Zhang Yafeng, Liao Xun, Luo Peihua, Peng Kui, Ye Wenyi, Lian Tengxiang, Ma Qibin, Nian Hai
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University; Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University;
Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University.
J Vis Exp. 2019 Jul 29(149). doi: 10.3791/60019.
In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate. In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot. By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.
在植物线粒体中，一些稳态转录本具有源自转录起始的5'三磷酸（初级转录本），而其他转录本则含有转录后产生的5'单磷酸（加工转录本）。为了区分这两种类型的转录本，已经开发了几种策略，其中大多数取决于5'三磷酸的有无。然而，初级5'末端的三磷酸不稳定，这妨碍了对这两种转录本的清晰区分。为了系统地鉴别和定位稳定积累在玉米线粒体中的初级和加工转录本，我们通过结合环化RT-PCR（cRT-PCR）、RNA 5'多磷酸酶处理、定量RT-PCR（RT-qPCR）和Northern印迹，开发了一种基于cRT-PCR的策略。作为改进，该策略包括一个RNA标准化步骤，以尽量减少不稳定的5'三磷酸的影响。在本方案中，富集的线粒体RNA用RNA 5'多磷酸酶进行预处理，将5'三磷酸转化为单磷酸。环化和逆转录后，来自5'多磷酸酶处理和未处理RNA的两种cDNA通过玉米26S成熟rRNA进行标准化，玉米26S成熟rRNA具有加工后的5'末端，对5'多磷酸酶不敏感。标准化后，通过比较从处理和未处理RNA获得的cRT-PCR和RT-qPCR产物来区分初级和加工转录本。通过对cRT-PCR产物进行克隆和测序来确定转录本末端，然后通过Northern印迹进行验证。通过使用该策略，已经确定了玉米线粒体中的大多数稳态转录本。由于一些线粒体基因的转录模式复杂，尽管在Northern印迹中检测到了一些稳态转录本，但仍有少数未被区分和/或定位。我们不确定该策略是否适用于鉴别和定位其他植物线粒体或质体中的稳态转录本。
