Transcriptome And MiRNA Analyses Of Papaya Sex Reversal Mutant

Papaya is an important diploid tropical herbaceous fruit tree which is perennial dicotyledon,and native to Central America.Now papaya is widely cultivated in the tropical and subtropical regions.Papaya has three types of sex:female,male and hermaphrodite,and the corresponding genotype are XX,XY,XYh.Y and Yh have a high degree of similarity.To study of sex chromosome evolution,papaya plants is the ideal material.In papaya,Sex chromosomes and sex determining genes play an important role in the process of sex differentiation.miRNAs can alter the sex of plants by regulating the expression of sex-related genes.In addition,plant hormones and environmental factors can also affect the growth and development of plants.In this study,hermaphrodite flower from EMS treatment plants and male flowers from normal phenotype plants were analyzed by RNA-seq sequencing.After compare the transcripts between male and hermaphrodite flowers,we identified important genes related to papaya sex differentiation.We found the regulatory miRNAs related by small RNA-seq technique.The results provide an theoretical basis for revealing the molecular mechanism of pistil development in male flowers and plant sex differentiation.The results are as follows:(1)Identification of important genes in sex differentiation by mRNA-seqWe took>5mm flowers as the materials from the mutant "MMH"and normal male plant "AU9".After high throughput sequencing,the changes of gene expression about sex change were compared and analyzed by technique of transcriptome sequencing.A total of 278 differentially expressed genes(DEGs)were detected between the mutants and control.By comparing the homologous genes with Arabidopsis thaliana,we obtained 26 transcription factors,25 hormone-related genes,and 14 transpoter proteins.The results showed that many of the ethylene-related genes were up-regulated in MMH,but the expression of GBF1 protein induced by light was down-regulated.In addition we also found that C2H2-type zinc finger protein was up-regulated in hermaphrodite flowers.Furthermore,Our GO,KEGG,COG enrichment results showed significant changes in the expression levels of genes related to glucose,sucrose and starch metabolism in bisexual flowers.These results suggest that light,phytohormones and carbohydrates may be involved in the regulation of papaya sex differentiation and abortion of pistil development.We also obtained a lot of enrichment results associated with metabolic enzymes,which indicating that the accumulation of endogenous hormones in plants was affected by changes in the expression levels of some important metabolic enzymes and regulated sex differentiation of papaya.(2)Identification of miRNA that regulate sex determining genesSimilarly,we also took more>5mm flowers as the materials from the mutant "MMH" and normal male plant "AU9",and then constructed and sequenced small RNA library.115 miRNAs were identified.including 59 homologous miRNAs from 23 miRNA families and 56 novel miRNAs.miR159,miR160,miR393,miR167 and other miRNAs related to flower organ development were found.We also analyzed the differential expression of these 115 miRNAs,48 differentially expressed homologous miRNAs and 18 differentially expressed novel miRNAs between MMH and AU9.We obtained 692 target genes from differentially expressed miRNAs.Finally,the target gene function was categorized by Arabidopsis thaliana database,GO,KEGG and COG enrichment.We found that transcription factor in the target gene contains a large number of genes related to protein kinase,and the GO enrichment of the target genes is also enriched with protein kinase activity,which may indicate that the sex differentiation are related to the expression of plant hormones in adversity.Many target genes about NAC are differentialy expressed,and NAC genes are associated with floral organ development.Our miRNA target genes also found that C2H2 type zinc finger protein which is mediated by miR394,which is the same as our analysis of differential expression mRNA results.miR394 may be an important factor regulating sex differentiation in papaya.The target genes were also enriched in the plant signal transduction pathway.Some of the above evidence suggests that phytohormone,plant hormones and C2H2 type zinc finger protein may be involved in the pistil development of male papaya.