Gene Expression Profiles In The Leaves Of Rubber Tree(Hevea Brasiliensis) Infected With Corynespora Cassiicola And Cloning Of Resistance Genes

Corynespora Leaf Fall Disease (CLFD) of rubber tree (Hevea brasiliensis) caused by the fungus Corynespora cassiicola has emerged as the most devastating disease after the South American Leaf Blight (SALB) over the recent two decades. This disease has caused significant losses to the production in the rubber growing regions in the world. Meanwhile it has become one of the serious potential risk of disease in our country natural rubber production. However, the resistant mechanism of rubber trees were rarely reported in the world. In that case, this study focused on the disease epidemic monitoring, investigation, resistant rubber varieties selection, the main defense enzyme activity and variability of gene expression after Corynespora cassiicola inoculated to the resistant and susceptible germplasms. Meanwhile cloning and identification of resistance genes and proteins, and further clarify its biological function. The main results are as follows:Based on disease epidemic monitoring and dynamic survey of the diseases, more than92%of the monitoring stations were found this disease and it refers to the aggravating trend year by year. Generally, the disease occurs during March to April, risen sharply during August to September and highest peak during October to November in the whole year and lack of relatively good disease resistant germplasm in field.Based on the pre-research, four kinds of methods were used on the resistant evalution of46Hevea brasiliensis germplasms. The similar results were obtained by these methods, and the differences between resistant and susceptible germplasms were very significant. For46germplasms.4of them were highly resistant (HR) and the ratio was8.70%,12of them were moderately resistant(MR) and the ratio was26.06%,11of them were slightly susceptible (S) and moderately susceptible (MS) and the ratio was23.92%separately,8of them were highly susceptible (HS) and the ratio was17.40%. Meanwhile, the excellent germplasms (Yunyan277-5) and resistant germplasms (IAN873) hybrid clones were evaluated by the indoor in vitro inoculation method. The result showed that there were highly resistant (70clones) and the ratio was26.12%. The result of defense enzyme activity determination that β-1,3-glucanase, peroxidase (POD), phenylalanine ammonia enzyme (PAL) and polyphenol oxidase (PPO) were significant differences between resistant and susceptible germplasms, and the resistant one was higher than susceptibleIn order to illustrate the resistant and susceptible response of rubber tree (Hevea brasiliensis) infected with Corynespora cassiicola at a transcriptional level, gene expression profiles in leaves of rubber tree, inoculated with fungal spore suspension were tested using cDNA-AFLP technology. Selective amplifications with96primer combinations allowed the visualization of4800transcript-derived fragments (TDFs) in infected leaves, and560of them were found to be differentially expressed (306induced and254repressed).218TDFs were cloned and sequenced, of which116were shown to have known functions and had predicted gene products by searching the GenBank database through BLAST. The116TDFs were homologous to genes involved in some means of physiological and biochemical such as metabolism (13.79%), protein synthesis (12.93%), disease defense (9.48%), signal transduction (6.90%), energy metabolism (6.03%),protein destination and storage (5.17%), signal transduction (6.90%), Carrier and transport in the cell (3.45%) and unknown protein (20.69%). The expressed patterns of six representative TDFs were further confirmed by real-time PCR analysis. It improved that the confirmed reliability of the cDNA-AFLP technique.Based on the conclusion of the cDNA-AFLP analysis, four resistance genes were cloned by RT-PCR and PCR technique. There are a plant resistance NPR gene family member HbNPRl, a haloalcanoic acid dehalogenase super family member HbVSP2and resistance gene analog members HbRGH2and HbRGA-1. Real-time PCR analysis showed that four resistance genes were expressed predominantly in corynespora cassiicola, jasmonic acid methyl ester (MeJA), salicylic acid (SA) and ethylene (ET). The biological functions, tissue-specific expression and resistance signaling pathways of four resistance genes were further confirmed with rubber tree.Comparative analysis with2-DE maps in the rubber tree leaves infected with Corynespora cassiicola, we found32proteins with good separation and obviously expressional changes by MALDI TOF and MALDI TOF/TOF MS identification, among which about16proteins were credible verified corresponding to13proteins. These proteins could be devided into5categories, such as Energy production and metabolism related proteins (31.25%), Defense and stress related proteins (37.5%), Carbohydrate synthesis and transport related proteins (18.75%), Amino acid transport related proteins (6.25%), Unknown function protein (6.25%).It also identified defense related enzymes: Superoxide dismutase and peroxidase ascorbic acid, etc.