葡萄CYP707A基因家族的鉴定及对果实成熟的功能验证

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

摘要/Abstract

摘要：

【目的】CYP707A是细胞色素P450家族的亚家族成员，所编码的ABA8'-羟化酶是ABA分解代谢的关键酶，对植物生长发育起着重要作用。鉴定葡萄CYP707A基因家族，可为进一步研究葡萄果实成熟机理和生物育种技术提供理论依据。【方法】利用生物学信息方法分析葡萄中CYP707A家族基因的基因结构、蛋白基序、染色体定位、共线性、系统进化关系及启动子区顺式作用元件等，并利用RT-qPCR分析VvCYP707A基因在葡萄不同品种（早熟和中熟）的时空表达模式、组织表达模式以及对外源ABA处理的响应模式，并利用果实瞬时表达技术对Vitvi07g01751进行功能验证。【结果】葡萄基因组鉴定出5个VvCYP707As基因，分布于5条染色体，其中Vitvi02g01269、Vitvi07g01751和Vitvi18g00792为片段复制基因。CYP707A基因家族高度保守，其中，基因结构和蛋白基序相似，亚细胞定位均位于内质网；VvCYP707A启动子区域富含与生长发育和激素响应相关的顺式作用元件，均含有ABA激素响应元件；通过RT-qPCR检测基因的表达模式发现，VvCYP707A基因具有时空特异性和组织特异性，主要在果实的幼果期与膨大期和茎、叶组织中高表达，其中Vitvi07g01751基因在早熟品种‘甬早红’膨大期果实中特异性高表达，且在两个品种间的表达模式一致，随着果实的成熟其表达先升高后降低。外源ABA处理下VvCYP707A分为两种表达模式，在浆果肉中外源ABA可诱导5个VvCYP707A基因高表达，在浆果皮中会显著抑制VvCYP707A基因的表达；在葡萄果实中瞬时表达Vitvi07g01751，葡萄的糖度积累缓慢，显著增加编码ABA的受体VvPYL4基因表达量，推测了Vitvi07g01751介导ABA调控葡萄果实成熟的机制。【结论】研究结果揭示了葡萄Vitvi07g01751在果实成熟中起着重要作用。

关键词: 葡萄, CYP707A基因, 全基因组鉴定, 生物信息学, 果实成熟, 脱落酸, PYL基因

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

龚丽丽, 余花, 杨杰, 陈天池, 赵双滢, 吴月燕. 葡萄CYP707A基因家族的鉴定及对果实成熟的功能验证[J]. 生物技术通报, 2024, 40(2): 160-171.

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.

图/表 11

表1 引物信息

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

表2 RT-qPCR所用引物

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

图1 葡萄CYP707A基因家族氨基酸比对（A）、共线性分析（B）与染色体定位（C）

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

表3 葡萄CYP707A蛋白相关信息

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

图2 CYP707A蛋白的系统进化树 MEGA X 软件构建24个CYP707A氨基酸序列系统进化树，其中包括5个葡萄、2个番茄、4个拟南芥、2个胡萝卜、4个水稻、2个苹果、2个马铃薯和4个柑橘CYP707A蛋白。葡萄的VvCYP707A蛋白用红色和红色星形标记

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

图3 拟南芥和葡萄CYP707As的系统发育关系（A）、基因结构（B）及保守基序分析（C）

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

图4 拟南芥和葡萄CYP707As基因启动子顺式作用元件

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

图5 两种葡萄果实发育的生理参数及VvCYP707As基因相对表达量 A：‘鄞红’和‘甬早红’不同发育时期，S1、S2幼果期，S3膨大期，S4转色期，S5成熟期，下同；B-D：‘鄞红’和‘甬早红’果实单重、果实硬度与可溶性糖含量，所用数据为3个生物学重复的平均值±SD，柱上*表示两个品种间差异显著，*：P<0.05，**：P<0.01，***：P<0.001；E：VvCYP707A在‘鄞红’和‘甬早红’中基因表达变化；不同小写字母代表显著差异（P<0.05）；n=3，下同

