Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.
Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.
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不同营养胁迫下嫁接西瓜植株 miRNA 表达标准化的合适参考基因的鉴定
DOI：
10.1371/journal.pone.0164725
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Zhang M
中科院分区：
文献类型：
--
作者：
Wu W;Deng Q;Shi P;Yang J;Hu Z;Zhang M
西瓜(Citrullus Lanatus)属于葫芦科，是一种重要的世界性作物。嫁接技术通常用于提高其抗逆性，以及增强其对养分的吸收和利用。据认为，miRNA最有可能参与其营养匮乏反应，作为一种可移植的信号。实时定量逆转录聚合酶链式反应是miRNA功能分析的首选方法，其中可靠的参考基因归一化是保证准确性的关键。本研究的目的是在西瓜嫁接植株的接穗(西瓜)和砧木(南瓜和丝瓜)正常生长和营养胁迫(氮磷饥饿)条件下筛选合适的参考基因。在营养饥饿条件下，geNorm发现miR167c和miR167f是西瓜叶片和南瓜根中最稳定的两个基因。GeNorm和NormFinder都推荐miR166b作为营养限制条件下葫芦根的最佳参照物。利用一个新的葫芦科miRNA miR85的表达来验证候选参考基因在营养饥饿条件下的可靠性。此外，通过比较qRT-PCR分析和RNA-seq数据中的几个靶基因的表达情况，认为miR166b和miR167c分别是最适合在正常生长条件下调节接穗和砧木组织miRNA表达的参考基因。本研究首次对葫芦科植物miRNA参考基因的稳定性进行了全面的研究，为进一步研究嫁接西瓜植株miRNA的准确表达提供了有价值的信息。
Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.
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