Molecular Identification, Genetic Diversity Analysis Of Tomato Yellow Leaf Curl Virus And Isolation Of Related Genes Induced By Tylcv

Tomato (Solanum lycopersicum Mill.) is one of the most important vegetables worldwide, with abundant nutrients and unique taste. The yields and quality of tomato are severely threatened by abiotic and biotic stresses. Tomato Yellow Leaf Curl Virus Disease (TYLCD) is one of the most serious diseases in tomato. To analyze the pathogenic mechanism, a full-length sequence of virus isolate was cloned in Zhejiang region. Structure feature, sequence homology and phylogenetic analysis of this virus isolate were conducted, to reveal genetic diversity of the whole TYLCV isolates, the representative TYLCV from48locations in29countries were searched in the NCBI website (http://www.ncbi.nlm.nih.gov/). Then, structure feature, GC content, homology comparison and phylogenetic relationship of these virus isolate were performed. In addition, absolute quantification of virus in different tomato accessions was analyzed by qRT-PCR technology. At last, different regulated genes associated with TYLCV disease resistance were isolated using cDNA-AFLP. As follows:1. Molecular identification and sequence analysis of tomato yellow leaf curl virusVirus isolate was cloned in Zhejiang region based on PCR amplification. Length of the virus isolate was2781nucleotides, it encoded six open reading frames (ORFs)(AV2, AV1, AC3, AC2, AC1, AC4), which were the typical structure of TYLCV. Nucleotide sequence analysis showed that the virus isolates had the highest homology (99.6%) with TYLCV in USA. ORFs analysis suggested that AV2was100%homology with that of TYLCV from other province in China, except for Yunnan. Phylogenetic analysis showed that virus isolate was grouped with that from Jiangsu, Beijing, USA, Mexico, suggesting the closest genetic relationship. 2. Genetic diversity analysis of tomato yellow leaf curl virusFourty-eight TYLCVs were searched in the NCBI website, these virus encoded six open reading frames (ORFs)(AV2, AV1, AC3, AC2, AC1, AC4), i.e., the typical structure of TYLCV. GC content analysis showed no obvious differences. Sequence alignment revealed that TYLCV were high conservation. Phylogenetic relationship revealed ten classes of TYLCV in tomato. Sequence homology showed large differences between the sequences (72.8%-98.6%).3. Quantification analysis of tomato yellow leaf curl virusAbsolute quantification of virus in different tomato accessions were analyzed by qRT-PCR technology. The results showed that virus content of resistant accessions were lower than the susceptible accessions in the same period. The number of virus in other resistant accessions were increased at first, then decreased, except for Ty-1+2+3+5. The number of virus and disease index of tomato accessions were coincident.4. Analysis of related genes associated with tomato yellow leaf curl virus disease by cDNA-AFLPWe isolated different expressed genes associated with tomato yellow leaf curl virus disease by cDNA-AFLP. The result revealed that287transcript derived fragments (TDFs) were obtained, of which156showed up-regulated and131down-regulated. Fifteen TDFs were selected for cloning, sequencing and identification. The result showed that11had known functions, which were involved in metabolism, signal transduction, transcriptional regulaation, DNA binding, stress defence. The remaining four TDFs had low identity or no match to known genes, therefore maybe represent novel tomato genes.