Construction And Analysis Of Pinus Massoniana SSH CDNA Library Inoculated With Bursaphelenchus Xylophilus

With the development of the global integration and international exchanging and trading, the invasive alien species dispersal causes a large damage to the ecology system of the country invaded. Pine wood nematode (Bursaphelenchus xylophilus), as an agent of pine wilt disease and the important quarantine target of many countries around the world, has been the most serious invasive pest in China. As an invasive alien species, this disease caused great destruction and impact to pine forest ecosystems since it had been found in China. Based on the classic and modern biology theory and method, many scientists focused on the biology, ecology and pathogenicity and so on from all over the world, but until recently there were no effective controlling measures to the pine wilt disease.The pine wood nematode disease is a complex system which would be influenced both by nematodes, host pine, environmental conditions, associated fungi, vector insects and other factors. The pathogenic process of this disease can also be influenced by a number of factors. Based on the prophase understanding of pine wilt disease, the genetic expression difference of native tree species Pinus massoniana seedlings before and after artificial inoculation had been analyzed with the method of molecular suppression subtractive hybridization in this study. A suppression subtractive hybridization (SSH) cDNA library was constructed with Pinus massoniana induced by Bursaphelenchus xylophilus. The branch was induced with 24 and 72 hour as tester, the non-induced branch as driver. The pine wood nematode strains for vaccination were isolated from Zhejiang Province in the South of China. The BLAST homology search revealed that these genes played varies functions in response to stress, signal transduction, energy metabolism and transportation etc. The results of the study could provide the scientific basis for demonstrating the pathology response mechanism of the disease as well as the molecular mechanism of pathogenicity. In addition, the results of this study could also give science data to provide an academic support for the sustainable management of pine wilt disease accordingly.Through preliminary tests, the results are as follows.Firstly, the population propagation ability of different B.xylophilus strains was analyzed. The population propagation ability of four B.xylophilus species was analyzed with culture method. The results of the test showed that both four B.xylophilus species have high propagation rate, suitable sex ratio and larvae/adult proportion on the Botrytis cinerea culture. In addition, four B.xylophilus strains have significantly different in propagating structure. B. xylophilus strain NXY03 exhibited the potential high propagating ability with ten days growth on the Botrytis cinerea culture. The propagation multiples of NXY03 reached a maximum of 357 times while the strain NXY14 is only 193 times as the lowest. Four species of pine wood nematode strains showed a similar trend of propagation in sex ratio mean 3.34. At the end, NXY03 lines a maximum of 3.3 on larvae/adult proportion, and NXY01 lines the minimum of 1.9.Secondly, the dispersal and propagation ability of different B.xylophilus species were compared.The dispersal and propagation ability of four B.xylophilus strains in Pinus thunbergii shoot section were analyzed with artificial inoculation. Compared with other strains, NXY03 could pass through the shoot section of P.thunbergii with a higher speed and less time. The reproduction multiple of NXY03 is 16 which was significantly higher than that of other strains in the section for 20 days. The final number of NXY03 strain was obviously different with other three strains as 3200±100.Thirdly, the virulence of different strains was compared. By comparison, the time of symptom appeared, infection rate and decease infection index of Pinus massoniana seedlings with the inoculation of four strains were all showed significantly different. NXY03 strain has the strongest virulence after analysis.The pilot experiment proved that the four nematode strains had significantly different in the reproduction ability, dispersal ability and virulence. The strain of NXY03 that isolated from Zhoushan city showed the highest ability in the population dynamics, proliferation ability, dispersal ability and pathogenic capabilities. With the analysis of the test result the author select the strain of NXY03 as the test sample in the latter part of the experiment.Tow suppression subtractive hybridization (SSH) cDNA libraries were constructed to Pinus massoniana seedlings that were induced with NXY03 selected from preliminary tests.Through testing, the genetic expression difference of Pinus massoniana seedlings before and after artificial inoculation had been analyzed. SSH libraries were successfully built with SMART method after the total RNA samples were extracted. A total of 665 positive clones were obtained with dot blot. And then 150 positive colons were filtered out for sequencing test which finally got 144 availed sequences. After the Sequencing Blast in the NCBI database, 141 effective results had been showed. Of these, 27 sequences have a high degree of similarity to the proteins that the function has been known which mostly got from Ricinus communi, Populus trichocarpa, Arabidopsis thaliana and Zea mays. There of them were conservative hypothetical proteins, and 111 of these were functional unknown or predicted protein.Features of functional known protein involves in: participating the disease resistance of plants, coding stress tolerance proteins, participation in structure and function metabolism, as well as participating signal transduction and energy transportation etc.In the SSH library that constructed in the experiment, many differently expressed gene were found. These genes involved cell division grows (60s ribosome protein L38), plant defense response (Chalcone synthase, Calcium-binding protein), signal transduction and adjustment (NPG1), stress tolerance (HSP, MT3, ribosome protein L15), structure and function metabolism (EMB1080, CAD) and so on.After artificial inoculation, the exciton recognized acceptor in very short time. The recognition response caused density changes of Ca2+ in the host cytoplasm. Then the Ca2+ stream enters the cell, and produces the ion channel, thus the calcium signal was sending out. The active oxygen suddenly increases, and activated cytoplasm transcription factor and the defense response gene. Afterward, the coercion expresses the gene, the disease-resistant gene expression also to change along with it. The host defense response mechanism was activated with the course of the disease, the expression quantity of the defense response gene also gradually rises in the same time. In addition, the difference expression of carbohydrate metabolism response gene showed that the host pine tree strengthen the respiration and sugar zymolysis process to strengthen cell's resistance after artificial inoculation.The above test result improved that, the host pine's gene expression had the varying degree expression difference on DNA duplication and translation, resistant gene participation, secondary metabolism control, signaling molecule control after artificial inoculation. The appearance of the difference expression result is likely associated with the interaction molecular mechanism between the host and the pathogen pine wood nematode. But whether the function of these difference expression genes are related to the disease-resistant also needs further research.The analysis results of secquences blast proved that the expression of disease resistance genes are up regulated in the course of disease after inoculated with pine wood nematodes. These genes all participate in the regulation and expression of the defense reaction-related signal transduction and energy metabolism mechanisms. The research results also provide the basic datas which are relevant to further study of the molecular interaction mechanism between pine wood nematode and the host pine trees, and could play an important role in clarifying the pathogenic mechanism of the disease.