Cloning And Expression Of Key Genes Encoding Carotenoids Cleavage Dioxygenase (LcCCD1,LcCCD4 And LcNCED1) In Lycium Chinense

The carotenoid can be cleaved by CCDs(carotenoid cleavage dioxygenases)to generate apocarotenoids,which fulfill a number of roles in plants development and stress responses.Although the CCDs have been studied in several plants,the function on fruit development and stress response still need to be identified.Lycium chinense is the best plant material for study of carotenoids metabolism because of the enriched expression of carotenoids in the fruits.In order to understand the functions of CCDs,the LcCCD1,LcCCD4 and LcNCED1 were isolated and characterized from L.chinense.E.coli expression vector pGEX-LcCCD1 and pGEX-LcCCD4 were constructed,and then expressed by heterologous complementation experiment in E.coli.The results strongly suggested that LcCCD1 can cleave ?-carotene and lycopene to generate ?-ionone and pseudoionone,respectively,while the LcCCD4 only can cleave ?-carotene.The expression pattern of LcCCD1,LcCCD4 and LcNCED1 were analyzed by Sq-RT-PCR technology.The results reveal that the expression level of LcCCD1 in fruits is in accordance with the contents of carotenoids and ?-ionone,while the transcription of LcCCD4 was decreased with the fruit development.These results indicate that LcCCD1 is mainly involved in formation of aroma compounds in fruit,while the LcCCD4 functions as regulation of carotenoids accumulation.In fruits,the expression level of LcNCED1 is associated with the changes of ABA contents,ethylene release and soluble sugar concentration,indicating that LcNCED1 may engage in regulating fruit ripening.In addition,ABA accumulation in leaves was associated with enhanced expression of LcNCED1 induced strongly by abiotic stresses(drought,salt).The transgenic tobacco overexpressing LcNCED1 and LcCCD1 were obtained by Agrobacterium-medicated genetic transformation system.The transgenic tobaccos overexpressing LcCCD1 showed enhanced contents of ?-ionone in leaves to certain extents compared with control.The transgenic tobacco overexpressing LcNCED1 obtained higher ABA content.Compared with the control plant,the transgenic lines exhibited higher activities of SOD and POD,and lower contents of H2O2 and O2-under drought stress.These results indicated that overexpression LcNCED1 in tobacco successfully improved its drought tolerance by increasing the ABA biosynthetic capacity.The transgenic tobacco have not affect on microbiologic population,enzyme activity,conductivity and pH in soil,indicating that LcCCD1 and LcNCED1 genes are safe to soil environment.