Piggybac Transposon-mediated Expression Of E.coli Cysteine Biosynthesis Genes In Transgenic Cashmere Goat

China is the world's largest producer of cashmere. Chinese cashmere enjoys a "softgold" reputation in the international community, but its output is low. Thus, it is of greatinterest to maximize the quantity of the cashmere fiber without affecting the fiber quality.Manipulation of the cashmere goat genome provides opportunities to improve cashmere woolproducts. Cysteine is one of the main ingredients of cashmere. Based on transgenictechnology, this study import cysteine synthesis genes to goats via piggyBac transposon, inorder to provide a more efficient pathway to cysteine synthesis in the cashmere goat andprovide the groundwork for cultivating cashmere goats with high cashmere production. Theresearch object, technology, methods, research purpose and results of this study were asfollows:1. The cysE (serine acetyltransferase)and cysM(O-acetylserine sulfhydrylase) genesare amplified by PCR from Escherichia coli genomic DNA. The cysE gene is822bp, whichencoding274amino acids, and cysM gene is912bp, which encoding304amino acids.Prokaryotic expression vectors pET-cysE and pET-cysM are successfully constructed andefficiently express in BL21(DE3) after induced.2. Two integrative fluorescent protein plasmids pIRES2-Red2-cysE andpIRES2-EGFP-cysM which containing cysteine biosynthesis enzymes gene cysE and cysMrespectively was constructed and co-transfected into cashmere goat fetal fibroblasts byLipofectamineTM2000. Cell clones stably co-expressed of cysE and cysM genes wereobtained after screening by G418. Subsequently, co-expression of the cysE and cysM genes incashmere goats embryos were gained by nuclear transfer oocytes.3. The objective of the experiment was to study the integration site of piggyBactransposon in Cashmere goat genome and piggyBac donor plasmid of pB-CMV-EGFP-NEOand helper-dependent plasmid of pcDNA-Trans were constructed and transferred intoCashmere goat fetal fibroblasts by lipofectamine2000. Cell clones stably transfected wereobtained after screening by G418. Transgenic cell genome was obtained and the integration sites of piggyBac transposons were detected by r-PCR. There were21integration sites ofpiggyBac transposon in Cashmere goat genome after r-PCR detection; Analysis of a flankingregion of TTAA flanking sequences from position+5showed that piggyBac3′tended toinsert into region rich in AT (58.35%) and the piggyBac5′tended to insert into region rich inGC (57.8%). The integration site information which is acquired from this research willprovide theoretical references for Cashmere goats study by piggyBac transposons.4. The3400bp of promoter sequences of small proline-rich protein II (PRD-SPRRII) wereobtained according to specifically expressed genes in rumen of sheeps. The trend is thatheterologous expression of some proteins in goat can be improved by altering codonpreference. New sequences of cysE and cysM which were more appropriate to express incashmere goats were synthetized by codons bias in goat. Then, the donor plasmidpB-EGFP-NEO-CMV-PRD-cysE-cysM was constructed with two genes driven by arumen-specific promoter from the goat small proline-rich protein gene (PRD-SPRRII).5. A piggyBac transposon system including donor vector ofpB-EGFP-NEO-CMV-PRD-cysE-cysM and helper vector of pcDNA-Trans were developed,and transgenosis cells differently transfected were obtained by G418. The expression levels ofpiggyBac-mediated EGFP and cysM increased7.78-fold and6.63-fold respectively comparedto control group, confirming that the transposition of foreign piggyBac transposons incashmere goats was highly efficient.29integration sites were obtained in the goat genome bysplinkerette PCR. Analysis of a flanking region of TTAA flanking sequences from position+5also revealed that piggyBac tended to insert into the region rich in AT (67.95%).6．A transgenic Shanbei white cashmere goat with E.coli cysteine synthesis gene cysEand cysM in its body was got through piggybac transposon.23integration sites were obtainedin the goat genome by splinkerette PCR and it was found that piggybac transposon tended toinsert into the region rich in AT (62.70%) of transgenic cashmere goat gene.In summary, this study confirms the function of piggyBac transposon on highly effectivetransposition of GFFs and on highly effective expression of foreign gene, which provides thegroundwork for cultivating cashmere goats with high cashmere production throughpiggyBac-mediated cysteine biosynthesis gene expression.