Molecular Response Of Chewing Cane (Saccharum Officenarum L.) To Infection By Gibberella Fujikuroi

In the south of China, chewing cane is an important economic crop which has high economic efficiency and rich nutritional value. It plays a significant role for the income of farmers. In the process of production and storage, various diseases threaten seriously the yield and quality of chewing cane, and caused tremendous economic losses. Germplasm resources of chewing cane are very rich in China and include the cultivated species contained a variety of disease resistant traits. However, there are few research and practical application in the interaction mechanism between chewing cane and pathogens, as well as the pathogenesis-related proteins and genes. In this study, differential proteomics, real-time fluorescence quantitative RT-PCR and plant physiology method were used to study the molecular mechanisms. Silico cloning and RT-PCR were used to clone genes of the signal transductional factor SGT1 and RAR1. The bioinformatics software was used to analyse the physical and chemical properties of chewing cane SGT1 and RAR1. Southern blot and RT-PCR were used to analyse the copies and transcription level of SoSgt1 and SoRar1 induced by shoot rot pathogen G. fujikuroi. The results were utilized to initially illustrate the molecular mechanism of interaction between chewing cane and shoot rot pathogen G. fujikuroi, and provide some material basis for the chewing cane breeding. The main results were as follows:1. The analysis of differential expression proteins in the leaves of chewing cane induced by shoot rot pathogen G. fujikuroiTen chewing cane cultivars were inoculated with G. fujikuroi, and showed different degree of symptoms. The cultivars Baishan, Dudu, Fenchenzipi and Badila had lighter symptoms than other cultivars. Xiamao, Wenlin, Ningde, Chengxian and Waigandan emerged more serious symptoms and had large area of lesion. The symptoms of Fuan were moderate. The cultivar Fuan was selected as the material for differential proteomics analysis. The proteins were extracted from the leaves inoculated with G. fujikuroi at 0h and 48h, and seperated by two-dimensional electrophoresis. 24 differential expression proteins were selected by the ImageMaster software and identified by the MALDI-TOF/MS. The 24 proteins were preliminarily divided into five categories which contained pathogenesis response-related proteins, antioxidant defense system-related proteins, resistance proteins, kinase-like proteins and others. These proteins may be involved in the pathogenesis response between the chewing cane and pathogen G. fujikuroi. However, the function and relation of these proteins must be further studied in the network of pathogenesis response.2. Studies on the characters of physio-biochemistry in the leaves of chewing cane inoculated with G. fujikuroiIn order to validate differentially expressed proteins in leaves of chewing cane inoculated with the G. fujikuroi, superoxide dismutase (SOD), peroxidase (POD) and the chitinase (CHIT) selected from the 24 proteins, and the related indicators of catalase (CAT) and malondialdehyde (MDA) were studied. The results showed that there were similar change trends between the five indicators, which displayed the increased first and then decreased pattern. In the leaves of different chewing cane cultivars inoculated with G. fujikuroi, the activities and contents of SOD, POD, CHIT, CAT and MDA were different. The results illustrated the response of different chewing cane cultivars to the G. fujikuroi were different at the physiological level, which limited the further expansion of G. fujikuroi in different degree.3. Validation of differentially expressed genes encoding pathogenesis-related proteins by qRT-PCRBased on the results of differentially expressed proteomics and physio-biochemistry, the genes encoding SOD, POD, CHIT and down-regulated TPS6P were selected as the object to study by qRT-PCR, and the degenerate primers were designed according to homologous genes. The sequences amplified from qRT-PCR were true through the bioinformatics analysis, and named as SoSOD, SoCHIT, SoPOD and SoTPS6P and submitted to the NCBI. The the accession number in GeneBank were GU219845~GU219848 respectively. SoSOD, SoCHIT and SoPOD induced by G. fujikuroi were up-regulated in leaves of different chewing cane cultivars, and SoTPS6P showed fist upregulated and then dow-regulated pattern. The results of qRT-PCR indicated pathogen G. fujikuroi induced the transcriptional diversification of genes encoding pathogenesis-related proteins.4. Isolation and characterization of defense-related genes SoSgt1 and SoRar1 in leaves of chewing caneThe degenerate primers were designed according to the conserved sequences of homologous SGT1 and RAR1 genes from other plants. The SGT1 and RAR1-like fragments were isolated from the cDNA in the leaves of Fuan and used as probes to isolate the complete cDNA sequence of chewing cane SGT1 and RAR1 genes through silico cloning and RT-PCR. The complete SGT1 and RAR1 genes were named SoSgt1 and SoRar1 respectively, and submitted to the NCBI. The accession number in GeneBank were GQ864255 and GQ864256. The length of SoSgt1 was 1086 bp open reading frame encoding 362 amino acids, which contained typical TPR, CS and SGS domains of SGT1 gene. SoRar1 contained 645 bp open reading frame encoding 215 amino acids with CHORD I, CCCH and CHORDâ…¡domain. The ailgement of SGT1 and RAR1 proteins from chewing cane with other plants showed that the amino acid sequence encoded by SoSgt1 and SoRar1 were highly conserved, and contained key functional domains. South Blotting results showed the SoSgt1 and SoRar1-related sequences were presented as 1-2 members in chewing cane genome. Semi-quantitative RT-PCR analysis showed the SoSgt1 and SoRar1 appeared to be induced by G. fujikuroi and displayed different transcriptional levels in the leaves of different chewing cane cultivars. The antisense eukaryotic expression vectors of SoSgt1 and SoRar1 were constructed and transformed into other sugarcane cultivar, and transformation frequencies were 5.4% and 4.2% respectively, which will be helpful to study their function and provide the material basis for further research on mechanism between the chewing cane and G. fujikuroi.