Tissue-specific And High-level Expression Of Silk Gland Serine Protease Inhibitors In The Silkworm, Bombyx Mori

Silk gland is a specific organ for synthesizing silk protein in the silkworm.Serine protease inhibitors are one of important protease inhibitors in silk gland,which may be concern with synthesizing and protecting silk protein.Regulation of serine protease inhibitors plays a key role in the high efficient silk protein-synthesis system,on which there is a little report at present.In 2003, the draft sequence for the genome of Bombyx mori had been finished successfully.In 2006,the first whole-genome microarray covering the presently known and predicted genes in the silkworm genome had designed and the study platform of function genome was constructed at Gene and Protein levels,which provide the reliable technology guarantee and study basic for analyzing systemically the types,structure characteristic and evolutionary relationships of serine protease inhibitors in the silk gland.In present study,we investigated the polymorphism of trypsin inhibitors of silk gland in silkworm with the method of study on protein,and identified the types of serine protease inhibitors by the genome-wide scanning with the methods of molecular biology and bioinformatics.To further study,we cloned and expressed serine protease inhibitors of the silk gland. The main results are as follows.1.Investigate the polymorphism of trypsin inhibitors of silk gland in the silkwormBy using Native-Polyacrylamide gel electrophoresis followed by TI activity staining,we investigated the polymorphism of trypsin inhibitors of silk gland in 25 silkworm strains.There are 6 TIs which were named TI-â… ,TI-â…¡,TI-â…¢,TI-â…£,TI-â…¤and TI-â…¥.We found that there are different TIs in different strains.Take silkworm strain DaZao for instance,we investigated the types of TIs in different tissues and found that there is the polymorphism of TIs in different tissues.The type of TIs in silk gland is different from the other tissues.Take silkworm strain DaZao and 21-872 for instance,We investigated the types of TIs in silk gland during the 5th instar silkworm larva,and found different type of TIs in different strains.We also discovered that the activity of the TIs was detected in different development times,the activity of TIs become stronge gradually with the silk gland development and get the peak of activity at the second day of spinning,then reduce gradually.2.Study on the physics and chemistry character of serine protease inhibitors in the silkworm cocoonUsing Native-Polyacrylamide gel electrophoresis followed by TI activity staining,we investigated the activity of TIs of the silkworm dated cocoon.We discovered that the activity of TIs of dated cocoon is lower than that of fresh cocoon and the activity of some TIs has disappeared in dated cocoon.We investigated the activity of TIs the silkworm cocoon treated with pH and temperature and found that different TIs has the different stability of pH and temperature,TI-VI is the most stable TI for pH and temperature.3.The types,structure characteristic and evolutionary relationships of serine protease inhibitors in the silk glandBased on the entire genome fine map of silkworm,microarray data and EST,we have identified 14 genes encoding serine protease inhibitors,which express specially and have a high expression in the silkworm silk gland.They were named as BmSPIs1-BmSPIs14 respectively and have the evidence of ESTs.We predicted their protein domains using BLAST in NCBI and SMART.We discovered that BmSPIs3,BmSPIs4,BmSPIs5,BmSPIs6,BmSPIs7 and BmSPIs8 have Serpin domain,BmSPIs1,BmSPIs2 and BmSPIs9 have TIL domain,BmSPIs12,BmSPIs13 and BmSPIs14 have Kunitz domain,BmSPIs11 has Kazal domain and BmSPIs10 has Serpin and TIL domains. Phylograms of between 14 serine protease inhibitors and those of other species was displayed by neighbor-joining analysis in ClustalW and MEGA.We found that the same domains gathered together,which suggested four structures of serine protease inhibitors in the silk gland are hidebound among species.4.Clonding and prokaryotic expression of serine protease inhiitors specifically expressed in the silk glandSemi-quantitative RT-PCR was employed for confirming the expression patterns of the five genes which are BmSPIs5,BmSPIs6,BmSPIs8,BmSPIs13 and BmSPIs14.The result showed that they do express highly and specially in the silk gland.The full ORF frames of these five genes were completed by TA cloning.In addition,we got a recombinant protein of BmSPIs6 by prokaryotic expression in vitro.We hope that the study of serine protease inhibitors of the silk gland could provide theories and dates support for elucidating the molecular mechanism of fibroin high efficiency synthesis.