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

图6 VvCYP707As在‘鄞红’不同组织不同时期表达模式 A：果肉；B：果皮；C：VvCYP707As转色期表达量组织特异性

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

图7 葡萄果皮（A）和果肉（B）VvCYP707As基因在ABA处理下相对表达量

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

图8 瞬时表达相关基因RT-qPCR检测与理化测定

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

参考文献 31

[1]	Chatterjee A, Dhal S, Pal H. Insight into the regulatory network of miRNA to unravel the ripening physiology of climacteric and non-climacteric fruits[J]. Plant Gene, 2021, 28: 100329. doi: 10.1016/j.plgene.2021.100329 URL
[2]	Li DM, Pang YJ, Li H, et al. Comparative analysis of the gene expression profile under two cultivation methods reveals the critical role of ABA in grape quality promotion[J]. Sci Hortic, 2021, 281: 109924. doi: 10.1016/j.scienta.2021.109924 URL
[3]	Kondo S, Sugaya S, Sugawa S, et al. Dehydration tolerance in apple seedlings is affected by an inhibitor of ABA 8'-hydroxylase CYP707A[J]. J Plant Physiol, 2012, 169(3): 234-241. doi: 10.1016/j.jplph.2011.09.007 URL
[4]	Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism[J]. Annu Rev Plant Biol, 2005, 56: 165-185. pmid: 15862093
[5]	Cutler AJ, Krochko JE. Formation and breakdown of ABA[J]. Trends Plant Sci, 1999, 4(12): 472-478. doi: 10.1016/s1360-1385(99)01497-1 pmid: 10562731
[6]	Rodriguez PL. Abscisic acid catabolism generates phaseic acid, a molecule able to activate a subset of ABA receptors[J]. Mol Plant, 2016, 9(11): 1448-1450. doi: S1674-2052(16)30217-9 pmid: 27693497
[7]	Weng JK, Ye ML, Li B, et al. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity[J]. Cell, 2016, 166(4): 881-893. doi: 10.1016/j.cell.2016.06.027 URL
[8]	Okamoto M, Kushiro T, Jikumaru Y, et al. ABA 9'-hydroxylation is catalyzed by CYP707A in Arabidopsis[J]. Phytochemistry, 2011, 72(8): 717-722. doi: 10.1016/j.phytochem.2011.02.004 URL
[9]	Brun G, Thoiron S, Braem L, et al. CYP707As are effectors of karrikin and strigolactone signalling pathways in Arabidopsis thaliana and parasitic plants[J]. Plant Cell Environ, 2019, 42(9): 2612-2626. doi: 10.1111/pce.v42.9 URL
[10]	Kushiro T, Okamoto M, Nakabayashi K, et al. The Arabidopsis cytochrome P450 CYP707A encodes ABA 8-hydroxylases: key enzymes in ABA catabolism[J]. EMBO J, 2004, 23(7): 1647-1656. doi: 10.1038/sj.emboj.7600121 pmid: 15044947
[11]	Sapkota S, Liu JY, Islam MT, et al. Contrasting bloom dates in two apple cultivars linked to differential levels of phytohormones and heat requirements during ecodormancy[J]. Sci Hortic, 2021, 288: 110413. doi: 10.1016/j.scienta.2021.110413 URL
[12]	Gonzalez HCB, Galli V. The CYP707A gene family in strawberry(Fragaria × ananassa)[J]. Braz Arch Biol Technol, 2021, 64: e21200133. doi: 10.1590/1678-4324-2021200133 URL
[13]	Ren J, Sun L, Wu JF, et al. Cloning and expression analysis of cDNAs for ABA 8'-hydroxylase during sweet cherry fruit maturation and under stress conditions[J]. J Plant Physiol, 2010, 167(17): 1486-1493. doi: 10.1016/j.jplph.2010.05.027 URL
[14]	Parwez R, Aftab T, Gill SS, et al. Abscisic acid signaling and crosstalk with phytohormones in regulation of environmental stress responses[J]. Environ Exp Bot, 2022, 199: 104885. doi: 10.1016/j.envexpbot.2022.104885 URL
[15]	吴月燕, 陈天池, 王立如, 等. 鲜食葡萄新品种‘甬早红’[J]. 园艺学报, 2022, 49(S2): 41-42.
	Wu YY, Chen TC, Wang LR, et al. A new table grape cultivar‘Yongzaohong’[J]. Acta Hortic Sin, 2022, 49(S2): 41-42. doi: 10.16420/j.issn.0513-353x.2022-0550
[16]	张友杰. 以蒽酮分光光度法测定果蔬中的葡萄糖、果糖、蔗糖和淀粉[J]. 分析化学, 1977, 5(3): 167-171.
	Zhang YJ. Determination of glucose, fructose, sucrose and starch in fruits and vegetables by anthrone spectrophotometry[J]. Chin J Anal Chem, 1977, 5(3): 167-171.
