Cloning And Functional Analysis Of CYP71B10L Gene In Tea Plant(Camellia Sinensis)

As a perennial evergreen plant,tea plant often suffers a variety of biotic and abiotic stresses during its growth and development.Therefore,cultivation,selection and popularization of tea cultivars with multi-and high-resistances is an important approach to minimize the impacts caused by the stresses on its growth and development as well as the yield and quality.Exploring the resistance-related genes and elucidating the resistance mechanism by using molecular biological technology will significantly promote the development of molecular breeding of tea plant.Based on our previous research,a highly up-regulated CYP71B10 L gene during fine manupilation of oolong tea processing was screened out.In this study,the CYP71B10 L and its promoter was cloned from the shoots of various tea cultivars to clarify the differences in organization structure and encoded products of the genes.Gene expression in various tissues harvested from different tea cultivars as well as in leaves under multiple stress conditions were conducted in order to illustrate the CYP71B10 L expression pattern.Subcellular localization and biological funcation of CYP71B10 L were investigated through fusion expression with fluorescent protein encoding gene and heterologous expression in E.coli expression system.The main results were as follows:(1)CYP71B10L promoter sequences were cloned from leaves of cultivar‘Hongyafoshou’,‘Fujianshuixian’,‘Fudingdabaich’ and ‘Zhenong139’,and the length of the promoter ranged from 1511 bp to 1698 bp.A large number of cis-elements such as light-responsive elements,hormone-responsive elements and stress-responsive elements were predicted in these promoters.A deletion varation around 200 bp was found in the CYP71B10 L promoter of ‘Hongyafoshou’ in comparion with the other three cultivars.The obtained full-length of CYP71B10 L genomic DNA sequence in tea cultivar ‘Hongyafoshou’,‘Fujianshuixian’,‘Fudingdabaich’ and ‘Zhenong139’ were from 2967 bp to 3309 bp.The CYP71B10 L genomic DNA in the four cultivars shared similar gene organizatin structure containing two exons and one intron,which led to a similar coding region.The length of exons were 906 bp and 627 bp and there was 336 bp insertion varation in the intron of ‘Fujianshuixian’.(2)CYP71B10L widely expressed in various tissues of tea plant,with highest level in fruits,followed by shoots and flowers,and lower level in mature leaves and roots,indicating the CYP71B10 L was involved in the regulation of the growth and development of tea plant.Expression of the CYP71B10 L was significantly up-regulated by the withering,fine manipulation,methyl jasmonate treatment and mechanical damage.Among them,expression level of the CYP71B10 L was the highest in the process of fine manipulation,followed by withering,and then methyl jasmonate treatment.The up-regulation was relatively low after mechanical damage.Much higher up-regulation expression of CYP71B10 L was found during treatment of withering and fine manipulation in cultivars suitable for producing oolong tea than that suitable for producing green tea.In comparison,CYP71B10 L intensely and sensitively responded to dehydration.(3)CYP71B10L was mainly located in the endoplasmic reticulum of tea plant cells.An expression vector was accurately constructed through consequently inserting the CYP71B10 L and cytochrome P450 reductase(CPR)into the two multiple cloning sites of the p ETduet-1,then transformed into E.coli BL21(DE3).CYP71B10 L and CPR were efficiently expressed and detected by westblotting under the optimized conditions(induced at 28℃ with 0.8 m M IPTG for 6 h).No enzyme activity had not been detected.