Identification and Analysis of Grape（Vitis vinifera L.）CYP707A Gene Family and Functional Verification to Fruit Ripening

doi:10.13560/j.cnki.biotech.bull.1985.2023-0856

Abstract

Abstract:

【Objective】 CYP707A is a subfamily member of cytochrome P450 family, encoding ABA8'-hydroxylase, which is a key enzyme in ABA catabolism and plays an important role in plant growth and development. Identification of grape（Vitis vinifera L.）CYP707A gene family may provide theoretical basis for further study of grape fruit ripening mechanism and biological breeding techniques.【Method】 Bioinformatics methods were used to analyze the gene structure, protein motif, chromosome localization, collinearity, phylogenetic relationship and cis-acting elements of promoter region of CYP707A family gene in the grape. RT-qPCR was used to analyze the spatiotemporal expression patterns of VvCYP707As gene in tissues, different grape varieties（early and medium maturity variety）and in response to the exogenous ABA treatment were analyzed by. Fruit transient transfection was applied to verify the function of Vitvi07g01751.【Result】 There were five VvCYP707As genes identified in grape genome, located on five chromosomes, among which Vitvi02g01269, Vitvi07g01751 and Vitvi18g00792 were fragment replicators. The CYP707A gene family had a high degree of conservation, sharing similarities in gene structure and protein motifs, and all subcellular localization were predominantly localized to the endoplasmic reticulum. The cis-acting elements of growth, development and hormone response were significantly enriched in the promoter regions of the CYP707A gene family, containing ABA hormone-corresponding elements. Furthermore, the RT-qPCR analysis demonstrated that VvCYP707A genes demonstrated both spatiotemporal- and tissue-specific expression patterns, with elevated expression levels during the early and expansion stages of fruit development as well as in stem and leaf tissues. Notably, Vitvi07g01751 gene was highly expressed in the fruits of the early maturity variety ‘Yong Zaohong’, and the expression pattern was consistent between these two varieties. The expression pattern of Vitvi07g01751first increased and then decreased with fruit development. Under exogenous ABA treatment, VvCYP707A showed two distinct expression patterns: it induced the high expression of 5 VvCYP707A genes in the grape flesh while significantly suppressing the expressions of these genes in the grape pericarp. Furthermore, upon transient expression of Vitvi07g01751 in the grape flesh, there was a slow accumulation of sugar content, accompanied by a significant upregulation of the ABA receptor VvPYL4 gene, suggesting that Vitvi07g01751 mediated ABA regulation of grape fruit ripens.【Cconclusion】Vitvi07g01751 plays an important role in fruit ripening of grape.

Key words: grape, CYP707A gene, genome-wide identification, bioinformatics, fruit ripening, abscisic acid, PYL gene

GONG Li-li, YU Hua, YANG Jie, CHEN Tian-chi, ZHAO Shuang-ying, WU Yue-yan. Identification and Analysis of Grape（Vitis vinifera L.）CYP707A Gene Family and Functional Verification to Fruit Ripening[J]. Biotechnology Bulletin, 2024, 40(2): 160-171.

Figures/Tables 11

Table 1 Primer information

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
Vitvi07g01751-F	ATGCTGCTGAAGAAGCCACA
Vitvi07g01751-R	TCAGCATTCCTTCCAAAACTTAGC
35S:VvCYP707A-F	ACGAGCTCGGTACCCGGGATGCTGCTGAAGAAGCCACAG
35S:VvCYP707A-R	GGGCGAATTGGTCGACGCATTCCTTCCAAAACTTAGCAGG

