Isolation And Identification Of Promoters From Tomato Yellow Leaf Curl China Virus And Its Associated Satellite DNA

Geminiviruses are a group of plant circular single-strand DNA viruses occurred worldwide. They have caused destructive diseases in many crops throughout the world and are emerging as great threat for crop production. In recent years, a novel satellite DNA, referred to as DNAβ, was found to be associated with some monopartite geminiviruses and it was required by geminiviruses for inducing typical symptoms in natural hosts. All reported DNAP molecules have a βCl gene on the complementary-sense, which is conserved on position and in size and is found to be a symptom determinant. Up to date, no study on promoter of βCl gene has been reported. Tomato yellow leaf curl China virus (TYLCCNV) was first identified in our laboratory to be associated with DNAp. In this dissertation, promoter fragments of βCl gene and bi-directional promoter fragments from TYLCCNV were isolated and identified. Regulation on promoters activity by proteins encoded on the complementary-sense of TYLCCNV or βCl protein was also studied.Promoter fragments of βCl ORF of DNAβ associated with TYLCCNV isolate Y10 were identified by Agrobacterium-mtdiated transient expression method. With the gus gene as a reporter gene, promoter activity was tested by bistochemical and fiuorometric assay. The results showed that a 955 nuclueotide (nt) fragment upstream of the translation start site of βC1 ORF has activity and the 5'- and A-rich region deletions of the fragment kept their activity, however, the 3'-deletions of the fragment lost their activity. The activity of the 955nt fragment was just about 10~16% when compared with a 35S promoter from Cauliflower mosaic virus. Histochemical assay revealed that the 955nt fragment can drive gus gene to be expressed specifically in phloem tissue of the stably transformed tobacco plants. Further studies indicated that a 173nt from 3' end of the 955nt fragment was responsible for basic promoter activity and its phloem-specific expression.Co-expression of proteins encoded on the complenmentary-sense of TYLCCNV-Y10 or βCl protein with promoter expression vector of satellite was carried out to study the regulation on promoter activity by those proteins. The results indicated that the proteins AC1, AC2 and AC4 can enhance promoter activity, but AC3 has not effect on regulation of promoter activity, and βCl protein can enhance promoter activity.