Gene Clone Of Thick Filament Gene In B. Mandarina And B. Mori

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.