青稞HvtAGO1基因的克隆及其在条纹病胁迫下的表达

Identification and Expression Analysis of HvtAGO1 Gene in Response to Barley Leaf Stripe in Tibetan Hulless Barley

AGO蛋白是RNA诱导沉默复合体的核心分子,在植物的生长、发育及胁迫响应中起重要作用。为探索青稞AGO基因在青稞抗条纹病病原菌过程中的作用机制,该研究以抗条纹病青稞品种‘昆仑14号’和感病品种‘Z1141’为材料,利用条纹病原菌侵染两品种,从感病前后的转录组测序结果中获得一个差异表达基因,克隆验证了该基因为HvtAGO1。结果表明:(1)HvtAGO1基因长3675 bp,其完整开放阅读框为3651 bp,编码1217个氨基酸;蛋白质序列分析表明,HvtAGO1为一个亲水性的不稳定碱性蛋白,具有高度保守的DUF1785、PAZ和PIWI结构域。(2)青稞HvtAGO1蛋白与山羊草、二穗短柄草、乌拉尔图小麦的AGO1蛋白序列相似性分别为96.64%、79.34%和80.07%,DUF1785、PAZ和PIWI结构域相似性分别为97.57%、98.44%和96.02%;系统进化树分析表明,青稞HvtAGO1蛋白与山羊草的亲缘关系最近,与玉米和粟的亲缘关系最远。(3)降解组测序结果表明,hvu-miR168-5p与HvtAGO1存在靶向关系。(4)qRT-PCR分析表明,条纹病胁迫下,与正常叶片相比,‘昆仑14号’与‘Z1141’感病叶片的hvu-miR168-5p和靶基因HvtAGO1表达量均极显著上调,且‘昆仑14号’表达量显著高于‘Z1141’。研究推测,hvu-miR168-5p和靶基因HvtAGO1在青稞抗条纹病的调控过程中发挥着重要作用。

AGO protein is the core molecule of the RNA-inducible silencing complex,which plays an important role in plant growth,development and stress response.In order to explore the mechanism of AGO gene in Tibetan hulless barley resistance to barley leaf stripe,we obtained a differentially expressed gene from the transcriptional sequencing results before and after infection with barley leaf stripe in resistant Tibetan hulless barley variety‘Kunlun14’and the susceptible variety‘Z1141’.The results showed that:(1)HvtAGO1 was isolated from leaves of‘Kunlun 14’and‘Z1141’.We obtained a 3675 bp sequence which contained a 3651 bp open reading frame(ORF),encoding 1217 amino acids in this study.Protein sequence analysis showed that HvtAGO1 was a hydrophilic unstable basic protein with highly conserved DUF1785,PAZ and PIWI domains.(2)The sequence similarity of the AOG1 protein between Tibetan hulless barely and Aegilops tauschii,Brachypodium distachyon and Triticum urartu was 96.64%,79.34 and 80.07%,respectively,and the domain similarity of DUF1785,PAZ and PIWI was 97.57%,98.44%and 96.02%,respectively.Phylogenetic analysis showed that the affinity of HvtAGO1 protein of Tibetan hulless barley was closest to that of A.tauschii,but far from that of Zea mays and Setaria italica.(3)The degradation sequencing proved that HvtAGO1 was the target gene of hvu-miR168-5p.(4)qRT-PCR results showed that the expression levels of hvu-miR168-5p and the target gene HvtAGO1 were significantly up-regulated in‘Kunlun 14’and‘Z1141’compaired with normal leaves,respectively.Meanwhile,the gene expression levels of‘Kunlun 14’were significantly higher than that of‘Z1141’under barley leaf stripe.Research speculated that hvu-miR168-5p and target gene HvAtGO1 play an important role in the regulation of resistance to barely leaf stripe in Tibetan hulless barley.