Cloning,Expression And PPO Activity Analysis Of Serpins Form Bombyx Mandarina

Serpin and PPO are involved in regulating many physiological responses in the organism.In order to study the expression of serpins and the activity of PPO in wild silkworm and silkworm,this paper has done the following research: The six genes of Bmmserpins were cloned and sequenced;Analysis of serpins transcription level of silkworm and wild silkworm at different developmental stages,different tissues;Analysis of the PPO activity at different developmental stages,different tissues of silkworm and wild silkworm.The results are as follows:1 Cloning and Analysis of serpins of Bombyx mandarinaThe open reading frame(ORF)sequences of serpins of wild silkworm were successfully cloned.GenBank accession numbers are: KR003729,KR003725,KR003726,KR003727,KR003728,KR003730,respectively.The ORF sequences of serpins of wild silkworm were 1137~1371 bp,encoding 378~458 amino acids,protein molecular weight were 42 ~ 51 kDa,isoelectric point were 4.65 ~ 7.32.Compared with serpins of silkworm,the homology of serpins of wild silkworm was very high,and the homology of serpin-3 was the highest,which was 99.27%;The lowest homology of serpin-6 also reached 97.83%.Gene structure analysis showed that there was a typical SERPIN domain in the same position of silkworm and wild silkworm,and the reactive center loop(RCL)was located at the C-terminus,whose sequence was highly conserved,and the P1 sites was the same.Cluster analysis showed that the relationship between serpins of wild silkworm and serpins of silkworm was the closest,indicating that serpin has better conservatived in evolution.2 Transcription level analysis of serpins in silkworm and wild silkwormThe transcriptional characteristics of fat body and epidermis from silkworm(The end of the fourth instar to mounting)and wild silkworm(5th instar)were studied with real-time PCR.The results showed that: The transcriptional level of serpin-1,2 and serpin-4,5,6 of fat body from silkworm were higher in the middle of fifth instar and mounting,and serpin-3 was higher in the early of fifth instar.The transcriptional levels of serpin-1,2 and serpin-4,5 of epidermis from silkworm were higher in the middle and the end of fifth instar,the transcriptional levels of serpin-3 and serpin-6 were higher in the mounting.The transcriptional levels of serpins in the body and epidermis from wild silkworm in the early of fifth instar were higher than those in the middle and the end of fifth instar.Comparing the transcriptional level of serpins from silkworm and wild silkworm in the fifth instar,the transcriptional level of serpins of fat body from wild silkworm were lower than those from silkworm;The transcriptional level of serpins of epidermis from wild silkworm were higher than that of silkworm in the early of fifth instar;but in the middle and end of fifth instar,the transcriptional level of serpins of epidermis from wild silkworm were lower than those from silkworm.The above results indicate that the transcription level of serpins from silkworm and wild silkworm are not the same.3 Comparison of PPO activity between silkworm and wild silkwormThe activity of PPO in different developmental stages and different tissues were determined Jingsong,Heigao,Dazao and wild silkworm.The results show that: The activity of PPO of hemolymph from Jingsong in the middle of fifth instar was higher than that in other stages of development,so was which in the epidermis.The activity of PPO of hemolymph from Heigao in the early of fifth instar was higher than that in other stages of development;The activity of PPO of epidermis from Heigao in the middle of fifth instar was higher than that in other stages of development.The activity of PPO of hemolymph from Dazao in the early of fifth instar was higher than that in other stages of development;The activity of PPO of epidermis from Dazao in the middle of fifth instar was higher than that in other stages of development.The activity of PPO of hemolymph from wild silkworm in the middle of fifth instar was higher than that in other stages of development;The activity of PPO of epidermis from wild silkworm in the end of fifth instar was higher than that in other stages of development.The PPO activity of hemolymph from high to low followed by Jingsong,Heigao,Dazao,wild silkworm;The PPO activity of epidermis from high to low followed by Dazao,Heigao,Jingsong,wild silkworm.Whether the hemolymph or epidermis,the PPO activity of wild silkworm were lower than silkworm.The activity of PPO of hemolymph from Jingsong was higher,but it was lower in the epidermis;To the opposite,the activity of PPO of epidermis from Dazao was higher,but it was lower in the hemolymph;This phenomenon was not significant in Heigao.In this paper,the six serpins of wild silkworm were cloned and analyzed,thetemporal and spacial of serpins in silkworm and wild silkworm were compared,the above research work provides a theoretical basis for the study of serpin from silkworm and silkworm.This paper also compared the activity of PPO in different tissues of silkworm and wild silkworm,and the results had some reference significance to clarify some key problems in the origin of silkworm.