| Poplar canker is one of disease which caused the stems cortical necrosis, the disease is one of the main obstacles for the development of poplar plantation in our country. There has been a large amount of research about the disease-resistant mechanism of poplar to canker and get a series of research progress in the induced resistant mechanism of host and pathogenic mechanism of pathogens. But the mechanism of interaction of plant-pathogen is very complexity. For probing the response mechanism of poplar to Botryosphaeria dothidea, there are lots of further research needed to do, especially in the regulation mechanism of gene, the expression mode and functional classification of protein, the type and content changes of secondary metabolites.Secondary metabolites are the ultimate products resulted of the genes and proteins expression. The various contents of secondary metabolites reflect the physiological and pathological state of plants. A number of research also indicated that the plant resistance depends on the existence of resistant genes, the speed and extension of gene expression, and the amount of disease-resistant substances which produced from the gene expression. And the different expression proteins and the contents and varieties reflect the speed and level of gene expression. In order to further understand molecular mechanism of the interaction between poplar-B. dothidea, this paper researched vary contents of secondary metabolites and different expression proteins of poplars infected with B. dothidea, the main results are as follows:(1) We have a qualitative analysis on the secondary metabolites of Populus tomentosa and P. beijingensis infected with B. dothidea using high performance liquid chromatography (HPLC). The results showed that there were significant differences in the second metabolites species. Compared with control 6 peaks were increased in P. tomentosa after inoculation with B. dothidea tomentosa,22 in P. beijingensis; and it could be found,30 peaks in completely not inoculation,11 peaks appeared only after inoculation with the medium,14 only peaks appeared only after inoculation with B. dothidea which divided on inoculation or not.(2) Standard content as a control,15 secondary metabolites were identified in P. tomentosa and 13 were identified in P. beijingensis, among which 11 secondary metabolites were identified in P. tomentosa and P. beijingensis, which respectively were coumaric acid, benzoic acid, gallic acid, ferulic acid, catechin, p-hydroxybenzoic acid, o-dihydroxybenzene, phenol, rutin, morin; and 6 species were identified that of resistance to the host, including 4 kinds only in P. tomentosa which were coffee acid, chlorogenic acid, salicylic acid and cinnamic respectively; 2 kinds only in P.×beijingensiswhich were isorhamnetin and kaempferol. (3) The quantitative analysis on the 10 main differential secondary metabolites In order to further understand the roles of the 10 main different secondary metabolites in interactions of poplar-B. dothidea, we investigated the content changes of the 10 main different secondary metabolites in 0,24,48,72,96,120,144,168h after inoculation with B. dothidea. The results showed that the difference of the variations of the 10 substance changed very significantly. In P. tomentosa, the contents of gallic acid, catechin, p-hydroxybenzoic acid, ferulic acid were accumulated quickly to peak value, the content of phenol, catechol and coumaric acid accumulated relatively slowly, the content benzoic acid didn't not change significantly in P. tomentosa after inoculation with B. othidea, while these changes in P. beijingensis were contrary. In one species of poplar, the content of the above phenolic compounds but morin in P. tomentosa changed very significantly. The contents of ferulic acid and coumaric acid in P. beijingensis were lower than the controls, and the contents of other phenolic compounds accumulated more quickly than the controls.(4) The variation analysis of the 10 main differential secondary metabolite (coumaric acid, benzoic acid, gallic acid, ferulic acid, catechin, p-hydroxybenzoic acid, o-dihydroxybenzene, phenol, rutin, morin) that identified in P. tomentosa and P. beijingensis showed that there were significant differences after inoculation in the same species or between P. tomentosa and P. beijingensis. Among which the contents of the other phenolic substances except o-dihydroxybenzene were improved in P. tomentosa after inoculation. The contents of coumaric acid and ferulic acid were reduced and the other phenolic substances were all increased in different degrees. Between the P. tomentosa and P. beijingensis, the contents of phenol, gallic acid, catechin P. tomentosa were significantly higher than in P. beijingensis; the content of o-dihydroxybenzene, ferulic acid, benzoic acid and p-hydroxybenzoic acid were significantly lower in P. tomentosa than in P. beijingensis; the content of morin and coumaric acid in P. tomentosa were also lower than in P. beijingensis but the difference was not obvious.