实时荧光定量PCR筛选荷叶离褶伞的内参基因及验证  

Screening and Validation of Reference Genes in Lyophyllum decastes by qRT-PCR

作　　者：梁力丹 李华君 兰玉菲 臧西哲 林嘉龙 韦勇琪 张佩锦 任鹏飞[2] 孟丽[1] LIANG Li-Dan;LI Hua-Jun;LAN Yu-Fei;ZANG Xi-Zhe;LIN Jia-Long;WEI Yong-Qi;ZHANG Pei-Jin;REN Peng-Fei;MENG Li(College of Plant Protection/Key Laboratory of Agricultural Microbiology of Shandong Province,Shandong Agricultural University,Tai'an 271018,China;Institute of Agricultural Resource and Environment/Key Laboratory of Wastes Matrix Utilization,Ministry of Agriculture,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Tai'an Academy of Agricultural Sciences,Tai'an 271000,China)

机构地区：[1]山东农业大学植物保护学院/山东省农业微生物重点实验室,泰安271018 [2]山东省农业科学院农业资源与环境研究所/农业农村部废弃物基质化利用重点实验室,济南250100 [3]泰安市农业科学院,泰安271000

出　　处：《农业生物技术学报》2024年第12期2915-2925,共11页Journal of Agricultural Biotechnology

基　　金：山东省重点研发计划项目(2022CXGC010610,2022LZGC023);山东省食用菌产业技术体系(SDAIT-07-06)。

摘　　要：选择合适的内参基因是qRT-PCR的关键步骤,但关于荷叶离褶伞(Lyophyllum decastes)内参基因的筛选研究还尚未见报道。本研究以荷叶离褶伞的不同后熟时间(45,55和65 d)、子实体不同发育时期(原基,幼菇,子实体)和不同菌盖形态(正常,畸形)为实验材料,选择在食用菌研究中6个常用的基因:细胞色素c氧化酶亚基1(cytochrome c oxidase subunit 1,Cox1)、ATP酶(ATPase)、葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase,PGI)、蛋白磷酸酶2A(protein phosphatase 2A,PP2A)、DNA指导的RNA聚合酶亚基2(DNA-directed RNA polymerase subunit 2,Rpb2)和泛素结合酶(ubiquitin-conjugating enzyme,UBC),以及4个鲜有报道的基因:含TCP1伴侣蛋白亚基-2(chaperonin containing TCP1,subunit 2,CCT2)、细胞色素b560亚基(cytochrome b560 subunit,Cyb)、羟基类固醇17-β-脱氢酶3(17 beta-hydroxysteroid dehydrogenase type 3,HSD17B3)和铜/锌超氧化物歧化酶(copper/zinc superoxide dismutase,SODC)作为候选内参基因,并借助ΔCt、geNorm、NormFinder、BestKeeper和RefFinder五种算法程序评估10个候选内参基因的表达稳定性,并以β-1,3葡聚糖酶为目的基因验证本研究筛选的内参基因。结果表明,HSD17B3在不同后熟时间和不同发育时期表达最稳定,Rpb2在不同菌盖形态中表达最稳定,而PP2A在不同后熟时间和不同菌盖形态中均最不稳定,在不同发育时期中,PGI的表达稳定性最差。本研究评估了荷叶离褶伞内参基因的稳定性,为荷叶离褶伞不同时期以及不同菌盖形态的研究提供参考。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 afterripening 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 byΔCt,geNorm,NormFinder,BestKeeper and RefFinder and validated byβ-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.

关 键 词：荷叶离褶伞 内参基因 实时荧光定量PCR 稳定性 

分 类 号：S646[农业科学—蔬菜学]