The Metabolic Regulation Mechanism Of Pods Color And Function Analysis Of Two Related Genes Of Caragana Intermedia

Caragana intermedia is a leguminous shrub widely distributed in the cold and arid regions and rich in secondary metabolites and naturally active substances,such as flavonoids and anthocyanin.It has good nutritional and medical value and its flowers and roots can be used as medicine.It is interesting that the pods of C.intermedia often show different colors,such as red and green,among individual plants.In this study,the metabolites of different color pods of C.intermedia were determined by LC-MS,the accumulation pattern of the significantly differential metabolites was studied systematically and the metabolic regulation mechanism of pods color formation was analyzed with metabolomics.The genes of chalcone synthase(CHS)and resveratrol synthetase(RS),which are important enzymes in phenylpropanoid metabolic pathway,were cloned and analyzed,and their functions were studied.Here below are the detailed results of this study.1.Red and green pods of C.intermedia at different growth stages were used to establish metabolomics.A total of 557 metabolites,including 348 annotated metabolites and 36 significantly different metabolites of which 11 were flavonoids,were detected in the pods extraction using liquid chromatography coupled with ESI-triple quadrupole-linear ion trap mass spectrometer(LC-ESI-MS/MS).2.The content of total flavonoids and proanthocyanidins in 30-day-old red pods was significantly higher than that in green pods.The insect feeding rate in red and green pods of C.intermedia was investigated,and the results showed that it was higher in green pods than in red ones.We speculated that the difference in feeding rate might be caused by the diverse content of flavonoids.3.The metabolites diversity in red and green pods at different growth stages of C.intermedia was analyzed by multivariate analysis.The results showed that the metabolites varied greatly between red and green pods,and the accumulation of metabolites was both gowth-stage and color dependent.Among the primary metabolites identified,the obvious difference in red and green pods mainly existed in amino acids and nucleotide derivatives,while the distinct secondary metabolites were flavonoids,phenolamines and polyphenols.Flavonoids are the most important differential metabolites that were up-regulated obviously in red pods.The majority of the distinct metabolites in red and green pods of different developmental stages are nucleotide derivatives and phytohormones.4.KEGG metabolic pathway enrichment analysis showed that widely targeted metabolites profiling of the 30-day-old green and red pods mainly encompassed in 21 metabolic pathways,in which four had significant difference.As for samples of different growth stages of the same color,the metabolites of 10-and 30-day-old green pods were distributed in 33 metabolic pathways,and 10 were significantly different.For red samples,there were 32 metabolic pathways and 7 showed significantly different.The most abundant compounds were metabolites in the shikimic acid pathway and the phytohormone metabolic pathway,indicating that these two pathways were the most active ones.5.Two essential genes of phenylpropanoid metabolic pathway,CiCHS and CiRS,were cloned from C.intermedia by degenerate primers and RACE technique,encoding 391 and 393 amino acids,respectively.The phylogenetic relationship among these two genes and their homologues was analyzed.The expression of CiRS gene was induced by drought,NaCl and ultraviolet(UV)stresses as revealed by quantitative real-time PCR(qRT-PCR).6.The over-expression vector of pCanG-CiCHS was constructed and was transformed into the wild type Arabidopsis thaliana.Further ananlysis showed that over-expression of CiCHS inhibited the expression of endogenous AtCHS in transgenic Arabidopsis.Total flavonoids experimental results showed that the total flavonoids content of the transgenic plants was significantly higher than that of the wild type.HPLC(High performance liquid chromatography)method was used to analyze the naringin content in transgenic plants which was higher than that of the wild type.The tolerance of the transgenic plants to UV stress was improved significantly,and the oxidation of membrane lipids was decreased.In addition,DPPH free radical scavenging ability of transgenic plants extraction was significantly higher than that of the wild type.Complemented CiCHS to Arabidopsis tt4 mutant partially restored the transparency seeds coat of the mutant to light brown seeds coat.7.The over-expression vector of pCanG-CiRS was constructed and was transformed into the wild type A.thaliana.Over-expression of CiRS gene inhibited the expression of endogenous AtCHS gene in transgenic Arabidopsis,meanwhile resulted in a significantly decrease in the total flavonoids content of the transgenic plants.But resveratrol was detected in the transgenic plants by HPLC.The tolerance of the transgenic plants to UV stress was improved,and the oxidation of membrane lipids was decreased.In addition,DPPH free radical scavenging ability of transgenic plants extraction was higher than that of the wild type.