Non-Starch Polysaccharides Enzyme Genes Selection And Coexpression Vector Construction With Phytase Gene

Non-starch polysaccharides(NSP) is anti-nutritional factors which widely found in plant cell walls,including cellulose, hemicellulose(arabinoxylan, ?- glucan, mannan, etc.), pectin and other substances.Some monogastric animals have low utilization efficiency for thus substances,so they can not absorb the nutrients within the cell,which resulted in not only a waste of feed, but also causes environmental pollution. In order to solve this problem, we usually employ microbial NSP enzyme as a feed additive. However, this practice is limited by cost, by inactivation at the high temperatures require for pelleting feed,and by loss of activity during storage.There is a novel strategy for solving the problem by pig and poultry endogenous digestive tract to produce NSP enzymes.We selecte some NSP enzyme genes from microorganisms and lower eukaryotes.After porcine codon optimization and signal peptide replacement,we cloned these genes into p CDNA3.1(+) eukaryotic expression vector and then transfected PK15 cells.After 48 h, we collected the cell culture medium which was measured for enzyme activity(Optimum p H,p H stability,pepsin/trypsin tolerance).We choose those NSP enzyme genes which expressing activity highly in PK15 cells for constucting dicistronic expression vector.Then we build a polycistron expression vector and a saliva-specific co-expression vector which expressed cellulase gene,xylanase gene,phytase gene and pectinase gene.This study laid the foundation for breeding diet-saving enviropig.The main results are as follows:(1) By analyzing the expression and enzymatic properties of cellulase gene from multiple microorganisms and lower eukaryotes source,we selected two cellulase gene expressed highly in pig cells: eg II and Te EGI.These genes have cellulase and ?-glucanase activity in PK15 cells.Cellulase and ?-glucanase activity of two cellulase gene expressed in PK15 cells are 0.20 U/m L(eg II),0.30 U/m L(Te EGI)(CMC-Na,p H4.00) and 0.61 U/m L(eg II),0.66 U/m L(Te EGI)(?-glucan,p H4.62).(2) We analyzed the expression and enzymatic properties of pectinase gene from multiple microorganisms and selected two pectinase genes expressed highly in pig cells: PG7 fn and PG. They expressed highest pectinase activity in PK15 cells:1.15 U / m L(polygalacturonic acid,p H4.0) and 1 U/m L(polygalacturonic acid,p H5.50),respectively.(3) By Comparing three xylanase genes from microbial: Asp-xyn, Xynl11, Penxyl.The maximum activity were respectively: 1.88 U/m L, 1.65 U/m L, 0.72 U/m L(xylan,p H5.00).Xylanase that Asp-xyn gene expressed in PK15 cell was highest,and the p H stability, tolerance of pepsin,tolerance of trypsin are preferred.(4)We constructed four bicistronic(xyn-flag-eg II-T2 A,xyn-flag-eg II,xyn-eg II-T2 A,xyn-eg II) through genetic recombination technology.After transfected PK15 cells, the results showed: xyn-eg II was best fusion.Compared with eg II and asp-xyn expression results, the xylanase activity of xyn-eg II is 57.35%(p H5.00); ?- glucanase activity is 46.90 %(p H4.5); CMC activity is 67.97%(p H6.00).(5) Constructed p CD-Es APPA-T2 A and p CD-Te EGI-T2 A expression vector Successfully. The enzyme activities was lower than that of the single gene expression in PK15 cells.(6) By optimizing the connection scheme of multi gene co-expression,we constructed PG7fn-asp-xyn-Es APPA-Te EGI polycistronic which co-expressed pg7 fn, asp-xyn, Es APPA, Te EGI four genes in PK15 cells successfully.(7) Constructed successfully transgene vector p PB-m PSP-PXAT-neo GFP which expressed in salivary gland specificly.