| Pleurotus eryngii is a rare edible fungus that combines edible,medicinal,nutritional,and health care values.Now it has become one of the most important edible mushroom species in China's factory cultivation.Light plays an important role in the growth of edible fungi throughout the life history.Now,there is no in-depth report on the mechanism of light affecting the fruit body formation and growth of P.eryngii.In this study,the differential expression of P.eryngii fruit body under light and dark conditions was analyzed using transcriptome technique.The effects of different light treatments on the fruit body formation and gene expression of P.eryngii were studied,and the function of blue light receptor gene wc-1 was verified by gene knockout technique.The main findings are as follows:(1)Transcriptome analysis of photo-induced genes associated with fruit body of P.eryngiiP.eryngii full bottle of mycelium was mushroomed in light and dark conditions,respectively.The results showed that:under light conditions,the fruit body of P.eryngii grew well,the morphology of the mushroom body was good,and the yield was high.But the fruiting body could not experience external stimuli in dark,or became a ball,or clustered,and almost could not form mushroom cap.The fruiting body was extremely small,and the color is white.The RNA-seq analysis of P.eryngii dark-treated fruiting body PE FDC and light-treated fruiting body PE_FLC revealed that there were 276 genes up-regulated and 317 genes down-regulated in FDC compared with FLC.In combination with differential genetic data,a total of 69 light-related genes were screened out,of which the blue-light receptor genes were two.KEGG metabolic pathway analyses results showed that the up-regulated genes were concentrated in peroxidase pathway,pyruvate pathway,fungal ribosome synthesis pathway and fatty acid metabolic pathway,while down-regulated genes were enriched in glycine,serine,and threonine metabolic pathways,PPAR signaling Pathways,fatty acid metabolism and synthesis of unsaturated fatty acids.It is concluded that the light affects the catalysis,protein synthesis,energy metabolism,cell growth,differentiation and apoptosis during the growth of P.eryngii fruit body,which is crucial for the growth and development of P.eryngii fruiting body.(2)Effects of different lights and light intensities on the fruit body growth and wc-11/wc-2 gene expression of P.eryngiiBy studying different lights(black,white,yellow,red,blue)and different light intensities(0,500 lx,1000 lx,2000 lx),the primordia formation,fruit body morphology,and influence of wc-1/wc-2 gene expression of P.eryngii were studied.The results showed that the 500 lx blue light promoted rapid and massive formation of the primordia,but was not conducive to fruit body growth;red light was not only unfavorable to the formation of primordium but also to fruit body growth;in dark conditions,the number of primordial formation was the least,abnormal growth,fruit body production is low.A short time(60 min)of blue light(500 lx)promoted the expression of wc-1 gene(5.85);and wc-2 gene was sensitive to 500 lx of yellow light,When light was irradiated for 30 min,the expression amount was 1.92;while white light at the time of irradiation,the wc-2 gene had the highest expression level(2.91)during the primordial period.Within a certain range of light intensity(<1000 lx),with the increase of light intensity,fruit body yield and wc genes expression of P.eryngii have also rapidly increased,and the color of the cap has also been significantly deepened,while the excessive light intensity(>2000 lx)is not conducive to the growth of fruit body.When the light intensity was 2000 lx,the expression of wc-1 gene increased to 1.25 after 15 min light irradiation,and when the light intensity decreased to 1000 lx,wc-1 gene expression increased only during the primordial phase(1.91).The expression amount of wc-2 has a certain lag effect.(3)wc-1 knockout and identification of transformantsUsing Split Marker method,we amplified and obtained the 712 bp and 571 bp non-coding sequences at the 5' and 3' ends of the target gene wc-1,and 764 bp and 931 bp for the upstream and downstream fragments of the hygromycin phosphokinase gene hph.Through recombination of SOE-PCR,recombinant fragments of 1476 bp and 1502 bp in size were obtained,which were purified to form a wc-1 knockout system.Using PEG4000 to mediate protoplast transformation,the regenerated strain was screened by further hygromycin resistance,and PCR sequence verification was performed.Knockout strains ?wc-A4,?wc-A5,?wc-A6,?wc-A 10,etc.were finally obtained.(4)Functional analysis of wc-1 gene in the formation of P.eryngii Fruit bodyMycelial growth rate and colony morphology observation showed that the mycelial growth of the knockout strains was extremely slow,the edges were irregular,the mycelium growth was weak;the scanning electron microscopy showed that the mycelia of the knockout strains were dry and had a fault,and the mycelia grew fragile.Antagonism experiments showed that the knockout strains and the original strains formed significant antagonistic lines,indicating that the knockout strains were distantly related to the starting strains and belonged to different strains.The results of blue light treatment experiments showed that the primordium number of knockout strains was very small(just 1-2),the yield of fruit body was extremely low,and even abnormal growth occurred,and the growth cycle lags behind that of the original strain.In the white mushroom experiment,during the primordium growth period,the growth of the knockout strain was similar to that of the blue light treatment.However,as the light and cultivation time increased,the fruit body growth gradually became normal,showing a certain light compensation effect,and finally the yield of fruit body,the biological efficiency(about 30%)were lower than the starting strain(61.17%).The results showed that wc-1 gene plays an important role in the fruit body morphogenesis of P.eryngii.The results of this study have important implications for the production of P.eryngii. |
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