Molecular Characteristics Of Heat Shock Protein 90 Genes From Bursaphelenchus Xylophilus, B. Mucronatus And B. Doui And The Research Of Temperature Adaption Of B. Xylophilus

The pine wood nematode, Bursaphelenchus xylophilus, could cause pine wilt, dehydration and chlorosis, which made the pine trees died rapidly. This is a kind of destructive forest disease called pine wilt disease. The disease is difficultly controled and causes serious economic losses, so B. xylophilus is listed as an important quarantine pest in many countries. B. xylophilus is widely distrbuted and has a good adaption to the temperature. Temperature adaptation is an important factor for determining the distribution and spread of B. xylophilus. The induced synthesis of heat shock protein 90 will change signifcantly at heat stress. As a target gene, heat shock protein 90 could be used to study the temperature adaptation of B. xylophilus. Therefore, the full length of heat shock protein 90 was isolated and characterized from B. xylophilus, B. mucronatus, B. doui, and preliminary research of temperature adaptation of B. xylophilus was made in this study.The cDNA sequences of heat shock protein 90 from B. xylophilus, B. mucronatus and B. doui were cloned by RT-PCR and RACE methods, and were named Bx-hsp90, Bm-hsp90, Bd-hsp90 (GenBank Accession Number: GU013561, HMO34943 and HM347331) respectively. The full cDNA length of Bx-hsp90, Bm-hsp90 and Bd-hsp90 were 2255 bp, 2193 bp and 2232 bp, and the ORF were all 2 127 bp encoding 708 amino acid residues, 5'-untranslated region (UTR) were 33 bp, 13 bp, 13 bp, and 3'-UTR were 95 bp, 53 bp, 92 bp, respectively. The full genomic length of Bx-hsp90, Bm-hsp90 and Bd-hsp90 were 2440 bp, 2388 bp, 2407 bp, and all genomic sequences contained 3 introns and obeyed the GT/AG rule in the splice-site. The amino acid sequence identities between Bx-hsp90, Bm-hsp90 and Bd-hsp90 was 98.78%. Evolutionary analyses showed that B. xylophilus, B. mucronatus and B. doui had a close relationship with Caenorhabditis elegans, which may be indicative of the low evolution of B. xylophilus, B. mucronatus and B. doui.The southern result showed that the heat shock protein 90 was presented in B. xylophilus and was a single copy gene. It was consistent with the results of heat shock protein 90 from C. elegans, Brugia pahangi and Heterodera glycines. Heat shock protein 90 is likely present in a single copy and is not easily confused with distantly related genes and is an evolutionarily conserved gene, so it is a good candidate gene for studying the biological evolution. The real-time PCR indicated that B. xylophilus showed an up-regulation of the level of heat shock protein 90 after 1h of heat exposure (35℃), this revealed that heat shock protein 90 from B. xylophilus played an important role in coping with environmental changes. The heat shock protein 90 had a close relation to the temperature adaptation of B. xylophilus, and this study had a practical significance for understanding the invasive mechanism of B. xylophilus.