Analysis On Resistance Differences Of Guanximiyou And Ponkan Upon CLas Infection Based On Bioinformatics

Citrus Huanglongbing caused by unculturable phloem-limited bacteria is one of the most destructiving diseases of citrus industry worldwide. In recent years, the disease has occurred and caused serious economic losses in many citrus producing areas in China. Candidatus Liberibacter asiaticus(CLas), the only pathogen responsible for HLB in China, is the most infectious, widespread and harmful pathogen in three HLB-causing ?-Proteobcateria species. In order to control HLB, citrus producing countries had conducted extensive researches on the disease. However, little research has been carried out about the resistance mechanism of HLB. In this paper, transcriptome changes in different resistant varieties of guanximiyou and pokan and at different time points were detected and comparatively analyzed by using microarray and bioinformatics. The aims are screening differentially expressed genes related to HLB resistance, understanding the resistence mechnisms of citrus against HLB, and finally laying theoretical foundation for controlling HLB on molecular level. The main results are as follows:1. Screening of HLB source plants. 30 plant leaf samples were collected from HLB endemic area,Jiangxi province. They were used for detecting CLas, Phytoplasma, and Citrus tristeza virus(CTV) by PCR. Results showed that two plants were infected with CLas, not with Phytoplasma and CTV. These two plants were considered as the ideal HLB source plants and used for the subsequent experiments.2. Indoor resistance observation of different citrus cultivars against HLB. Buds with CLas were grafted on plants of different citrus varieties. HLB Infection status among them were evaluated timely by PCR and the symptoms were observed. It showed that to HLB, variety Ponkan was highly susceptible, and variety Guanximiyou was resistant.3. Analysis of differentially expressed genes. Using cDNA microarray, detection of gene expression was conducted from CLas-inoculated different variety citrus leaves four and six months after inoculation, and function of the differential genes were analyzed by Gene Ontology(GO) and MapMan.It showed that amount of up-regulated GO terms at both time interval in Guanximiyou(resistant variety)was much larger than in Ponkan(susceptible variety), and as for Guanximiyou, amount of up-regulated GO terms six months after inoculation was larger than four months after inoculation. In transcriptome data of Guanximiyou at six month, GO terms of biotic stress, immunity and defense were obviously up-regulated. The result of SEA showed, most of genes of plant growth, carbohydrate metabolism,phenylpropane synthesis, and biotic stress in Guanximiyou were increasingly expressed. Meanwhile,through pathway analysis, it was found that most of genes of cell wall, salicylic acid and jasmonic acid synthesis, disease resistance related substances and transcription factors were in up-regulated expression in Guanximiyou. Different from Ponkan, Guanximiyou made a positive plant defence response upon CLas infection and so improved their ability to resist the CLas infection.4. Five resistence candidate genes were selected for qRT-PCR test. Results showed that the relative transcript level of these genes in qRT-PCR were basicly consistent with the transcript level in microarray, validating the reliability of our microarray test.