Cloning And Sequence Analysis Of Bm-TSSK Gene From Silkworm, Bombyx Mori

Post-translational modifications of proteins through the phosphorylation of serine,threonine, and tyrosine residues by protein kinases play a role in many cellularprocesses such as transduction of extracellular signals, intracellurlar transport, and cellcycle progression. Considering the integral role that protein kinases play in the controlof cellular mechanisms and signal transduction, it is not surprising that several proteinkinases have been shown to be involved in spermatogenesis. Nevertheless, only a fewkinases have been characterized, whose expression is restricted to either germ cells or tothe testis.TSSK (testis-specific serine kinase) was first found in mammals which plays animportant role in the spermiogenesis.By now we have found that the TSSK family has6members.We use the animo acid sequence of Homo sapiens TSSK(TSSK2、TSSK4、TSSK6） and Mus musculus TSSK (TSSK1、TSSK3、TSSK5) to search against the silkworm(Bombyx mori) genome database with BLAST program. The homologous sequences wereclustered, and through the electronic cloning, a testis-specific serine kinase sequence ofBombyx mori was got. By using the primers based on the predicted sequence, we cloned theTSSK gene of Bombyx mori, termed it as Bm-TSSK (GeneBank Number: JN159476). The lengthof the Bm-TSSK cDNA was1402bp, contains an open reading frame (ORF) Which encoded346anima acid. The Bm-TSSK gene contains1exon and surprisedly none intron. The deduced aminoacid has81%identity to Tribolium castaneum and86%identity to Bombus impatiens,85%identity to Acromyrmex echinatior. Bm-TSSK in testis on day3of the5th instar larvawas highly expressed based on the data analysis of real-time PCR and gene chip.Analysing the expression pattern on the4th nad5th instar larval testis, the expressionlevel was highest in the five days of the4th instar. This period was supposed to be thefastigium of initialing spermatocyto differentiation. It was predicted that Bm-TSSKprobably invovled in spermgenesis by interferecing the spermatocyto differentiationprocess. In this study, RNAi was employed to restrain the expression of Bm-TSSK, and thetranscription level was detected by real-time PCR. In the results, Bm-TSSK was greatlyrestrained at larval and pupal stages, which amounted to58%and27%of the controlgroup respectively. The pupe restrained were hybridized and then investigated forfertility rate and hatching rate of F1newly hatched silkworm. The fertility rates of F1were97%and95%. The hatching rates of graine in experiment groups was impaired,namely68%and95%. The results demonstrated that the impairment of Bm-TSSKexpression would not greatly influence the fertility rate of sperms, but did influence thehatching rate. Therefore, the effect of Bm-TSSK on the mechanism of silkwormreproduction was encouraged for further investigation.