cry2两类,果蝇只有Cry1,蜜蜂等膜翅目昆虫只有Cry2。为了研究鳞翅目模式昆虫家蚕Bombxy mori的昼夜生物钟分子调控机制和昆虫CRY蛋白的进化,本研究克降了家蚕Bmcry1与Bmcry2基因的全长cDNA序列,长度分别为2 166 bp和2 389 bp(GenBank登录号分别为HM747059和HM747060),拼接了全基因序列(GenBank登录号分别为HM747057和HM747058)。Bmcry1基因具有12个外显子和11个内含子,Bmcry2具有9个外显子,8个内含子。染色体定位表明Bmcry1和Bmcry2分别位于第17号和15号染色体。通过同源建模获得了Bmcry1和Bmcry2蛋白的三维结构,其FAD入口大而深,这与CRY不与嘧啶二聚体结合相符;Bmcry1和Bmcry2表面多为负电荷,只在FAD入口位置有正电荷寓集。多序列比对、蛋白质基序和功能域分析、聚类分析等结果显示,Bmcry1和Bmcry2分属昆虫的CRY1和CRY2,与柞蚕Antheraea pernyi等鳞翅目昆虫中CRY蛋白的亲缘关系最近。家蚕的两类CRY与其他昆虫CRY相似,也都具有DNA光解酶和FAD结合功能域,但保守位点和蛋白基序位点不同。本实验为进一步研究家蚕CRY1和CRY2的分子进化机制和功能创造了条件。

Cryptochrome gene（Cry） is one of the major biological clock genes which were widely distributed in bacteria and eukaryotes.Cry genes of insect species are clearly divided into two types,Cry1 and Cry2.Only Cry1 is expressed in Drosophila,while only Cry1 was expressed in bees and other hymenopteran insects.In order to explore the molecular mechanism of circadian clock in lepidopteran model insect Bombyx mori and the evolution of CRY proteins in insect species,we cloned the cDNA sequences of Bmcry1（2 166 bp,GenBank accession no.HM747059） and Bmcry2（2 389 bp,GenBank accession no. HM747060）,and obtained their gene sequences（GenBank accession no.HM747057 and HM747058, respectively） by sequence alignment and assembly.Bmcry1 have 12 exons and 11 introns,while Bmcry2 have 9 exons and 8 introns.Chromosome mapping showed that Bmcry1 and Bmcry2 were located on chromosome 17 and chromosome 15,respectively.We predicted the three-dimensional structure of Bmcry1 and Bmcry2 by homology modeling.The results showed that the FAD entrances are large and deep, consistent with the fact that CRY proteins do not bind with pyrimidine dimers,and the surfaces of Bmcry1 and Bmcry2 are more negatively charged,only FAD entrances have accumulated positive charge.Moreover, we researched the molecular evolution of Bmcry1 and Bmcry2 by multiple sequence alignment,protein motif analysis,functional domain analysis and cluster analysis.The results showed that Bmcry1 and Bmcry2 belonged to insect CRY1 and CRY2,respectively,and were closely to the corresponding proteins in other lepidopteran insects like Antheraea pernyi.Similar to the CRY proteins in other insects,Bmcry1 and Bmcry2 have DNA photolysis enzyme domain and FAD binding domain.However,the two domains have different conservative sites between CRY1 and CRY2 in insect species,and their protein motifs are also different.Our experiment provided a basis for further investigating the mechanism of molecular evolution and function of CRY1 and CRY2 in B.mori.