Molecular Identification Of Tomato Yellow Leaf Curl Virus In Yangling And Genetic Transformation Of Its CP And Rep Interference Vectors To Tomato

Tomato yellow leaf curl virus（TYLCV）, one of the most devastating viral diseases ofcultivated tomato(Solanum lycopersicon), have caused serious influence on tomato productionin China even all over the world. The best way to control this virus is to develop a newvirus-resistant tomato variety. RNA interference technology has attracted more concerns as akind of effective mechanism of silence in virus-resistant breeding.Firstly, tomato yellow leafcurl virus in Yangling, Shaanxi, China, was identified. then, tomato genetic transformation oftomato was conducted by interference coat protein gene(CP) and repliction associated proteingene(Rep), respectively. The main results are as follows:1. The identification of tomato yellow leaf curl virus in YanglingWith geminivirus general primers PA／PB，PCR amplification with the total DNAtemplate extracted from the growing point of tomato plant with type TYLCV symptomshowed a500bp specific fragment, which confirms this islates belongs to geminivirus. Thetotal DNA was extracted using CTAB method and the diseased tomato sample were plantedin the experiment station of Horticulture of College, Northwest A&F University. PCRamplified with the total DNA template and F1／R1and F2／R2primers and the nearly fullnucleotide sequence length of the isolate was gained by PCR amplication with two pairsprimers of F1／R1and F2／R2designed according to the sequence of TYLCV(NCBIAccession number:X15656). Tomato yellow leaf curl virus in Yangling was named tomatoyellow leaf curl virus-Yangling(TYLCV-Yl, NCBI accession number is KC293824)It is found tha tomato yellow leaf curl virus–Yangling is monopartite begomoviruseswithout DNA-B and contain beta satellite molecular(DNAβ) and belongs to Israel strain bycomparing TYLCV-Yl sequence with other TYLCV sequence from18different courtries orregions.2. Cloning and sequence analysis of tomato yellow leaf curl virus coat protein inYangling and its core tCP The coat protein gene about780bp of TYLCV-Yl was one part of the1400bp sequencefragment, which was cloned with total DNA template and CP-F／CP-R primers.TYLCV-Yl-CP gene sequence were compared with TYLCV-CP from18different countries andregions and found that the core sequence tCP was conservative and located on the5′end ofCP. The core sequence tCP can encode137amino and containing a transmembrane domainbetween79amino and97amino, so it can be used as a target of RNAi.3. The construction of interference vectors of TYLCV coat proten(CP) and replicationassociated protein(Rep)The conservative sequenc tCP of coat protein and tRep of replication associated proteinof TYLCV-Yl were connected respectively in both sides of carrier introns in oppositedirections. Interfenence carrier of pFGC5941-tCP-rtCP and pFGC5941-tRep-rtRep wereconstructed successfully, CaMV35S as a promoter. The size of tCP or rtCP is420bp and thesize of tRep or rtRep is300bp.4. The optimization of tomato regeneration systemThe main factors influencing the tomato regeneration were studied, such as thedifference of varieties, the different organs of material, the difference of flask，the length ofpreculture time and acetosyringone.The results showed that the regeneration rate of tomatovariety(TTI1201B-2-1) was higher than others. The budding rate of the middle cotyledon washighest among four tomato materials. When preculture time was two or three days, which canguarantee explants wound do not heal and the browning rate was relatively low. The buddingrate of explants cultured in conical flask was twice of that in the culture flask. The growth ofcallus could be inhibited on medium add to acetosyringone. The concentration of bastaherbicide was more than0.5mg.L-1, which could be effective on the selective forresistance-Baste explants5. Tomato genetic transformationThe vector pFGC5941-tCP-rtCP and pFGC5941-tRep-rtRep were transformed inagrobacterium GV3010and EHA105, respectively. After agrobacterium-mediatedtransformation,23transformation plants were gained by using leaf-disc method. The resultsshowed that two of them were transgenetic plants with tRep and rtRep and two of them justwere transgenetic plants with tCP. The transformation rate is about8.7percentage.