Cloning of CsGR-RBP3 and Its Functional Roles in Cold Tolerance of Harvested Cucumber

doi:10.13560/j.cnki.biotech.bull.1985.2024-1144

Abstract

Abstract:

Objective Cucumber (Cucumis sativus L.) is a cold-sensitive vegetable prone to chilling injury during postharvest storage, which limits the application of cold storage technology in postharvest cucumbers. Cold treatments induce the expression of CsGR-RBP3 (glycine-rich RNA-binding protein 3) in harvested cucumber fruit. Cloning CsGR-RBP3 and studying its functional roles in chilling tolerance of postharvest cucumber can provide candidate gene for the cultivation of new cold-resistant cucumber varieties. Method The coding sequence of CsGR-RBP3 was cloned using cDNA from cucumber peels as templates. Virus-induced gene silencing (VIGS) technology was used to knock-down CsGR-RBP3 expression in postharvest cucumbers to study the effects of the reduced expression on chilling injury in cold-stored cucumbers. RNA-Seq was used to identify potential pathways modulated by CsGR-RBP3 regulating chilling tolerance of cucumber fruit. Result Cucumber CsGR-RBP3 encoded a protein spanning 168 amino acid residues with a conserved RRM (RNA-recognition motif) domain, which might be a mitochondria-related protein and had low sequence similarity with Arabidopsis AtGR-RBPs. Reducing cold-inducible CsGR-RBP3 expression aggravated chilling injury of postharvest cucumbers during cold storage, downregulated the expression of cold-inducible genes, and reduced mitochondrial antioxidant enzyme activity and gene expression. Furthermore, differentially expressed genes (DEGs) potentially regulated by CsGR-RBP3 were significantly enriched in the phenylalanine metabolism, phenylpropanoid biosynthesis and plant-pathogen interaction pathways. Moreover, the expressions of genes in these three pathways were overall downregulated when cold-inducible expression of CsGR-RBP3 was restricted. These results indicated that the CsGR-RBP3 expression was positively correlated with cold resistance in cold-stored cucumbers, and it might maintain mitochondrial redox balance by regulating activities of mitochondrial-related antioxidant enzymes and integrating multiple defense pathways in cucumber fruit. Conclusion CsGR-RBP3 genehad important roles in cold tolerance of harvested cucumber fruit, which may might regulate cold tolerance through multiple defense pathways.

Key words: cucumber, chilling injury, chilling tolerance, glycine-rich RNA-binding protein, defense pathway

WANG Bin, LI Jian-rong, ZHAN Zhao-xia, YUAN Xiao. Cloning of CsGR-RBP3 and Its Functional Roles in Cold Tolerance of Harvested Cucumber[J]. Biotechnology Bulletin, 2025, 41(6): 155-166.

Figures/Tables 9

Table 1 Primer sequence

基因 Gene	正向序列 Forward sequence （5′‒3′）	反向序列 Reverse sequence （5′‒3′）	用途 Usage
GR-RBP3	ATGCAATTATTCCCCACACGA	TCAGTTTTTGTCACCACCACCA	TA克隆 TA cloning
GR-RBP3	atttggagaggacagggtaccATGCAATTATTCCCCACACGA	tctagaggatccccgggtaccGTTTTTGTCACCACCACCATAGC	亚细胞定位载体构建 Construction of subcellular localization vectors
GR-RBP3	agaaggcctccatggggatccTTCCAAGCTGTCTATCTTTCGAAC	cgtgagctcggtaccggatccGTTTTTGTCACCACCACCATAGC	VIGS实验载体构建 Vector construction of VIGS experiment
GR-RBP3	GCAGCCTTCCAAGCTGTCTA	ACTTGCTGAACGCTACCCTC	荧光定量PCR RT-qPCR
LEA5	CTCCATTCTCTTCAGGCGGG	GTAACCGGTAACGGGGTCTG
DREB1D	GCAGCTCACACGCTCTAAGT	GCGTTTGAGGAGGAGGTGTT
ERD15	CAAAGCTAAACCCGAACGCC	CCATGTCGAGGTTGTCACCA
DLP4	CTGAGGATGCGGGTTCAAGT	AGCTTTCTTGCACAGCTCCA
NAA3	GGAGAGGCTCAAACTTGGCT	ATGCCTCCAACCAGTGATGG
CAT1	GATCCTTACAGGCACCGACC	CCAACGGTCAACGAGGAGTT
CAT3	GAGAAGCTTTGCGTATGCGG	GGTGAGGACATTTGGGAGCA
APX3	TGGCCCAAAGGATGAGCTTT	GGGAGGGCGTTTGATTCGTA
APX4	TCCAGACCTGAAAACGCCAA	TTAACGCCACTTTGTGCTGC
APX6	GCCAAACTCAGCAACCTTGG	TCTGATAGCTCTCTCTTTCCGT
POD41	TTGCTTAGTGGGAGGCTGTG	GAGGTTGATTTCCGCATCGC
POD42	GTAGACCCTGTGCTGAACCC	CGTACTGTACAGCCTTGGGG
POD64	CTGTCAGGGCTGCAGCTTAT	GCCACGTTGTTCCCTACTGA
Actin	AGGCCGTTCTGTCCCTCTAC	AGCAAGGTCCAAACGGAGAA

Fig. 1 Electrophoresis images of PCR products of CsGR-RBP3 coding sequencesA: The PCR product amplified by cucumber cDNA as template. B: The PCR product amplified by recombinant plasmid as template. M: DNA marker; 1‒6: PCR products

Fig. 2 Multi sequence comparison (A) and RRM domain conservation analysis (B) of CsGR-RBP3 and Arabidopsis AtGR-RBP proteinsRRM conserved domain is indicated in blue box

Fig. 3 Phylogenetic (A) and subcellular localization (B) analysis of CsGR-RBP3 protein

Fig. 4 Effects of reducing cold-inducible CsGR-RBP3 expression on the chilling injury of cold-stored cucumber fruitA: Chlorophyll fluorescence of cucumber fruit. B: Chlorophyll fluorescence parameter, Fv/Fm. C: Chlorophyll fluorescence parameter, Y(NO). D: Relative electrical conductivity. * indicates significant difference at P<0.05 level. The same below

Fig. 5 Effects of reducing cold-inducible CsGR-RBP3 expression on the expressions of cold-responsive genes in cold-stored cucumber fruit

Fig. 6 Effects of reducing cold-inducible CsGR-RBP3 expressions on mitochondrial antioxidant enzyme activity and gene expression in cold-stored cucumber fruit

Fig. 7 Functional enrichment and expression pattern analysis of differentially expressed genes caused by reducing cold-inducible CsGR-RBP3 expressionA: Venn diagram of differentially expressed genes (DEGs). B: KEGG enrichment of overlapped DEGs from two comparison groups. C: Expression patterns of DEGs in the phenylalanine metabolism pathway. D: Expression patterns of DEGs in the plant-pathogen interaction pathway. E: Expression patterns of DEGs in the phenylpropanoid biosynthesis pathway

Fig. 8 GSEA analysis of differentially expressed genes caused by reducing cold-inducible CsGR-RBP3 expressionA: Phenylalanine metabolism pathway. B: Phenylpropanoid biosynthesis pathway. C: Plant-pathogen interaction pathway

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