Li Dayong, Liu Huizhi, Zhang Huijuan, Wang Xiaoe, Song Fengming
State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University-Huajiachi Campus, Hangzhou, Zhejiang 310029, People's Republic of China.
J Exp Bot. 2008;59(8):2133-46. doi: 10.1093/jxb/ern072. Epub 2008 Apr 25.
DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.
DEAD盒蛋白构成了一个庞大的蛋白家族,其成员来自所有生物界,在RNA代谢的各类过程中发挥着重要作用。在本研究中,克隆并鉴定了一个编码DEAD盒RNA解旋酶蛋白的水稻基因OsBIRH1。预测的OsBIRH1蛋白包含一个DEAD结构域以及所有保守基序,这些都是DEAD盒RNA解旋酶的共同特征。从大肠杆菌中纯化得到的重组OsBIRH1蛋白在体外显示出具有RNA依赖性ATP酶和ATP依赖性RNA解旋酶活性。在用防御相关信号化学物质(例如苯并噻二唑、水杨酸、l - 氨基环丙烷 - 1 - 羧酸和茉莉酸)处理后,水稻幼苗叶片中OsBIRH1的表达被激活,并且在抗性水稻基因型与稻瘟病菌Magnaporthe grisea的不亲和互作中也上调。构建了过表达OsBIRH1基因的转基因拟南芥植株。抗病性表型分析表明,过表达OsBIRH1的转基因植株对链格孢和丁香假单胞菌番茄致病变种DC3000表现出增强的抗病性。同时,防御相关基因(例如PR - 1、PR - 2、PR - 5和PDF1.2)在转基因植株中显示出上调表达。此外,OsBIRH1转基因拟南芥植株对氧化胁迫的耐受性也增强,并且氧化防御基因AtApx1、AtApx2和AtFSD1的表达水平升高。结果表明,OsBIRH1编码一种功能性的DEAD盒RNA解旋酶,在对抗生物和非生物胁迫的防御反应中发挥重要作用。