烟草黑胫病胁迫相关miRNA鉴定及功能分析

Identification and Functional Analysis of miRNA Relatedto Tobacco Black Shank Stress

MicroRNAs是一类非编码RNA分子,通过RNA沉默在转录后水平调控基因表达,在响应非生物和生物胁迫中扮演重要角色。本研究为了阐明烟草黑胫病抗病分子机制,构建了抗病品种云87接菌前0 h和接菌后12 h、24 h、48 h和72 h茎部组织的5个小RNA文库。运用solexa高通量测序技术鉴定出565个已知miRNAs（代表63个miRNA家族）和187个新miRNAs。在黑胫病菌胁迫下,总共有156个miRNA差异表达,大部分差异表达miRNA聚类到profile 18、profile 15和profile 9等3种显著性动态表达模式,并用qRT-PCR方法验证部分差异表达miRNA表达模式。靶基因预测和功能分析表明,差异表达miRNA主要参与调控防御响应、转录因子或信号级联途径和不同生理过程相关基因表达。本研究初步揭示了烟草与黑胫病非亲和互作间在转录后水平上的调控机制,为培育抗黑胫病烟草品种提供miRNA水平的理论依据。

Micro RNAs（miRNAs）, a type of non-coding RNA molecule, regulates gene expression through RNA silencing at the post-transcriptional level, and play an important role in response to abiotic and biotic stress. In this study, in order to clarify the molecular mechanism of disease resistance of tobacco black shank, 5 small RNA libraries were constructed from stem tissues of disease-resistant variety Yun 87 before inoculation and 12 h, 24 h,48 h, 72 h after inoculation with the pathogen Phytophthora parasitica var. nicotianae. Using solexa sequencing technology, 565 known miRNAs（representing 63 miRNA families） and 187 novel miRNAs were identified. Under the stress of black shank, a total of 156 miRNAs were differentially expressed, andmost of the differentially expressed miRNA were clustered into 3 significant dynamic expression patterns, including profile 18, profile 15 and profile 9. Furthermore, the expression patterns of several differentially expressed miRNAs were validated by qRT-PCR. The target gene prediction and functional analysis showed that the differentially expressed miRNA was mainly involved in the regulatio n of defense response, transcription factor or signal cascade pathway and gene expression in different physiological processes. The study preliminarily revealed that the regulatory mechanisms of incompatible interaction in the post transcriptional level between tobacco and black shank, and might provide a theoretical basis of miRNA for the cultivation of tobacco varieties resistance to black shank.