实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

Genome size estimation of Rhizoctonia cerealis with quantitative real-time PCR

摘要:

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础，本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列，Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani) AG1-IA融合群菌株GD118为对照，采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小，研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。

关键词:禾谷丝核菌，基因组大小，翻译延伸因子A，单拷贝，实时定量PCR

Abstract:

[Objective] Understanding the genome size of Rhizoctonia cerealis is the basis for the genome sequencing and assembly. In this study, we estimated the genome size of R. cerealis via the quantitative real-time PCR method. [Methods] The partial translation elongation factor A gene (tef A) of R. cerealis strain R0301 was cloned and sequenced. Southern blot analysis indicated tef A was a single copy in the genome. Finally, we used the quantitative real-time PCR method to calculate the genome size of R. cerealis strain R0301 with the sequenced R. solani AG1-IA strain GD118 as the control. [Results] The quantitative real-time PCR method could accurately determine the genome size of R. solani and the genome size of R. cerealis R0301 was between 32.2-36.6 Mb. [Conclusion] Quantitative real-time PCR was a fast, highly accurate and reliable method for the genome size estimation of Rhizoctonia.

Key words:Rhizoctonia cerealis, Genome size, Translation elongation factor A, Single copy, Real-Time quantitative PCR