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通过高通量测序鉴定和表征鹰嘴豆中对枯萎病和盐胁迫响应的微小RNA

Identification and characterization of Wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing.

作者信息

Kohli Deshika, Joshi Gopal, Deokar Amit Atmaram, Bhardwaj Ankur R, Agarwal Manu, Katiyar-Agarwal Surekha, Srinivasan Ramamurthy, Jain Pradeep Kumar

机构信息

NRC on Plant Biotechnology, IARI Campus (PUSA), New Delhi, India.

Department of Botany, North campus, University of Delhi, Delhi, India.

出版信息

PLoS One. 2014 Oct 8;9(10):e108851. doi: 10.1371/journal.pone.0108851. eCollection 2014.

Abstract

Chickpea (Cicer arietinum) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus Fusarium oxysporum f.sp. ciceris is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5' RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress.

摘要

鹰嘴豆(Cicer arietinum)是全球种植范围第二广的豆类,也是印度次大陆最重要的豆类作物。鹰嘴豆的生产力受到大量生物和非生物胁迫的不利影响。微小RNA(miRNA)参与了植物对多种生物和非生物胁迫的反应调控。本研究首次尝试鉴定与生物和非生物胁迫相关的鹰嘴豆miRNA。由尖孢镰刀菌鹰嘴豆专化型(Fusarium oxysporum f.sp. ciceris)引起的枯萎病感染是严重影响鹰嘴豆产量的主要病害之一。近来,土壤盐渍化加剧已成为实现这些潜在产量的主要问题。利用受真菌感染、盐处理和未处理的幼苗构建了三个鹰嘴豆文库,并使用下一代测序技术进行测序。共获得12,135,571条独特 reads。除了属于25个不同家族的122个保守miRNA外,还鉴定出59个新的miRNA及其星序列。在所有文库中均发现了四个豆科植物特异性miRNA,包括miR5213、miR5232、miR2111和miR2118。基于poly(A)的qRT-PCR(定量实时PCR)用于验证11个保守miRNA和5个新miRNA。miR530在真菌感染时高度上调,其靶向编码锌指和微管相关蛋白的基因。许多miRNA在生物和非生物胁迫下的反应方式相似,表明参与调控这些胁迫的途径之间存在相互作用。基于序列同源性预测了保守和新miRNA的潜在靶基因。miR166靶向一个HD-ZIPIII转录因子,并通过5' RLM-RACE进行了验证。本研究在鹰嘴豆中鉴定出了几个与枯萎病和盐胁迫后基因调控相关的保守和新miRNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/4190074/dd251f68a343/pone.0108851.g001.jpg

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