| Recently, Heterodera avenae and its relative species Heterodera filipjevi are known to cause damage to wheat plant and economic yield losses in China. Only few researches have been reported about these two nematodes, however their pathogenic mechanism is still unclear. Thus, their controls are very inefficient. In order to find a safe and effective method to control H. avenae and H. filipjevi, a full descriptive understanding of their parastic genes is very importante. In this study, two fatty acid and retinol binding protein(far) genes(Ha-far-1 and Ha-far-2) from H. avenae and one far gene(Ha-far-1) from H. filipjevi were cloned for the first time. Their sequences, protein binding activities, gene expression sites and partern expressions were analyzed by bioinformatics analysis, fluorescence-based ligand binding analysis, in situ hybridization, and q TR-PCR, respectively.Ha-far-1 consists for a large Open Reading Frame(ORF) with 576 nts, encoding 191 amino acids. Software prediction showed that Ha-FAR-1 is rich in α-helix, carried a 21 amino acids signal peptide at the N-terminus and possessed two potential casein kinase II phosphorylation sites at residues 71 and 81. Ha-FAR-2 consists of 280 amino acids and also riched in α-helix. Three potential glycosylation sites were present at residues 32, 84 and 95. The secondary structure of the longer peptide at the C- terminus of Ha-FAR-2 was randon coil. In addition, the signal peptide of Ha-FAR-2 was 22 amino acids. The ORF of Hf-far-1 encoded for 191 amino acids. There is a 21 amino acids signal peptide at the N-terminus of Hf-FAR-1, and a potential casein kinase II phosphorylation site at residue 71. Multiple sequence alignment and phylogenetic tree analysis showed that Ha-FAR-1 and Ha-FAR-2 were very similar, and they were significantly similar to Gp-FAR-1 protein from Globodera pallid with the ID number(CAA70477) from NCBI. Ha-FAR-2 was significantly different from Ha-FAR-1 and Ha-FAR-2. The Blast analysis result showed that Ha-FAR-2 have the highest identity to FAR from Radopholus similis(AFI80890.1). The Ha-far-1 g DNA consists of 1212 bp from ATG to TAA, containing 7 introns and 8 exons, while the g DNA of Ha-far-2 contain 2174 bp from ATG to TAG, and 6 introns and 7 exons are identified in this sequence.The recombinant proteins Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 were obtained by prokaryotic expression, and they all could bind to DAUDA and retinol, but the binding activity of Ha-FAR-2 was much weaker. This may be caused by the difference of Ha-FAR-2 structure. Competition experiments showed that oleic acid displaced DAUDA and retinol from binding sites, implying the binding sites of fatty acid and retinol were the same or interactional.q RT-PCR showed Ha-far-1 and Ha-far-2 expressed at all stages, although the expression levels of Ha-far-2 were very low, and the highest expression level was in second-stage juveniles. Ha-far-1 had higher expression levels than Ha-far-2 at all stages, and its highest expression level was in fourth-stage juveniles. The m RNA of Ha-far-1 and Ha-far-2 were detected by in situ hybridization in pre-parasitic juveniles of H. avenae. This result showed that m RNA of Ha-far-1 and Ha-far-2 were both in the hypodermis.The results above indicated that Ha-FAR-1, Ha-FAR-2, Hf-FAR-1 might be secreted into host tissue through the body wall of nematode, and bind to the fatty acid and retinol, help nematode to acquire lipid nutrient, and promote the development of nematode. Moreover, decrease of relevant substance will reduce host defense response, and better for the maintenance of syncytium and parasitism of nematode. |
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