【作者】 陈默；

【导师】 刘彦群； 王振东；

【作者基本信息】 沈阳农业大学 ， 细胞生物学， 2013， 硕士

【摘要】 柞蚕(Antheraea pernyi)属于鳞翅目大蚕蛾科,是我国特有的一种产丝和食用昆虫。本学位论文分为两个部分。第一部分报道柞蚕线粒体DNA中A+T丰富区的结构特征与长度多态性。第二部分报道柞蚕两个12kDa FKBP基因的分离与表达模式。测定了柞蚕19个样品(包括16个放养型和3个野生型)的线粒体A+T丰富区的全长序列。从16个放养型柞蚕样品中鉴定出9种单倍型,序列长度在550bp到554bp之间,序列之间具有很高的相似度,序列变异仅为0.18-0.91%。3个野生型柞蚕样品分为2种单倍型,序列长度为516bp或517bp。与放养型柞蚕的A+T丰富区序列相比,野生型柞蚕A+T丰富区序列显示出了长度多态性和相对较多的碱基差异。系统进化分析发现,柞蚕A+T丰富区序列明显被分为两种类型：放养型和野生型。串联重复序列数量的变化造成了放养型与野生型柞蚕A+T丰富区之间的长度多态性。在柞蚕A+T丰富区也发现了已在其他昆虫中鉴定出的一些保守结构元件。同时,在柞蚕A+T丰富区中还发现了2个类tRNA序列。克隆出的两个柞蚕12kDa FK506结合蛋白(FKBP12)基因分别被命名为ApFKBP12A和ApFKBP12B。柞蚕FKBP12A和FKBP12B都含有108个氨基酸残基,且氨基酸序列一致性为82%。系统进化分析显示,柞蚕(A.pernyi)、家蚕(Bombyx mori)、黑脉金斑蝶(Danaus plexippus)的FKBP12A与FKBP12B被明显分开,表明昆虫的FKBP12A与FKBP12B可能已经开始独立进化。RT-PCR分析表明两个ApFKBP12在柞蚕不同发育时期以及5龄幼虫的不同组织器官中均有表达。实时荧光定量RT-PCR分析表明,ApFKBP12A的表达水平显著高于ApFKBP12B。热激处理后,两个ApFKBP12的相对表达量分别在不同时间点显著上调。研究结果表明,ApFKBP12A和ApFKBP12B可能在柞蚕生长发育过程中发挥不同作用。更多还原

【Abstract】 Chinese oak silkworm, Antheraea pernyi, belonging to the family Saturniidae of Lepidoptera, is an important silk-producing and edible insect in China.There are two main parts in this dissertation. In the part Ⅰ, length polymorphism and structural organization of the A+T-rich region of mitochondrial DNA in A. pernyi are analyzed. In the part Ⅱ, two12kDa FK506-binding protein (FKBP12) genes are isolated and characterized in A. pernyi.The complete A+T-rich region of mitochondrial DNA had been cloned and sequenced from19samples of A. pernyi. Nine haplotypes were obtained from16inbred strains of the domesticated type with the range from550to554bp in size, exhibiting a low degree of sequence divergence from0.1S to0.91%. Two haplotypes were observed from three individuals of the wild type with516or517bp, respectively, showing a length polymorphism and a relatively high degree of sequence divergence compared with those of the domesticated type. Phylogenetic analysis showed the presence of two clearly differentiated mitochondrial clades:the domesticated type and the wild type. The length polymorphism between the two types was caused by the presence of a variable number of tandemly repeated units. Several conserved structural elements previously described in other insects were also present in the A pernyi A+T-rich region. Moreover, two tRNA-like structures were observed in this region.Two12kDa FK506-binding protein (FKBP12) genes cloned in A. pernyi were designated as ApFKBP12A and B, respectively. Both ApFKBP12A and B contained108amino acids with82%sequence identity. Phylogenetic analysis showed that FKBP12B sequences of A, pernyi, B. mori and D. plexippus were clearly separated from FKBP12A sequences of these three species, suggesting that FKBP12A and FKBP12B of insects may have evolved independently. RT-PCR analyses revealed that two ApFKBP12genes were expressed during the four developmental stages and in all tested tissues of fifth instar larvae. qRT-PCR analyses indicated that the mRNA expression level of the ApFKBP12A gene was significantly higher than the ApFKBP12B gene. After heat shock treatment, relative expression of the two FKBP12genes were up-regulated, but at different time points. Our results suggested that each paralogue of the FKBP12genes may play a distinct functional role in the development of A. pernyi.更多还原