向日葵锈病抗性相关microRNA的挖掘及其靶基因预测  

Excavation of sunflower rust-related microRNAs and target gene prediction

作　　者：龚静 路妍 宋阳[1] 景岚[1] GONG Jing;LU Yan;SONG Yang;JING Lan(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010019,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,内蒙古呼和浩特010019

出　　处：《中国油料作物学报》2020年第4期687-700,共14页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(31760509)。

摘　　要：前期利用高通量测序技术及生物信息学分析方法,在向日葵上预测筛选到10个差异表达的新microRNA(miRNA)可能与锈病抗性相关。本试验利用实时荧光定量PCR(qRT-PCR)技术,对筛选到的10个miRNA做进一步的定量验证。结果表明,相比于水处理的健康叶片(对照组A),接种锈菌300生理小种的叶片中(抗病组B)HanmiR21、Han-miR25、Han-miR42表达量呈上调趋势,Han-miR11、Han-miR34、Han-miR43、Han-miR47、Han-miR67表达量呈下调趋势;接种锈菌737生理小种的叶片中(感病组C)Han-miR25、Han-miR42的表达量呈上调趋势,Han-miR11、Han-miR21、Han-miR34、Han-miR43、Han-miR47、Han-miR67表达量呈下调趋势。qRT-PCR的检测结果与前期高通量测序结果 80%相一致。利用miRanda软件预测靶基因,10个miRNA共预测到117个靶基因,其中86个靶基因获得其生物信息学功能注释。运用miRBase数据库,对10个新预测miRNA进行同源性分析发现,新预测的10个miRNA与拟南芥、烟草、水稻等作物中参与病害逆境胁迫响应的miRNA有较高相似性(62%~100%)。定量检测和验证miRNA作用的靶基因时发现,抗病组表达量上调的Han-miR21对应的靶基因有1个下调、表达量下调的Han-miR43对应的靶基因有3个上调,符合miRNA与其靶基因互作的规律。Previously 10 differentially expressed new microRNAs(miRNAs) on sunflower rust resistance were predicted and screened by high-throughput sequencing technique and bioinformatics analysis. This paper continiusly foucused on their expressions by qRT-PCR. Results showed that expression levels of Han-miR21, Han-miR25,and Han-miR42 in leaves inoculated Puccinia helianthi race 300(resistant group B) were up-regulated compared with healthy leaves treated with water(control group A) and the expression levels of Han-miR11, Han-miR34,Han-miR43, Han-miR47, Han-miR67 showed down-regulated. Expression levels of Han-miR25 and Han-miR42 in leaves inoculated Puccinia helianthi race 737(susceptible group C) were up-regulated, and expression levels of Han-miR11, Han-miR21, Han-miR34, Han-miR43, Han-miR47, and Han-miR67 were down-regulated. Results of real-time PCR were consistent with 80% of the previous high-throughput sequencing results. By software miRanda prediction, a total of 117 target genes were predicted, of which 86 had bioinformatic functional annotations. By miRbase database, these 10 new predicted miRNAs revealed high similarity(62-100%) to miRNAs in Arabidopsis,tobacco, rice and other crops, among which Han-miR42 and Han-miR67 were involved in disease stress response.Expression of 1 target genes of Han-miR21(which was up-regulated in resisitant group) was down-regulated.While expression of 3 target genes of Han-miR43(which was down-regulated in resistant group) were up-regulated. These contrary expressions were obviously in accordance with the regulation of miRNA and its target genes.

关 键 词：向日葵锈病 抗锈相关miRNA 靶基因预测 QRT-PCR 

分 类 号：S432.2[农业科学—植物病理学] S435.655[农业科学—农业昆虫与害虫防治]