Selection Of Reference Gene For QRT–PCR In Wolfiporia Cocos

Wolfiporia cocos belongs to Polyporaceae,Wolfporia.The undefground growth selerotium often as a edible resources with irreplaceable medical value in China.Of late years,it has become the current research frontier of Chinese fungus to application of modern molecular biotechnology.This paper using real-time quantitative technique to obtain the appropriate internal gene of poria cocos to correction target gene expression by fitering poria cocos reference gene under different cinditions.It provides a theoretical basis for the study of gene expression regulation,reveal sclerotium biological characteristics and scientific guidance poria cocos cultivation.This study selected 10 reference gene : 28 S rRNA,18 S rRNA,GAPDH,RPL4(ribosomal protein L4),EF1-α,EF1-β,RPB2(DNA-direct RNA polymerase subunit 2),ACT,α-TUB,β-TUB.The stability of the expression levels of ten candidate reference genes under different stress treatments of high-salt,high-sugar and high pH as well as three different growth stages and two different tissues of Poria cocos are assessed by using qRT-PCR.Three commonly used statistical programs-geNorm,NormFinder and BestKeeper are used to comprehensive analysis of ten candidate genes expression of stability.Accordiing to published 10 reference genes design primers and amplification 10 internal genes fragment by PCR.After BLAST on NCBI we can see that 10 reference genes sequence having a high degree of similarity with homologous genes from other species.After adding sodium acetate and beta-mercaptoethanol in traditional Trizol method steps to optimization reaction system,extracting total RNA from poria cocos different tissues on different growth stages and different stress conditions of mycelium.The results showed that 18 S and 28 S two bands sapareted clear and tidy.In the different growth stages,geNorm software analysis shows that the expression stability of 10 candidates reference gene in order was 28S>α-TUB>18S> RPB2>GAPDH>EF1-α>β-TUB>RPL4>EF1-β>ACT.Expression stability in order analyzed by Norm Finder was GAPDH>28S>18S>RPB2>α-TUB>RPL4>EF1-α> β-TUB>EF1-β>ACT.BestKeeper shows that the most stable reference gene are 28 S and GAPDH.Comprehensive analysis of the results obtained by the three software,in the different growth stages,it is recommended to use 28 S as reference gene.In the different tissues,geNorm software analysis shows that the expression stability of 10 candidates reference gene in order was α-TUB>GAPDH>28S> RPL4>EF1-α>β-TUB>18S>EF1-β>RPB2>ACT.Expression stability in order analyzed by NormFinder was 28S>α-TUB>18S>β-TUB>EF1-α>GAPDH>EF1-β>RPB2>RPL4> ACT,BestKeeper shows that the most stable reference gene are 28 S and α-TUB.Comprehensive analysis of the results obtained by the three software,in different tissues,it is recommended to use α-TUB and 28 S as reference gene.Under different stress conditions,geNorm software analysis shows that the expression stability of 10 candidates reference gene in order was GAPDH>ACT> 28S>EF1-α>α-TUB>18S>RPB2>β-TUB>EF1-β>RPL4.Expression stability in order analyzed by NormFinder was ACT>28S>GAPDH>RPB2>EF1α>18S>α-TUB>EF1-β> β-TUB>RPL4.BestKeeper shows that the most stable reference gene is GAPDH and ACT,Comprehensive analysis of the results obtained by the three software,in different stress conditions,it is recommended to use GAPDH and ACT as reference gene.In all samples,geNorm software analysis shows that the expression stability of 10 candidates reference gene in order was GAPDH>α-TUB>28S>RPB2>18S>β-TUB> ACT>EF1-β>RPL4>EF1-α.Expression stability in order analyzed by NormFinder was α-TUB>RPB2>28S>GAPDH>18S>EF1α>ACT>β-TUB>RPL4>EF1-β,BestKeeper shows that the most stable reference gene are α-TUB and RPB2.Comprehensive analysis of the results obtained by the three software,in all samples,it is recommended to use α-TUB as reference gene.