雌雄异型异熟胡桃楸性别分化关键miRNA的鉴定和分析

[Objective]The dioecious heterozygous maturation is an intermediate transition form from monoecious to dioecious plants.The rare tree species Juglans mandshurica Maxim.is a dioecious heterozygous heteromorphic plant,which is divided into two types:female-first and male-first,and it is a good economic and ecological tree species in Northeast China.microRNA(miRNA)is a class of endogenous eukaryotic non-coding RNAs with a length of about 18-24 nt.In plants,they mainly regulate the expression of target genes by targeting complementary sequences,thus controlling many biological functions.However,the formation of miRNA-mediated sex differentiation and dioecious heterozygous maturation mechanisms in J.mandshurica is still unclear.[Result]In this study,we used high-throughput sequencing technology to analyse the microRNA sequencing of male and female flower buds of the male and female phenotypes of J.mandshurica,constructed 12 sRNA libraries and compared them with the reference genome,and identified a total of 310 known and 278 unknown miRNAs,and counted the lengths,families and other information of the miRNAs.129 significantly differentially expressed miRNAs were screened using the DEG package.these differentially expressed miRNAs were hierarchically clustered.638 miRNA target genes were predicted using the PSRobots package,and these target genes were subjected to GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analyses.these target genes were mainly enriched in the metabolic pathways,and were significantly enriched in flower-forming related pathways,such as the reproduction,floral development,and phytohormone signaling pathways.It was hypothesised that miR159c-3p,miR156d-3p,miR172d,miR171b,miR164a,miR167a,miR319a,miR399a,miR396t might be the key genes affecting sex differentiation in J.mandshurica,with miR164a,miR167c,and miR171c-5p probably being the key genes in J.mandshurica miR164a,miR167c and miR171c-5p may be the key genes for sex differentiation in J.mandshurica.These key genes regulate phytohormones such as growth hormone and gibberellin,respectively,and target many flower-forming key genes and transcription factors.A predictive regulatory network was established by combining these key miRNAs and their target genes.Nine miRNAs with significant differences were randomly selected for real-time fluorescence quantitative PCR detection and jointly analysed with transcriptome sequencing results to establish a differential miRNA-differential mRNA primary co-expression regulatory network.[Conclusion]These miRNAs were further uncovered to play an important role in sex differentiation in J.mandshurica,revealing the regulatory roles between miRNAs and some important transcription factors related to flower formation.The joint analysis with transcriptome data lays the foundation for multi-omics to reveal the regulatory network of sex differentiation in J.mandshurica.It is hypothesised that these key miRNAs mainly affect sex differentiation by regulating the expression level of target genes in Nutmeg,which in turn regulates its hormone secretion,such as the miR159 family regulates the GAMYB gene family to control the secretion of gibberellin,which induces sex differentiation through the gibberellin pathway.And the formation of dioecious heterozygous maturation which is an intermediate excessive morphology between monoecious and dioecious.This study lays the research foundation for the final discovery of the molecular mechanism of sex differentiation and dioecious heterozygous maturation in J.mandshurica.