| Apostichopus japonicus is one of the most important economic breeding species in northern coastal areas of China.Temperature is an important environmental factor affecting the growth and breeding of A.japonicus.The frequent occurrence of high temperature in summer has a serious impact on the cultivation of A.japonicus.A large number of A.japonicus skin rot and die,causing huge economic losses.A.japonicus needs to resist and adapt to high temperature stress through its own defense mechanism.Epigenetic modification plays an important role in this regulation process.At present,the main sources of the relative ginseng in the seedling industry are "wild" and the artificial culture of sea cucumber.The basic population of the seedling breeding is composed of the phenomenon of slow growth of offspring,large individual difference,poor stress and weak resistance to resistance to disease,which seriously affects the healthy development of the industry.Therefore,it is an important way to cultivate new varieties of A.japonicus with good stress resistance to solve the problem of lack of good germplasm and safe summer.It is important to explore the regulation mechanism of epigenetic.The main results are as follows:1.Using MSAP technology to investigate and analyze the effect of selection activities on genomic DNA methylation from an epigenetic perspective.The muscle tissue of A.japonicus was taken as experimental tissue,the genetic diversity of control population,selected population F1,and selected population F4 were analyzed using methylation-sensitive amplification polymorphisms(MSAP).The results showed that698 of 806 loci were polymorphic in both selected populations.Based on the genetic analysis of non-methylated loci,the Shannon polymorphism index of the F4 was 0.3981 and Nei gene diversity was 0.2264.Based on the analysis of methylation sensitive sites,the Shannon polymorphism index of the F4 was 0.5873,and Nei gene diversity was0.2598,both of which were higher than in the parent population.Moreover,the epigenetic diversity was higher than the sequence genetic diversity caused by variation in nonmethylated loci,indicating that the frequency of epigenetic variation was higher than that of sequence genetic variation.Analysis of the MSAP methylation patterns revealed some changes in the methylation levels and patterns in the selected F1 and F4 after breeding,which indicated that the genome methylation status of the A.japonicus population was changed by breeding under temperature stress.The number of type Ⅱ bands in the F4 was161,which was significantly higher than that of control population of A.japonicus and may have acquired epigenetic characteristics during breeding.Collectively,our results revealed the genetic changes and progress of the breeding population from the perspective of a genetic material basis and provide a reference for the study of epigenetics in the breeding of new stress resistant varieties.2.The respiratory tree,digestive tract,muscle and internal wall of A.japonicus were used as experimental tissues.The experimental conditions were acute warming and chronic warming were used to analyze the expression levels of the gene hsp70 and the complement C3 gene region by q RT-PCR.The methylation rate was measured and analyzed,and the correlation between the gene expression level and promoter methylation rate was analyzed.The expression of hsp70 was significantly up-regulated in the respiratory tree,digestive tract,muscle,and body wall at 30 ℃ and 34 ℃,and significantly higher than the T18、T22、T26 groups.During the process of chronic heating of A.japonicus,the expression of hsp70 gene in different tissues was up-regulated at the stage of maintaining26 ℃,and the expression level decreased when the water temperature returned to the initial temperature.Different experimental tissues of hsp70 showed different methylation patterns: in respiratory tree,T34 group had the highest methylation rate,in digestive tract,T26 group,in muscle,T26 and T34 groups had the highest methylation rate,and in body wall,T22 group had the highest methylation rate.The correlation coefficient between hsp70 gene expression and promoter methylation rate was 0.883 under acute warming condition,and there was significant relevant between them(P<0.05).In the digestive tract,muscle and body wall,the correlation coefficients between gene expression and promoter methylation rate were negative,indicating that there was a negative correlation between them,but there was no significant difference.During the chronic warming of A.japonicus,the methylation rate of hsp70 in four experimental tissues was the highest in 23℃,24 h group.The gene expression of hsp70 was negatively correlated with methylation rate in respiratory tree,muscle and body wall.But positively correlated with methylation rate in digestive tract,and the correlation coefficient was 0.064.Under the condition of acute warming,the expression pattern of complement C3 in digestive tract and longitudinal muscle was the same,the expression level was first down regulated to a lower level,and then up-regulated significantly under the stimulation of34 ℃;the expression pattern of respiratory tree was up-regulated at 22 ℃,then down regulated to a lower level,and then up-regulated to a higher level at 34 ℃.Under the condition of acute warming,the correlation coefficients of C3 gene expression and promoter methylation rate in the respiratory tree,digestive tract and muscle were 0.130,0.102 and 0.541,showing a positive correlation,but no significant.During the chronic warming of A.japonicus,the expression of complement C3 was low in all tissues,and there was no obvious trend.The correlation coefficient between the expression of complement C3 and promoter methylation rate in respiratory tree was-0.960 under this temperature condition,and the two had a very significant negative correlation(P<0.01).The correlation coefficient between C3 gene expression and promoter methylation rate in digestive tract was-0.641,which indicated that there was a negative correlation,but the correlation was not significant.In muscle,the correlation coefficient between the two was low,and there was no significant.The results showed that there was different correlation between gene expression and methylation rate in different tissues under different conditions,which indicated that the relationship between DNA methylation and gene expression was regulated by many factors.At the same time,it could provide a certain direction for the study of epigenetics and gene expression of sea cucumber. |
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