Cloning Of The Seed Globulin Gene Promoter From Citrus Sinensis Osbeck Cv. Jincheng And Analysis In Transgenic Tobacco

Seedless character has an important value in citrus breeding for fresh fruit and processing. China is very rich in resource of citrus fruits and there are many local varieties. However, many cultivars in citrus fruits produce numerous seeds, which is disadvantage for orange commercial value and for competition in the international market.In this study, the promoter of the seed globulin gene (citrin gene) from Citrus sinensis cv. Jincheng was obtained. To define the function of promoter, a set of vectors containing different deleted sequences of the citrin promoter were constructed. The promoter can be used for artificial intervention and blocking of the embryo development.By using the total DNA of the Citrus sinensis Osbeck cv. Jincheng as template, and by using the chromosome walking technology with linker, together with the feature of nested-PCR, touchdown-PCR, hot-start PCR and two stages PCR, 2054bp fragment was cloned, which contained a partial sequence of the orange seeds globins gene and its 5'-upstream regulatory sequence and was called "ssp".It was demonstrated by plant CARE analysis that 5'-upstream sequence from ATG had the feature of promoter: TATA-box and CAAT-box involved in the transcriptional start and the translation respectively, and other regulatory sequence related to seed-specificexpression. It was speculated that the regulatory sequence would have function specifically in the seed development.The 5'-upstream sequence of the seed globulin gene (citrin gene) was deleted in series of 0bp, 389bp, 846bp, 1186bp and 1525bp from the ATG. Five plant expression vectors, p-A-gus , p-B-gus , p-C-gus , p-D-gus and p-N-gus, were constructed with the pCAMBIA3301.By means of Agrobacterium tumefaciens-mediated, the fused genes from plant expression vectors p-A-gus were transformed into cells of Nicotiana tabacum L. cv. Wisconsin 38. Some transgenic tobacco plants demonstrated by molecular analysis were obtained. It is proved that 5'-upstream sequence of citrin gene was a seed-specific expression promoter. This is the first report about obtaining the tissue-specific expression promoter from Citrus sinensis Osbeck.