【作者】 孙卫忠；

【导师】 王彦文； 李斌； 鲁成；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2004， 硕士

【摘要】 家蚕是重要的经济昆虫,人们利用家蚕已经有五千多年的历史。同时,家蚕也是用于科学研究的模式生物之一。2002 年 10 月家蚕 10 万条 EST数据库的构建及 2003 年 11 月家蚕基因组框架图的相继完成,不仅对于蚕业科学研究及蚕业生产具有重大意义,而且对于生命科学的研究也具有重要的促进作用。低分子量热激蛋白(small heat shock protein, sHSP)是一种普遍存在的蛋白,广泛存在于生物界,不但在生物体受到各种理化刺激作用下表达,在生物体的正常生长发育过程中也有表达。因其在序列上具有较高的保守性,是用做进化分析,探索物种系统演化的重要基因之一。为了系统地了解 sHSP 基因在家蚕中的分布特点、基因结构、表达时间特异性、表达组织特异性、基因位置特异性等多方面信息。本研究对家蚕基因组数据库进行同源检索,同时对已报道家蚕低分子量热激蛋白HSP20.8A 进行克隆表达及荧光原位杂交研究。实验结果如下:1 家蚕 sHSP 的数量及基因结构 利用来自人类 10 种、鼠 8 种、果蝇 11 种及已知家蚕的 6 种 sHSP 序列在家蚕基因组中进行同源性检索。一共得到 14 个 sHSP 基因,6 个与已知的序列相同,8 个为新发现基因。14 个 sHSP 基因中,有 3 个基因含有内含子结构:其中 Bmb018979_Bmb18980 基因含有 4 个内含子,其余两个基因(Bmb007190 和 Bmb026351)各有两个内含子。分析结果表明所有基因编码的蛋白质在二级结构上具有共同结构域,即均含有一个 N-端的可变区、一个α-晶体结构的保守区和一个较短的 C-端可变区。α-晶体保守区长度约 60～90 个氨基酸。2 家蚕 sHSP 基因的串联重复现象 根据 sHSP 基因在家蚕基因组序列中的分布位置,发现有 8 个基因分别位于 3 个 scaffold 内,表现出串联重复的特点,同一 scaffold 中的 sHSP相邻基因间无其它结构基因存在。根据基因在 scaffold 中的预测方向,发现每个 scaffold 中含有的基因序列方向两两相对,这种现象在其它物种中未见报道。3 家蚕 sHSP 基因表达 EST 证据 利用预测的sHSP 基因的CDS 序列与家蚕 EST数据库进行同源检索, - 1 -<WP=11>家蚕低分子量热激蛋白的基因组学分析与 HSP20.8A 的表达及 FISH 研究结果发现除 1 个基因(Bmb036757)未发现 EST 证据外,其余 13 个基因在家蚕的正常生长发育时期均有表达。但不同基因在不同的表达时期和表达部位其表达量不同。基因 Bmb002839 发现的 EST 最多,有 250 条。其次为 Bmb018979_Bmb018980 和 Bmb002837,分别为 159 条和 77 条。在不同的组织器官中 sHSP 基因的表达量有所不同,其中在未受精卵中表达量最多,共有 295 条,在精巢、卵巢、丝腺及脂肪体中表达量在 53～71条之间。同一组织器官中,不同 sHSP 的表达量也有所差异。在表达量最多的未受精卵中,基因 Bmb002839 发现的 EST 数目最多,有 219 条,占总表达量的 74%。4 家蚕 sHSP 系统进化分析 对 14 种家蚕 sHSP、人类的 10 种 sHSP 及果蝇的 11 种 sHSP 基因编码的α-晶体保守区进行系统进化分析显示:低分子量热激蛋白在 3 个物种中存在 3 个主要分支即人类、果蝇、家蚕,表现出各个物种的 sHSP 基因具有独立进化的方式。同时,三个物种之间存在相互交叉的分支。5 HSP20.8A 的克隆与表达 根据已公布的 HSP20.8A 的 cDNA 序列设计引物,从家蚕基因组中钓取基因序列构建测序载体。经测序证明该基因无内含子序列,最长开放阅读框为 561bp,共编码 186 个氨基酸残基。将此基因序列两端用限制性内切酶酶切后构建表达转移载体,然后与线性化杆状病毒共感染草地贪夜蛾细胞系 sf21,对表达的重组蛋白进行变性聚丙烯酰胺凝胶电泳检测,其分子量大小约 20kDa。6 HSP20.8A 基因的染色体荧光原位杂交 利用随机标记法对 HSP20.8A 进行地高辛荧光标记,构建杂交探针。与家蚕生殖腺染色体进行荧光原位杂交,杂交点均在染色体近中部区域。且杂交信号只有一个,说明该基因在家蚕基因组中为单拷贝基因。根据各个时期产生的杂交信号来看,细胞间期染色体比较容易获得杂交信号,而中期染色体及粗线期染色体较难获得杂交信号。更多还原

