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Functional analysis of the Theobroma cacao NPR1 gene in arabidopsis

机译:拟南芥可可NPR1基因的功能分析

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Background The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. Results A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica , Arabidopsis and Carica papaya . The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Conclusion Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1 , and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.
机译:背景拟南芥NPR1基因编码转录共激活因子(NPR1),在调节植物防御反应的机制中起主要作用。在病原体感染并响应水杨酸(SA)积累后,NPR1从细胞质转移到细胞核中,与其他转录因子相互作用,导致2000多种植物防御基因的表达增加,从而促进了病原体的抗性反应。结果利用简并引物,根据芸苔属,拟南芥属和番木瓜的同源序列,通过简并引物RT-PCR分离到了可可可可NPR1 cDNA。该cDNA用于从可可酵母中分离出含有推定的TcNPR1基因的基因组克隆。 DNA测序表明存在一个包含三个内含子并编码591个氨基酸的多肽的4.5 kb编码区。预测的TcNPR1蛋白与拟南芥NPR1具有55%的同一性和78%的相似性,并且包含指示此类转录因子的高度保守的功能域(BTB / POZ和锚蛋白重复蛋白-蛋白相互作用域和核定位序列( NLS))。为了在功能上定义TcNPR1基因,我们将TcNPR1转移到高度易受植物病原体丁香假单胞菌pv感染的拟南芥npr1突变体中。番茄DC3000。在组成型CaMV35S启动子的驱动下,可可TcNPR1基因部分补充了转基因拟南芥植物中的npr1突变,在叶片感染试验中细菌生长减少了100倍。用SA诱导后,显示TcNPR1以与拟南芥NPR1相同的方式转移到叶和根细胞的核中。可可NPR1也能够参与SA-JA信号的串扰,这在过表达TcNPR1的转基因植物中JA响应基因表达的抑制中得到了证明。结论我们的数据表明TcNPR1是拟南芥NPR1的功能直系同源基因,可能在可可的防御反应中起主要作用。这些基本知识可以通过应用分子标记或使用转基因策略来促进抗病性可可品种的选育。

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