在地钱中构建简便、高效的基因表达检测体系

Constructing a Simple and Efficient System for Analysis of Gene Expression in Marchantia polymorpha

实时荧光定量聚合酶链式反应(qRT-PCR)广泛应用于基因转录水平的差异分析.选择稳定表达的参比基因,设计理想的参比引物扩增参比基因进而对样本进行标准化处理是得到可靠的qRT-PCR分析结果的前提.地钱具有基因组小、生长周期短、易于培养繁殖、转基因效率高等优势,已成为一种新兴的合成生物学模式植物.植物合成生物学的研究产生了大量的转基因和突变系等地钱材料,同时也提出了在地钱底盘中提升qRT-PCR的效率和可靠性,从而快速准确分析外源以及内源基因表达水平的现实需求.对地钱基因组进行生物信息学分析发现,地钱中参比基因ACT7存在两个高度同源的基因序列,其中任一基因的表达均不太稳定,但两个基因的表达存在一定的互补性.为了在地钱中构建简易、高效的qRT-PCR分析体系,利用数学模型,分析qRT-PCR误差传导机制,建立参比引物的综合评价指标体系.研究发现,能够同时扩增两个ACT7高度同源基因的共退火引物对应的表达水平更为稳定,其中编号为3～6的引物在扩增效率、表达水平及稳定性等多个指标上表现均衡优越,从而开发了一种在地钱中快速准确检测基因表达水平的qRT-PCR平台技术,同时也为在其他物种中如何优化qRT-PCR检测体系指明方向.

Real time fluorescence quantitative polymerase chain reaction(qRT-PCR)has recently become widely used in the differential analysis of gene transcription.Stable expression of the reference genes,ideal reference primers designed to amplify the reference genes,and standardized treatment samples are the necessary preconditions for reliable qRT-PCR.With the advantages of having a small genome,short growth cycles,easy cultivation and reproduction,and high efficiency in transgenesis,Marchantia polymorpha is one of the new model plants used in synthetic biology.A large number of transgenic and mutant lines have been produced for the study of plant synthetic biology.Meanwhile,improvements to efficiency and reliability of qRT-PCR on Marchantia polymorpha have been proposed through improvements to the speed and accuracy of analyzing the exogenous and endogenous gene expression levels.Bioinformatics analysis of the Marchantia polymorpha genome showed that the reference actin gene ACT7 had double highly homologous gene sequences,and the expression of the two genes was complementary.However,the expression of either gene was not very stable.In order to construct a simple and efficient system to analyze gene expression in qRTPCR for Marchantia polymorpha,a mathematical model was used to analyze the error conduction mechanism.A comprehensive evaluation index system for the reference primers was also introduced.We found that the co-annealing primer expression level was made more stable by amplifying the two highly homologous ACT7 genes.Primers numbered3—6 showed overall superiority on multiple indicators,such as amplification efficiency,expression level,and stability.In this way,a qRT-PCR platform for rapid and accurate detection of gene expression level was developed in Marchantia polymorpha.This method can guide future research aiming to optimize the qRT-PCR detection systems in other species.