Screening and Validation of Reference Genes in Lyophyllum decastes by qRT-PCR
LIANG Li-Dan1, LI Hua-Jun1, LAN Yu-Fei3, ZANG Xi-Zhe1, LIN Jia-Long1, WEI Yong-Qi1, ZHANG Pei-Jin1, REN Peng-Fei2*, MENG Li1*
1 College of Plant Protection/Key Laboratory of Agricultural Microbiology of Shandong Province, Shandong Agricultural University, Tai'an 271018, China; 2 Institute of Agricultural Resource and Environment/Key Laboratory of Wastes Matrix Utilization, Ministry of Agriculture, Shandong Academy of Agricultural Sciences, Jinan 250100, China; 3 Tai'an Academy of Agricultural Sciences, Tai'an 271000, China
Abstract：Choosing an appropriate internal reference gene is an essential requirement for qRT-PCR. However, Lyophyllum decastes has no internal reference genes reported. In this study, mycelia at different after-ripening times, fruiting bodies of different developmental stages and different cap morphologies were selected as the experimental materials. qRT-PCR was used to quantitatively amplify 6 commonly internal reference genes which were cytochrome c oxidase subunit 1 (Cox1), ATPase, glucose-6-phosphate isomerase (PGI), protein phosphatase 2A (PP2A), DNA-directed RNA polymerase subunit 2 (Rpb2), and ubiquitin-conjugating enzyme (UBC) and 4 rarely internal reference genes which were chaperonin containing TCP1, subunit 2 (CCT2), cytochrome b560 subunit (Cyb), 17 beta-hydroxysteroid dehydrogenase type 3 (HSD17B3) and copper/zinc superoxide dismutase (SODC). The stability of 10 internal reference genes were evaluated by ΔCt, geNorm, NormFinder, BestKeeper and RefFinder and validated by β-1,3-glucanases, which was the key enzyme in starch and sucrose metabolism. The results showed that HSD17B3 were the most stable gene in different after-ripening times and different developmental stages; Rpb2 was the most stable gene in different cap morphologies. However, PP2A was the most unstable gene in different after-ripening times and different cap morphologies samples. Moreover, PGI was the most unstable in different developmental stages. The study evaluate the stability of internal reference genes in L. decastes and expect to offer reference for researches about different stages and different cap morphologies in L. decastes.
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