VvAGAMOUS Regulates Carpel Development through VvCRABS CLAW in Grape

doi:10.13560/j.cnki.biotech.bull.1985.2024-1127

Abstract

Abstract:

Objective The expression characteristics of grape carpel development related genes VvAGAMOUS (VvAG) and VvCRABS CLAW (VvCRC) were analyzed. This work aims to investigate the molecular mechanism by which VvAG regulates grape carpel development via VvCRC gene, and to provide a theoretical basis for analyzing the molecular mechanism of grapefruit formation. Method The inflorescences of different lengths of 'Musct Hambourg' grapes were used as materials, and were made into paraffin sections for observing the development of carpels. The expressions of VvAG and VvCRC at different developmental stages was analyzed using real time fluorescence quantitative PCR (RT-qPCR). The VvAG and VvCRC genes were cloned and subjected to bioinformatic analyses, including homology sequence alignment and phylogenetic relationships. The subcellular localization of proteins encoded by two genes were conducted by transient injection of tobacco leaves. Finally, the relationship between VvAG and VvCRC was verified using the yeast one-hybrid assay and dual luciferase assay. Result Anatomical observations indicated that the stamen primordia were basically fully developed and the carpel primordium was ready to form when the inflorescence length of 'Musct Hambourg' grapes was 1‒2 cm. As the inflorescence developed to 2‒3 cm and 3‒4 cm, the carpel continued to develop and the ovule primordia began to form. Fluorescence quantitative analysis revealed that the expressions of VvAG and VvCRC were low when the inflorescence length was 1‒2 cm, while the expression of VvCRC gene increased rapidly with the increase of VvAG gene during the two stages of inflorescence length of 2‒3 cm and 3‒4 cm. Phylogenetic analyses showed that VvAG and AtAG, and VvCRC and MdCRC clustered into a small branch with close genetic relationships. The results of the subcellular localization assay indicated that both VvAG and VvCRC proteins were located in the nucleus. Yeast one-hybrid and dual luciferase assays showed that VvAG can directly bind to the VvCRC promoter and activate its transcriptional activity. Conclusion The VvAG transcription factor may further regulate carpel development by activating the expression of the VvCRC gene, thereby affecting the development of grape ovary.

Key words: grape, carpel, VvAGAMOUS, VvCRABS CLAW, growth and development, expression characteristics, transcriptional regulation

ZAN Shu-wen, XIE Huan-huan, ZHANG Yu-qin, WANG Wen-Juan, ZHANG Peng-fei, LIANG Jin-jun, WEN Peng-fei. VvAGAMOUS Regulates Carpel Development through VvCRABS CLAW in Grape[J]. Biotechnology Bulletin, 2025, 41(5): 208-217.

Figures/Tables 8

Fig. 1 Anatomical structure of grape ‘Musct Hambourg’ at different developmental stagesA: 0‒1 cm; B: 1-2 cm; C: 2-3 cm; D: 3-4 cm; E: 4‒5 cm. s: Sepals; f: flower caps; st: stamens; c: carpels; o: ovules. Scale bar=100 μm

Fig. 2 Expressions of VvAG and VvCRC in different inflorescence development stagesDifferent lowercase letters indicate significant differences of each gene at the 0.05 level during the five periods of inflorescence development

Fig. 3 Amplified results of VvAG (A)and VvCRC (B)A: 1-2: PCR amplification bands of VvAG gene; B: 1-2: PCR amplification bands of VvCRC gene. M: DL2000 marker

Fig. 4 A Multiple alignment of homologous amino acid sequences of VvAG and VvCRCA: Multiple alignment of homologous amino acid sequences of VvAG.B: Multiple alignment of homologous amino acid sequences of VvCRC. Black: 100% homology. Purple: Homology≥75%. Blue: Homology≥50%

Fig. 5 Phylogenetic tree of VvAG (A) and VvCRC (B) homologous proteins from different specie

Fig. 6 Subcellular localization results of VvAG and VvCRC

Fig. 7 Analysis of the regulatory role of VvAG on the VvCRC promoterA: Schematic diagram of the vector for the dual luciferase assay. B: Y1H validation of the interaction between VvAG and the VvCRC promoter. C: LUC assay to validate the effect of VvAG on VvCRC promoter activity. Asterisks indicate significant differences (* P<0.05, ** P<0.01, t-test used)

Fig. 8 Model diagram of VvAG regulation of grape carpel development through VvCRC

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