Molecular Cloning And Functional Analysis Of An Ankyrin Protein Gene OsBIHN-N22 And An Acyl-CoA Synthetase Gene OsBNHN-N7 In Rice

Benzothiadiadiazole (BTH) which could induce rice resistance against blast, bacterial leaf blight and sheath blight has been demonstrated in the previous studies of our lab. And based on these studies over 200 differential expressed cDNAs associated with the BTH-induction through suppression subtractive hybridization were cloned and identified. Among them, two cDNA clones J023125M12 of 1529 bp and J013000M2 of 11475 bp were both inferred to be a full-length sequence of rice gene encoding ankyrin-repeat proteins based on the similarity search against the GeneBank database. This study cloned and identified a full-length cDNA of a rice gene, OsBIHN-N22, encoding an ankyrin-repeat protein.To obtain the full-length sequence of OsBIHN-N22 for analysis, we designed a pair of specific primers according to the sequence of J023125M12 for PCR amplifying, using phage DNA prepared from a rice cDNA library as a template. The full-length cDNA of the OsBIHN-N22 gene is 1574 bp with a predicted 990 bp open reading frame (ORF). OsBIHN-N22 gene encodes a protein of 329 amino acid , the deduced OsBIHN-N22 protein contains three conserved ankyrin-repeat domains. The OsBIHN-N22 gene located on chromosome 9 of the rice genome as a more-copy gene and consisted of eight exons and seven introns. Subcellular localization analysis revealed that the OsBIHN-N22 protein was localized in the cytoplasma membrane in the plant cells. Take together, we concluded that OsBIHN-N22 gene encodes an ankyrin repeat protein belongs to the ankyrin protein family.To illuminate the function of OsBIHN-N22 gene in rice disease resistance, the expression patterns of OsBIHN-N22 gene in BTH-induced disease resistance response and in the compatible/incompatible interactions between rice and the blast fungus Magnaporthe grisea were analyzed. Northern blotting analysis showed that the OsBIHN-N22 gene has a relatively low level of basal expression in rice leaf tissue. However, expression of OsBIHN-N22 gene was activated upon induction of BTH treatment, which is capable of inducing disease resistance. The expression of OsBIHN-N22 gene was up-regulated in BTH-pretreated rice seedling at Oh after inoculation with M.grisea, and which was also in the incompatible interaction between M.grisea and a resistant genotype H8R during the first 6h after inoculation. These results suggested that tile inducible expression of OsBIHN-N22 was associated with disease resistance response in rice.Among the differential expressed cDNA, another clone 002-112-H10 contained 1897 bp showed high level of similarity to genes encoding known plant AMP-binding proteins. So in order to obtain the full-length sequence for studying this AMP-binding protein gene, a pair of specific primers according to the sequence of 002-112-H10 were designed to amplify this sequence by PCR using phage DNA prepared from a rice cDNA library. and so the full-length sequence was named OsBNHN-N7 gene after sequencing. The OsBNHN-N7 gene contained an open reading frame (ORF) of 1677 bp and encoded a protein of 558 amino acid which contained a Caic Acyl-CoA synthetases (AMP-fonning)/AMP-acid lignases II) domain. The OsBNHN-N7 gene was a single copy gene in rice genome.In the study of the possible involvement of OsBNHN-N7 gene in rice disease resistance responses, northern blot analysis showed that expression of OsBNHN-N7 gene was activated rapidly after BTH treated, and increased gradually until reached a peak at 24h. In the water-treated seedlings, only a weaker expression of OsBNHN-N7 was detected. Moreover, expression of OsBNHN-N7 gene was further induced by infected with M.grisea, the rice blast fungus, as compared with those in water-treated seedlings. The further study about the expression of OsBNHN-N7 in incompatible interaction between M.grisea and H8R showed that this expression was actived rapidly. The level of expression was much higher relative to that in compatible interaction between M.grisea and H8S (a genotype susceptible). The expression in H8S was hardly detected in the whole experiment time. All these results indicated that OsBNHN-N7 gene was involved in the rice disease resistance response.