Analysis Of Maize R. Solani AG1-IA-induced Genes Differential Expression And Defense Enzymes Detection

Maize banded leaf and sheath blight(BLSB), caused by Rhizoctonia solani Kühn, is a destructive disease that results in significant yield loss in most maize-growing areas in China. Although this is a regional maize disease mainly occurring in China and Southeastern Asia, for the weather of these regions, it is possible that this disease may spread to other parts of the maize BLSB, the understanding of resistance mechanism is of great importance. In this research, cDNA-AFLP was utilized to analysis gene expression profile in maize induced by AG1-ⅠA. The objective of the present research was to explore efficient methods to analyze high-throughout gene expression, and to obtain a primary gene expression profiling of the disease resistance in maize, and to understand the resistance mechanism against maize BLSB at transcriptional level.By use of cDNA-AFLP, a pathogen inducible gene expression profile was obtained from high tolerance maize inbreed lines of R15 and high pathogenic fungus anastomosis groups AG1-ⅠA of Rhizoctonia solani Kühn in Southwestern of China. At the jointing stage, two wheat seeds colonized by AG1-ⅠA were placed into the third sheath of the maize plants. At 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs of the post-inoculation, the inoculation and non-inoculation leaves was picked down for total RNA extraction to reverse transcribing and analysised genes differential expressed. We also analysised the changes of several resistant enzymes in high tolerance maize inbreed lines R15 and high sense maize inbreed lines 478, at Ohrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs,72hrs of the post-inoculation in sheath and leaves. The obtained main results were as follows:1. By 56 combinations AFLP primer screening, 87 transcript derived fragments showed differential expression, we tried to reclaim 60 TDFs of them, 35 TDFs were succeed, then we jointd these 35 TDFs in pMD18-T vector and transformed in E coli DH5α. 18 clones were selected to sequence.2. All of the sequences of insertion fragments were analyzed using bioimformatical methods. 13 ESTs sequence had significant homology sequence in Genbank, the remaining 5 ESTs had no significant similarity sequence, in the 13 ESTs,11 ESTs were known genes, one mRNA sequence and one genome sequence: Zea mays rust resistance protein rp3-l(rp3-l) gene, putative senescence-associated protein, gag protein, SNF2-related, NADH dehydrogenase subunit K, NADH dehydrogenase subunit 2, Ser/Thr protein phosphatase family protein, serine/threonine kinase protein, polyprotein, teosinte glume architecture 1, Zea mays PCO118792 mRNA sequence, Zea mays subsp. parviglumis mitochondrion complete genome.3. The activities of peroxidase(POD), phenylalanine ammonia lyases(PAL), Ascorbate Peroxidase, superoxide dismutase(SOD) and catalase(CAT) were measured in R15 and 478 both in sheathes and leaves inoculated with AG1-ⅠA or not inoculated. The results showed that, as to resistant material(R15), the activities of POD, CAT, SOD, APX increased and PAL kept stable in sheaths, in leaves the activity of POD,CAT and APX increased, SOD,PAL decreased; as to high susceptible material(478), the activities of POD and CAT increased in both sheaths and leaves, SOD increased in leaves but decreased in sheathes, both PAL and APX had no striking change. Generally speaking, the POD and CAT activities of maize were positively related to its resistance. Experimental results suggested that these defense enzymes functioned in differential areas and courses in plant accomplished defense reactions.