Cloning And Characterization Of Two New Heat Shock Protein Genes (Dd-hsp90-1 And Dd-hsp70-1) From Ditylenchus Destructor

Ditylenchus destrctor is a kind of important plant quarantine pests in China. D. destructor is native to North America and now it distributes in more than 50 countries and regions. In China, it widely distributes in North China, East China, Northeast China and shows good environmental adaptability to overcome biotic and abiotic stress. In this research, two new Hsp90 and Hsp70 genes related to stress resistance were cloned from D. destructor. Bioinformatics analysis and southern blot analysis were carried out. The research lay a foundation for studying the stress resistance and ecological adaptability of D. destructor and had important significance to control this nematode in the future.A new Hsp90 gene was cloned from D. destructor by RT-PCR and RACE, which was named Dd-hsp90-1, with Genebank accession number HQ901595. The full length of cDNA was 2347 bp. It contained a 2 160 bp open reading frame (ORF) with 9 introns and obeyed the GT/AG rule in the splice-site junctions. There was a 117 bp untranslated region (UTR) at 5'-end, a 70 bp UTR at 3'-end, a tailing signal of AATAAA and poly (A) in 3'-end region. The Hsp90 gene encoded a protein containing 719 amino acids with molecular weight 82.79 kD. Signal peptide prediction concluded that there was no signal peptide in the N-terminal, which indicated Dd-Hsp90-1 as a kind of cytoplasmic proteins. Through multi-sequence alignment of Hsp90s from 7 different organisms, some conserved structures were found, which were Leucine zipper sequence, LXXLL, GXXGXG, five characteristic sequences and MEEVD. The hsp90s phylogenetic analysis showed that D. destructor and Bursaphelenchus xylophilus belonged to the same evolutional branch, which suggested that the hsp90s of the two species may be homologous. Southern blotting hybridization with genomic DNA showed that Dd-hsp90-1 is present in single copy. The gene has potential value in classification and identification of plant parasitic nematodes.A new Hsp70 gene was cloned from D. destructor by RT-PCR and RACE, which was named Dd-hsp70-1 with Genebank accession number HQ386232. The full length of cDNA was 2087 bp. It contained a 1938 bp open reading frame (ORF) with 11 introns and obeyed the GT/AG rule in the splice-site junctions. There was a 48 bp untranslated region (UTR) at 5'-end, a 101 bp UTR at 3'-end, and poly (A) in 3'-end region. The Hsp70 gene encoded a protein containing 645 amino acids with molecular weight 70.45 kD. Signal peptide prediction concluded that there was no signal peptide in the N-terminal, which indicated Dd-Hsp70-1 did not have secretory function. Through multi-sequence alignment of Hsp70s, three characteristic sequences and EEVD were found. The hsp70s phylogenetic analysis showed that the phylogenetic tree could not reflect the genetic relationship of the organisms, maybe the similarity of biological functions was revealed. Southern blotting hybridization with genomic DNA showed that Dd-hsp70-1 is present in multiple copies. This is the first report that the copy number of hsp70 is multiple in plant parasitic nematodes.