| Serine protease(SP) is one important member of the protease,mainly functioning in the degradation and synthesis of protein,under the regulation of the serine protease inhibitor(SPI).Both SP and SPI serve as essential components in keeping the normal functions of an organism,as were widely reported in animals,plants and microorganisms.Likewise,the silkworm cannot live and develop in its normal way without SP and SPI,which plays very important roles in controlling the development and ecdysis and are even involved in the enhancement of self-defensive ability.The SPI was first purified in the silkworm by Morita in 1961,and was at last classified into tens of types about 40 years later,including Kunitz,Serpin and Kazal types,among which the chymotrypsin inhibitor(CI) has been extensively studied.More than 16 kinds of CI have been identified and nominated by Fujii group.Furthermore, they also demonstrated the inheritance law of the CI and their localization of linkage groups in the silkworm,and especially specified their classification,purification, physicochemical property,and gene structures,so as to pay the way for the further study of SPI in this animal and even in many other insects.However,almost all references on SPI so far were still centering on biochemics and genetics,leaving much unknown about their genetic polymorphism,expression patterns,and evolution and functions. Based on our silkworm genome data therefore,this study aims to present a systemic study on the SPI,pinpointing at CI and trypsin inhibitor(TI) from their genetic polymorphisms,isolation and purification to gene cloning,expression and biological functions.The main results are as follows.1.Study on polymorphism of CIs in Bombyx moriWe investigated hemolymph from 434 silkworm strains conserved in Silkworm Genetic Resource Center of Southwest University in order to learn polymorphism and distribution character of CI in Bombyx mori.We found all 18 kinds of CIs coded by five genes Ict-A,Ict-E,Ict-D,Ict-B,Ict-H.CI-9 and CI-10,less reported previously,were detected for many times.Moreover unknowed CI-5,CI-11 and CI-12 were found in a few of strains.The investigation showed that all strains express one of CIs coded by Ict-A and Ict-D,and that CIs coded by genes Ict-B and Ict-H were not found in 9 silkworm strains. These suggested that Ict-A and Ict-D,but not Ict-B and Ict-H,are essential for development and growth of silkworm.In previous point Ict-E was considered to be necessary for silkworm.In the study,we found no CI-3 and CI-4 coded by Ict-E in some strains.Our results suggested that silkworm may be able to lack Ict-E.Investigation of 52 pure strains showed that most of CIs distribute in different geographical strains,and only individual CIs were detected in special strains.For instance,CI-4 and CI-9 appear in Chinese strains,and not in 12 Japanese strains.CI-8 was found in rare Chinese local strains.Cluster analysis indicated that we did not found the distribution characteristic of CIs thought strains with same origin cluster together.Our experiment also showed that CIs express in special tissues and stages,such as CI-8 and CI-13.They express in all stages and tissues of silkworm.So they should be involved in embryogenesis,growth,ecdysis and metamorphism of silkworm.2.Study on polymorphism of TI from Bombyx moriThe first investigation of silkworm TI found 9 kinds of TI in 375 strains.We found that there are TIs and no CI in silk gland.And TI can be detected in middle silk gland from 5th instar to spinning stage.This cues that TI may protect silk protein against protease activity,or play roles in the process of tissue dissolution.Analysis on distribution of TI from 80 Chinese and Japanese strains indicated that most of TI-4 appears in Chinese strains and TI-b3 in Japanese strains.This suggested that there was original trend in distribution of some TIs.3.Purification,characterization,and cloning of CI 9 from Bombyx moviIn order to purify CI-9 we tried for several times and built a set of effective method. Homogeneous CI-9 was purified from b40 by ammonium sulfate precipitation,Butyl Toyopearl hydrophobic chromatography,gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography.Activity of CI-9 increases 1819 folds and its absolute recovery is 4%.CI-9 was determined to be 7176 Da with the Voyager TOFMass analyzer.The pI value for CI-9 was 4.3.CI-9 exhibited inhibitory activity at a temperature as high as 100℃and a stability against a wide range of pH (1-12).In N-terminal amino-acid analysis of CI-9,40 amino acid residues were obtained,and the active site is AYF.We confirmed it is a novel Kunitz type of CI from above.The rest unknowed sequence of CI-9 was gained by cloning of gene fragment of 598 bp including an intron.So the whole CI-9 consists of 62 amino acid residues.4.Genome-wide identification and analysis of SPs in Bombyx moriBased on the entire genome fine map of silkworm,we have identified 143 genes encoding SPs in silkworm and named as BmSP1-BmSP143.Among them,80(55.9%) genes have the evidence of ESTs,and 20 genes have been reported or submitted online, other 123 genes were newly identified.The information analysis showed that these genes' molecular weights were varied from several kDa to hundreds kDa and their isoelectric points were ranging from 3.5 to 11.0.In these 143 genes,51 genes have included the Ser,His and Asp residue complete active position spot;others have the non-complete active position spot.The chromosome localization results showed that 128 genes were located to 26 chromosomes and distributed non-uniform.The 