【作者】 张新春；

【导师】 郑重； 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2006， 博士

【摘要】 在本实验室前期研究中，证明了苯并噻二唑(benzothiadiazole，BTH)能诱导水稻对稻瘟病、白叶枯病和纹枯病等病害的系统抗病性，并在此基础上运用抑制性差减杂交法分离鉴定了200多个与水稻抗病反应相关的差异表达cDNA。同源性搜索发现，其中一个差别表达克隆BIHN-n22含有623bp的插入片段，该插入片断编码的蛋白与植物中ankyrin(锚蛋白)类型蛋白具有高度相似性。进一步搜索发现GenBank数据库中的水稻cDNA序列发现有一个长度为1529 bp的差别表达cDNA克隆J023125M12，(GenBank accession NO．AK121370)和一个1475by的cDNA克隆J013000M21(GenBank accession NO．AK098858)(Kikuchi，S et al.,2003)，它们与本序列的相似性都达到99％，与植物中ankyrin(锚蛋白)类型蛋白具有高度相似性。为研究探讨这个可能的锚蛋白基因的生物学功能以及在水稻抗病性中的作用，我们以这个已知序列设计引物，我们以前期构建好的水稻全长cDNA文库为模板克隆了这个基因，并命名为OsBIHN-N22。OsBIHN-N22基因编码蛋白由329个氨基酸组成，含有3个保守的ANK重复。这个蛋白属于ANK家族。细胞定位分析表明这个蛋白定位在细胞膜上。对其基因结构分析表明OsBIHN-N22基因包含八个外显子和七个内含子。Southern杂交表明OsBIHN-N22基因在水稻基因组中以单拷贝的形式存在于水稻的第九条染色体上。通过对OsBIHN-N22基因在水稻中的特异性表达分析发现，OsBIHN-N22能被苯并噻二唑(BTH)诱导表达，并且在BTH处理后的24h达到很高水平，在随后的60h内一直维持在相当高的水平上。而在水处理的对照中发现OsBIHN-N22的表达水平在整个实验检测时期都较低。在BTH处理的水稻幼苗中，OsBIHN-N22基因的表达在受到稻瘟病菌M.grisea侵染后被快速激活。稻瘟病菌接种后0h即可检测到OsBIHN-N22基因的激活表达，而且在随后的18h内强度增加，并在48h内维持在一个相当高的水平上。在没有BTH处理，只有稻瘟病菌接种的水稻叶片中直到6h才有诱导表达，然后在18h内可以检测到比较明显的表达水平，随后迅速降低。在水稻与稻瘟病菌的非亲和性互作中，OsBIHN-N22基因在接种后的6h就能检测到其快速表达，而在亲和性互作中则只有在36h才有微弱的表达。在与水稻抗病反应相关的差异表达cDNA中，另外一个长度为1897bp的差别表达克隆002-112-H10(GenBank accession NO．AK106615)。这个克隆与植物中的AMPBP(AMP-binding protein)蛋白家族中的酯酰辅酶A合成酶类(acyl-CoA Synthetases)有高度的相似性。我们以该cDNA克隆的已知序列设计了一对特异性引物，从水稻的cDNA文库中通过PCR扩增到这个序列，并把这个基因命名为OsBNHN-N7。OsBNHN-N7基因编码一个含有558个氨基酸的蛋白，分子量为60.4kD，等电点为7.87，含有一个Caic(Acyl-CoA synthetases(AMP-forming)／AMP-acid ligasesⅡ)结构域。Southern杂交显示OsBNHN-N7基因以多拷贝的形式存在于水稻基因组内。通过细胞定位实验也证明OsBNHN-N7编码蛋白定位在细胞质膜上。Northern杂交表明OsBNHN-N7基因在BTH处理后的24小时后有一个很高的表达水平；而在水处理的对照中只有较低的本底表达水平。另外我们在研究中发现在BTH处理的水稻幼苗在受到M.grisea侵染后，OsBNHN-N7基因在接种后的6小时就被快速的激活表达，其后表达水平继续增强并持续稳定一个高水平上。在水处理的对照中，稻瘟病菌接种后36小时内只有很微弱的低水平诱导表达。在利用水稻的一对近等位基因系H8R和H8S研究与稻瘟病菌的亲和与非亲和性互作中发现，与H8R的非亲和性互作中，OsBNHN-N7基因能被快速激活表达，而与H8S的亲和性互作中，只有极微弱的表达。更多还原

【Abstract】 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.更多还原