Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying.

Shuangshuang Gao,Gangzheng Wang,Yinbing Bian,Zhicheng Huang,Ying Liu,Xiaoyu Lei,Wen Huang

doi:10.3390/molecules24010136

Shuangshuang Gao, Gangzheng Wang + Show 5 more

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https://doi.org/10.3390/molecules24010136

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Abstract

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

Highlights

Lentinula edodes, an edible mushroom well known for its nutritional and pharmacological values as well as the bioconversion of agricultural wastes, ranks only second to Agaricus bisoporus in production and cultivation in the word [1,2]
The stability of internal reference genes is a prerequisite for the reliability of quantitative real-time polymerase chain reaction (qRT-PCR) results
Several housekeeping genes such as Actin and GAPDH as well as TUB were selected as the internal reference genes because of their functions involved in the basic cytoskeleton of organelles and basic biochemical metabolic processes of organisms [15,16]

Summary

Introduction

Lentinula edodes (shiitake mushroom), an edible mushroom well known for its nutritional and pharmacological values as well as the bioconversion of agricultural wastes, ranks only second to Agaricus bisoporus in production and cultivation in the word [1,2]. L. edodes mushrooms have been highly prized since ancient times, due to their higher amount of nutrients and greater umami flavor than fresh mushrooms [3,4]. The L. edodes mushrooms can be dried by hot air, freezing, vacuum, and microwave. Hot-air drying (HAD) is widely used because of low cost and easy operation as well as obvious improvement of the content of sulfur compounds essential for the unique aroma of L. edodes [3,5]. The molecular mechanism for the hot-air drying of. For the sake of its high sensitivity and good repeatability as well as wide dynamic quantification range [6,7], qRT-PCR (quantitative real-time polymerase chain reaction) is widely used to analyze and verify the expression patterns and levels of genes regulating the metabolism of sulfur compounds after the drying process

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