Regulatory Mechanism Of Fibroinase In Silk Gland Development In Silkworm,Bombyx Mori

Silkworm,Bombyx mori,is an important economic insect.Its greatest economic value comes from the silk gland,which is one of the most concerned parts of research in silkworm.The silk gland shows a periodic development process throughout the larval stage,that is,the silk proteins and inner cell layer degrade during the molt period and rebuild during the feeding period.There has evidence that the degradation process of silk proteins is likely to be regulated by fibroinase.Therefore,understanding the mechanism of fibroinase involved in the development of silk gland is conducive to explain how silk proteins stability or degradation during the silk gland development,which is of great significance for studying the mechanism of tissue degradation during the development of insect metamorphosis.In this study,we determine the physiological functions of fibroinase and its regulation mechanism in the silk gland development from in vitro to in vivo,from cells to individual levels through the methods of bioinformatics analysis,genetic manipulation,biochemistry,and molecular biology.The main results obtained in this study are as follows:1.Fibroinase is involved in the silk gland developmentRNA interference experiments indicated that the fibroinase was participating in the degradation of the silk gland and the contents in the lumen.The spatiotemporal expression of fibroinase showed that it was involved in the development of silk gland:from 2^nd instar larval to 4^th instar larval,the expression of fibroinase is lower at feeding periods and it is mainly involved in the degradation of old tissue and preventing the lumen blockage of silk gland;the expression of fibroinase becomes higher in molt stages to renew the inner cell of silk gland and digest the residual silk proteins in the silk gland lumen.In 5^th instar larval,the fibroinase expression decrease drastically to ensure the rapid growth of the silk gland and the accumulation of silk proteins,and prepare for the spinning.At the wandering stage and the spinning stage,the expression of fibroinase increases to prepare for metamorphosis.At the pre-pupa period,the expression of fibroinase is greatly increased to assist the apoptosis of the silk gland.2.Inhibitors are involved in the development of silk gland by regulating the activity of fibroinaseTo further study fibroinase and its physiological inhibitors,we obtained fibroinase by eukaryotic expression.The activity assays showed that the optimum pH value of fibroinase was acidic,which was similar to the internal environment of the silk gland.Fibroinase showed the highest activity at 42°C,but the growth environment of silkworm usually did not exceed 28°C.It is indicated that fibroinase usually functions at 50%of its maximum vitality.Previous studies showed that Serpin 18 is one of the physiological inhibitors of fibroinase in the silk gland.The proteomic data of the silk gland showed that the BCPI,a cysteine protease inhibitor,was identified also.Thus,we purified recombinant BCPI and determine its inhibitory activity towards fibroinase.The results showed that BCPI is another physiological inhibitor of fibroinase,and the inhibitory molar ratio of BCPI to fibroinase is 1.2.The spatiotemporal expression characteristics of the inhibitors,Serpin18 and BCPI,showed that its main function is to prevent the excessive activity of fibroinase at 5^th instar larval,wandering stage and early spanning stage.And Serpin 18mainly expressed in the 5^th instar larval,and BCPI mainly expresses from the end of 5^thh instar to the early spinning stage.These two inhibitors are involved in the silk gland development by regulating fibroinase activity.3.The regulation of fibroinase on the silk gland development is affected by ecdysoneHere,we demonstrated that the expression of fibroinase is up-regulated by ecdysone but does not respond to the juvenile hormone.After cloning,truncating and analysis of the fibroinase promoter,we found that BmAbd-B,BmAntp,BmBrc-Z2,and BmPOU-M2 are most likely involved in the regulation of fibroinase.Further research shows that ecdysone could induce an increase in BmAbd-B and BmPOU-M2 and a decrease in BmBrc-Z2,which up-regulating the expression of fibroinase and leading to periodic developmental changes in the silk gland.