目的 基于转录组测序数据鉴定栝楼Trichosanthes kirilowii ERF基因家族（TkERFs）成员，解析其在栝楼生长发育中的生物学功能，为深入研究栝楼性别分化机制提供理论依据。方法 采用生物信息学方法系统鉴定栝楼ERF基因家族成员，并筛选性别分化关键候选基因TkERF2进行功能解析。利用实时荧光定量PCR（qRT-PCR）技术分析其组织表达模式；构建TkERF2过表达载体，通过亚细胞定位及拟南芥遗传转化实验，结合表型观察、相关基因表达量检测及乙烯含量测定，探究其生物学功能；整合转录组与small RNA组学数据，筛选靶向调控TkERFs的miRNAs，完善其调控网络。结果 基于转录组数据，经开放阅读框（open reading frame，ORF）及保守结构域分析，共鉴定出54个TkERF基因（TkERF1～TkERF54）。其编码蛋白序列长度为101～389个氨基酸，相对分子质量为11 580～43 310，理论等电点（pI）为4.66～10.60。亚细胞定位预测显示74%的TkERFs定位于细胞核，26%分布于叶绿体及细胞质。蛋白质保守基序分析鉴定出10种motif。系统发育树将该家族划分为8个亚族。筛选鉴定出TkERF2、TkERF20和TkERF45受特异性miRNA靶向调控。转基因拟南芥实验表明，TkERF2显著调控乙烯合成途径，且生长素合成基因（AtYUC2、AtYUC6）、生长素响应因子（AtARF8）及生长素转运基因（AtPIN2）的表达量均显著高于野生型（P＜0.05），证实TkERF2通过正向调控生长素合成与转运参与发育进程。结论 完成栝楼ERF基因家族的系统鉴定与功能初探，为解析栝楼性别决定机制及遗传改良提供了理论基础。

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.

R286.12

山东省自然基金面上项目（ZR2024MH046）;山东省大学生创新创业项目（202510452030）