CmCRC基因影响甜瓜性别分化的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2026-0068

摘要/Abstract

摘要：

目的研究甜瓜（Cucumis melo L.）CmCRC基因在花器官发育，尤其是性别分化中的功能，为解析甜瓜的性别决定机制及创制甜瓜全雄株提供依据。方法以甜瓜品种‘IVF05’为材料，利用CRISPR/Cas9基因编辑技术靶向敲除CmCRC基因，获得突变体株系并进行全生育期的表型观察；通过自交获得未发生基因编辑植株的T₁代种子，对其进行基因编辑鉴定和表型分析；采用实时荧光定量PCR（RT-qPCR）检测T₁代植株花器官中CmCRC基因的表达。结果与对照相比，CmCRC突变体营养生长正常，但生殖生长表现为全雄株，无法形成雌花，不能正常结实。未发生编辑的T₀植株（CR-CmCRC-23和CR-CmCRC-48）在T₁代发生编辑后，表型与T₀代突变体一致，完全丧失两性花形成能力，仅产生雄花。RT-qPCR显示，T₁代突变体雄花花药中CmCRC表达量显著下调，表明CmCRC功能缺失及表达下调是导致性别表型改变的直接原因。结论 CmCRC基因调控甜瓜两性花的形成，在性别决定机制中发挥关键作用。

关键词: 甜瓜, CmCRC, 遗传转化, 基因编辑, 阳性鉴定, 性别决定, 全雄株

Abstract:

Objective This study is aimed to investigate the function of the CmCRC gene in melon (Cucumis melo L.) during floral organ development, especially sex differentiation, so as to provide a theoretical basis for elucidating the sex-determining mechanism and creating all-male melon lines. Method Using the melon cultivar ‘IVF05’ as material, the CmCRC gene was targeted and knocked out via the CRISPR/Cas9 genome-editing system. Mutant lines were obtained and subjected to phenotypic observation throughout the whole growth period.T₁seeds derived from self-pollinated non-edited plants were used for gene-editing identification and phenotypic analysis. The expression of CmCRC in floral organs of T₁ plants was detected by quantitative real-time PCR (RT-qPCR). Result Compared with the wild-type control, CmCRC mutants exhibited normal vegetative growth but showed an all-male phenotype during reproductive growth, failing to produce female flowers and set fruits normally. When T₀ non-edited plants (CR-CmCRC-23 and CR-CmCRC-48) produced edited T₁ progeny, the T₁mutants displayed a phenotype consistent with that of T₀ mutants: they completely lost the ability to form bisexual flowers and only produced male flowers. RT-qPCR analysis showed that CmCRC expression was significantly downregulated in the anthers of male flowers from T₁ mutants, indicating that loss of function and reduced expression of CmCRC directly caused the alteration of sexual phenotype. Conclusion These results demonstrate that the CmCRC gene regulates the formation of bisexual flowers in melon and plays a critical role in the sex-determining mechanism.

Key words: melon, CmCRC, genetic transformation, genome editing, positive identification, sex determination, all-male line

袁梦博, 赵光伟, 贺玉花, 黄祥, 徐永阳, 张健, 孔维虎, 田小琴, 户克云, 唐伶俐. CmCRC基因影响甜瓜性别分化的功能研究[J]. 生物技术通报, 2026, 42(3): 294-301.

YUAN Meng-bo, ZHAO Guang-wei, HE Yu-hua, HUANG Xiang, XU Yong-yang, ZHANG Jian, KONG Wei-hu, TIAN Xiao-qin, HU Ke-yun, TANG Ling-li. Functional Study of CmCRC Gene Influencing Sex Differentiation in Melon[J]. Biotechnology Bulletin, 2026, 42(3): 294-301.

图/表 7

表1 基因表达量分析引物

Table 1 Primers for gene expression analysis

基因 Gene	引物序列 Primer sequence（5′‒3′）
Bar-F	AACCACTACATCGAGACAAGC
Bar-R	CTGAAGTCCAGCTGCCAGA
CR-CmCRC-F	CCCTCTCCATATTTCCTAATTT
CR-CmCRC-R	TAGAGGATGATCAATGCATT
RT-CmCRC-F	GAGGAAATTCAACGTATCAAAG
RT-CmCRC-R	ACCCAGCAGCTGAATTAGGA
RT-ACTIN-F	CAGGTGTTATGGTCGGAATG
RT-ACTIN-R	GAGCGACACGAAGTTCAT

图1 基于PRJNA803327转录组项目的CmCRC组织特异性转录水平

Fig. 1 Tissue-specific transcription levels of CmCRC based on the transcriptome project PRJNA803327

图2 甜瓜表达谱网站的CmCRC组织特异性表达情况外果皮和果肉

Fig. 2 Tissue-specific expression pattern of CmCRC in melon from the expression profile databaseEpicarp (ep) and flesh (fl)

图3 甜瓜T0代CmCRC基因编辑植株靶点区域PCR扩增WT：野生型材料；1‒70：70株阳性T0代株系

Fig. 3 PCR amplification of the target region in CmCRC-edited T0 melon seedlingsWT: Wild type. 1‒70: 70 positive T0 transgenic lines

图4 甜瓜T0代CmCRC基因编辑植株编辑情况鉴定

Fig. 4 Identification of editing events in CmCRC-edited T0 melon seedlings

图5 甜瓜T0代转基因植株花性别分析-：全雄花；+：有两性花分化；编号为突变体植株号

Fig. 5 Sex differentiation analysis of T0 transgenic seedlings in melon-: Male flower. +: Bisexual flower. Numbered as mutant plant ID

图6 甜瓜T0代植株表型

Fig. 6 Phenotypes of T0 melon plants

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