Expression And Function Of Phytase PhyA Gene In Mycelium Of Cordyceps Militaris

Phytic acid,an anti-nutritional factor,is the main source of phosphorus,which plays a prominent role in hindering the absorption of minerals by plants.Research and development of phytase can effectively solve the problem.Currently,phytase is widely produced by recombinant expression systems of bacteria,fungi,and plants,however,it was still required more natural,quality and efficient by Phytase expression systems.Cordyceps militaris,as a kind of medicinal fungi,is considered to be a good receptor material for recombinant expression of phytase because its components have the advantages of regulating immune function and anti-inflammatory activity,short growth cycle,and good safety.However,it has not been reported that Cordyceps militaris is used as an expression vector for recombinant phytase.In this experiment,Cordyceps militaris was used as an expression vector,fungal expression vector pCB130 NG containing phyA phytase gene was transferred into Cordyceps militaris strain.The expression and activity of phytase in Cordyceps militaris recombinant expression were determined.At the same time,the strain producing phytase Cordyceps militaris was obtained,and a large number of fermentation experiments were carried out to feed broilers.144 one-day-old AA broilers were randomly divided into six treatment groups,each with 3 replicates,and each replicate had 8 broilers.Treatment group T0 was normal diet group,treatment group T1 was a normal diet with wild Cordyceps militaris,treatment group T2 was a normal diet with 500 U/kg phytase,and treatment group T3-T5 was a normal diet with 250 U/kg,500 U/kg and 1000 U/kg phytase-producing Cordyceps militaris.The experimental date was 42 days,which was divided into 1-21 days in the early stage and 22-42 days in the late stage.The effects of phytase-producing Cordyceps militaris on growth performance,tibia development,nutrient utilization and immune organ index of broilers were studied.The results are as follows: 1.The recombinant plasmid pCB130NG-phyA was transformed into Cordyceps militaris grassland protoplasts.The Cordyceps militaris strains transformed with the phyA gene were obtained by PCR and Western Blot detection.The highest expression of phyA gene was determined by ELISA,which accounted for about 0.91(+0.064)of total soluble protein.Determination of phytase activity in the freeze-dried powder of Cordyceps militaris strain producing phytase per gram by Ammonium Molybdenum vanadate colorimetry is 402.2 U.2.In terms of growth performance of broilers,the treatment groups T2,T4 and T5 at 1-21 and 22-42 days could significantly increase the average weight of Broilers(P < 0.05),while the treatment groups T2,T4 and T5 had significant improvement in average daily gain and average daily feed intake(P<0.05).At the age of 1-42 days,the feed weight ratios of broilers in treatment groups T2,T4 and T5 were significantly different from those in the control group(P < 0.05).3.In terms of tibial index,compared with the control group,the ash content of the tibia in the treatment group T2,T4 and T5 increased significantly at 21 and 42 days of age(P < 0.05),and the phosphorus content in the tibia also increased significantly(P < 0.05).At the age of 21 days,the calcium content in the tibia of broiler chickens in treatment groups T2,T4 and T5 increased significantly(P < 0.05),and at the age of 42 days,the calcium content in the tibia of broiler chickens in treatment group T5 increased significantly(P < 0.05).4.In terms of nutrient utilization rate,at 21 and 42 days of age,the crude protein utilization rate of groups T2,T4 and T5 treated with phytase was significantly higher than that of control group T0(P < 0.05).In terms of phosphorus utilization rate,there were significant differences in T2-T5 between the four treatments with phytase at 21 and 42 days of age compared with the control group(P < 0.05).In terms of calcium utilization rate,T2-T5 of the four treatments with phytase at 21 days of age significantly increased the calcium utilization rate of Broilers(P < 0.05).At 42 days of age,T2,T4 and T5 of the treatments significantly increased the calcium utilization rate of Broilers(P < 0.05).5.In terms of immune organ index,compared with the control group,the immune organ index of broilers increased with the addition of phytase-producing Cordyceps militaris in the diet(P > 0.05).