Studies On The Meiosis Arrest Gene And The RNA And Export Factor Binding Protein Gene In Silkworm, Bombyx Mori

The Drosophila always early (aly) gene ,which is required for G2-M cell cycle control and of spermatid differentiation,is one of meiosis arrest genes. In order to address the functions of Bombyx mori aly in germlines developmental events, we used the amino acid sequenceds of Drosophila melanogaster to blast the silkworm sequences of genomic and EST sequence ,the cDNA sequences of Bmaly gene were obtained Via silico cloning. We designed special primers based on the silico cloned sequence, and cloned the Bmaly gene via RT-PCR. It was showed that the Bmaly cDNA sequence was consistent with the predicted seqence, and the open reading frame of Bmaly is 1713bp in length, encoding 570 amino acid residues. The structure prediction displayed that BmAly contains Domain in Rb-related Pathway(DIRP), which exist in other homologues from other species.Phylogenetic analysis showed that the different insect'Aly were closed related to each other, however, the Aly of different insects was not gathered to a cluster according to its insect orders, so it was deduced that the different insect aly gene had its own evolutionary way. The Bmaly gene was inserted into expression vector pGS21a (+),the recombinant protein was expressed in Escherichia coli and used to immunize mice to prepare the antibody against BmAly. The results of real-time PCR revealed an obvious diversity in transcriptional level of Bmaly gene among different tissues. The transcriptional level in testes was higher than that of in ovaries, and almost undect in other tissues. Immuno-fluorescence examination showed that BmAly was distributed in both cytoplasm and nucleus. In order to further explore the function of Bmaly , we injected Bmaly siRNA within silkworm at the beginning of 3th and 4th instar ,and surveyed the resulting influence of sperm cells develop and fertilitied eggs rate. The results revealed that the speed of development of sperm cells markedly decreased after siRNA injection. Most of sperm cells at the first day of 5th instar went into the early stage of primary spermatocyte after aly657 siRNA injection, when those of normal control already entered into the mid-late stage of primary spermatocyte. The average unfertilized egg rate was 22.20% within aly657 siRNA injected silkworms. By comparsion, only 1.76% within normal controls. All the results showed that Bmaly was meiosis arrest gene in Bombyx mori, and expression defect could block the development of sperm cells.RNA and export factor binding proteins(REF)plays important roles in RNA stability, processing and nuclear export. Referring to the sequence of Bombyx mori ref gene (DQ497195.1) deposited in GenBank, the ref cDNA gene (Bmref)from Bombyx mori was cloned via RT-PCR. The sequencing result showed that the open reading frame(ORF) of Bmref is 765 bp in length, encoding 254 amino acid residues, the amino acid sequence of BmREF showed 49.7% and 52.7% identities with those of Drosophila melanogaster and Mus musculus, respectively. The structure prediction displayed that BmREF contains RNA binding domain (RRM), REF-N and REF-C motif of REF subfamily. Phylogenetic analysis showed that the insects REF clustered to a group and that the BmREF closed to those of Apis mellifera and Tribolium castaneum. The Bmref gene was inserted into expression vector pGS21a (+),the recombinant protein was expressed in Escherichia coli and used to immunize mice to prepare the antibody against BmREF. Immuno-fluorescence examination showed that BmREF was distributed in both cytoplasm and nucleus, but mainly located in nucleus. Microarray data analysis showed that Bmref gene was highly expressed in tissues at the day-3 old of 5th instar larvae of the silkworm. The results suggested that BmREF is likely to exert important role in RNA nuclear export, which lays a good foundation to further investigate function of BmREF.