[17]	Ren C, Zhang Z, Wang Y, et al. Genome-wide identification and characterization of the NF-Y gene family in grape(Vitis vinifera L.)[J]. BMC Genomics, 2016, 17(1): 605. doi: 10.1186/s12864-016-2989-3 URL
[18]	李敬蕊, 王育博, 解紫薇, 等. 甜瓜PIN基因家族的鉴定及高温胁迫表达分析[J]. 生物技术通报, 2023, 39(5): 192-204. doi: 10.13560/j.cnki.biotech.bull.1985.2022-1196
	Li JR, Wang YB, Xie ZW, et al. Identification and expression analysis of PIN gene family in melon under high temperature stress[J]. Biotechnol Bull, 2023, 39(5): 192-204.
[19]	Rehal PK, Tuan PA, Nguyen TN, et al. Genetic variation of seed dormancy in wheat(Triticum aestivum L.)is mediated by transcriptional regulation of abscisic acid metabolism and signaling[J]. Plant Sci, 2022, 324: 111432. doi: 10.1016/j.plantsci.2022.111432 URL
[20]	Gupta A, Upadhyay RK, Prabhakar R, et al. SlDREB3, a negative regulator of ABA responses, controls seed germination, fruit size and the onset of ripening in tomato[J]. Plant Sci, 2022, 319: 111249. doi: 10.1016/j.plantsci.2022.111249 URL
[21]	高真真, 徐功勋, 王东岭, 等. 桃ABA 8'-羟化酶基因PpeCYP707As在拟南芥中过表达的功能分析[J]. 园艺学报, 2018, 45(2): 239-249. doi: 10.16420/j.issn.0513-353x.2017-0350
	Gao ZZ, Xu GX, Wang DL, et al. Functional analysis of peach PpeCYP707As gene in Arabidopsis thaliana overexpressing plants[J]. Acta Hortic Sin, 2018, 45(2): 239-249.
[22]	Sun WJ, Ma ZT, Liu MY. Cytochrome P450 family: Genome-wide identification provides insights into the rutin synthesis pathway in Tartary buckwheat and the improvement of agricultural product quality[J]. Int J Biol Macromol, 2020, 164: 4032-4045. doi: 10.1016/j.ijbiomac.2020.09.008 pmid: 32896558
[23]	Wang H, Umer MJ, Liu F, et al. Genome-wide identification and characterization of CPR5 genes in Gossypium reveals their potential role in trichome development[J]. Front Genet, 2022, 13: 921096. doi: 10.3389/fgene.2022.921096 URL
[24]	Zhu PP, Cai YX, Yu J, et al. Characterization and expression of abscisic acid signal transduction genes during mulberry fruit ripening[J]. Acta Physiol Plant, 2017, 39(7): 149. doi: 10.1007/s11738-017-2442-5 URL
[25]	Saito T, Thunyamada S, Wang SS, et al. Exogenous ABA and endogenous ABA affects ‘Kyoho’ grape berry coloration in different pathway[J]. Plant Gene, 2018, 14: 74-82. doi: 10.1016/j.plgene.2018.05.001 URL
[26]	董昳伶, 肖旭腾, 张敏, 等. 环割对葡萄VvNCED基因的表达和果实成熟的影响[J]. 核农学报, 2022, 36(7): 1339-1349. doi: 10.11869/j.issn.100-8551.2022.07.1339
	Dong YL, Xiao XT, Zhang M, et al. Effect of girdling on the expression of VvNCEDs and fruit ripening in grapes[J]. J Nucl Agric Sci, 2022, 36(7): 1339-1349.
[27]	邓昌哲, 秦于玲, 李开绵, 等. 外源ABA对木薯叶片β-胡萝卜素合成通路相关基因表达的影响[J]. 热带作物学报, 2017, 38(4): 667-672.
	Deng CZ, Qin YL, Li KM, et al. Effects of exogenous ABA on the expression of genes associated with β-carotene synthesis pathway in cassava leaves[J]. Chin J Trop Crops, 2017, 38(4): 667-672.
[28]	王东岭, 杜培勇, 郇蕾, 等. 桃CYP707A家族基因的克隆以及表达分析[J]. 植物生理学报, 2016, 52(5): 659-668.
	Wang DL, Du PY, Huan L, et al. Molecular cloning and expression analysis of CYP707A gene family in peach[J]. Plant Physiol J, 2016, 52(5): 659-668.
[29]	Saito S, Hirai N, Matsumoto C, et al. Arabidopsis CYP707As encode(+)-abscisic acid 8'-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid[J]. Plant Physiol, 2004, 134(4): 1439-1449. doi: 10.1104/pp.103.037614 URL
[30]	Mou WS, Li DD, Bu JW, et al. Comprehensive analysis of ABA effects on ethylene biosynthesis and signaling during tomato fruit ripening[J]. PLoS One, 2016, 11(4): e0154072. doi: 10.1371/journal.pone.0154072 URL
[31]	刘雪梅. 新疆玛纳斯河流域酿酒葡萄成熟度指标与葡萄酒质量关系的研究[D]. 杨凌: 西北农林科技大学, 2008.
	Liu XM. The research on wine qulity and grape maturity in the basin of the manas river, Xinjiang[D]. Yangling: Northwest A & F University, 2008.