Turn Pgh Gene Silkworm Build Research And Transfer Of Gfp And The Neo ~ R Silkworm, Subculture

Silkworm (Bombyx mori) is an important economic insect and model organism for genetics study. Transgenic research of silkworm plays an important role in molecular biology theory study and bioengineering production practice.In this context, porcine growth hormone gene(PGH) as exogenous gene from pSMTPGH was linked with fibrion promoter from plasmid pFblOO.pFbPGH, the construct was transformed into silkworm race^QA FANG by sperm-mediated method to analyze integration and expression of PGH by the action of fibrion promotenAt the same time,the expressional plasmid pSMTPGH which was constructed with PGH and sheep metallothionein promoter (SMT) by Chen Qingxuan is transformed into silkworm to analyze the integration of PGH. hi addition, in order to probe into the stabilization of foreign gene transformed into silkworm, GFP and neor are analyzed by PCR screening in transgenic silkworm.Based on above research^ can provide some experimental bases for transgenic research of silkworm.1. The Construction of Plasmid pFbPGHThe plasmid pFbPGH was constructed with porcine growth hormone gene(PGH) and silkworm fibrion promoter(Fb).The resulting plasmid,pFbPGH ,with PGH structural gene removed by promoter, is fit for transgenic silkworm research.2. construction and detection of transgenic silkworm of pFbPGH and of pSMTPGH2.1 transformation of silkworm with plasmid pFbPGH or pSMTPGHThe summer-autumn rearing race, XIA FANG, was transformed with plasmid pFbPGH and pSMTPGH ,by sperm-mediated method. Plasmid pFbPGH or pSMTPGH (5 P L,2n g/u L) with gene桺GH is injected into copulatory pouch by glass needdle before mating. Eggs of the treated moths were collected and reared for screening.2.2 Molecular screening2.2.1 PCR screeningFrom every batches, 10 of the second instar larva were random selected and mixed for specific amplifiction of PGH(1460bp).Positive fragment was obtained at certain rate(transgenic silkworm of pFbPGH:20%; transgenic silkworm of pSMTPGH :20%) and corresponding positive batches (GO) were selected out.2.2.2 Southern blottingIn order to get rid of PCR false positive, Southern blotting was applied. 15 u g DNA of 10 from GO generation were digested with Hindlll and electrophoresised on 1% agarose gel and then transferred to nylon membrane.Plasmid pSMTPGH was digested with Smal,and the small fragment (about ISOObp) was labeled with DIG, which was used for probe.Positive signals of southern blotting were gotEggs of positive parent were kept for further study.2.2.3 Northern blottingTo analyze the transcription of PGH gene in the transgenic silkworm,Northern blotting was applied. From every batches, 10 silkworm larvae were random selected and mixed forextraction of RNA. 10-20 u g RNA was electrophoresised on agarose gel with formaldehydeand transferred onto nylon membrane by capillary transfer method.The small fragment which of pSMTPGH digested with Smal was DIG labled,which was used for probe.But no positive signals of Northern blotting were detected.Probable reasons are the following.The first one is that the transcription product maybe existed in some transgenic silkworm ,but the sample which was random selected had no positive individuals.The second one was that in the course of Northern blotting,mRNA had been degraded.3. Analysis of transgenic silkworm transformed with GFP or neor Positive individuals both in the G2 and G3 generation of transgenicsilkworm of green fluorescent protein(GFP) by sperm-mediated method and in the Gl and G2 generation of transgenic silkworm of neomycin resistance gene(neor) by gene gun method were obtained by using PCR screening.The results showed that transgenic silkworm by sperm-mediated method and gene gun method were relatively stable transmission.