Cloning And Sequence Analysis Of The Gene Encoding The Precursor Protein Of DH-PBAN Of Chinese Wild Silkworm, B.mandarina China

Diapause hormone (DH) is the hormone that can induce diapause of egg. DH activity may be widely distributed, only in Bombyx mori (silkworm), among the insects studied, has this peptide been captured as a regulator of diapause (Denlinger,2002). Therefore the studies on DH not only will help ascertain the mechanism of diapause, but also have great significance for the molecular evolutionary studies of DH. The studies on the mechanism of diapause will play basic role in utilizing beneficial insects such as reforming the diapause type of silkworm, and in biological control.It is verified that pheromone biosynthesis activating neuropeptide (PBA.N) is encoded together with DM by the same gene. PBAN is a kind of neuropeptide that controls the biosynthesis of sex pheromone. Studies on PBAN have the theoretic signification find the latent practical prospect.B. mori has origined from Bombyxmandarina China (Chinese wild silkworm), so the studies on DH-PBAN gene of B. mandarina C. have great significance to ascertain function and evolution of DH-PBAN gene of B. mori that is regarded as the model organism and to study origin and evolution of B.mori and so on.This study aims to obtain DH-PBAN gene of B. mandarina C. , then analyze sequence and phylogenetics with databases online to study the function of DH, PBAN and DH-PBAN genes and the evolution of wild silkworm and silkworm.According to the DH-PBAN gene sequences of B.mori , the primers are designed to amplify the DH-PBAN gene of B. mandarina C. in this study. The results are as follows:1. The better reaction volumn to amplify aimed gene or gene fragment (especially the single-copy gene ) from genomic DNA of B. mandarina is established. This result will help amplify other genes or gene fragments from genomic DNA of B. mandarina C. with specific primers. The purity and content of genomic DNA and the contents of primers are the main factor that affecting the result of amplification.2. The specific primers are designed to amplify DH-PBAN of B. mandarina C. from its genomic DNA. The DH-PBAN sequence of 5520bp from B. mandarina C. is obtained. The alignments and structure analysis result shows that this gene .which is consist of 6 exons and 5 introns, encodes DH, PBAN, a - SGNP, P - SGNP and Y-SGNP . Identity of DH-PBAN genes between B.mori and B. mandarina C. is 94%. Compared with DH-PBAN of B. mandarina C., 289bp-long-insertion is found in the 3rd intron of DH-PBAN of B. mori and 64bp-long-deletion is found in the 5th intron of it. It should be further studied whether it is significance for the study of DH-PBAN gene' s evolution of insertion/deletion. Alignment of DH-PBAN genebetween B. mori and B. mandarina show that extrons play important role on keeping the function of DH-PBAN gene.3. The sequence and biochemical character are analyzed with Protean (Nishikawa & Ooi, 1987). This polypeptide is consist of 192 aa, 22271.46 m. w., 1A(280)=1. 29mg/mL Its isoelectric point is 9.12, and the charge at PH7 is 4. 23. The structure of protein is analyzed .The result shows that the leading peptides lies at the beginning of more than 20 amino acids of N-end.4. Sequences of DH, PBAN and cDNA, DNA of their genes are aligned, the results shows that the functional region of DH lies in the six-peptide of C-end; and the functional region of PBAN lies in five-peptide of C-end.5. The phylogeny analysis are investigated using the sequences of DH, PBAN and cDNA, DNA of their genes. The clusters are based on all kinds of sequences, and they can be divided into two kinds: one is B. mandarina China is clustered with B. mori first, then clustered with B. mandarina Japan, and the other is B. mandarina China is clustered with B. mandarina Japan first, then clustered with B. mori . It can be referred that B. mandarina China and B. mandarinaJapan origin from the ancestor in common----old wild silkworm, and B. mori diverges fromB. mandarina China, the sketch map of evolutionary route is showed below.6. The phylogenetic trees are reconstructed based on the sequences of...