黄瓜蜡质基因CsCER1调控因子CsCOL5的筛选及其功能分析

doi:10.16420/j.issn.0513-353x.2023-0232

摘要/Abstract

摘要：

以黄瓜（Cucumis sativus L.）蜡质合成基因CsCER1启动子序列中的70 bp片段作为诱饵进行酵母单杂文库筛选，结合转录组数据分析筛选得到HSPA2、ZFP、COL5、GLO1、PGK共5个基因，并将CsCOL5作为候选基因进一步研究。荧光定量结果显示，CsCOL5在黄瓜的各个组织部位均有表达，其中在雄花中表达最高。克隆CsCOL5并通过CRISPR/Cas9技术创制敲除CsCOL5的株系col5，荧光定量结果表明敲除CsCOL5使得CsCER1的表达降低，并且col5敲除株系的蜡质总含量比对照下降了84% ~ 86%。据此推测CsCOL5可能通过与CsCER1启动子结合来调控黄瓜果实蜡质的合成。

关键词: 黄瓜, 蜡质调控, CsCOL5, 基因编辑

Abstract:

A 70 bp promoter sequence of wax synthesis gene CsCER1 was used as bait to screen the upstream regulators in Cucumis sativus L. by yeast one hybrid assay. Combined with transcriptome data analysis，HSPA2，ZFP，COL5，GLO1 and PGK were considered as possible candidate genes，and CsCOL5 selected as a candidate gene for further study. qRT-PCR showed that CsCOL5 was expressed in all tissues of cucumber，with the highest expression in male flowers. Then，CsCOL5 was cloned and col5 mutants were generated by CRISPR/Cas9 technology. qRT-PCR showed that knockout of CsCOL5 reduced CsCER1 expression，and the total wax content of col5 knockout lines decreased by 84%-86% compared with the control. Based on the findings above，it is concluded that CsCOL5 might act as an upstream regulator of CsCER1 to regulate the wax content in cucumber.

Key words: cucumber, wax regulation, CsCOL5, gene editing

王文娇, 邢军杰, 申成丞, 李斌. 黄瓜蜡质基因CsCER1调控因子CsCOL5的筛选及其功能分析[J]. 园艺学报, 2024, 51(5): 1005-1016.

WANG Wenjiao, XING Junjie, SHEN Chengcheng, LI Bin. Screening and Functional Analysis of CsCOL5，the Upstream Regulator of CsCER1，in Cucumber Pericarp Wax Synthesis[J]. Acta Horticulturae Sinica, 2024, 51(5): 1005-1016.

图/表 10

图1 黄瓜CsCOL5基因和靶点的示意图 A：黑色方框和线条分别代表外显子和非编码区，红色竖线表示靶点位置和设计的gRNA名称。B、C：载体示意图，将B图中红色虚线部分用内切酶切下转入C图的polylinker上。

Fig. 1 Schematic diagram of CsCOL5 gene and target in cucumber A：The black boxes and lines represent exons and non-coding regions，respectively. The red vertical bars indicate the target location and the gRNAs name of the design. B，C：Carrier diagram. The red dotted line in B was cut off with endonucliase and transferred to the polylinker of C.

图2 CsCER1的启动子顺式作用元件分布位置（A）及诱饵选择（B）启动子元件包括：厌氧诱导（ARE）、光响应相关元件（AE-box、Box-4、TCT-motif、G-box、L-box、LAMP-element），蛋白结合位点（Box-III），MYB参与干旱诱导的结合位点元件（MBS），赤霉素响应（P-box、TATC-box），参与防御和应激反应（TC-rich），水杨酸反应（TCA-element），昼夜节律控制元件（Circadian）。

Fig. 2 Location of promoter cis-acting element distribution（A）and decoy selection（B）of the CsCER1 gene The promoter elements include anaerobic induction（ARE），light responsive element（AE-box，Box-4，TCT-Motif，G-box，L-box，LAMP-element），protein binding sites（Box-III），MYB binding sites involved in drought-inducibility（MBS），gibberellin response（P-box，TATC-box），defense and stress responsiveness（TC-rich），salicylic acid response（TCA-element），circadian control（Circadian）.

图3 酵母单杂交验证COL5、PERK1与CSCER1启动子互作 10-1、10-2、10-3和10-4为酵母Y1HGold（pAbAi-CsCER1）溶液的稀释梯度。

Fig. 3 The result of interaction between COL5 and CsCER1，PERK1 and CsCER1 10-1，10-2，10-3 and 10-4 were the dilution gradients of yeast Y1HGold（pAbAi-CsCER1）solution.

