Study On Sulfolobus Solfataricus β-Glycosidases Gene Transgenic Dairy Cattle By PiggyBac Transposon

Milk as a nutritious whole protein food, plays an important role in the supplement nutrition, promote absorption of calcium, phosphorus and enhance human immunity. However, in our country as much as92%of the crowd has the obstacle to digestion and absorption of lactose in milk, which termed as "lactose intolerance ". How to reduce the content of lactose in the milk, and then reduce "lactose intolerance" should be anxious solved in dairy cows breeding research area. Sulfolobus Solfataricus β-Glycosidases has a function on catalytic hydrolysis glycosidic bond and hydrolysis of lactose. So, the "lactose intolerance" could be fundamentally solved through the genetically modified method to expression of Sulfolobus Solfataricus β-Glycosidases in the milk. Piggybac transposon as a new transgenic technology, because of the advantages such as high integration rate and load capacity, single copy integration, easy simulation endogenous gene expression environment, which has an important application prospect. At present, the piggybac transposon mediated transgenic cattle research have not been reported success. So, in this study, we constructed a transgenic vector which piggybac transposon mediated mammary gland specific expression of Sulfolobus Solfataricus β-Glycosidases, combined with somatic nuclear transfer technology (SCNT) to produce transgenic cattle, in order to reduce lactose content in the milk, and do the exploratory research for low lactose dairy cows breeding.Experiment1, the Sulfolobus Solfataricus β-Glycosidases was codon optimized and synthetized based on the codon usage frequency of bovine. The bovine lactoglobulin and piggybac transposon element was cloned, digest and ligated to complete the mammary specific expression of Sulfolobus Solfataricus β-Glycosidases vector, which one do not contain the transposon element as a negative control; Another one contain the transposon element. All of them have the GFP and neomycine marker gene anchoraged by LOXP sequence, in order to enhance the screen efficiency and safety of transgenic. Experiment2, verification of expression vector by mammary gland cell. The PCR and RT-PCR analysis result showed that, the BLG promoter could direct the codon optimized Sulfolobus Solfataricus β-Glycosidases expressed in mammary gland cells, and there are no mutations in coding region of Sulfolobus Solfataricus β-Glycosidases verified by sequencing. Experiment3, piggybac transposon and transposase were co-transfected to fibroblasts at proportion of5:1. After48h, cells were passaged at1:30,1:60,1:100proportion and screened with G418. Gimasa and cell counting result showed that, the cell colonies was177/5、134/0.2and79/0under add with transposase or without transposase condition at above three passage proportion. These results showed that piggybac transposon could enhance integration rate greatly. However, the high integration rate may have some adverse effect on cell shape and growth condition.40available sequencing data of tailPCR results of colonies showed that, the piggybac transposon mediated gene integration is more tend to intron and intergenic of bovine chromosome. Experiment4, transfection and screening of fibroblasts with vector which with or without transposon element, then, SCNT with single colonies. Result showed that, the embryo cleavage rate of plox-EGF-LacS is slightly higher than ZGL-LacS-EN (83.2%/75.7%), and the blastosphere rate was no obvious differences between them (15.3%/14.9%), but both of them was obvious higher than non-modified embryos (cleavage rate88.2%, blastosphere rate36.7%). The blastosphere with green fluorescent were transfered to uterus of three receptor cattle treated with synchronization, no pregnant of them.In this study, we first constructed piggybac mediated a double marker gene vector, which specific expression of Sulfolobus Solfataricus β-Glycosidases in mammary gland. Our result showed that the piggybac transposon could mediated transposition of target gene in fibroblasts efficiently, and the integration of targeting gene have more tend to intron and intergenic of bovine chromosome. Result of SCNT showed that, there are no obvious differences between plox-EGF-LacS and ZGL-LacS-EN of blastosphere rate. The above study do an exploratory research at cultivating of low lactose dairy cow new species, meanwhile, lays a foundation on more efficiency and safety of genetically cattle breeding use of piggybac transposon technology.