【作者】 徐升胜；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养学， 2009， 博士

【摘要】 昆虫纲在生物多样性中有着重要地位,它在进化中的成功与飞行能力的获得有着密切的关系。而有着水解ATP并将化学能转换为动能的重要功能的粗肌丝是飞行的基础。昆虫的粗肌丝主要由肌球蛋白/肌球杆蛋白、副肌球蛋白/小副肌球蛋白、Myofilin和Flightin等蛋白组成。肌球蛋白是由一对重链和两对轻链组成的六聚体蛋白,有着将化学能转变成机械能和形成粗肌丝的双重作用。肌球蛋白重链在大多数昆虫中由单基因编码,通过选择性编辑产生所有亚型的肽链。肌球杆蛋白和肌球蛋白重链有同一基因编码,它仅包括肌球蛋白重链的杆状区域和一个与轻链1同源的较小的N-末端。副肌球蛋白和小副肌球蛋白由同一基因编码,通过选择性启动子的使用产生的2个有着相同C-末端的蛋白。副肌球蛋白同源二聚体在肌肉的组装过程中,有着形成肌原纤维核心的作用,小副肌球蛋白的作用尚不明确。Flightin是仅在间接飞行肌中存在的一个小分子量的粗肌丝结构蛋白,可以调节间接飞行肌的性能。Myofilin是最近确认的一个粗肌丝结构蛋白,它可能位于肌原纤维的表面,功能尚不明确。随着家蚕基因组测序的完成,必将极大地促进家蚕分子生物学的研究,确立家蚕在鳞翅目昆虫研究中的地位。而粗肌丝结构的研究,无论是对于探讨昆虫飞行机制的理论基础,还是在害虫扩散、迁移的预测,或资源昆虫的养殖驯化中,都有着重要的意义。但家蚕飞行能力的丧失,使它不能有效代表野外昆虫,因此我们以家蚕和与家蚕有着共同祖先的野桑蚕为材料,研究其粗肌丝结构基因。在本试验中,我们通过RT-PCR、PCR、RACE和基因步移技术,对家蚕和野桑蚕的粗肌丝结构基因进行了研究,克隆到家蚕和野桑蚕肌球蛋白重链长度分别为2 2710 bp和2 3055 bp的基因组序列,包含转录起始位点上游约1.8 kb和poly(A)信号下游约1.0 kb。并通过生物信息学方法,确定了相关基因的结构。结果显示家蚕和野桑蚕肌球蛋白重链由单一基因编码,该基因在家蚕和野桑蚕中分别存在37和38个外显子,家蚕中的外显子24在野桑蚕中被1个大小约为300 bp的内含子分割。翻译起始密码子位于外显子2,在末端和次末端外显子中各有一个终止密码子。其中外显子3、8、11、14、17和22是含有多个外显子的相互排斥型选择性外显子组,在mRNA中存在且仅存在这些选择性编辑外显子组中的1个;可能含有,也可能排除次末端外显子。通过选择性编辑,这一基因最多可以转录780个亚型。其中在外显子13和选择性外显子3a、8b中存在导致氨基酸序列变化的差异。副肌球蛋白/小副肌球蛋白基因含有17个外显子,在使用最上游启动子时转录编码副肌球蛋白的mRNA,而在使用位于外显子10后面的外显子时转录编码小副肌球蛋白的mRNA,两者共用3’-末端的7个外显子。克隆到的家蚕和野桑蚕副肌球蛋白mRNA长度分别为3 250 bp和3 321 bp,可以编码877个氨基酸残基,其中第106和135位氨基酸不同。家蚕Myofilin基因含有9个外显子,可以产生3’-末端不同的5个亚型。所有这5个亚型的都使用5’-末端的3个外显子,且翻译起始密码子位于外显子2中。本实验从家蚕和野桑蚕成虫中分别获得3个亚型,分别编码113、193和345个氨基酸残基,其中家蚕和野桑蚕在亚型A和B中的125位存在一个氨基酸残基得差异。在成虫中野桑蚕可以产生2个不同的5’-末端,而家蚕仅使用其中的1个转录起始位点。家蚕的Flightin基因与黑腹果蝇基本相似,都是由4个外显子组成,且翻译起始密码子位于外显子2中。我们克隆到的家蚕和野桑蚕cDNA长度分别为661 bp和663 bp,编码158个氨基酸残基,其中在53位存在一个氨基酸的差异。通过对比分析家蚕和野桑蚕的粗肌丝结构基因,结果表明,家蚕和野桑蚕之间不含有在黑腹果蝇中鉴定的影响飞行能力的突变。这2种生物间粗肌丝结构基因存在的差异,是否与家蚕和野桑蚕的飞行能力有关,还有待进一步深入研究。另外,在实验过程中,应用家蚕的研究信息和技术方法可以在野桑蚕中得到理想的结果,因此推测野桑蚕可以作为联结家蚕和野外鳞翅目昆虫研究的纽带。更多还原

【Abstract】 Increasing data have demonstrated that hexapoda plays a very important role in biodiversity. Gaining such a success in evolution, the insect is widely believed to be strongly associated with the obtaining of flying ability. And the flying ability of insects based on myosin filament, the functions of which are transferring chemical energy to kinetic energy as well as hydrolyzing ATP. The insect myosin filament is composed of myosin/MRP, Paramyosin/ miniParamyosin, Myofilin and Flightin.Myosin comprises a couple of heavy chains and two couples of light chains; it is a hexamer protein, which could transfer chemical energy to mechanical energy by conformational change. In most of insects, the heavy chain is encoded by single gene, and its hypotype peptide is formed by alternative editing. MRP, encoded by the same gene which encoded myosin, is composed by the rod-shaped region of the heavy chains of myosin, and a small N-terminal which is homologous with light chain 1. Paramyosin and mini-paramyosin are also encoded by one gene, and produce the same C-terminal but different N-terminal protein by using the alternative promoter. Paramyosin probably acts as a nuclear of forming myofibrilla in the process of installing muscular. Whereas the biology function of mini-paramyosin is unclear so far. Flightin is a myosin filament structural protein with small molecular weight. It only exists in the indirect flight muscle in Drosophila melanogaster and it is important in regulating the function of indirect flight muscle. Myofilin is a newly confirmed structural protein of myosin filament. It is probably on the surface of myofibrilla with unclear function.The accomplishment of B. mori genomic sequencing deeply improved the molecular biology research of B. mori, and firmly established the position of B. mori in Lepidoptera insect research. The study of myosin filament structure is meaningful