Study On Genetic Transformation Of Tea

The leaf of Camellia sinensis cv. Longjing43plantlet in vitro was taken as explants toconstruct the genetic transformation system via Agrobacterium. Conservative domain of TeaCaffeine Synthase（TCS）gene was used to construct RNAi vector. Factors influencing teatransformation mediated by Agrobacterium tumefaciens have been studied through GUSexpression. The free radical scavenging capacity and antioxidant activity of Puerarialobata callus extract were detected, which were helpful to select the new and efficientnatural antioxidants.1. Construction of RNAi vector of TCS（Tea caffeine synthase）gene.TCS is a key enzyme of the tea biosynthesis of caffeine. Inhibition of the expressionactivity of TSC gene is an effective method to cultivate low caffeine tea plant. The totalRNA was isolated from tea. A specific fragment with predicted size was amplified fromcDNA of TCS （Tea Caffeine Synthase） by RT-PCR with degenerate oligonuclectideprimers designed from the conserved domain of other plant lipoxygenases. The amplifiedfragment was350bp.Two pairs of specific primers with different restricted enzyme siteswere designed according to the sequence. Antisense and sense TCS gene were amplified,respectively, and then ligated with the YYT gene, which was related to carotenoidsynthesis in Subcocci as interval sequence. The fusion gene was digested by two restrictedenzymes and cloned into plant expression vector to construct the RNA interference vectorof TCS gene.2. Agrobacterium tumefaciens-mediated transformation.The genetic transformation system via Agrobacterium was obtained with leaf as acceptor. Onthe basis of GUS transient expression, parameters were optimized.The suitable system ofAgrobacterium-mediated transformation is suggested that tea plant leaf was pre-culturedthree-day, infected10min by engineering bacteria of OD600about0.8followed byco_culture of4days.15days later, the rate of GUS expression is63.3%.40mg·L^-1hygromycin is the suitable concentration for expalnt survival.3. The ability to eliminate free radical of the Pueraria lobata callus extract.Using an organic free radical system of1,1-dipHenyl-2-picrylhydrazyl （DPPH·） andtwo kinds of inorganic free radical systems of hydroxyl free radical and superoxide anionfree radical, the free radical scavenging capacities and antioxidant activities of Puerarialobata callus extract were examined in vitro, and were compared with P. Radix extract.Theresults showed that:the Pueraria lobata callus extract The results showed that theDPPH· free radical scavenging capacity of P. lobata callus extract was similar to those of P. Radix extract, but the capacity to clean up hydroxyl free radical is weaker than P. Radixextract.When the concentration is0.2mg·mL^-1,P. lobata callus extract played the significantscavenging roles on the superoxide anion free radical, which were better than P. Radixextract. But the ability to inhibit pyrogallol autoxidation of P. lobata callus extract wassimilar to P. Radix extract.When the concentration is1.0mg·mL^-1, the scavenging roles of P.lobata callus extract about the superoxide anion free radical were consistent with P. Radixextract.But the inhibition rate on pyrogallol autoxidation of P. lobata callus extract was3.48times higher than P. Radix extract. All above indicates that the P. lobata callus extracthas a comparatively strong ability of cleaning up free radical.But analyzing and comparingP. lobata callus extract and P. Radix extract by HPLC, the results showed that componentand content of isoflavone are differences.