The Relationship Bewteen Sex Differentiation And DNA Methylation In Sphaeropteris Lepifera

The Sphaeropteris lepifera(Sphaeropteris lepifera(Hook.)R.M.Tryon)belongs to the only tree ferns of the Jurassic period,the tree ferns,which play a unique role in the evolution of the plant system.In recent years,the growth environment of the wild population of Sphaeropteris lepifera has been destroyed,the number of seedlings has decreasedand the survival rate is low.Therefore,it is of great significance to study the reproductive mechanism of Sphaeropteris lepifera to solve their reproductive disorders.As an important part of epigenetics,DNA methylation plays a key role in the whole process of plant growth and development.At present,there is no unified view on whether DNA methylation patterns can be stably inherited and whether they act as inducing factors in sex differentiation.However,in the process of reproduction,many genes are expressed by demethylation.It can be seen that DNA methylation is essential for plant growth and development.S-adenosylmethionine(SAM),as a methyl donor of DNA methylation,plays an important role in plant growth and development and resistance to stress.5-Azacytosine nucleoside(5-azaC)can bind to methyltransferase by intercalating DNA,thereby inhibiting the maintenance of methylation status.Therefore,we used these two chemical substances to treat the gametophyte of Sphaeropteris lepifera,in order to explore the relationship between DNA methylation and sex differentiation of the Sphaeropteris lepifera.The results are as follows:(1)The Sphaeropteris lepifera gametophyte was treated with 5-azaC and SAM.By counting the seedling rate,the ratio of male to female gametophyte and the number of gametophytic spermatozoon,it was found that 5-azaC had little effect on the seedling rate of the Sphaeropteris lepifera.SAM treatment Sphaeropteris lepifera holder the seedling rate of the Sphaeropteris lepifera;5-azaC promoted the differentiation of the male gametophyte,especially at a concentration of 100 ?mol;SAM can promote the differentiation of male and female gametophytes,and reduce the ratio of asexual gametophytes(undifferentiated).To a certain extent,SAM can promote the early development of gametophyte.5-azaC can greatly promote the development of male gametophytes,including the increase in the number of spermatozoa and the expansion of the spermatozoon distribution.(2)The total cytosine methylation rate of 5-azaC and SAM treated Sphaeropteris lepifera gametophytes was studied by LC-MS/MS method.The results showed that5-azaC could increase the methylation rate of gametophyte in Sphaeropteris lepifera,and reached about 18.80%,18.04%,23.10% and 29.45% at 3h,24 h,48h and 96 h.The methylation rate of the Sphaeropteris lepifera gametophyte was treated by SAM,and the methylation rate increased first and then decreased,reaching about 11.80%,5.06%,4.82%,and 4.92% at 3h,24 h,48h,and 96 h,respectively.(3)Changes in DNA methylation of the 5-azaC and SAM-treated Sphaeropteris lepifera gametophyte DNA were determined using the MSAP method.Conclusion:selective amplification were amplified polymorphism bands,2411,the total number of methylation stripe between 940 ~ 1166,the control group total methylation of DNA CCGG point Sphaeropteris lepifera at a rate of 38.99%,the methylation rate of 16.76%,half the methylation rate was 22.23%,the external single-strand DNA methylation,double-stranded DNA methylation internal and external double-stranded DNA methylation accounted for 18.08%,24.89% and 57% respectively.The 5-azac treatment could improve the total methylation rate(42.51%),reduce the total methylation rate(16.26%),and increase the semi-methylation rate(26.26%)of the DNA CCGG sites in the Sphaeropteris lepifera,and the methylation polymorphism rate was 84.25%,with significant demethylation rate(52.91%)and small hypermethylation rate(31.34%).SAM treatment could increase the total methylation rate(48.36%),increase the total methylation rate(31.36%),reduce the semi-methylation rate(19.33%),increase the polymorphism rate of methylation(73.53%),decrease the proportion of demethylation(33.21%),and increase the proportion of hypermethylation(40,32%).