in many ways, including the rationale of flying mechanism of insect, the prediction of pest extension and migration, as well as the cultivation and training of resource insect. But the B. mori have losed the ability of fly, so it should not reflect the nature of wild Lepidoptera insects. Therefore we studied two kinds of creature, B. mori and B. mandarina, that have a common ancestor, to investigate their myosin filament structural genes.In the present study, a number of experimental methods consist of RT-PCR, PCR, RACE, and genome walking, were used to address the myosin filament structural gene of B. mori and B. mandarina. Further more, we have confirmed the structure of relative genes by the means of bioinformatics. The genomic sequeces of B. Mori and B. Mandarina Mhc gene cloned in this exprement are 22 710 bp and 23 055 bp, respectively. The result indicates that the MHCs of B. mori and B. mandarina are encoded by a single gene, which contains 37 and 38 exons in B. mori and B. mandarina, respectively. The sequence correspond to Bombyx mori Mhc gene exon 24, is divided to 2 exon by a intron in Bombyx mandarina. These exons include six clusters of alternatively spliced exons and one differentially included penultimate exon. Thus, 780 combinations of alternatively exons are possible. In addition, 3 amino acid encoded by exon 3a, 8a, and 13, respectively, are different between Bombyx mari and Bombyx mandarina.Paramyosin/mini-paramyosin gene contains 17 exons. When the upstream promoter is active, it transcripts the mRNA that encodes paramyosin, while the promoter which located after exon 10 carried out its function, it transcripts the mRNA that encodes mini-paramyosin, however, both of them share 7 exons that located at 3’-terminal. The lengths of cDNA, acquired from B. mori and B. mandarina, are 3 250 bp and 3 321 bp, respectively. Of the 877 amino acids encoded, the 106th and 135th amino acids showed diversity between the two species.The Myofilin gene of B. mori contains 9 exons and it can bring 5 different isoforms. All of these isoforms share 3 exons of 5’-terminal, and the start codon of these isoforms locates in exon 2. We acquired three isoforms from the moth of B. mori and B. mandarina, which could encode 113, 193, and 345 amino acids, respectively. The 125th amino acid of isoform A and B is different between B. mori and B. mandarina. Alternative start sites give rise to two transcripts that differ in their 5’noncoding region but share a single open reading frame in the moth of B. mandarina, whereas only one form of the start sites could be recognized in the process of transcription in the moth of B.mori. The Flightin gene of Bombyx mori is extremely similar to the gene of Drosophila melanogaster, which comprised 4 exons, and the start codon of the gene locates in exon 2. The lengths of cDNA, acquired from B. mori and B. mandarina, are 661bp and 663bp, respectively. Of the 158 amino acids encoded, the 53th amino acid showed diversity between the two species.After comparing the structural gene of myosin filament of B. mori with the genes of B. mandarina, no difference of the mutations that can affect flying ability in Drosophila melanogaster was found between B. mori and B. mandarina. Therefore, the mechanisms, that contribute to the difference of the movement between the two species, pending further study. In this study, the research information and technology used on the studies of B. mori are used on the studies of B. mandarina similarly, and achieved the ideal results finally. On the basis of the conclusions above, B. mandarina can be considered as a bridge that links the studies of B. mori and wild Lepidoptera insects.更多还原