Screening,Cloning And Expression Analysis Of Sex Differentiation-related Genes In Sphaeropteris Lepifera

The sex differentiation of ferns has always been a hot topic of research.5-azacytidine(5-aza C)is a DNA methylation inhibitor.Previous studies have confirmed that 5-aza C promotes the formation of male gametophytes in Sphaeropteris lepifera,indicating that DNA methylation is associated with sex differentiation of Sphaeropteris lepifera.N6-methyladenine(6m A)is a DNA methylation modification existing in eukaryotic genomes,and 6m A methyltransferase(N6AMT1)is one of the most important enzymes in 6m A modification.In this study,the full-length of Sl N6AMT1 gene was obtained by RACE method by screening the transcriptome data of gametophytes treated with 5-aza C.The bioinformatics analysis and expression pattern analysis were carried out to reveal the molecular mechanism of sex differentiation of gametophytes and lay a foundation for improving the relationship between DNA methylation and sex differentiation.The findings are as follows:(1)The stability of expression levels of five commonly used reference genes at different developmental stages of Sphaeropteris lepifera and different developmental stages of Sphaeropteris lepifera gametophytes was evaluated,and relatively stable reference genes were screened.The stability of Actin was the best when evaluating different developmental stages of the Sphaeropteris lepifera,while the stability of EF-1a was the best when evaluating different developmental stages of the Sphaeropteris lepifera gametophyte,followed by Actin.Therefore,it is more appropriate to use Actin as an internal reference gene when analyzing the expression pattern of key genes in the Sphaeropteris lepifera gametophyte period.(2)In this study,the transcriptome data of gametophytes under 5-aza C treatment were analyzed.Combined with GO analysis and KEGG analysis,16 genes were screened from differentially expressed genes.q RT-PCR analysis was performed to screen out the genes c61872.graph＿c0 and c62234.graph＿c0 with significant increase in gene expression during a large number of sex differentiation periods in the tree.According to the gene annotation,the N6 DNA methyltransferase gene(N6AMT1)was selected for subsequent experiments.(3)The SlN6AMT1 gene was cloned by RACE technique.Finally,a full-length gene sequence of 1097 bp was obtained and named Sl N6AMT1.(4)The amino acid sequence of Sl N6AMT1 gene was analyzed.The bioinformatics analysis of Sl N6AMT1 protein by online software showed that Sl N6AMT1 protein was a hydrophilic protein and did not contain a transmembrane domain.MEGA5.0 was used to compare the homologous proteins of the pencil tree and the phylogenetic tree was constructed.It was found that the Sl N6AMT1 protein was a conserved protein,which had the highest homology with algae.(5)The expression changes of Sl N6AMT1 gene in different developmental stages,gametophyte development stages and gametophytes of different sexes were analyzed by q RT-PCR.It is speculated that Sl N6AMT1 gene can promote the production of female gametophytes,further promote the sex differentiation of Sphaeropteris lepifera and play a crucial role in sex differentiation.