ObjectiveTo identify the members of the ERF gene family (TkERFs) of Trichosanthes kirilowii based on transcriptome sequencing data, and to analyze their biological functions in the growth and development of T. kirilowii, so as to provide a theoretical basis for further studying the mechanism of sex differentiation in T. kirilowii. Methods Bioinformatics methods were used to systematically identify the ERF gene family members of T. kirilowii, and the key candidate gene for sex differentiation, TkERF2, was screened for functional elucidation. The tissue expression patterns of the tissues were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The TkERF2 overexpression vector was constructed and its biological function was explored through subcellular localization and genetic transformation experiments of Arabidopsis thaliana, combined with phenotypic observation, related gene expression detection and ethylene content determination. Transcriptome and small RNA omics data were integrated to screen miRNAs that target the regulation of TkERFs and improve their regulatory networks. Results Based on transcriptome data, a total of 54 TkERF genes (TkERF1—TkERF54) were identified by open reading frame (ORF) and conserved domain analysis. It encodes a protein sequence length of 101—389 amino acids, a molecular weight of 11 580—43 310, and a theoretical isoelectric point (pI) of 4.66—10.60. Subcellular localization prediction showed that 74% of TkERFs were localized in the nucleus and 26% were distributed in chloroplasts and cytoplasm. Protein conservation motif analysis identified 10 motifs. The phylogenetic tree divides the family into eight subfamilies. TkERF2, TkERF20 and TkERF45 were identified by specific miRNA targeting. Transgenic A. thaliana experiments showed that TkERF2 significantly regulated ethylene synthesis pathway, and the expressions of auxin synthesis genes (AtYUC2, AtYUC6), auxin response factor (AtARF8) and auxin transport gene (AtPIN2) were significantly higher than those of wild type (P < 0.05), confirming that TkERF2 was involved in developmental process by positively regulating auxin synthesis and transport. Conclusion This study has completed the systematic identification and preliminary exploration of the ERF gene family, which provides a theoretical basis for analyzing the mechanism of sex determination and genetic improvement of T. kirilowii.

Trichosanthes kirilowii Maxim.  /  ERF gene  /  gender differentiation  /  ethylene  /  auxin