黑木耳实时荧光定量PCR内参基因的筛选

黑木耳Auricularia heimuer是我国主要栽培的食用菌之一,具有较高的经济价值和生态价值。本研究以黑木耳不同菌株（A14、A137和A12）和不同生育期的样品（菌丝体、原基和子实体）为实验材料,提取RNA,反转录成cDNA,在全基因组注释结果的基础上,选择12个候选内参基因（APRTase、β-TUB、RPL2、EF-1a、EF-2、PGI、PGM、H ⁺-ATPase、Tspd、TUB-1a、18S rRNA和28S rRNA）并设计跨内含子的引物,采用实时荧光定量PCR（qRT-PCR）技术进行扩增,利用geNorm、NormFinder、BestKeeper和ΔCt算法以及综合评价软件RefFinder,筛选适宜的内参基因。结果表明18S rRNA、β-TUB、EF1-a和28S rRNA适宜作为不同菌株的内参基因,APRTase、18S rRNA和28S rRNA适宜作为不同生育期的内参基因。

Screening of reference genes for qRT-PCR amplification in Auricularia heimuer

Auricularia heimuer is one of the main edible fungi cultivated in China, with high economic and ecological value. RNA was extracted from different development stages (mycelium, primordium and fruiting stages) of A. heimuer straius (A14, A137 and A12) and subsequently reverse-transcribed into cDNA. Based on the whole-genome of A. heimuer, 12 candidate reference genes (APRTase, β-TUB, RPL2, EF-1a, EF-2, PGI, PGM, H ⁺-ATPase, Tspd, 18S rRNA, 28S rRNA) were selected. The stability of each candidate reference gene, and the reference genes that could be stably expressed were selected for amplification using real-time fluorescence quantitative PCR (qRT-PCR) technology, and evaluation using geNorm, NormFinder, BestKeeper, and ΔCt algorithm and comprehensive evaluation software RefFinder. It was found that 18S rRNA, β-TUB, EF1-a, and 28S rRNA were suitable for regarding as internal reference gene combinations of different strains, and APRTase, 18S rRNA, and 28S rRNA as internal reference gene combinations of different growth stages.