Studies On Luohanguo-infecting Zucchihi Yellow Mosaic Virus And Genetic Transformation System For Luohanguo

Zucchini yellow mosaic virus (ZYMV), a member of genus Potyvirus, family Potyviridae, is one of the most economically important viruses of cucurbit crops. ZYMV alone or mixed with other virus can cause viral diseases to Luohanguo (Siraitia grosvenorii). To understand the epidemiology of Luohanguo-infecting ZYMV (ZYMV-LHG) and its primary inculum source, surveys were conducted in fields for ZYMV-induced mosaic disease of Luohanguo throughout the growing seasons and the plants collected from and around the fields were detected for ZYMV by ELISA. To elusidate the genetic dynamics of ZYMV-LHG, coat protein gene sequences of viral isolates collected from different Luohanguo-growing regions were compared for divergence. Finally, Luohanguo genetic transformation by apploying RNA interference (RNAi) strategy for resistance to ZYMV was attempted.Surveys in field locations for the viral disease were conducted in Yongfu and Rong’an counties of Guangxi in May, July and September,2006. In the early stage of growing, all of the ratoon Luohanguo showed severe mosaic leaves but tissue culture Luohanguo were symptomless. In the middle stage of growing, the disease incidence of the field with ratoon Luohanguo was up to100%, and the growth of plants in this field was worst. The adjacent field had higher disease incidence than other fields without ratoon Luohanguo around. In the latter stage of growing, the virus disease symptom appeared in all fields, the incidence ranged from0.8%to100%.The purified ZYMV-LHG preparation was injected into New Zealand rabbits and gained antiserum which titer was1:16000assayed. The ZYMV-LHG antiserum was used to detect the plants collected from and around the Luohanguo fields with indirect enzyme-linked immunosorbent assay (ELISA). Part plants of9families (Cucurbitaceae, Amaranthaceae, Leguminosae, Compositae, Gramineae, Polygonaceae, Malvaceae Commelinaceaeand Rubiaceae) showed positive rates from114species,96generas,46families. The result suggested that ZYMV-LHG had wide natural host range. The rate of ZYMV-LHG transmitted through seeds was detected, the results indicated that ZYMV can not be transmitted via seeds of Luohanguo, or the transmission rate was very low.To study the genetic dynamics of ZYMV-LHG, the coat protein (CP) gene sequences of isolates from typical mosaic Luohanguo collected from Yongfu, Longsheng and Xing’an, Guilin, GX were analysed.17complete CP gene sequences from independent ZYMV-LHG isolates were obtained by RT-PCR. The homologous rate of nucleotide and deduced amino acid sequences was100%-92.1%and100%-96.4%respectively. Genetics analysis based on the17nucleotide sequences was conducted by Dnasp. The result indicated that the5’terminus of ZYMV CP was more variety. The Tajima’s D was0.44664, sugessted that ZYMV-LHG didn’t occurrence population expansions. The analysis revealed that mutation was not occurrence on the conserved DAG motif among sequences, the result implied that all of these17isolates can be transmit by aphid. The result of antigen forcast analysis assistanted the analysis by Dnasp, the antigen determin which located in N terminus of ZYMV-LHG CP was more variety. There were6kinds of antigen forecast analysis profile among this17ZYMV-LHG isolates and I contained6isolates:6,7,8,9,12, N10-1and N10-4.Markov Chain Monte Carlo (MCMC) methods were employed to estimate dates of ancestors for groups of ZYMV-LHG. Bayesian coalescent analysis estimated the date of the most recent common ancestor for the ZYMV in this study to be about1983; for the ZYMV-LHG to be1999. The phylogenetic tree based on nucleotide sequences of ZYMV CP gene was constructed using the neighbor-joinging method in MEGA5. Phylogenetic tree showed that the isolates of ZYMV-LHG were clusted to two subclades except ST-15. This analysis revealed that ST-15infected luohanguo later and had higher evolutional rate than other ZYMV-LHG isolates.There were3kinds of explants was studied on regeneration efficienty. The result suggested that differentiation ability of the regenerated shoot of different explant, the cotyledon with the super regenerated frequency was the best choice for luohanguo genetic transfomation system. And regenerated plants were obtained from cotyledon explants with kanamycin selection on60mg/L were effectively. This development lays a foundation for genetic engineering of Luohanguo for viral disease-resistance breeding.Conserved region of ZYMV NIb and CP were obtained and the secondary structure of ZYMV NIb and CP were predicted by RNAstructure. The target fragments were cloned into pBI121vector with kinds of restriction endonuclease. The RNAi expression vector which harboured ZYMV NIb or CP were designated with pBI121ZYMVCPRNAi and pBI121ZYMVNIbRNAi respective. The sense and antisense of ZYMV NIb and CP were introduced into luohanguo using Agrobacterium tumefaciens EHA105. A total of258plantlets were confirmed to be positive by PCR analysis, of which139plants were introduced with ZYMV CP and119plants were introduced with ZYMV NIb.