The Study On Gene Differential Expression In Disease Resistance Of Populus Tomentosa Induced By Chitosan-Oligosaccharides

Populus is a common street tree and a windbreak tree species in northern China. It has a good ecological and economic value for it's fast growth speed and good wood color. But more types of pest and pathogenic fungi often cause serious economic losses. Chemical pesticide used on controlling pests and diseases had lower effect, did great harm to the environment and promoted the resistance to the pesticide; Times of improvement breeding increase the difficulty of experiment and practice. Many study showed that Chitosan-oligosaccharides could induce plant's resistance to disease as exogenous elicitors and have a great prospect of biological pesticides. The main purpose of this study is to find out the oxidation process of preparing the Chitosan-oligosaccharides of most biological activities and the effects on differential gene expression induced by this oligosaccharides. Through this study, we did the basic work for the further research on molecular biological mechanism of resistance to disease induced by Chitosan-oligosaccharides on Populus tomentosa .This research usesd chitosan and Populus tomentosa callus as the research objects. Decomposed chitosan with H₂O₂ and 2% acetic acid and got four Chitosan-oligosaccharides of different average polymerization degree by controlling the reaction conditions. Induced the callus with different concentrations of each oligosaccharide and tested the effection on PAL activity and chitinase activity. Got the Chitosan-oligosaccharides which have the most biological activity after comparation. Tested the differential gene expression of Populus tomentosa callus after induced by this Chitosan-oligosaccharides at different time.The results of this research as follows:(1)These four oligosaccharides all have inducing activity. 75mg/L, the average degree of polymerization of oligosaccharide at 10.02 has the best biological activity after comparison.(2)The best oxidation process of preparing the Chitosan-oligosaccharides: The ratio of the amount of substance of H2O2 to glycosyl unit was 0.8.The ratio of chitosan's quality and 2% acetic acid's volume was 1:20, the decompose reaction continued at 60℃for 6 hours with stirring. After the reaction, adjusted the PH to 8.5, added 3 times the volume of pre-cold ethanol to the filtrate, filtrated after precipitating 12 hours. Used 75% ethanol to wash the precipitation, centrifuged at 3500rpm for 15min; washed the precipitation by ethanol, centrifuged at 4000rpm for 15min(three times), freeze-dring the sediment.(3)Treated the populus tomentosa callus with 75mg/L oligosaccharides which the average degree of polymerization is 10.02, collected total RNA of callus samples at 4th, 6th, 24th, and 48th hour. Then used DDRT-PCR and fast silver staining to analyze different expression genes. 6% denaturing polyacrylamide gel electrophoresis analysis of various treatment times on callus proved that oligosaccharides caused hundreds of different fragments. 144 fragments appeared when induction time reached 4 hours. 74 fragments appeared when induction time reached 6 hours. 108 fragments appeared when induction time reached 24 hours. 94 fragments appeared when induction time reached 48 hours. However, the result also need Reverse Northern blotting to ensure the real different expression genes.