5th,7th and 16th chromosome have more genes,with 11,13,10 respectively.Especially,the silkworm serine protease genes have the tandem repeat distribution phenomenon,such as BmSP74,BmSP143,BmSP18,BmSP142,BmSP103,BmSP40,BmSP53,BmSP39 and so on,they were located on the 16 chromosome with tandem repeat distribution and these gene sequences were similar,suggested that they maybe originate in the gene duplication event.5.Genome-wide identification and analysis of serine protease inhibitors in silkwormOur analysis found that there are at least 56 serine protease inhibitor genes in silkworm,named as BmSPI1～BmSPI56.In these genes,49(87.5%) genes have the evidence of ESTs,18 gene sequences are consistent or highly similar with reported genes,other 38 genes are new genes.A total of 49 genes can be located to the chromosomes;the distribution is not uniform also.The 3rd,22nd and 28th chromosome, have 10,6,7 genes respectively,and the 2nd,3rd,7th,15th,22nd and 28th chromosome have found the tandem repeat group,including 2～8 sequences in a similarity separately, which were members of gene duplication.Most importantly,the information analysis found the genomic sequence which were correspond to five genetic locus Ict-A,Ict-B, Ict-D,Ict-E and Ict-H identified by Fujii.The corresponding relationships were BmSPI48 to Ict-A,BmSPI54 to Ict-B,BmSPI40 to Ict-D,BmSPI38 to Ict-E,BmSPI55 to Ict-H.In particular,the genetic analysis showed that the three sites Ict-A,Ict-B and Ict-H were from the same ancestor,and each had at least 2,2 and 3 reiterated genes respectively.Based on genome information,we speculated that the variation of Ict-H site was most active in all sites,of which functions were taking place differentiation, and Ict-A must be the only essential gene locus of the 3 sites which had the oldest evolutionary history.6.The expression patterns of SPs and SPIsAmong the 56 SPIs identified by the genome-wide scanning,there are 39 inhibitors have been covered by the silkworm genome-wide microarray.A systematic expression pattern analysis of these inhibitors was carried out.Some CIs(CIs) were found specifically expressed in some tissues or during certain developmental stages.CIs which contain domains such as KunitzBPTI,napellucida and Thyroglobulin1 all expressed extremely lower in the tested ten tissues,while BmSPI22,a SPI containing I-set domain,has a high expression abundance among all the tested tissues.Five SPIs with Kazal domain mainly expressed in the head,epidermis and hemocyte.In addition, CI-8 was found specifically expressed in the malpighian tubule,while BmSPI24, BmSPI19,BmSPI9 and BmSPI29 specifically expressed in the silk gland,which implicated they were closely related to immunity and fibroin synthesis separately. Mostly important,we found that genes duplicated from the same ancestor had a similar expression pattern and this phenomenon further confirmed that the polymorphism of CIs due to gene duplication.So,we deduced that genes coming from the same duplication cluster might share a similar role.Similarly,a systemic expression analysis of 107 SPs was carried out and there were totally five representative expression patterns.Two main expression patterns of these SPs during developmental stages were characterized:some SPs expressed constantly during the whole developmental course and some SPs expressed only before pupation. However,unlike the expression pattern of SPIs,there was no similar expression pattern of SPscoming from the same duplicated cluster,or vice versa.7.Induced expression patterns of SPs and SPIs In order to analyze the possible relationship between SPs and defense immune response,four kinds of microbes,E.coli,B.thuringiensis,B.bassiana and BmNPV, were used to challenge silkworm Bombyx mori.Induced expression patterns of SPs and SPIs were also analyzed by microarray.The results showed that 25 SPs and 15 SPIs were up-regulated after microbe challenging.Interestingly,BmSPI9 was sensitive to the challenge of E.coli and B.bassiana,while BmSPI17 was sensitive to the challenge of BmNPV.8.Cloning and prokaryotic expression of SPIs specifically expressed in the silk glandThree SPIs(BmSPI19,BmSPI9 and BmSPI29),which were specifically expressed in the silk gland,were selected for further study.Semi-quantitative RT-PCR was employed for confirming the expression patterns of the three genes.The results showed that BmSPI9 and BmSPI19 expressed mainly in the whole silk gland and posterior part of middle silk gland separately,while BmSPI19 expressed exclusively in the anterior part of middle silk gland.The full ORF frames of these three genes were completed by T-A cloning.In addition,we got a recombinant protein of BmSPI29 by prokaryotic expression in vitro.We hope that the study of SPIs will be helpful for elucidating the molecular mechanism of fibroin synthesis.9.Analysis on polymorphism of CIs and TIs from main Lepidoptera insectsIn our study,we primarily detected TI and CI in hemolymph from more than 20 kinds of Lepidoptera insect and from 18 kinds of eri-silkworm.We found that pI of CI from wild Lepidoptera is between 4.5 and 9.0 and that rich polymorphism occurs,after compared with TI and CI from Bombyx mori.We detected 11 kinds of CIs and 12 kinds of TIs in eft-silkworm,and no obvious polymorphism of CI was observed in 18 kinds of eri-silkworm.But sort of TI is more than Bombyx mori and there is rich polymorphism in them.These results are valuable clue for further studying function of CI and finding the way to controlling Lepidoptera insects.
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