In Vitro Resistance Of Transgenic Plants With Cecropin B Gene To Citrus Canker Caused By Xanthomonas Axonopodis Pv. Citri

Citrus canker disease caused by Xanthomonas axonopodis pv.cilri.(Xac.) is a disease in the world citrus industry. The disease causes defoliation, dieback, fruit drop and vitality decline,and lesion on the fruits that greatly degrades the economic value of the citrus. There is no effective method to control citrus canker. Conventional disease resistance breeding is difficult because of limitation of reproduction barrier among different species. Bactericide can solve the problem in some context, but some bactericide can cause heavy environmental pollution. Therefore, resistant gene cloning and transgenic approach is the key to fight against the bacterial pathogens.The preliminary research showed that the antibacterial peptides from tussah could greatly improve citrus resistance to canker. In this study, resistance of77transgenic plants with Cecropin B to Xac has been investigated by in vitro leaf inoculation tests. Pin-puncture inoculation was selected to investigate resistance to citrus canker disease. The number and size of the lesion, disease index and bacterial growth Xac. in transgenic leaf were investigated in detail. Expression and integration of Cecropin B gene in transgenic plants was analyzed by Real time q-PCR and Southern blotting. Transcript profiles of PR-1、MKK4、GST、PrxA genes in transgenic leaf was also analyzed by Real time q-PCR. Our study provides some references for further greenhouse and field evaluation of resistance. The main results are as follows:1. Citrus canker pathogen separation and purificationXac from the typical canker disease leaves is islated by dilute separation. Typical symptoms were observed in the wild types after pin-puncture inoculation.2. Selection of pathogen infection methodsTo select an efficient pathogen infection method to evaluate resistance of transgenic plants to Xac, wild-type Jingcheng Orange leaves were tested. Results showed that visible lesions was detected in80%leaves4days of inoculation using needle-puncture inoculation, while it was observed in only10%leaves after10days of infection when using spray inoculation. Thus, needle-puncture inoculation was used for the following experiments.2. Evaluation of resistance of transgenic plants to Xac.To investigate resistance of transgenic plants to Xac, the number of lesions in transgenic leaves were counted after4d inoculation.17out of77transgenic plants showed45.54%-67.86%of incidence of needle-punctured spots, significantly lower than that of non-transgenic plants. Further tests showed that disease indexes (0.8%-35.5%) of these17transgenic plants were significantly lower than that (73.3%) of non-transgenic plants. The number of Xac. cells in transgenic leaf was only0.07-0.80folds of that in non-transgenic leaf indicating bacterial growth of pathogen was greatly inhibited in transgenic plants. These data showed that Cecropin B gene can significantly enhance resistance of transgenic citrus plants to Xac.3. Molecular analysis of transgenes in transgenic plantsTotal RNA were isolated from transgenic plants and the expression of cecropin B was investigated by quantitative real-time PCR using actin gene as reference. High levels of expression of Cecropin B gene were detected in transgenic plants tested, and its expression was not detected in non-transgenic plants. The transgenic plants were further analyzed by Southern blotting, and the results showed that most of transgenic plants had one copy of transgene while the cXC-PR11、XC-PR21、PR12、PR8、 CB12and CB20plants had2copies of transgene, and CB13had3copies of transgene. No blot signal was detected in non-transgenic plants.5. Analysis of expression profiles of plant resistance-related PR-1、MKK4、GST and PrxA genes in transgenic plantsTo invstigate expression profiles of PR-1、MKK4、GST and PrxA gene in transgenic plant, total RNAs were isolated from needle-punctured spots in infected leaves after0h、2h、6h、24h、48h and72h of pathogen inoculations and were amplified by quantitative real-time PCR. Among pathogen inoculation, the similar expression profiles of these genes were detected in transgenic and non-transgenic plants. Interestedly, the inducible expression levels of these genes in transgenic plants were much higher than that in non-transgenic plants, suggesting that PR-1、MKK4、 GST and PrxA genes-related response systems could be activated in transgenic plants with cecropin B gene. This might improve resistance of transgenic plants to Xac.