The Construction For TYLCCNV Infectious Mutant With Genetic Marker

Geminiviruses have circular single stranded (ss) DNA genomes. Previous studies demonstrated that the population structure of a geminiviruse is quasispecies-like and the recombination is one of the important evolutionary forces that drive the evolution of the geminivirus population. To understand the population change during the systemic movement within a plant and horizontal transmission among plants, a genetically marked artificial population (quasispecies) is expected. In this thesis, we used infectious clone of Tomato Yellow Leaf Curl China Virus (TYLCCNV) isolate Y10 (TYLCCNV-Y10 pBinPLUS-1.7A) as a parental clone and the technique of overlapping PCR, replaced C with T at nt45 to create Mlu I restriction enzyme site (RES) in IR5; replaced A with T at 277 as well as replaced C with T at 281 to create Sac I RES in AV2 region; replaced G with C at nt575 to create another Mlu I RES in AV1 region, and replaced G with A at ntl675 to create Sma I RES in AC1 region. The presence of these restriction enzyme sites in respective mutant was confirmed by both digestion and sequencing. Preliminary results indicated that these mutants are infectious to Nicotiana benthamiana. The test for their individual accumulation level and neutrality in both N. benthamiana and Solarium lycopersicom plants is on-going.