Preparation Of An Environmentally Eriendly Transgenic Pig Model With The Coexpression Of Several Enzymes

With the rapid development of pig industry,the impact of large amounts of nitrogen and phosphorus in excreta on the environment has become increasingly serious.Although feed added with enzyme can effectively reduce the emissions of nitrogen and phosphorus,high temperature and pressure in the feed processing leads to low activity and their catalysis are unstable.The currently enzyme preparations mainly include non-starch-polysaccharides(NSP)enzyme and phytase.The two enzymes in environmental friendly transgenic pig can continue to function stably in the form of endogenous enzymes,that cannot be synthesized by the animals themselves,which not only eliminate the influence of processing technology,but also reduces the use of enzyme preparations and inorganic phosphorus..It is of great significance to reduce feed cost and minimize negative impact of nitrogen and phosphorus on the environment.In this study,a polycistron expression vector is constructed with four enzyme genes by using 2A-like sequence which had the capable of self shearing and independent expression.And the enzyme genes included PG7fn(pectinase gene),XynB(xylanase gene),EsAPPA(phytase gene)and TeEGI(cellulase and ?-glucanase gene).After comparing the transfection efficiency,the U-023 program of Nucleofector 2b was the ideal condition for transfecting pig fetal fibroblasts(PFFs)with large plasmid.With this optimized condition,PXAT was used to generate piggyBac-medicated transgenic cells,and then deleted fluorescent labeling gene by cyclization recombination(Cre)enzyme.Finally,PXAT-transgenic pigs without fluorescent labeling were successfully obtained by the somatic cell clone technique(SCNT).The results shown that four enzyme genes were integrated into the genome of cloned pigs,included 920001,920005 and 920307.All of them were double copies of PXAT.Meanwhile,the mRNAs of the four genes were high in the three salivary glands,especially in the parotid gland.However,only XynB and EsAPPA protein-specific bands were detected by Western blot,and the activity of enzyme were 0.07 U/mL to 0.43 U/mL and 0.43 U/mL to 3.40 U/mL in saliva samples,respectively.In this study,the piggyBac system was used to randomly integrate a plasmid up to 20 kb into PFFs,and PXAT-transgenic pigs with the coexpression of several enzyme genes were successfully generated.This study has reference significance for the preparation of environmentally friendly transgenic pigs with large fragments.