Expression Of Arginine Deiminimide In Monascus

L-citrullinine is an ?-amino acid,which has the activity of anti-aging,antioxidation,enhancing immunity and promoting male sexual function.It is widely used in medicine and health care industry.L-citrulline can be converted to L-arginine by Arginine Deiminase(ADI).Monascus is an important industrial microorganism fermentative bacteria.It has great potential in the field of food,medicine and health care industry.The fermentation product has the function of regulating blood lipid and blood pressure.It has good preventive and therapeutic effects on many diseases such as diabetes and Parkinson's disease.In this study,the Agrobacterium tumefaciens-mediated transformation(ATMT)was used to random insert the exogenous of ADI gene into the genome of Monascus,and constructed an intracellular constituent expression of Monascus.This engineering bacterium can be used to production L-citrulline and development of related health industry of Monascus.This research uses seamless cloning technology to connect the ADI gene of Mycoplasma with the linearized micro Ti plasmid: p CAMBIA-Pgpd A-Tcbh1-hph-Ptrp C.And transform the product into the cloned host Escherichia coli E.coli DH5?.Than extract the plasmid,the positive transformants are screened by gene sequencing.A miniature Ti vector p CAMBIA-Pgpd A-adi-Tcbh1-hph-Ptrp C for intracellular expression has been constructed.A variety of methods were used to prepare Agrobacterium tumefaciens competent cells,and the expression vector was transformed into Agrobacterium tumefaciens.The results showed that the number of Agrobacterium receptive cell transformants prepared by 50 mmol/L Ca Cl2 was the best.The micro Ti plasmid was transformed into Agrobacterium tumefaciens,and identified by PCR.The Monascus spores suspension liquid and Agrobacterium tumefaciens transformants co-cultured at 24 ?,and then transferred to the screening medium after 48 h culture.The resistant colony of Monascus in the medium was cultured in PDA liquid medium,and the contect of L-citrulline was detected by TLC and two acetyl monoxime method.The genome of Monascus transformants,which can produce Lcitrulline,was extracted,and PCR was used to identification.In the end,two transformants were obtained: bacteria No.6 and No.7,similar to wild Monascus.Through PCR identification,ADI gene had been successfully transformed into the genome of Monascus.At end,an engineered strain of Monascus,which was used to express ADI in the cell,was successfully constructed.