Identification And Function Analysis Of Short Interspersed Repetitive Sequence(BmSer_SINE) Of Silkworm, Bombyx Mori

Silkworm(Bombyx mori) as an important economic insect which can produce silk and spin cocoons had been domesticated long ago.Silk was synthesized in silk gland.When larva develops to five instar it starts to synthesize mass protein and secret to cocoon.The total protein is 0.5 gram.After the silkworm transgenic technique has been constructed,people focus on how to utilize the silkgland as bioreactor to effectively synthesize foreign protein.The silk are mainly composed of sericin and fibroin.Foreign protein fusion expression with partial 3'light-chain fibroin would be segregated by high salt and high denaurant. The main reason is that fibroin can not dissolve in water.But sericins which are composition of floss are sluble in water.The solution characteristics of sericin is that sericin₁ protein is predominant and can be firstly dissolved in water.At the initiation of the study is to clone sericin₁ promoter and utilize the promoter to drive foreign gene to synthesize foreign protein specially in middle silkgland.When the sericin1 promoters were cloned,the length of sericin1 promoter is the same with the length of upstream regulatory sequence of sericin-1 in GenBank when dazao genomic DNA was used as template to amplify, the length has 449bp decrease when N4 genomic DNA was used.The investigation result indicate sericin1 promoter has some fragment insertion in some breeds.The insertion fragment is presumed as a short interspersed repetitive sequence(SINE) by whole genome and GenBanK data analysis.After that,the origin,insertion site,function,the mechanism of function and others of the SINE were studied.1.Polymorphism of sericin1 promoter of silkworm,Bombyx moriThe sequence of sericin1 promoter(AB007831.1) was used to design primer to amplify sericin1 promoter.PCR product of sericin1 promoters of 78 breeds of Bombyx mori and Bombyx mandarina were investigated.The results showed that the length of the productions amplified from 7 breeds are all 1kb,which is consistent with the length in GenBank;the fragments amplified from 46 breeds are 660bp,which is 449bp shorter than length in GenBank;both of the 1kb and 660bp fragments can be amplified from 9 breeds;meanwhile there are some of the breeds have no production.The cloned sequences of sericin1 promoter were used CLUSTAL.X 1.83 to analyze,the result is tâ‘ 660bp sequences are highly similarity except a GCTA and a poly(A) insertion mutation;â‘¡1kb sequences have extremely similar except poly(A) insertion mutation;(3)Alignment between 660bp sequence and 1kb sequence reveals a 449bp fragment insertion in 1kb sequence;â‘£The same characteristic of the two sericin1 promoter with different length is that they have three cis-regulatory elements:SA,SB and SC site.⑤The length of sericin1 promoters amplified by PCR has no relationship and regularity with the genome DNA from ecology type, geosystem and oltinism of Bombyx mori or Bombyx mandarina.2.Identification of BmSer_SINEUsing the assembled 9x genome data to analyze the insertion sequence in sericin1 promoter,The result shows thatâ‘ the insertion sequences have 4770 copies in silkworm genome and distribute evenly on 28 chromosomes;â‘¡the insertion sequences integrate on genome at TA_rich region.They maybe originate from L1(LINE_BM) of silkworm by analyzing the inserted sequence with retrotransposon data.The inserted sequence was presumed as SINE and named as BmSer_SINE according to its distribution among genome sequence,insertion sites in genome and its origin.The BmSer_SINE was definitely confirmed present in all of genome of Bombyx mori.3.Research of the activity of BmSer_SINE by transfected cellThe truncation and deletion sericin1 promoters and BmSer_SINE were cloned into pGL3_basci vector to generate sericin1 promoters and BmSer_SINE driven luciferase vectors.The recombinant vectors were transfected into Sf9 cell and BmE cell by Cellfectin(?)â…¡Reagent.Using Dual Luciferase Reporter Gene Assay System to detect the activity of truncation and deletion sericin1 promoters and BmSer_SINE. The result indicate that luciferase driven by BmSer_SINE has relative activity both in Sf9 and BmE cell.But luciferase driven by truncation and deletion sericin1 promoters has no activity in BmE and Sf9 cell except luciferase be driven by sericin1 with intact SINE in Brae cell.The result indictas intact BmSer_SINE has promoter activity.4.Identification the enhancer activity of BmSer_SINEThe truncation and deletion sericin1 promoters,DsRed,terminal sequence of SV40 were connected to generate truncation and deletion sericin1 promoters driving DsRed expression cassette.The expression cassettes were cloned into pBac[3XP3-EGFP]vevtor to generate pBac[3XP3-EGFP,Ser-DsRed].The pBac[3XP3-EGFP,Ser-DsRed]plasmids were injected into eggs and integrated into genome to generate transgenic germline.The activity of truncation and deletion sericin1 promoters were detected using quantitative PCR by detecting the transcription level of DsRed.The result indicates sericin1 promoters containing BmSer_SINE sequence have much more activity than that have none.DsRed synthesized in sericin was detected by Western blot,the result indicates BmSer_SINE contain some sequence can increase protein expression.In addition,the silkgland and fat body with generation cocoon expressing DsRed were detected by DsRed fluorescence.The result indicated DsRed only expressed in middle silkgland,which indicates-148bp sequence of sericin1 promoter control the sericin specially expressed in middle silkgland.5.Exploring the mechanism of BmSer_SINE as enhancerThe cis-regulatory elements of BmSer_SINE sequence were predicted byhttp://www.cbil.upenn.edu/cgi-bin/tess/tess?RQ=WELCOME.Typical region of RNA polymeraseâ…¡transcription factors binding site was labeled with Cy3.The Cy3 and nuclear protein complex detected by native PAGE were identified by LC/MS/MS.LC/MS/MS results indicated the nuclear factors are translation initiation factor,elongation factor,ATPase,GTP binding protein and other nuclear protein.The BmSer_SINE was analyzed with GenBank data,CDS,EST and microarray data.The result reveals no coding sequence of 19 identified gene and coding sequence of 46 predicted gene were integrated by BmSer_SINE.19 identified gene and 22 predicted gene in total of 46 predicted gene have signal value. It has been confirmed that serum promoter and first intron of light-chain fibroin gene contain partial sequence of BmSer_SINE can enhance the promoter activity.So,the signal value(18.9) of serum protein as criterion,the signal value of 16 gene from 42 gene with signal value are about or higher than 18.9,which indicates that BmSer_SINE may have universal enhancer effect.