The Role Of Salicylic Acid In Camptotheca Acuminata Suspension Cell Culture And Preliminary Construction Of Transgenic CaSAT Camptotheca Acuminata

Camptotheca acuminata is a unique tree species in China.Through tissue culture,especially through genetic transformation and genetic engineering methods,it is of great significance to increase the camptothecin content of the medicinal plant.In this experiment,the young leaves and stem segments of Camptotheca acuminata were used as materials to investigate the induction of the callus and the subculture of the basic medium of MS and WPM and the matching of different hormones(2,4-D,IBA,6-BA and IAA).The effects of the culture and selection of the optimum medium formulation for the suspension cell culture system of Camptotheca acuminata.The suspension cell system was constructed using callus of Camptotheca acuminata,and different concentrations of SA were used to detect the expression level of key enzyme genes in suspension cells.A genetic transformation system for the CaSAT gene of the Camptotheca saccharate transporter mediated by Agrobacterium tumefaciens GV3101 was initially established.The results showed that:1.The callus culture of young leaf explants of Camptotheca acuminata was optimal with WPM+2,4-D 0.15 mg/L+6-BA 0.6 mg/L+sucrose 30 g/L,pH 5.8.For subculture,MS+2,4-D 0.15 mg/L+6-BA 0.6 mg/L was the most suitable.2.Through callus induction on Camptotheca acuminata,the callus suspension system of Camptotheca acuminata was successfully established,and the culture medium was modified to confirm the suspension cell culture of Camptotheca acuminata as MS+2,4-D 0.15 mg/L+ 6-BA 0.6 mg/L+0.25 mg/L IAA +Sucrose 30 g/L,pH 5.8.3.The content of camptothecin in different parts of Camptotheca acuminata was detected by HPLC.The results showed that the suspension cells stored in the liquid medium contained almost no camptothecin,and the suspension cells preserved in the solid medium and the callus of Camptotheca acuminata contained.A small amount of camptothecin,but contains a lot of camptothecin in roots,stems and leaves,of which the highest content of camptothecin in leaves.4.By real-time quantitative PCR detection of different concentrations of SA and different treatment durations on the key enzyme genes of Camptotheca acuminata,it was found that high concentration of SA significantly inhibited the expression of the key enzyme genes of Camptotheca acuminata.SA treatment improves the expression of CaSTR and CaSAT.5.The genetic transformation system of Agrobacterium tumefaciens using cotyledon as the transforming receptor was initially established.We also conducted comparative experiments on the effects of cefotaxime(Cef)and Timentin on the adventitious buds of leaf differentiation.The results confirmed that the best choice of Cef was 500 mg/L.