Study On Cellulases Of Bursaphelenchus Xylophilus And Endo-β-1,4-glucanase CDNA Gene Cloning And RNAi

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. 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. Based on the understanding of cellulase from pine wood nematode secretion, the component of cellulase from B. xylophilus and B. mucronatus was analyzed while the relationship between cellulase activity and dispersal ability of the nematode was also discussed. And cellulase encoding gene was isolated and identified with RNAi. This will try to demonstrate the molecular mechanism of cellulase in the pathogenicity, and give science data to provide an academic support for the sustainable management of pine wilt disease.The population propagation ability of B. xylophilus and B. mucronatus was analyzed. The results showed that both B. xylophilus and B. mucronatus reproduced very fast on the Botrytis cinerea culture which produced large number of mixed stage nematodes. Three index including propagation rate, sex ratio and larvae/adult were used to comparing the propagation ability. B. xylophilus strain Bx02, Bx03 and B. mucronatus Bm01 exhibitted the potential high propagating ability.The secretion cellulase of B. xylophilus and B. mucronatus was analyzed while CMC, MC and SC were used to be the celllose resouces for the nematodes secretion degrading. The results showed that in vitro endo-β-1,4-glucanase, exo-β-1,4-glucanase andβ-glycosidase activity could be detected in the secretion of both B. xylophilus and B. mucronatus. And the three cellulase activity from B. xylophilus secretion was higher than that from B. mucronatus. It is inferred that the pathogenicity of B. xylophilus was more serious than B. mucronatus for the reason of the cellulase activity. The dispersal and propagation ability of B. xylophilus and B. mucronatus in Pinus thunbergii shoot section were analyzed with artificial inoculation. Compared with B. mucronatus, B. xylophilus passed through the shoot section of P. thunbergii with a higher speed and less time. The numbers of nematode passing through the section at first were not significantly different among the most strains of nematodes including B. mucronatus, although the numbers of most strains of B. xylophilus were much more than that of B. mucronatus. The reproduction numbers of B. xylophilus were more than that of B. mucronatus in the section for 20 days and all the numbers of B. xylophilus strains were significantly different with the numbers of three strains of B. mucronatus. Considering the same phenomena in the investigation of the cellulase activity of both species nematodes above, the cellulase probably was one of the driving for the nematode dispersal inside the host tree.The cDNA fragment coding endo-β-1,4-glucanase of B. xylophilus was isolated and identified from total RNA with RT-PCR techniques. The cDNA fragment was 678 bp in length and contained an open reading frame of 225 amino acids (GenBank accession number EU660207). The cDNA could eccode a protein similar to glycosyl hydrolase family 45. The Blast result showed that the cDNA shared 95% identity with endo-β-1,4-glucanase from Japanese strain of B. xylophilus. The characteristic of the putative protein were analyzed with biological information method. The alignment of several selected sequences similar to this cDNA in GenBank suggested that the cellulase gene from Chinese strain Bx02 of B. xylophilus was acquired by horizontal gene transfer from fungi.The function of gene encoding endo-β-1,4-glucanase was identified with RNAi. The siRNA was introduced into the body of pine wood nematodes with the fluorescence microscope investigation. The effect of inhibition on the expression of gene encoding endo-β-1,4-glucanase was detected with fluorescence quantity real time PCR. siRNA15 and siRNA275 showed more higher effective inhibition than other siRNA. The biology characteristic of pine wood nematodes after RNAi was demonstrated and analyzed. The cellulase activity decreased to a low level and the reproduction rate was also reduced. Furthermore, it took some more time to pass through the shoot section of P. thunbergii for pine wood nematodes after RNAi. This indicated that some functions related to the development or dispersal ability of pine wood nematodes were likely to be influenced while siRNAs interferenced the gene encoding endo-β-1,4-glucanase.