| Caffeine(1,3,7-trimethylxanthine)which has a variety of physiological effects,such as dispelling the effects of alcohol,aiding digestion,and refreshing,is the main component of purine alkaloid in tea plant.Due to its varieties of healthy functions,caffeine is widely used in medical,food,chemical and other fields,and the demand for caffeine has increased.However,the certain groups of people,such as young kids,pregnant women,or some patients would suffer from side effects when they consume caffeine,so the tea products with low-caffeine are also in great demand.At present,commercial caffeine,which mainly produced by chemical synthesis methods,have many defects,such as redundant additional product and environmental contaminations,etc.Conventional methods of breeding low-caffeine or high-caffeine tea varieties are tim-consuming and low adaptability of new varieties.While methods of extracting or removing caffeine from tea and coffee are too costly and easy to remain harmful residues.Therefore,the methods above are not the effective ways of regulating the content of caffeine in the long term.However,genetic engineering technology has the advantages of shorter cultivation cycles,lower cost,higher yield and higher quality.Through the construction of engineering bacteria,caffeine could be biosynthesisd in vitro.Besides,to study on the biological function of key enzyme gene during the caffeine biosynthesis can realize the artificial regulation of caffeine production,which lays the foundation for further genetic transformation and optimization.The caffeine biosynthetic pathway mainly includes four steps as follows:xanthosine(XR)→ 7-methylxanthosine(7mXR)→ 7-methylxanthine(7-MX)→theobromine(Tb)→Caffeine(Cf).In the present study,the key enzymes involved in the biosynthesis of caffeine have been cloned from coffee.However,the main report of tea plant is about research of tea caffeine synthase(TCS1)that catalyzed the final two steps reaction.The genes of the first two steps have not been cloned from tea leaves and never been reported.This paper will study the expression of suspected N-Methynucleosidase screened from tea plant genomic library in Escherichia coli,find the N-methylnucleoside that could catalytic second step reaction in the caffeine synthesis pathway,and ultimately regulate the biosynthesis of caffeine.The results are asfollows:1.Based on the ORF of suspected N-Methynucleosidase(STx),the recombinant expression vectors pMAL-ST1 、 pMAL-ST2 and pMAL-ST3 we obtained.The recombinant protein was expressed by induction with IPTG.The results show that induced by optimizing the conditions,we obtained the best expression condition of the recombinant protein-soluble protein expression is expressed in high quantity as:28 ℃,6 h.The testing results were suggesting that pMAL-ST2 and pMAL-ST3 can directly catalyze the conversion form XR to X,and pMAL-ST3 can directly catalyze the conversion from XR to 7-MX.2.The multi-gene fusion expression vector pMAL-ST3-CaXMT is successfully constructed by ligating the digested products of pMAL-ST3 and pMAL-CaXMT,which were produced by the SalI & SbfI restriction enzyme digestion.The results show that induced by optimizing the conditions,we obtained the best expression condition of the recombinant protein-soluble protein expression is expressed in high quantity as: 16℃,20 h.The activity of the fusion proteins were assayed in vitro,the more content of 7-MX from pMAL-ST3-CaXMT is produced.Based on the testing results of HPLC,according to standard curve of 7-MX,pMAL-CaXMT produce more content of 7-MX than pMAL-ST3.The results show that the enzyme activity of CaXMT is more reactive than ST3 when they catalyze the conversion from XR to7-MX. |
PDF Full Text Request