Studies On The Reproductive Development-Related Gene BmSTAT In Silkworm, Bombyx Mori

In order to address the functions of Bombyx mori signal transducer and activator of transcription (Bm-STAT) in developmental events, we used the amino acid sequences of Drosophila melanogaster and Spodoptera frugiperda to blast the silkworm sequences of genomic and EST sequence, the cDNA sequences of Bm-STAT gene were obtained by silico cloning. It was found that Bm-STAT cDNA has two isoform by alternative splicing. The ORF of long-form Bm-STAT was 2313nt in size, encoding 770 amino acid residues and the ORF of short-form Bm-STAT was 2202bp in size, encoding 733 amino acid residues. The cDNA of short-form Bm-STAT gene was cloned by RT-PCR with a pair of designed special primers based on the silico cloned sequence. It was showed that the short-form cDNA sequence was consistent with the predicted sequence; the amino acid sequence was consisted of STAT_int, STAT_alpha, STAT_bind and SH2 domains. The genomic sequence of Bm-STAT was finally assembled based on the sequence of PCR products and the deposited genomic sequence of silkworm in the GenBank database. The results showed that the genomic sequence corresponding to the long and short-form Bm-STAT ORF was 28777,22752bp in size, respectively. the long-isoform was consisted of 1-18 exon and 20 exon, the short was sonsisted of 1-19 exon. Phylogenetic tree was constructed based on the amino acid sequences of STATs, it was found that the insects'STAT were closely related to each other, however, the STAT of different insects was not gathered to a cluster according to its insect orders, so it was deduced that the different insect STAT gene had its own evolutionary way. The cDNA of short isoform was inserted into a prokaryotic expression vector pET-28a (+) and the recombinant protein was expressed in E. coli. The mice were immunized with the protein purified by nickel chelate affinity chromatograph to prepare polyclonal antiserum. Western blotting for silkworm gonads protein was performed with the antiserum. Two special protein bands with molecular weight of 70 kDa and 130 kDa, respectively, were detected in testes, but no special signal band was detected in ovary samples. Immunofluorescence assay with the FITC-labeled antibody showed that the green fluorescence was observed in both nucleus and cytoplasm of cultured BmN cells and haemocyte of silkworm, but the emitted green fluorescence was primarily located in nucelus, so we deduced that Bm-STAT was located primarily in nucelus. The results of real-time RT-PCR revealed an obvious diversity in transcriptional level of Bm-STAT gene among different tissues. The transcriptional level in testes was about 10 times higher than that of in ovaries, while low expression level in malpighian tubule, silk gland and midgut. These results laid a foundation for further addressing the functions of Bm-STAT.