Genetic Cloning And Protein Structural Studies Of A Glycoside Hydrolase Family 16 Enzyme Gene From Aphelenchoides Besseyi

The Plant-Parasitic Nematode Aphelenchoides besseyi is an obligate parasite which can infect many kinds of plant. According to the record, there are 35 genera plants as the host of Aphelenchoides besseyi. The cellulase are produced by nematodes to hydrolyse the plant cell wall during nematode entry into plant tissues and infect into plant. Our study try to found genes about infect and to know the pathogenic processes. We also want strengthens the argument of Horizontal gene transfer.Total RNA was extracted from mixed-stage nematodes using TRIzol regent and then cleaned using an RNeasy Minikit column according to the manufacturers’instructions. The extracted RNA was assessed for quality and quantified using a BioPhotometer D30. A total of 46,826,350 raw reads were obtained by Solexa RNA paired-end sequencing, and 36,905,372 high-quality clean reads were obtained after a strict filtering process. The average length of the clean reads was 89.89bp. The ratio of clean reads was 78.81%. Using the clean reads, Trinity produced 51,270 transcripts with length>200bp. The average length and N50 of these transcripts were 1,241bp and 1,857bp, respectively. A total of 35,180 unigenes with an average length of 1,050bp and N50 of 1,626bp was generated after further clustering and assembly. Unigenes were determined by performing BLASTX searches against the NR protein database. A total of 17.338 orthologs were identified.524 contigs were assigned to the following four classes of CAZymes:136 contigs with GH domains to 10 families of GHs,328 contigs to 28 families of GTs,42 contigs to 2 families of CEs and 18 contigs to 3 families of CBMs.The first strand cDNA was synthesized by RNA reverse transcription. The primers were designed according to AbGH16 sequences which we blasted before. Complete reading frame sequence acquired by PCR amplification with the first strand cDNA as template. The lengh of ORF sequence is 779bp. Eight homology sequences of GH16 protein was obtained by BLASTX searches against the NCBI. Multiple sequence alignments were performed using ClustalW, and phylogenetic trees were constructed based on those sequences using the MEGA 5.05 program showed two major clusters. Phylogenetic analyse to all GH16 protein sequences in NCBI showed that AbGH16 protein have closer affinity with fungus. After ORF analyse we know the AbGH16 protein contain 259 amino acids, Relative molecular mass is 29.066, isoelectric point is 8.11. SignalP 4.1 Server was used to analysed the transmembrane domain of AbGH16 protein. It showed that the transmembrane domain at N end of the 1-20 amino acids in the protein. Hphob./Kyte & Doolittle analyse showed that hydrophobicity of this sequence is strong. The AbGH16 protein is secretory protein. Analyse the three-dimensional structure of protein showed the AbGH16 protein belongs to the endoglucanase. Further analys showed that the structure of AbGH16 protein from Aphelenchoides besseyi is similar with the structure of GH16 protein from Bursaphelenchus xylophilus and more similar with the GH16 protein from Penicillium oxalicum. The two proteins all have small alpha helical structure in Continuous reverse parallel beta folding. The GH16 proteins from bacteria have four beta folding differenc. Several lines of evidence demonstrate the endogenous origin of the AbGHl 6.In situ hybridization was used to determine the tissue specificity of AbGH16 cellulase gene transcription. The anti-sense probe stained the esophageal gland cell area. The gland region was strongly stained by probes against the AbGH16 cellulase gene, whereas the sense probes gave no signal.A semi-quantitative RT-PCR on different pathogenesis nematode was performed to study the expression of GH16. It is more abundantly expressed in highly pathogenic nematode compared to others.Our study can provide the foundation of the further study of nematode infection mechanism and provide theoretical basis for how to prevention and treatment of infection of nematodes. It also strengthens the argument that HGT has played a key role in evolution of plant parasitism.