(5) The different peaks of unknown compounds which changed significantly in P. tomentosa but not in P. beijingensis after inoculation with B. dothidea, were separated by semi-preparative chromatography, then were identified by magnetic resonance imaging (NMR) and high resolution mass spectrometry (HRMS), as well as confirmed 5 different compounds through comparing the structure with documentation. These compounds were determined as salicin,2'-O-Acetylsalicorti, Tremuloidin, Populin, Tremulacin respectively. These 5 substances belong to phenolic nucleoside compounds and showed the important different between P. tomentosa and P. beijingensis. Furthermore, the 2'-O-Acetylsalicorti and Tremuloidin were100% in the relative difference of between P. tomentosa and P. beijingensis, it was to say that 2'-O-Acetylsalicorti and Tremuloidin were not found in P.×beijingensisunder the same conditions. In additional, we speculated the other 10 derivatives of same series substances based on the structure and mass spectrometry of the five compounds. Nine different compounds of the ten speculated compounds were 100% of the relative difference between P. tomentosa and P. beijingensis. (6) The preliminary research on the different protein expression of P. tomentosa and P. beijingensis after 72h inoculation with B. dothidea by two-dimensional gel electrophoresis. A result from gel electrophoresis maintained high reproducibility. This indicated that quality of protein extraction were better. Compared the two-dimensional gel electrophoresis profiles with the software of imageMaster 2D Platinum,35 different protein spots with 3 folds were obtained between P. tomentosa and P. beijingensis. In the same species,14 different protein spots with 3 folds were observed in the P. tomentosa while 18 different points with 3 folds were found in P. beijingensis compared with the control.(7) To identify more resistant related proteins, we analyzed the different proteins by the quantitative methods of Label-free. The results showed that a total of 606 different proteins were identified, and classified according to their functions. Among them included 205 up-regulated proteins and 186 down-regulated proteins in P. tomentosa,84 up-regulated proteins and 302 down-regulated proteins in P. beijingensis after inoculating with B. dothidea. Between the P. tomentosa and P. beijingensis, there also were 190 proteins which contents in P. tomentosa were higher than in P. beijingensis,192 proteins which contents in P. tomentosa were lower than in P. beijingensi; and there were 352 proteins which contents in P. tomentosa were higher than in P. beijingensis,153 proteins which contents in P. tomentosa were lower than in P. beijingensis infected with B. dothidea. In these different proteins, there were 18 proteins of plant-pathogen interaction procession,10 proteins of peroxidation,31 proteins relative with phenylpropanoid biosynthesis and phenylalanine metabolism. Finally, we got functional positions and pathways through analyzing the pathway of the proteins relative with plant-pathogen interaction procession, phenylpropanoid biosynthesis and Phenylalanine metabolism.In conclusion, these studies were first the systematic and comprehensive study about the differential secondary metabolites and protein expression in the poplar after which were infected with B. dothidea. First qualitative analyzed on secondary metabolites of P. tomentosa and P. beijingensis, and quantitative analyzed a part of them used standard as control, then determined the changes of ten secondary metabolites which varied in P. tomentosa and P. beijingensis after inoculation with B. dothidea; Finally, isolated and identified the peaks of different and unknown compounds between P. tomentosa and P. beijingensis, and confirmed the structure. Through these studies not only identified a number the kinds, contents and their variations of some different secondary metabolites, but also reflected physiological and pathological state of P. tomentosa and P. beijingensis after inoculation with B. dothidea. Another the isolation and identification of the unknown secondary metabolites was a breakthrough progress, and the phenolic nucleosides had been identified were inferred as resistant compounds of P. tomentosa according to the literature. To the different proteomics research, on the basis of the preliminary analysis with a two-dimensional electrophoresis technology, we achieved a high-throughput different protein analysis with label-free quantitative methods, got a lots of different proteins and made functional classification of these proteins, which represent different functions of different types and extents of genes expression, and which also explains the content changes of the secondary metabolites that previous statement. Finally made the genes, differential protein expression, differential secondary metabolites come together through the signal pathways analysis of the plant-pathogen interaction, the phenylpropanoid biosynthesis and phenylalanine metabolism, which revealed the molecular mechanism of poplar resistance to B. dothidea, while the results of these studies laid the foundation for further research and brought a broad idea, which may greatly promote the studies the mechanism of interaction between poplar and B. dothidea.
|
PDF Full Text Request