| Hi-tree is a unique woody plant in China.It is important to improve the content of camptothecin in this medicinal plant by cell culture.The maintenance of various hormones in the body within a suitable concentration range can regulate its growth and development,and also has an effect on the synthesis of alkaloids in the body.Therefore,the addition of exogenous hormones can regulate the biosynthesis of alkaloids.In this experiment,the constructed regeneration of seedlings and callus were studied,and the effects of different species and concentrations of exogenous hormones on the biosynthesis of camptothecin were studied.Five common exogenous hormones were selected: 6-BA,2,4-D,NAA,GA and KT,which were treated with different materials of Camptotheca acuminata.After 45 days of growth and treatment,they were physiologically The morphology,the content of camptothecin and the relative expression of genes were analyzed.The results of HPLC analysis showed that the CPT content in different vegetative organs of Camptotheca acuminata was: leaf>stem>root>stem callus,and the CPT content in leaves was 36.16 times that of callus.CPT was mainly in Synthesized and accumulated in the leaves.When treated with different hormones,6-BA had the most obvious effect on the leaves and roots of the plants,and KT had prominent effects on the stems and callus of the plants.In the callus system of Camptotheca acuminata,the callus of 2,4-D treatment was red and bright and loose;GA treated callus was dense and the color was light green.In the callus,only the 6-BA treatment group detected CPT,and the CPT content of the 6-BA 0.05 mg/L group and the6-BA 0.1 mg/L group increased by 0.32 times and 0.36,respectively,compared with the stem-derived callus.Camptothecin was detected by TLC and TLC thin layer chromatography.Other treatments were insensitive to HPLC detection due to too little content and were not detected,also,were not detected by TLC thin layer chromatography.Real-time quantitative fluorescent PCR(q RT-PCR)analysis showed that the expression levels of Ca CYC1,Ca TDC1 and Ca7 DLGT in the leaves were increased by 3.37 times,2.27 times and 3.02 times,respectively,compared with the control group when treated with 0.05mg/L 6-BA.At 0.1 mg/L 6-BA treatment,the expression levels of Ca CYC1,Ca TDC1 and Ca7 DLGT in the leaves were 1.92 times,2.91 times and 1.32 times higher than those in the control group,respectively.The main key enzyme gene of camptothecin content in dry weight,the expression of Ca STR in 0.05mg/L 6-BA and 0.1mg/L 6-BA was 1.55 times and 1.61 times higher than that in the control group,respectively;0.05mg/L in root system.The expression of Ca TDC1 in the 6-BA treatment was significantly increased by 11.03 times compared with the control group,and the expression of Ca TDC1 was increased by 3.43 times in the treatment of0.1 mg/L 6-BA.The expression of Ca STR and Ca7 DLS was mainly regulated in stem-derived callus.The content of camptothecin in the callus of Camptotheca acuminata was increased by2.38 times and 1.29 times,respectively,and the expression of Ca7 DLS was increased by 3.59 times and 2.35 times compared with the control group when treated with 1 mg/L KT and 1mg/L GA,respectively.In the constructed leaf source callus,the expression of Ca STR and Ca7 DLS increased by 7.25 times and 5.75 when treated with 0.1 mg/L 6-BA.,Consistent with regulation stems source acuminata callus key genes.The results showed that 6-BA has multiple effects in regulating the growth of camptotheca acuminata and camptothecin.Low concentration can induce the growth of cluster buds to facilitate rapid propagation,while high concentration can increase the content of CPT in camptotheca acuminata and callus.Moreover,the relative expression levels of several key enzyme genes in the camptothecin synthesis pathway are also increased,which provides a theoretical basis for the 6-BA regulation of camptothecin synthesis. |
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