Expression Profile Analysis Of Serpin-6 Gene Of Bombyx Mori

Serpin genes comprise a large gene family,and their protein products regulate a wide variety of proteinse-depent physiological functions. In order to study the function of BmSerpin-6 gene in Bombyx mori,the transcriptional level of BmSerpin-6 at different developmetal stages and larvae challenged by LPS or Bacillus thuringiensis at different periods was determined by semi-quantitative RT-PCR. Bac-to-Bac system was used to express the BmSerpin-6 protein.The main results are as follows:1 Haemolymph was collected from the 4th molting larva,newly moulted 5th instar larva and day-3 fifth instar larva challenged by LPS or B.thuringiensis,respectively. The results revealed that the transcriptional level of BmSerpin-6 of different developmetal stages showed a trend of rise first,then fall. BmSerpin-6 of the 4th molting larva expressed highest at 6h post-infection with LPS and 3h post-infection with B.thuringiensis. BmSerpin-6 of newly moulted 5th instar larva expressed highest at 9h post-infection with LPS and 6h post-infection with B.thuringiensis. BmSerpin-6 of day-3 fifth instar larva expressed highest at 9h post-infection with B.thuringiensis. BmSerpin-6 was all highly induced and highly expressed in haemolymph of lavae at different developmetal stages.and the time to arrive the highest transcriptional level was different. It is further inferred that this Serpin gene may play an important role in immunity of Bombyx mori.2 The transcriptional level of BmSerpin-6 gene is different at different developmetal stages. The results are as follows:(1)The transcriptional levels of BmSerpin-6 of newly moulted larva of 1st to 5th instar is almost the same,but that of 4th molting larva is much higher than 5th instar newly moulted larva.(2)There is significant difference between the transcriptional levels of BmSerpin-6 gene at each day of Bombyx mori 5th instar larvae,which decreases to the lowest in the 4th day and then increases to the highest in the 6th day.(3)The transcriptional levels of BmSerpin-6 gene at 12h of the spinning stage is ?lower than that of mature larva,but it then increases. (4) During the pupal stage, the transcriptional levels of BmSerpin-6 gene increases to the highest in the 6th day and then decreases to the lowest in the 13th day. (5)The transcriptional levels of BmSerpin-6 gene of testes and ovaries showes a trend of rise first,then fall. The results indicated that transcription of BmSerpin-6 is stage-specific. It was suggested that BmSerpin-6 plays different roles in Bombyx mori during different developmental stages. BmSerpin-6 could be involved in the regulation of cuticle degradation during the insect molting cycle and protecting fibroin from degradation.3 Bac-to-Bac baculovirus expression system was used to obtain the recombinant protein. SDS-PAGE and Western blot results of the expression of recombinant protein showed that a specific band was detected around 46 kD,which is consistent with the expected size of the fusion protein. The result showed that BmSerpin-6 was expressed successfully with Bac-To-Bac system.The result of this study provides reference for studing immune defense of Bombyx mori and other Lepidoptera insects.The eukaryotic expression and purification of BmSerpin-6 protein provided materials for further studying functions of Serpin in vivo and in vitro.