Study On Molecular Ecological Mechanism Of The Pine Wood Nematode (Bursaphelenchus Xylophilus) In Adaptation To Temperature Stress

The pine wood nematode Bursaphelenchus xylophilus, the causal agent of pinewilt disease, is a kind of important alien invaded pest in many countries. It issupposed to have originated in America. Now, the area damaged by this disease hascontinued to spread to many Asian countries. With the effect of human activities onnature is getting much greater, the problems of biological invasions havebecome focus in the world. From native range to new habitats, the ecologicalenvironments alien invasive species meet are certain different. For stressfactor in environment, how alien species exert the potential adaptability is thekey of invasion in new habitats. Therefore, the research on the ecologicaladaptability and mechanisms of B. xylophilus to environment stress factor isnecessary, which will give a better understanding of mechanisms of biologicalinvasion, and is essential and significant in understanding of invasion progressand control work. In ecological physiology, temperature has been recognizedas a dominant environmental factor. In this paper, cold and heat tolerance of B.xylophilus were analyzed in cold and heat stress. With this base, themechanisms of heat tolerance of B. xylophilus were discussed at thephysiological and molecular level. Meanwhile, the functions of heat shockprotein 70 gene and cellulose gene were more investigated by the applicationof RNAi technology. It is the first time that the ecological adaptability andmechanisms of B. xylophilus has been studied systematically and overally.Now the major results studied in this paper are abstracted as follows:1. Cold and heat tolerance of B. xylophilus during temperature stress werestudied. At cold stress, B. xyIophiIus males could survive at 4℃for 2 days,and perish after being transitorily exposed to subzero temperatures, whichindicated nematode is chill-susceptible. At heat stress, heat tolerance washigher in B. xylophilus than in B. mueronatus. Significant differences wereobserved in 3 populations of B. xylophilus in china. Acclimation couldenhanced the heat tolerance of B. xylophilus. When acclimated at 35℃for 2-3hours, survival of B. xylophiIus increased by 4 times. These findings providedreference for studing mechanism of themotolerance of B. xylophilus.2. The analyse method of trehalose concentration in B. xylophilus was formed.At the physiological level, the mechanism of adaptation to temperature stresswas studied from trehalose accumulation in nematode. But, a positivecorrelation between trehalose accumulation and thermal tolerance was notobserved, suggesting that trehalose synthesis is not required for the inductionfor themotolerance for B. xylophilus.3. Heat shock protein 70 (Hsp70) is one of the important heat shock proteins.Overexpression of Hsp70 may obviously improve the survivability of cell,enhance tolerance to stress or injury. Here, rapid amplification of cDNA ends(RACE) method was used for the isolation of full length cDNA (2061bp) ofHsp70 from B. xylophilus (GenBank Accession number: DQ785812). It encodes a 642-amino acid protein with a calculated molecular weight of 70 kD,which carries three important and intact Hsp70 signature sequences. The resultof sequence similarity analysis revealed that the translated molecule showedhigh homology to other eukarya, and it is the likely orthologue of theCaenorhabditis elegans protein, HspTOA, also known as hsp-1. So, we haddesignated Bx-hsp-1 for the B. xylophilus Hsp70 gene. The complete ORFencoding Bx-hsp-1 was inserted into pEASY-E1 and an expression vectorBx70pEASY-El was constructed. The Bx-hsp-1 protein was induced toexpress by 0.4-0.8mmol/L IPTG. This study provided a fundamental conditionsupporting research on mechanisms of the ecological adaptability of B.xylophilus.4. Recombinant Bx-hsp-1 was overexpressed in Escherichia coli to study itspossible function under temperature stress. The result suggested that Bx-hsp-1provided thermotolerance to the transformed E. coli. With the heat shockresponse of B. xylophilus, the dynamic expression of Bx-hsp-1 induced bydifferent prior temperature treat was examined by quantitative PCR, suggestedthe Hsp70 mRNA increased with continuance of higher temperature treat(increase by 2-3 times). This resultes explained Bx-hsp-1 is the molecularmechanisms of heat tolerance in their responding to heat stress. Furtherinvestigation was conducted to analyse the function of Bx-hsp-1 in normaltemperature, indicated Hsp70 also play role in fetation and move of worm.5. RNAi was of value in determing gene function. We firstly described theapplication of RNAi to B. xylophitus for the knock-down of Bx-eng-1 geneconfirmed as a functional cellulose in vitro. Result showed B. xylophilus canuptake without a neurochemical octopamine, which is different from RNAiresearch of other plant parasite nematodes. After 24h of soaking, dsRNA canbe effectively ingested by nematode, causing post-transcriptional silencing ofBx-eng-1 gene and reduction of cellulase activity. In this process, rotation tosuspend nematodes played a key important in showing advantage of soaking.By analysis of RNAi phenotype, silence of Bx-eng-1 reduced fecundity andheat tolerance of nematodes, indicating this gene is essential for the adaptationof B. xylophilus to heat stress.