Effects Of Smut Pathogen On Photosynthetic Characteristics And Gene Differential Expression In Sugarcane At Seedling Stage

Sugarcane is an important sugar and biofuel crop with high capability to assimilate carbonand high productivity. Sugarcane smut is caused by basidiomycotina fungus Ustilago Scitaminea Syd., and is a common problem causing serious sugar content and cane yield diminution worldwide. The mechanism of sugarcane response to smut pathogen is not clear,especially for the molecular mechanism at the beginning of the pathogen infection into sugarcane seedlings.In order to provide useful information for developing rational strategies to control smut at early stage of disease development, acupuncture inoculation of smut pathogen into young plants was done. The photosynthetic and fluorescent parameters in the plants during the initiation of the pathogen infection were detected, the differential gene expressions at both transcription and translation levels were investigated, and whole lenth of4genes related to smut pathogen stress were cloned. The main results were as follows.1. The net photosynthetic rate (Pn), stomatal conductance (Gs), Transpiration rate(Tr) are all declined before3d inoculation, while the intercellular CO2concentration (Ci) is increased in sugarcane seedlings. The variablefluorescence (Fv), maximalfluorescence (Fm), photochemical quantum efficiency of PSII (ΦPS II), maximal photochemical efficiency of PS II (Fv/Fm) and photochemicalefficiency of PS II in light (Fv’/Fm’) were declined obviously, but the increased rapidly after inoculation3d. There were significant positive correlations between There were significant positive correlations (>0.800) between Pn and Gs, water use efficiency (WSE), between Ci and Fv’/Fm’, ΦPS II, between Fo and Fv/Fm, Fs, ETR, between Fv/Fm and Fv’/Fm, between Fs and Fv’/Fm’, and between Fv’/Fm’ and Φ PS Ⅱ.2. At the translation level, a total of thirty-four differentially expressed protein spots were found significantly (p<0.05) change in intensity after3d inoculation, as compared to the control. Twenty-six of the differential proteins were up-regulated and eight were down-regulated. Protein identification was performed with mass spectrometry MALDI-TOF/TOF and eighteen protein spots were successfully identified. Based on their putative biochemical role, the proteins were classified into7categories as follows:defense response (spot3,12,21,27and30) accounting for29%, metabolism (spot8,9,10, and18) accounting for23%, photosynthesis (spot6,11. and32) accounting for18%, signal transduction (spot24) accounting for6%, protein processing (spot2and29) accounting for12%, cell growth/division (spot19) accounting for6%, and unclassified (spot33) accounting for6%. It should be noted that any given protein may be responsible for multiple biological processes and, therefore, could be classified into more than one biological process.3. At the transcriptional level, a forward SSH library was successfully constructed.224differentially expressed genes (ESTs)reponded to smut were sequenced and functional annotations were done for199of them successfully.The results of cellular component classfication at GO level2showed that152unigenes were annotated241times and could be divided into8categories. Those responsible for cells, organelles and macromolecular complexes occupied the largest proportion of36.7%, while those responsible for symplasm, envelope and extracellular domain account for19.3%. This indicated smut pathogens mainly impact on the interior of cell. Molecular function classfication at GO level2showed that129unigenes were annotated for132times and could be divided into8categories. The largest proportion of the unigenes was found to be those for binding and catalytic activity, and they were annotated for49times and61times respectly. The major role of these two groups of proteins is regulatory. This indicated the differential gene expression is regulated by certain factors. Biological process classfication at GO level2showed that139unigenes were annotated for273times and could be divided into18categories. The largest proportion of the unigenes was found to be those for metabolic process and celluar process, and they were annotated for85times and74times respectly. This indicated the plants have basic life activities. Furthermore, the unigenes for stimulus response and biological regulation were also annotated for28times and17times respectly.4. The full length of ScPAL, ScSAM, ScXTHand ScNudix were obtained using the method of RT-PCR and RACE. The expression of these genes in biotic stress (smut) or abiotic stress (PEG,4℃, NaCl, H2O2) were analyzed using the qRT-PCR. The results provided theoretical basis for further application of these genes in molecular assisted resistance breeding of sugarcane.