小麦K-型细胞质雄性不育系COI1基因的克隆与表达分析

Cloning and Expression Analysis of COI1 Gene from a Wheat K-Type Cytoplasmic Male Sterility Line

COI1(coronatine-insensitive 1)基因是茉莉素信号传导中起关键作用的基因,与植物的抗病性、花粉育性密切相关。为探讨COI1与小麦K型细胞质雄性不育性之间的关系,本研究从小麦K型细胞质雄性不育系豫麦3号中同源克隆得到COI1基因的三个拷贝。该基因大小为2.8 kb,开放阅读框为1 761 bp,编码586个氨基酸残基的蛋白,含有COI1蛋白典型的F-box和LRR保守结构域。进化分析结果表明COI1基因的三个拷贝分别与来自小麦祖先种乌拉尔图小麦、拟斯卑尔脱山羊草和粗山羊草、大麦以及短柄草的COI1亲缘关系较近,而与拟南芥、大豆、黄瓜等的COI1亲缘关系较远。利用中国春缺体四体系统进行染色体定位分析,发现它们分别定位于小麦4A、4B、4D染色体上。对COI1基因在小麦品种中国春的根、茎、叶和幼穗中的表达模式分析发现三个拷贝呈组成型表达,但幼穗中Ta COI1A的表达量明显高于Ta COI1B和Ta COI1D。在不育系、保持系和F1代不同发育时期的花药中,COI1基因的三个拷贝在不育系单核期花药的表达量明显高于保持系,在二核期至三核期(不育系花粉败育的关键时期),Ta COI1B和Ta COI1D在不育系和保持系中的表达趋势一致,而Ta COI1A在不育系中的表达呈上调趋势,在保持系和F1代中的表达呈下调趋势,说明Ta COI1A基因在不育系和可育系(保持系和F1代)花药中的表达有明显差异,可能跟不育系的花粉败育有关。

COI1（coronatine-insensitive 1） gene plays a crucial role in jasmine signal transduction and is closely related to disease resistance and pollen fertility in plants. To clarify the relationship between COI1 and wheat K-type cytoplasmic male sterility（CMS）, three copies of COI1 gene were isolated from a wheat K-type CMS line, Yumai 3by homologous cloning. The full length of COI1 was 2.8 kb, which open reading frame was 1 761 bp, encoding 586 amino acids of proteins that had typical F-box and LRR conserved domains of COI1. Phylogenetic analysis indicated the three copies of COI1 had close relations with COI1 genes from diploid wheat relative species, Triticum urartu, Aegilops speltoides, and Aegilops tauschii, Hordeum vulgare, and Brachypodium distachyon, but had distant relations with that of Arabidopsis, Glycine max, and Pisum sativum. Using nulli-tetrasomes of Chinese Spring DNA as PCR templates, the three copies of COI1 were respectively localized on wheat chromosomes 4A, 4B, and 4D,thus they were named Ta COI1 A, Ta COI1 B, and Ta COI1 D. Their expression patterns in Chinese Spring roots,stems, leaves, and spikes were analyzed using quantitive RT-PCR. The results showed that the three copies of COI1 were constitutively expressed in all the tested tissues, but the expression levels of Ta COI1 A were significantly higher than Ta COI1 B, and Ta COI1 D in spikes, indicating Ta COI1 A may play a major role in wheat spikes. Expression analysis of COI1 genes in anthers of three lines（CMS line, maintainer line and F1 line） at different developmental stages showed that transcripts of the three copies of COI1 in CMS line at uninucleate stage were higher than those of the maintainer line. Ta COI1 B and Ta COI1 D had same trends in CMS line and maintainer line,but Ta COI1 A up-regulated from dinucleate stage to trinucleate stage（pollen sterilizing stage） in CMS line, and downregulated in maintainer line and F1 line. These results indicate that Ta COI1 A has obvious different expression patterns in CMS line and fertile lines（maintainer line and F1 line）, it may play a role in the male sterility of wheat K-type CMS line.