【Abstract】 The silkworm is the one of the most important economic insects in theworld. It has been domesticated for about 5000 years. The finished silkwormEST database in October 2002 and the finished whole genome sequencing inNovember 2003 provide abundant information which is of great importancenot only for sericulture but also for fundemental research of life science. Thesmall heat shock protein (sHSP) is a ubiquitous protein which can be foundnot only in prokaryote organism but also in eukaryote organism. Functionally,the sHSPs are involved in a variety of cellular processes, notably relate tocytoskeleton rearrangements and apoptosis. It endows cells with a transient,increased tolerance to heat and other stress. Prevent in vitro aggregation ofunfolding proteins. The sHSP bound to denatured protein and transferred thecomlex to adenosine triphosphate (ATP) dependent chaperones (such asHSP70) and refolded. In order to get an inventory about the sHSP of B.mori,the known sHSP sequences of human, mouse, D.melanogaster and B.moriwere used as query in B.mori database, the results was analyzed by comparinghethod. The known HSP20.8A’s cDNA that accessed in NCBI (access numberAF315317) was used to construct into the baculovirus expression vector andhave it expressed in sf21 cell lines. The HSP20.8A gene was also constructedas the probe and hybridized it with the chromosome of B.mori. All the resultsare listed as below.1 The sHSP gene number and gene structure Use the 10 sHSPs of H.sapiens, 11 sHSPs from D.melanogaster, 8 sHSPfrom Mus and the 6 known sHSPs of B.mori as queries. A BLASTP researchwas performed in the silkworm genome database. The result is that there total - 85 -<WP=95>家蚕低分子量热激蛋白的基因组学分析与 HSP20.8A 的表达及 FISH 研究14 sHSPs in B.mori. Six genes are identical with the known sHSP sequences.The other 8 genes were the new found which have no identical sequence inNCBI. All the sHSP of B.mori have α-crystallin domain after scanned theconservative Domain of the sHSP genes, and the peptide length of conserveranges from 60 residues to 90 residues. Only three sHSP genes of B.mori haveintrons and the intron number for different sHSP gene ranges from 2 to 4. Theother 11 genes are intronless. A multi alignment was made regarding the 14sHSP. the result shows that the structure of the 14 sHSPs are mainly composedof three domains: the N-terminal domain which have poorly conservesequence and its length obviously variant; the alpha crystallin domain whichhave high similarity with the known conserve domain of most sHSPs, and theresidues length of this domain in B.mori ranges from about 60 residues to 90residues; The last domain is the short C-terminal domain.2 The tandem duplication phenomena about the sHSP It is interesting that 8 sHSP genes which have the character of tandemduplication locate at three scaffolds (Bmb002837, Bmb002838, Bmb002839and Bmb002840 locate at scaffold000419; Bmb007189 and Bmb007190locate at Scaffold001353; Bmb016798 and Bmb016799 locate atscaffold004261), and there no other functional genes in the same scaffold thatlocate between these sHSP genes. Another interesting thing is that there arefour genes of which the orientation is different from other four genes. Thereare no report about this special character.3 EST found of the sHSP gene Of the 14 genes, 13 have confirmed EST except for one gene, which geneID is Bmb036757. The EST number is different when it was transcripted bydifferent sHSP genes. Such as the EST found of Bmb002839 is 250, thenumber of which is much more than any other genes. The EST found ofBmb018979_Bmb018980 and Bmb002837 is 159 and 77 respectively. Indifferent tissues or organisms the transcription products of sHSP genes aredifferent such as of the total EST found 295 ESTs in unfertilized eggs, 71 in - 86 -<WP=96>山东农业大学硕士论文ovary, 58 in testis, 55 in silk gla更多还原