DNA Methylation Variation And Regulatory Genes Research During The Pleurotus Tuoliensis Primordium Formation Induced By Low Temperture

Pleurotus tuoliensis is a kind of edible fungus with independent intellectual property rights and clear origin in China.It is distributed in Asia and Southern Europe.They are showed earliest Xinjiang of China's?mainly distributed in Yumin,Tori,Qinghe and other places?domesticated cultivation,is a new rare medicinal bacteria varieties.Pleurotu tuoliensis belongs to the medium-low temperature type variable temperature and the edible fungus.The leaching stage needs the low temperature and the temperature difference to stimulate,and the low temperature can force the primordial differentiation and the mushroom buds.This growth characteristic has become the bottleneck of the industrial development of Pleurotus.The aim of this experiment is to study the mechanism of DNA methylation in the process of low temperature stress treatment of P.tuoliensis,and to provide theoretical basis and experimental guidance for high yield cultivation and low temperature resistance breeding of Pleurotus.In this paper,P.tuoliensis was selected as the experimental material under different temperature conditions induced by low temperature stress.The methylation level and ultrastructure of P.tuoliensis genomic DNA induced by low temperature treatment were studied by methylation sensitive polymorphism?MSAP?/Low methylation mode,and the distribution and regulation of DNA methylation were studied by modern bisulfite-sequencing?BS-seq?sequencing.The main findings are as follows:1.Extraction of genomic DNA from P.tuoliensis and establishment of MSAP analysis methodThree methods were used to extract the genome of P.tuoliensis,and the result was improved by CTAB method.Through the design of single factor and orthogonal test,it was found that the optimal digestion time was 5 hours at 37?,and the reaction was more appropriate at 16??ie,12 h?.The pre-amplification products were 10 times dilution The dilution factor for the connected product.According to the pre-amplified product in the 1%agarose gel was the principle of dispersion distribution,intuitive pre-amplification product electrophoresis diagram,while the best consideration for the use of frugal,so the choice of pre-amplification PCR reaction system identified as 10×PCR Buffer 2.5?L,dNTP 2.5mmol·L-1,Primer 200 ng·?L^-1,0.2?L of Taq polymerase,2.0?L of DNA template.2.Changes of DNA methylation in different growth and development stages of P.tuoliensis induced by low temperature stressThe optimized MSAP method was used to study the methylation of P.tuoliensis induced by low temperature stress.First,a total of 100 pairs of primers were screened from 420 pairs of primers.The amplified products produced 2060 bands.The methylation level was 24.3%at25?,and the methylation level was 32.2%after 3 days of low temperature stress treatment at4?.The methylation level was up to 41.4%.After the formation of the fruiting body,The baseline level was reduced to 38.7%.DNA methylation pattern analysis showed that the frequency of methylation of CG and CHG methylation sites was higher than that of their corresponding methylation.The frequency of mutation of CG was higher than that of CHG The frequency of variation.3.Genetic function analysis of DNA methylation mutations in P.tuoliensis induced by low temperature stressThe study found that low-temperature treatment showed a slightly higher DNA methylation variation,and full-methylated levels were significantly higher than hemi-methylated levels,full-methylated from 12.62%to 19.10%,hemi-methylated from 3.84%to 9.21%.Low temperature stimulation causes CG hypermethylation to cause even greater changes while CG hypo-changes are minimal.?Day 3?and 13??Day 3?,most of the genes showed significant changes,calcium ion signal and osmotic regulation gene and low temperature defense protein-related gene expression in the analysis of related gene regulation expression For down-regulation.4.DNA methylation modification of Bisulfite-sequencing induced by total genome of P.tuoliensis under low temperature stressThe genomic DNA methylation average of CG and no-CG motifs of P.tuoliensis were CG54.65%,CHG 8.48%and CHH 36.86%,respectively.In the P.tuoliensis genome,the CG locus is generally at a high methylation level?most CG methylation sites are at 10%-20%methylation level,or are not methylated?,the trend of the CG site Relatively slow,CHG site methylation level is relatively low.After the low temperature treatment of P.tuoliensis,the motifs of genomic methylation were CG 47.14%,CHG 10.28%and CHH 42.58%.The methylation level of intron was higher than that of exon methylation,and the promoter-Exons-Introns showed a tendency of depression.Three motifs,CG is always higher than CHG and always higher than CHH,and although the molecular mechanism is different,their distribution on the chromosome is consistent.