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【目的】克隆小麦条锈菌细胞分裂基因PsCdc2,分析该基因在条锈菌接种小麦后不同时间点的表达特征。【方法】利用PCR和RT-PCR技术克隆PsCdc2的cDNA序列和基因组序列,采用生物信息学技术预测分析该基因编码蛋白的保守结构域及基本特性,对该蛋白进行系统发育分析,构建进化树;运用实时荧光定量RT-PCR技术,以PsCdc2在夏孢子时期的表达情况为对照,分析该基因在亲和及非亲和互作中不同时间点的表达特征。【结果】PsCdc2基因组序列长2279 bp,由11个外显子和10个内含子构成,开放阅读框为885 bp,编码294个氨基酸,分子量为33.14 kDa,等电点为6.26。编码蛋白含两个保守的激酶特征位点,一个跨膜螺旋区域。PsCdc2基因编码蛋白与小麦秆锈菌、新型隐球菌、玉米瘤黑粉菌等多种真菌的Cdc2高度相似,其中与小麦秆锈菌的Cdc2亲缘关系最近,序列相似性达73.1%。实时荧光定量RT-PCR结果表明,在亲和组合中,该基因在条锈菌接种小麦的前期上调表达,其中接种后12 h时表达量最高,约为夏孢子中表达量的1.62倍,接种后24-268 h,基因表达基本呈下调趋势,其中96 h基因表达量最低,仅为夏孢子时期的0.07倍。在非亲和组合中,该基因表达基本呈下调趋势,在接种后各个时间点的表达量均低于在夏孢子中的表达量,其中接种后12 h时表达量最高,但仅为夏孢子中表达量的0.34倍;接种后96 h表达量最低,为夏孢子中表达量的0.02倍。【结论】PsCdc2可能通过调控条锈菌的细胞周期循环参与了侵染前期初生菌丝生长和吸器母细胞的形成,与条锈菌的致病性相关。本文首次报道了小麦条锈菌的Cdc2基因,为进一步揭示条锈菌细胞周期调控的本质及研究开发靶向Cdc2的新型农药,以及实现对小麦条锈病的新型药剂防治提供了理论基础。
【Objective】 The objective of this study was to clone the PsCdc2 gene of wheat stripe rust and to analyze its expression pattern at different time points after wheat stripe rust inoculation. 【Method】 The cDNA sequence and genomic sequence of PsCdc2 were cloned by PCR and RT-PCR. Bioinformatics was used to predict the conserved domains and basic characteristics of the encoded protein. The phylogenetic tree was constructed and phylogenetic tree was constructed. Real-time fluorescent quantitative RT-PCR was used to compare the expression of PsCdc2 in the sporulation period and the expression of the gene at different time points in both affinity and non-affinity interactions. 【Result】 The sequence of PsCdc2 was 2279 bp in length, consisting of 11 exons and 10 introns. The open reading frame was 885 bp, encoding 294 amino acids with a molecular weight of 33.14 kDa and an isoelectric point of 6.26. The encoded protein contains two conserved kinase sites, a transmembrane helix. The Cdc2 protein of PsCdc2 was highly similar to Cdc2 of many fungi such as wheat rust, Cryptococcus neoformans, maize tumor, and so on. Cdc2 was closely related to Cdc2 of wheat straw rust, and its sequence similarity was 73.1%. The result of real-time fluorescence quantitative RT-PCR showed that the gene was up-regulated during the early stage of inoculation of wheat stripe rust in the affinity combination, and the highest expression was at 12 h after inoculation, which was about 1.62 times of the expression in Uospora. After 24-268 h, the gene expression showed a downward trend, of which 96 h gene expression was the lowest, only 0.07 times of the summer sporulation period. In non-affinity combinations, the gene expression showed a downward trend, the expression levels were lower at each time point after inoculation than in the spores, of which the highest expression was reached 12 h after inoculation, 0.34 times the amount of expression; 96 h after inoculation the lowest expression of the amount of 0.02 times the amount of expression in the summer spores. 【Conclusion】 PsCdc2 may be involved in the growth of primary mycelium and the formation of astrocytes in the early stage of infection through regulating the cell cycle of stripe rust, which may be related to the pathogenicity of stripe rust. This paper reports for the first time the Cdc2 gene of wheat stripe rust, which provides a theoretical basis for further revealing the nature of the cell cycle regulation of Stripe rust and the development of a new type of pesticide targeting Cdc2, as well as the realization of a new type of chemical control of wheat stripe rust.