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The Landsberg erecta (Ler) Arabidopsis and the crc-1 mutant were obtained from Zhang's lab. The Arabidopsis were cultivated in a growth chamber under 16/8 h light/dark at 22 °C. Cucumber inbred line R1461 were grown in the glasshouse of Inner Mongolia University in Hohhot under standard environment control. The GUS reporter gene recombinant construct and CsCRC overexpression vector were transferred into Agrobacterium and transformed into Ler and crc-1 mutant.
RT-qPCR
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The young ovaries of R1461 were used for RNA extraction. The synthesized cDNA was used as the template for qPCR. The SYBR Premix Taq Mix (Takara) was used for RT-qPCR assay and an Applied Biosystems 7500 real-time PCR system was used for statistical analysis. Arabidopsis ACTIN2 and cucumber UBI were used as reference genes, respectively. For each RT-qPCR analysis, three biological replicates and three technical replicates were performed.
Yeast One-Hybrid assay
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CsPIF1 coding sequence was cloned into the pGADT7 vector (Clontech, CA, USA). The putative PIFs binding sites were found at the promoter of CsCRC and then cloned with the pAbAi vector (Clontech, CA, USA). The resultant constructs were introduced into the yeast Y1H Gold strain and selected by different AbA (Aureobasidin A) concentration on the SD/-LEU (Synthetic Dropout Medium/-Leucine) medium (Clontech, CA, USA).
LUC/REN assay
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The 2038 bp upstream sequence of CsCRC was inserted into pGreenII 0800 vector and used as reporter. The coding sequence of CsPIF1 was cloned and constructed with pGreenII 62-SK vector. The constructs were introduced into Agrobacterium tumefaciens strain GV3101 together with pSUPER and p19 plasmid[20]. The bacterial fluid of effector: reporter (9:1) construct cell mixture was injected into young leaves of Nicotiana benthamiana. The LUC/REN ratio was measured by luciferin (Promega, USA). A strain of the proCsCRC and empty effector was used as a negative control.
Yeast Two-Hybrid assay
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Coding sequence of CsCRC, CsSPT1, and CsINO genes were inserted into pGADT7 and pGBKT7 vectors. All constructs were transformed into yeast strain AH109 for preparation. Yeast two-hybrid assays were performed according to the description of Matchmaker TM GAL4 Two-Hybrid System 3 & Libraries (Clontech, CA, USA). Combination of each gene with blank vector pGADT7 and pGBKT7 were used as negative control. The primers used for Y2H vector construction are listed in Supplemental Table S1.
Bimolecular Fluorescence Complementation (BiFC) assay
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The coding sequence of CsCRC, CsSPT1, and CsINO which are lacking a stop codon were inserted into the fluorescence vectors pSPYCE-35S and pSPYNE-35S. Those constructs were transformed into Agrobacterium GV3101. Each combination of two proteins was injected into the young leaves of tobacco. The infected tobacco leaves were sampled and observed through Zeiss LSM 510 Meta confocal laser microscopy under 488 nm excitation wavelength. AtIND-YFPC and AtSPT-YFPN interaction was used as a positive control[21].
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About this article
Cite this article
Che G, Song W, Zhang X. 2023. Gene network associates with CsCRC regulating fruit elongation in cucumber. Vegetable Research 3:7 doi: 10.48130/VR-2023-0007
Gene network associates with CsCRC regulating fruit elongation in cucumber
- Received: 09 December 2022
- Accepted: 04 January 2023
- Published online: 02 March 2023
Abstract: In cucumber (Cucumis sativus L.), fruit length is a crucial agronomic trait that affects appearance quality and crop yield. Different cucumber germplasms exhibit a large variation in fruit length, and the underlying candidate genes and regulatory mechanisms need to be further characterized. Recently we showed that an essential gene in modulating the cucumber fruit length, the YABBY transcription factor CRABS CLAW (CsCRC), activates its downstream target gene auxin responsive protein CsARP1 to promote cell expansion and fruit elongation. Here, we show that auxin activity and three fruit development-related genes are involved in the CsCRC-regulated gene network in cucumber. We found that a PHYTOCHROME INTERACTING FACTOR (CsPIF1) protein elevates CsCRC transcription by directly binding to its promoter. The protein interaction assays showed that a reproductive YABBY transcription factor INNER NO OUTER (CsINO) and a bHLH protein SPATULA (CsSPT1) have direct interaction with CsCRC, suggesting their redundant role in cucumber fruit development.
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Key words:
- CsCRC /
- gene network /
- cucumber /
- fruit development