| Auricularia heimuer is one of the main edible fungi cultivated in China,with high economic and ecological value.In recent years,with the rapid development of omics,studying differential expression of A.heimuer under different test conditions,screening and mining related functional genes has become the important contents of genetics and breeding.However,due to the lack of research on the internal reference genes,the research on the expression of important functional genes related has been seriously affected,and the genetic breeding of A.heimuer has been hindered.RNA was extracted form A.heimuer strains of AR1,AR2,AR3 and different development stages of mycelium,primordium,fruiting and subsequently reverse-transcribed into c DNA.Based on the whole-genome of A.heimuer,10 candidate reference genes(APRTase,?-TUB,RPL2,EF-1a,EF-2,H~+-ATPase,Tspd,18S r RNA,28S r RNA)were selected.The stability of each candidate reference gene,and the reference genes that could be stably expressed were selected for amplification using Quantitative Real-time PCR(qRT-PCR)technology,and evaluation using ge Norm,Norm Finder,Best Keeper,and?Ct algorithm and comprehensive evaluation software Ref Finder.Then,using the mycelium of AR2 strain cultured on PDA medium at 25?as control to analyze the differential expression of lignin-degrading enzyme genes and polysaccharide synthesis key enzyme genes under different test conditions for amplification using qRT-PCR technology.This experiment will lay the foundation for studying gene expression under different test conditions,and promote the mining and utilization of related functional genes,thereby promoting the research of genetics and breeding of A.heimuer.Main findings are as follows:1.In this study,the qRT-PCR technology was used to screen of reference genes.It was found that 18S r RNA,?-TUB,EF1-a,and 28S r RNA were suitable for regarding as internal reference gene combinations of different strains,APRTase,18S r RNA,and28S r RNA as internal reference gene combinations of different growth stages,APRTase??-TUB and EF1-a as internal reference gene combinations of different nutrition condition,and APRTase and 18S r RNA as internal reference gene combinations of different stress condition.2.In this study,the expression of 10 lignin-degrading enzyme genes(Lac1,Lac2,Lac3,Lac4,Lac5,POX1,POX2,POX3,POX4,POX5)were corrected and standardized by internal reference genes(APRTase,18S r RNA,28S r RNA)of different growth stages to analyze their differential expression at different growth stages.The results showed that:(1)Lac1,Lac3,POX1 and POX4 had the highest relative expression in the mycelium stage,which was significantly higher than the primordial stage and the fruiting body stage.(2)The relative expression of Lac2 genes in the mycelium stage and the primordium stage was significantly higher than that in the fruiting body stage.(3)The relative expression of POX2 and POX3 in the primordium stage is significantly higher than that in the mycelium stage and fruiting body stage.(4)The relative expression of Lac4 and Lac5 in the mycelium stage and the fruiting body stage were significantly higher than that in the primordium stage.(5)POX5 have high expression levels throughout the growth period in Auricularia heimuer.3.In this study,the mycelium of AR2 strain cultured on PDA medium at 25?as control,and qRT-PCR technology was used to analyze the differential expression of polysaccharide synthesis key enzyme genes(PGI,PGM and UGPase)under different test conditions of Auricularia heimuer.The results showed that:(1)At different growth stages,the relative expression level of UGPase in the fruiting body stage was higher than that in the primordial stage and the mycelium stage,and the relative expression of PGI and PGM in the mycelium stage was significantly higher than that in the primordial stage and the fruiting body stage.(2)Under different nutritional conditions(including glucose,starch,sucrose,yeast and peptone),the relative expression of PGI was 41 times,10 times,14 times,103 times and 5 times of that of the control group respectively;The relative expression of UGPase was significantly higher than the control group under the conditions of glucose and peptone conditions,which were 1 times and 5 times respectively;The relative expression of PGM in different nutritional conditions was significantly lower than that in the control group.(3)Under different stress conditions(4?,30?and p H=9),the relative expression of PGI was 10 times,4 times and 6 times that of the control group respectively.The relative expression of UGPase and PGM were significantly lower than the control group. |
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