Table 2 Primers for RT-qPCR

基因Gene	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
Vitvi02g01269	F: GTGTTCTGCAAGCTGTCACG	R: TGGCATCTTCCCAAGTCAGAG
Vitvi03g00508	F: ATCGGCCTTTGACATGGGAT	R: AGTGGCATCACCTTCCAACC
Vitvi06g00498	F: GCAGCTTCTCAACGAGACTTTCAGG	R: GAATTCCCAGCAATCCACCTCCTTG
Vitvi07g01751	F: CTCTGCGGCTCTATTCCCAG	R: CTTGGTGGGTATGTGGGCTT
Vitvi18g00792	F: TTGTCCTGGAAATGAGCTTGCCAAG	R: CAATTCCACCTTGTGATCCCACCAC
Actin	F: CAAGAGAAACCATCCCTAGCTG	R: TCAATCTGTCTAGGAAAGGAAG
VvPYL1	F: CAAGAGAAACCATCCCTAGCTG	R: TCAATCTGTCTAGGAAAGGAAG
VvPYL2	F: TCCCTACTGTCTGGTCCGTC	R: CCAATCTCTCCGTGCTGGTC
VvPYL3	F: GACGAGTACATCCGCAGACA	R: TGATGTGCTTGACGAGAGAGG
VvPYL4	F: GTGCTCTTCCCTACTCGCTC	R: GAAGCGATGAACGACGGAAC
VvPYL5	F: ATAGACGGCTGTGATGGGTG	R: TGAACTGCACTGGTTCTCCC
VvPYL6	F: CCACTACCAGCACTGAAAGGT	R: TCGTCCTTGGTGTTTCCCTC

Fig. 1 Amino acid alignment（A）, collinearity analysis（B）and chromosome localization（C）of grape CYP707A gene family

Table 3 Information of CYP707A proteins in grape

基因ID Gene ID	氨基酸长度 Amino acid length/aa	分子量大小 Molecular weight/kD	等电点 pI	亚细胞定位 Subcellular location
Vitvi02g01269	470	53 118.2	9.03	内质网
Vitvi03g00508	479	54 538.9	9.68	内质网
Vitvi06g00498	488	55 496.0	10.00	内质网
Vitvi07g01751	447	50 689.3	9.50	内质网
Vitvi18g00792	470	53 448.7	10.08	内质网

Fig. 2 Phylogenetic tree analysis of CYP707A protein The phylogenetic tree was constructed with the full-length amino acid sequences of the 24 CYP707A using MEGA X. The analysis included 5 Vitis vinifera VvCYP707A proteins, 2 Solanum lycopersicum SlCYP707A proteins, 4 Arabidopsis thaliana AtCYP707A proteins, 2 Daucus carota DcCYP707A proteins, 4 Oryza sativa OsCYP707A proteins, 2 Malus domestica MdCYP707A proteins, 2 Solanum tuberosum StCYP707A proteins and 4 Citrus CsCYP707A proteins. The VvCYP707A proteins in V. vinifera were marked with red color and red stars

Fig. 3 Phylogenetic relationships (A), gene structure (B) and conserved motif analyses (C) of CYP707As from Arabidopsis and V. vinifera

Fig. 4 Cis-acting elements of AtCYP707As and VvCYP707As gene promotors in Arabidopsis and V. vinifera

Fig. 5 Physiological parameters and VvCYP707As related expression related to fruit development in two kinds of V. vinifera A: ‘Yin Hong’ and ‘Yong Zaohong’ at different developmental periods; S1-S2: young fruit stage; S3: expansion stage; S4: veraison stage; S5: mature stage. The same below. B-D: ‘Yin Hong’ and ‘Yong Zaohong’ fruit single weight, fruit hardness and soluble sugar content. The data used are the mean ±SD of the three biological replicates. * on the column indicates significant differences between the two varieties, *: P<0.05, **: P<0.01, ***: P<0.001. E: VvCYP707A gene expression changes in ‘Yin Hong’ and ‘Yong Zaohong’. Different lowercase letters represent a significant difference（P<0.05）; n=3, the same below

Fig. 6 VvCYP707As expression patterns in different tissues and different periods in ‘Yin Hong’ A: Flesh; B: pericarp; C: tissue specificity of VvCYP707As expression in veraison stage

Fig. 7 Relative expressions of VvCYP707As gene in grape pericarp（A）and grape flesh（B）under ABA treatment

Fig. 8 Transient expression related genes were detected by RT-qPCR and determination of fruit sugar

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