Artificial Breeding And The Study Of RNA Interference Of The Pine Wood Nematode(Bursaphelenchus Xylophilus)

The pine wood nematode (Bursaphelenchus xylophilus) disease is a kind of pine tree's destructive disease, it has such characteristics: dangerous, spread fast, difficult to control, and it is a global quarantine object. The predecessors have done a lot of research on the disease resource of nematode, pathogenesis and prevention technology. Since the pine wood nematode disease was discovered, the domestic and foreign have invested large amounts of human, material and financial resources to fight the disease. But so far the disease has not been effectively controlled, and it still has the tendency of continued spread. In this study, based on previous research, preliminary researches has been done on the establishment of simple, effective, artificial feeding method of pine wood nematode and inhibit the reproduction using RNA interference, the results show that:1.Culture 9 days, the 50 pine wood nematodes entered the large population of the exponential phase of growth, the number can expand 300 times by culture them 10 days, the number of nematode can expand a few thousand times after 15 days'culture.2.In the traditional classification of form, distinguish the nematodes and B.mucronatus rely mainly on the tail characteristics of the adult female Larvae are harder to identify. According to the small difference in the ITS section , primer was designed in this experiment, by electrophoresis after PCR reaction, the emergence of objective bands about 340bp length with SF+R primers is the pine wood nematode, while objective bands with NF+R primers is B.mucronatus .This can accurately distinguish two related species with quite similar morphological characteristics.3.The pine wood nematode single-vivo culture not only operated simply but also has relatively high yields. Botrytis cinerea is cultivated with PDA plate, after Botrytis cinerea is paved with plate, pine wood nematode is inoculated, and 25℃constant temperatures for 15 days, then funnel collection is a simple and effective method of artificial feeding.4.By using the highly conservative nature of critical genes between different species, RNA polymerase gene fragment was cloned with the base of C. elegans RNA polymerase gene, at the same time, vector LITUMS38i with double promoters was chosen, the recombinant vectors which can express double-stranded objective fragments was successfully constructed.5.Soaking with double-stranded RNA (dsRNA) can make double-stranded RNA (dsRNA) interfere the normal development of the pine wood nematode, and then it can effectively restrain the reproduction of pine wood nematode. And the effect of soaking with double-stranded RNA(dsRNA) interference of the pine wood nematode at 20°C was better than t soaking at 4℃.