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【目的】克隆小麦条锈菌几丁质合成酶基因PstChsⅡ,分析其在小麦条锈菌不同发育时期的表达水平。【方法】利用RT-PCR和PCR技术克隆PstChsⅡ的cDNA序列和基因组序列,利用不同的生物信息学软件对序列进行分析,运用实时荧光定量技术分析基因在孢子、芽管以及不同侵染时间的表达水平。【结果】PstChsⅡ基因(GenBank登录号GQ329851)编码区存在15个内含子,开放阅读框长2727bp,编码908个氨基酸。PstChsⅡ蛋白C端含有7个跨膜螺旋区,N端含多个保守结构域和“QXRRW”、“GXGPL”、“DXD”等motifs,蛋白序列与小麦杆锈菌的PgtChsⅡ相似性达94%,系统进化分析表明PstChsⅡ属于Ⅱ类Chs蛋白亚家族,与担子菌亚门真菌同源序列的相似度高于子囊菌亚门真菌。基因在小麦条锈菌夏孢子萌发时期明显上调(约10倍),而在其他发育阶段表达基本恒定。【结论】PstChsⅡ可能参与了小麦条锈菌夏孢子萌发时芽管细胞壁的合成;PstChsⅡ的克隆与表达分析为进一步研究该基因在小麦条锈菌专性寄生生活中的功能奠定了基础。
【Objective】 The objective of this study was to clone the PstChs Ⅱ gene of the stripe rust of wheat stripe rust and to analyze the expression level of PstChs Ⅱ at different developmental stages of wheat stripe rust. 【Method】 The cDNA sequence and the genomic sequence of PstChs Ⅱ were cloned by RT-PCR and PCR. The sequences were analyzed by different bioinformatics software. The expression of PstChs Ⅱ gene in spores, germ tubes and different infection times were analyzed by real-time fluorescence quantitative analysis Level. 【Result】 There were 15 introns in the coding region of PstChs Ⅱ gene (GenBank accession number GQ329851). The open reading frame was 2727bp and encoded 908 amino acids. PstChsⅡprotein contains seven transmembrane helix regions at the C-terminus and several conserved domains at the N-terminus and motifs such as “QXRRW”, “GXGPL” and “DXD” The phylogenetic analysis showed that PstChs Ⅱ belongs to the class Ⅱ Chs subfamily, which is more similar to the homologous sequences of Basidiomycotina, than the Ascomycotina. The gene was significantly up-regulated (about 10-fold) during the spore germination of the stripe rust of wheat and remained essentially constant at other developmental stages. 【Conclusion】 PstChsⅡis involved in the synthesis of germ cell wall during the spore germination of wheat stripe rust. The cloning and expression analysis of PstChs Ⅱ laid the foundation for further study on the function of this gene in the virulent parasitism of wheat stripe rust.