| Terpenoid compounds play an important role in plant,including primary metabolites and secondary metabolites.Due to the wide types and functional diversity of terpenoids,it is very important to study of terpenoid metabolism.Apple ?-farnesene synthase(MdAFS)is the terminal enzyme ?-farnesene metabolism,directly determine the generation of ?-farnesene.MdAFS not only has the ?-farnesene synthase characteristic,also has the isopentenyl transferase and monoterpene synthase characteristics.? and ?-farnesene produced with FPP,linalool,?-ocimene and ?-myrcene produced with GPP,and ?-farnesene produced with GPP and IPP as substrate.In this study,?-farnesene synthase fusion expression vector was constructed,transformed into cytoplasm and chloroplasts in Arabidopsis thaliana,and to understand the regulation of ?-farnesene synthase and effect of volatiles product release from transgenic Arabidopsis.The main results as follow:(1)Promoter(rbcS1)of rubisco small subunit gene from Chrysanthemum leaves,chloroplast signal peptide(pla)from Arabidopsis leaves,and ?-farnesene synthase gene(MdAFS)from white winter pearmain apple peels promoter were cloned,constructed expression vector pBI122-GFP,pBI122-rbcS1-pla-MdAFS-GFP,the results of transient transformation of Nicotiana benthamiana show that MdAFS was located in chloroplast,and rbcS1 promoter activity was proved.(2)Cytoplasm and chloroplast eukaryotic expression vector of MdAFS were constructed and transgenic Arabidopsis was generated by Agrobacterium-mediated genetic transformation Analysis of PCR and qRT-PCR revealed that the MdAFS protein was localized in cytoplasm and chloroplast of Arabidopsis.(3)Analysis of the effect of ABA(100?M/L),SA(500?M/L),ETH(50mg/L),MeJA(100?M/L),MV(100?M/L),the high temperature(45?),light(1200?mol/m2/s),and low temperature(4?)on the expression MdAFS indicated that the expression of MdAFS was induced by many factors.(4)The quantification of photosynthesis-related isoprenoid metabolites in all plants at the rosette and flowering stages revealed that total chlorophyll was higher in the transgenic lines as compared to wild-type control.Especially,at the flowering stage,the increase of chlorophyll a and carotene content is more significant.(5)Different genes in the MVA and the MEP pathway were tested using qRT-PCR.There were no significant difference is detected the genes of terpenoid metabolism between the lines of cytoplasmic localization of overexpression and wild type in the rosette stage,except upregulation of AtIPI2 and AtDXS,however,AtAACT2,AtIPI1,AtHMGR1 and AtDXS were up-regulated in the flowering stage.At the rosette stage,the transcript levels of AtHMGR1 and AtDXS were significantly higher in the chloroplast localization plants.AtIPI1,AtIPI2,AtHMGR2,AtDXR and AtDXS expression was significantly increased in the flowering stage.These results indicated that overexpression of MdAFS affected the changes of terpenoid metabolism in Arabidopsis thaliana.(6)The volatiles emitted from the wild-type and transgenic plants were collected at the flowering stage and analyzed by coupled GC-MS.There were two new kinds of volatile terpenes volatiles(D-limonene and geranylacetone)detected in the leaves of transgenic lines.The volatiles in flower of results showed that the transgenic lines were significantly higher than the wild type,(E,E)-?-farnesene was detected in f1 and P3 plants,farnesol were detected in f6 and P4,(Z,Z)-?-farnesene was detected in P2,the linalool was detected in f4,f6 and P4.(7)Free and bound sterol in leaves,and bound sterol in flower of transgenic and wild-type Arabidopsis thaliana were detected showed that the sterol of transgenic lines had decreased compared to the sterol of wild type Arabidopsis thaliana.(8)Compared with the wild type Arabidopsis thaliana,the transgenic plants had a lower degree of chlorosis and less ROS accumulation,higher antioxidant enzyme activities and higher membrane stability after oxidative stress.The results showed that overexpression of MdAFS could improve the antioxidant ability of plants... |
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