Heterologous Expression And Characterization Of A Bilirubin Oxidase Gene From Myrothecium Verrucaria In Arabidopsis Thaliana

Bilirubin oxidase(BOD)is a copper-containing polyphenol oxidase which catalyzes the formation of bilirubin and other substances.BOD,isolated and purified from microorganisms and plants,could oxidize many substrates of laccase and have a better application prospect In this study,we modified a BOD gene from Myrothecium verrucaria and expressed in Arabidopsis thaliana.The characterization of the heterologous expressed enzyme was detected,and its function on dyes decolorization was determined.The research laid a foundation for building a plant bioreactor for recombinant BOD.The specific research contents and results are as follows:1.A bilirubin oxidase gene Mv BODI from M.verrucaria was modified accoding to the plant basis codon and the mechanism of RNA stabilization and N terminal translation.The Mv BODI gene was synthesized and ligated into plant vector p CAMBIA1301.2.Using floral dip method,Mv BODI genes were introduced into A.thaliana and positive transformations were selected with hygromycin B.And the stable transformation lines,which can secrete BOD,were successfully obtained from T2 by screened under the condition of hygromycin.3.By genomic DNA PCR,reverse transcription PCR and native PAGE,it can be confirmed that Mv BODI gene has been integrated into the genome of A.thaliana.4.The characterization of BOD expressed in A.thaliana was detected using bilirubin or ABTS as the substrate.When bilirubin was used as substrate,the optimum temperature and p H are 60 °C and 7.0,respectively.When bilirubin is replaced withABTS,the optimum temperature and p H are 60 °C and 4.0,respectively.5.The crude enzyme extract of transgenic A.thaliana can degrade crystal violet and malachite green.And the degradation effect was obviously improved using ABTS as mediator.It was also found that the transgenic plants could degrade crystal violet and malachite green dyes efficiently in liquid culture,and the degradation rate reached more than 90% after the addition of ABTS.The above results showed that A.thaliana could efficiently express BOD with the ability to decolorize dyes.