Functional Study of CmCRC Gene Influencing Sex Differentiation in Melon

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

Fig. 6 Phenotypes of T0 melon plants

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