CDNA Cloning Of Ace-2 Gene From Bursaphenchus Xylophilus And Its Sequence Analysis

Acetylcholinesterase (AChE,EC3.1.1.7)is one of the key enzymes in biological nerve conduction, it can degrade acetylcholine(ACh) specifically,stop neurotransmitter's excitation over postsynaptic membrane,guarantees the nerve signal correctly transmising in organism. The organophosphorus and carbamate compounds are strong inhibitors of nematodes'AChE,they can bate AChE's catalytic activity by combine with Serine residue in AChE's active site. The acetylization is terminated and nerve conduction is destroyed, finally causes nematode's death.AChE-2 is the most important of four kinds AchEs,and its coding gene named ace-2. ace-2 from Bursaphelenchus xylophilus was researched in this paper,the main results are as follows:1. Bursaphelenchus xylophilus used in this paper came from Guangzhou,divided Bursaphelenchus xylophilus and Botrytis cinerea by collecting the nematodes which living in water adhere to culture dish's lid.Isolated the total RNA from Bursaphelenchus xylophilus, achieved two cDNA squences by techniques of reverse transcriptional PCR and RACE (Rapid amplification of cDNA ends) and method of touchdown PCR and nested PCR,one is the ace-2 gene's cDNA sequence contains full 3'sequence,it was registered at GenBank as number FJ947083,the other is partial cDNA sequence of AChE.2. Analysised the former sequence by Bioinformatic softwares.The length of this segment was 1880bp,compared it with ace-2 gene cDNA sequences from other nematodes, it was discovered that its template RNA was't mature mRNA but mRNA precursor.The nematodes collected for this paper contained all growth stages from egg to imago,and were freezed with liquid nitrogen when they were living,they induced the existence of mRNA precursor. After splicing form and three introns were hypothesized, its remnant was 1767 bp and coded 554 amino acids,there was 100bp non-coding nucleotides after the termination codon TAA,it contained a 18bp poly(A). Compared AChE-2 of different nematodes with Vector NTI 10.3,it was deduced that there were about 70 amino acids from N terminal unknown.3. Compared these AChE-2's amino acids,it was founded that the boundary of conserved regions and hypervariable regions were distinct,the three hypervariable regions sited 360-410bp,640-700bp and after 720bp.There was highest homology between Bursaphelenchus xylophilus AChE-2 and Ditylenchus detructor AChE-2.