海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证

在对黄萎病菌胁迫处理的海岛棉Pima 90-53根组织全长c DNA文库分析中,筛选到一个与黄萎病胁迫相关的杂合富含脯氨酸蛋白(hybrid proline-rich protein)基因,将其命名为Gb Hy PRP1。该基因c DNA序列全长1747 bp,开放阅读框945 bp,编码一个由314个氨基酸残基组成的蛋白,包含信号肽、N端富含脯氨酸域及C端Pollen Ole e I域。同源序列分析显示,Gb Hy PRP1与来自雷蒙德氏棉、陆地棉和亚洲棉的Hy PRP1蛋白序列相似性最高,分别为95.95%、93.87%和91.34%。q RT-PCR分析结果显示,受黄萎病菌胁迫后海岛棉根部Gb Hy PRP1表达显著下调。将Gb Hy PRP1基因克隆至植物超表达载体,农杆菌介导转化拟南芥获得转基因植株。病指统计分析表明Gb Hy PRP1过量表达显著降低了拟南芥对黄萎病的抗性。据此推测Gb Hy PRP1参与棉花抗黄萎病,可能是一个重要的负调控因子。

Cloning of GbHyPRP1 from Gossypium barbadense and validation of Verticillium wilt resistance in transgenic Arabidopsis

In a full-length cDNA library of roots from Gossypium barbadense Pima90-53 inoculated with Verticillium dahliae, we identified a gene, named GbHyPRP1, which encodes a hybrid proline-rich protein. The nucleotide sequence of the 1747-base pair (bp) cDNA includes a 945 bp open reading frame (ORF), which could encode a protein of 314 amino acids. GbHyPRP1 has a signal peptide, a proline-rich repetitive domain at the N-terminus and a Pollen Ole e I domain at the C-terminus. Homologous analysis found that GbHyPRP1 had a similarity with HyPRP1 proteins from G. raimondii, G. hirsutum and G. arboreum as high as 95.95%, 93.87% and 91.34%, respectively. qRT-PCR (quantitative real time polymerase chain reaction) analysis indicated that the GbHyPRP1 mRNA level was drastically down-regulated in roots of G. barbadense seedlings inoculated with V. dahliae. Furthermore, the ORF of GbHyPRP1 was cloned into the plant expression vector and then introduced into Arabidopsis by Agrobacterium-mediated transformation. Statistical analysis of disease index showed that overexpression of GbHyPRP1 compromised transgenic Arabidopsis plants resistance to V. dahliae. Overall, our results suggest that GbHyPRP1 involves in cotton resistance to V. dahliae and may be an important negative regulator.