摘要
【目的】克隆小麦条锈菌产孢相关基因PsCon1,分析其在病菌不同发育时期的表达水平。【方法】利用RT-PCR和PCR技术克隆PsCon1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的DNA序列结构及其编码蛋白的保守域等基本特性,运用实时荧光定量RT-PCR技术分析PsCon1在夏孢子,芽管以及不同侵染时间的表达水平。【结果】PsCon1由3个外显子和2个内含子构成,开放阅读框长为252bp,编码83个氨基酸;编码的蛋白PSCON1不含跨膜区,无信号肽,定位在细胞质,具有2个conidiation-specific protein6保守结构域。PsCon1与小麦秆锈菌核苷酸序列的一致性在外显子区为78%,内含子区为43%,与小麦秆锈菌(Puccinia graminis)的亲缘关系最近,与烟曲霉(Aspergillus fumigatus)和费氏新萨托(Neosartorya fischeri)的亲缘关系次之。PsCon1在小麦条锈菌萌发夏孢子时期基因表达量为新鲜夏孢子中的1.69倍。在亲和组合中,PsCon1在小麦条锈菌接种小麦后6h和24h表达量最高,分别为在新鲜夏孢子中表达量的3.21倍和2.91倍;在接种后24h至168h,基因表达基本呈下调趋势,在接种后168h的表达量最低,仅为夏孢子时期的0.0004倍,在接种后216h和264h,表达量有所增加,表达量约为接种后168h的15倍。在非亲和组合中,PsCon1在小麦条锈菌接种小麦后36h表达量最高,但仅为新鲜夏孢子基因表达量的0.13倍,基因表达总体呈下调趋势。【结论】PsCon1参与了小麦条锈菌对小麦的侵染,可能作为一个致病相关基因影响了条锈菌芽管和吸器的形成,同时促进了条锈菌在侵染过程中的产孢。PsCon1的克隆为进一步研究该基因在小麦与条锈菌互作过程中的功能奠定了基础。
[ Objective] To clone a conidiation-related gene PsConl from Puccinia striiformis f.sp. tritici （Pst） and analyze its expression profile. [Method] The cDNA and genomic DNA of PsConl were isolated by using RT-PCR and PCR and the gene expression level at different morphological stages was analyzed via real-time RT-PCR. [ Result] PsConl comprised of 3 extrons and 2 introns. Open reading frame （ORF） of PsConl was 252 bp in length, encoding 83 amino acids containing two conserved domains of conidiation-specific protein 6. PsCon1, without transmembrane domain or signal peptide sequence, was predicted to be located in cytoplasm. The nucleotide acid identity between PsConl and PtCon extrons was 78%, much higher than the 43% between the introns. Phylogenetic analysis indicated that PSCON1 had high similarity to Con protein from Puccinia graminis, Aspergillus fumigatus and Neosartoryafischeri. Real-time PCR analysis showed that the transcripts of PsConl of germinating urediospore was 1.69 fold over that of urediniospore. In compatible interaction of Pst and wheat, PsConl was up-regulated as early as 6 h postinoculation （hpi）. The maximum induction occurred at 6 and 24 hpi, whose transcripts were 3.21 and 2.91 fold over that of urediospore, respectively. From 24 to 168 hpi, the accumulation of transcripts decreased steadily. However, the amount of PsConl transcripts was increased at 216 and 264 hpi, which was 15 times as much as that of 168 hpi. In incompatible interaction of Pst and wheat, the accumulation of PsConl transcripts decreased steadily. [Conclusion] PsConl might have influence on germ tube elongation, haustorium and urediospore formation, so it might play a role in the process of stripe rust infection on wheat. The cloning of PsConl served as a good foundation for further analysis of its role in the pathogenic process.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第6期1156-1163,共8页
Scientia Agricultura Sinica
基金
公益性(农业)行业科技专项(200903035-02)
国家"973"计划项目(2006CB100203)
教育部科学技术研究重点项目(107104)
现代农业产业技术体系建设专项资金资助
高等学校学科创新引智计划资助项目(B07049)
西北农林科技大学青年学术骨干支持计划
