Cloning And Identification Of Citrus Fruit Specific Promoter

The promoter is an important element of gene expression regulation, for gene function in plants, constitutive promoter, such as Ca MV 35 S is most widely used; however, the constitutive promoters may lead to a massive accumulation of target gene products in the plant which may impair plant’s growth and even lead to its death. Tissue specific promoters(e.g. root, stem, flower, fruit specific expression promoter) can control efficient expression of foreign genes in the special tissues and organs, on the one hand, it avoid unnecessary accumulated of foreign genes in other tissues and organs, hence is better for gene faction identification. On the other hand, it leads efficient expression of target gene in the target tissues and organs, so it’s better for efficiently transgenic breeding. Our studies based on citrus genome RNA-seq data, used different tissues for q RT-PCR and obtained seven potential genes with remarkably high expression level in fruit. Then begin the genetically modified verification work.This study carries out from the following several aspects:1) Based on citrus genome RNA-seq data, obtained seven potential genes with remarkably high expression level in fruit. The expression patterns for the seven high-expressed genes were analyzed in different tissues and genotypes; and then one specific-high-expressed gene(p Cit PDILP) was targeted, its expression level in fruit is higher(110) than in leaf and flower.2) The cloning of the promoter and the GUS fusion expression vector constructed. In order to study p Cit PDILP promoter, p Cit PDILP promoter was cloned and this promoter is 1913 bp long; both p Cit PDILP promoter and 35 S promoter GUS fusion expression vectors were constructed by PMV2-DR5 vector which has both GUS and YFP reporter gene.3) Gus transient expression to identification the promoter. GUS fusion expression vector with p Cit PDILP promoter and another one with 35 S promoter were transferred into tomato by Agroinfiltration; the availability of the PMV2-DR5 vector and p Cit PDILP promoter’s fruit specific traits was checked by GUS staining.4) Agrobacterium-mediated transferred early flowering Poncirus trifoliate. Finally, the GUS fusion expression vector with p Cit PDILP promoter was transferred into early flowering Poncirus trifoliata by Agrobacterium-mediated, therefore the positive plant was prepared for the further research on p Cit PDILP promoter’s fruit specialty and other related traits.In conclusion, this study aimed at screening and identifying the fruit specific promoter, in order to lay a solid theoretical foundation to the identification of the gene which related to citrus fruit quality and to help the fruit quality breeding.