青稞新基因HvMEL1 AGO的克隆和条纹病胁迫下的表达

Isolation and expression of a new gene HvMEL1 AGO in Tibetan hulless barley under leaf stripe stress

为筛选与青稞条纹病相关的AGO类基因,本研究以青稞抗病品种昆仑14号和感病品种1141为材料,从感病和正常叶片的转录组测序结果中获得一个差异表达的AGO家族新基因,克隆验证了该基因为青稞HvMEL1 AGO。HvMEL1 AGO基因全长3462 bp,其中蛋白质编码区(CDS,coding domain sequence)在昆仑14号和1141品种中的一致性为100%,无内含子,全长3161 bp,包含一个3129 bp开放阅读框,编码1043个氨基酸,理论等电点为9.33,预测蛋白分子量为115,865.58 Da。蛋白质序列分析表明,HvMEL1 AGO为亲水性的不稳定酸性蛋白,具有高度保守的DUF1785、PAZ和PIWI结构域,属于AGO基因家族成员。进化树分析表明,HvMEL1 AGO与大麦AGO家族中HvAGO12、HvAGO18、HvAGO1D、HvAGO1B在拟南芥AGO家族系统发育树上属于AGO1一类;与HvAGO12的亲缘关系最近。蛋白质互作预测结果表明,在水稻中与MEL1作用密切的已知蛋白为DCL类,分别为DCL1、DCL2A、DCL3A、DCL3B和DCL4。半定量和定量PCR结果表明,条纹病胁迫下,抗病品种昆仑14号与感病品种1141的HvMEL1 AGO基因表达量显著下降;且1141显著高于昆仑14号(P<0.01)。推测青稞HvMEL1 AGO基因在青稞抗条纹病过程中发挥重要作用。本研究为探索HvMEL1 AGO基因在青稞抗条纹病过程中的作用及调控机制奠定基础。

To explore AGO genes related to leaf stripe in Tibetan hulless barley(BLS),we obtained a differentially expressed HvMEL1 AGO gene from the transcriptional sequencing in normal and diseased leaves from resistant Tibetan hulless barley vari-ety Kunlun 14 and the susceptible variety 1141.The HvMEL1 AGO gene was 3462 bp in length,of which the CDS(coding do-main sequence)was 100%consistent in Kunlun 14 and 1141 varieties.The length of HvMEL1 AGO gene was 3161 bp without intron,contained a 3129 bp open reading frame,encoded 1043 amino acids,had a theoretical isoelectric point of 9.33,and has a predicted molecular weight of 115,865.58 Da.Protein sequence analysis showed that HvMEL1 AGO was a hydrophilic unstable acidic protein with highly conserved structural domains of DUF1785,PAZ and PIWI,belonging to AGO gene family.Phyloge-netic tree analysis showed that HvMEL1 AGO belonged to the AGO1 class in Arabidopsis AGO family phylogenetic tree with HvAGO12,HvAGO18,HvAGO1D and HvAGO1B in barley AGO family,and were closely related to HvAGO12.The predicted protein interactions showed that the known proteins that acted closely with MEL1 in rice were the DCL classes,DCL1,DCL2A,DCL3A,DCL3B and DCL4,respectively.Semi-quantitative and quantitative PCR results indicated that the relative expression levels of HvMEL1 AGO gene were significantly down-regulated in 1141 and Kunlun 14 under BLS.We hypothesized that HvMEL1 AGO gene played an important role in the streak resistance mechanism of barley.This study lays the foundation for ex-ploring the role and regulatory mechanism of HvMEL1 AGO in the process of BLS resistance.