表1 酵母单杂交筛选结果阳性克隆定位分析

Table 1 Y1H screening results positive clonal localization analysis

基因库ID Gene bank ID	基因名 Gene name	基因组ID Genome ID	功能描述 Description	亚细胞定位 Subcellular localization	转录因子 TF
KAA0042485.1	HSPA2	CsaV3_5G026450.1	热休克同源的70 kD蛋白2-like Heat shock cognate 70 kD protein 2-like	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
KAA0053034.1	ZFP	CsaV3_3G001590.1	推定的锌结合蛋白 Putative zinc-binding protein	细胞核 Nucleus	是 Yes
KAE8637537.1	hypothetical protein	CsaV3_UNG165240.1	假定的蛋白质CSA_017393 Hypothetical protein CSA_017393	细胞核、线粒体 Nucleus，mitochondria	是 Yes
XM_004139789.3	PGK	CsaV3_7G022930.1	黄瓜磷酸甘油酸激酶 Cucumis sativus phosphoglycerate kinase	细胞核、细胞质、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XM_011659861.2	UBQ14	CsaV3_6G049240.1	黄瓜的聚泛素 Cucumis sativus polyubiquitin	细胞质、细胞核、液泡 Cytoplasm，nucleus，vacuole	是 Yes
XP_004134215.1	TCTP	CsaV3_3G014730.1	翻译控制肿瘤蛋白 Translationally-controlled tumor protein homolog	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004135973.1	PP2C49	CsaV3_7G030300.1	蛋白磷酸酶2C 49 Probable protein phosphatase 2C 49	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004136304.2	AIG2	CsGy3G042330.1	类似AIG2的蛋白质D的异构体 X1 AIG2-like protein D isoform X1	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004144847.1	HIPP26.1	CsaV3_7G001010.1	与重金属相关的异戊烯酸化植物蛋白26 Heavy metal-associated isoprenylated plant protein 26	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
XP_004146297.1	COL5	CsGy1G019700.1	锌指蛋白CONSTANS-LIKE 5-like Zinc finger protein CONSTANS-LIKE 5-like	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004146881.1	DHNA2	CsaV3_4G004540.1	二氢蝶呤醛缩酶2 Dihydroneopterin aldolase 2	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
XP_004150052.1	PERK1	CsaV3_1G024290.1	富含脯氨酸的受体样蛋白激酶PERK1 Proline-rich receptor-like protein kinase PERK1	细胞核 Nucleus	是 Yes
XP_004152924.1	DEAD32	CsaV3_3G005200.1	依赖型DEAD-box RNA解旋酶 DEAD-box ATP-dependent RNA helicase 32	细胞质、细胞核 Cytoplasm，nucleolus	是 Yes
XP_008439666.1	DNA-binding protein	Csa6G522140.1	DNA结合蛋白 DDB_G0278111 DNA-binding protein DDB_G0278111	细胞核、胞质溶胶 Nucleus，cytosol	是 Yes
XP_011658294.1	GLO1	CsaV3_6G051820.1	GLO1乙醇酸氧化酶 (S)-2-hydroxy-acid oxidase GLO1	细胞质、细胞核、线粒体 Cytoplasm，nucleus，mitochondria	是 Yes

表1 酵母单杂交筛选结果阳性克隆定位分析

Table 1 Y1H screening results positive clonal localization analysis

基因库ID Gene bank ID	基因名 Gene name	基因组ID Genome ID	功能描述 Description	亚细胞定位 Subcellular localization	转录因子 TF
KAA0042485.1	HSPA2	CsaV3_5G026450.1	热休克同源的70 kD蛋白2-like Heat shock cognate 70 kD protein 2-like	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
KAA0053034.1	ZFP	CsaV3_3G001590.1	推定的锌结合蛋白 Putative zinc-binding protein	细胞核 Nucleus	是 Yes
KAE8637537.1	hypothetical protein	CsaV3_UNG165240.1	假定的蛋白质CSA_017393 Hypothetical protein CSA_017393	细胞核、线粒体 Nucleus，mitochondria	是 Yes
XM_004139789.3	PGK	CsaV3_7G022930.1	黄瓜磷酸甘油酸激酶 Cucumis sativus phosphoglycerate kinase	细胞核、细胞质、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XM_011659861.2	UBQ14	CsaV3_6G049240.1	黄瓜的聚泛素 Cucumis sativus polyubiquitin	细胞质、细胞核、液泡 Cytoplasm，nucleus，vacuole	是 Yes
XP_004134215.1	TCTP	CsaV3_3G014730.1	翻译控制肿瘤蛋白 Translationally-controlled tumor protein homolog	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004135973.1	PP2C49	CsaV3_7G030300.1	蛋白磷酸酶2C 49 Probable protein phosphatase 2C 49	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004136304.2	AIG2	CsGy3G042330.1	类似AIG2的蛋白质D的异构体 X1 AIG2-like protein D isoform X1	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004144847.1	HIPP26.1	CsaV3_7G001010.1	与重金属相关的异戊烯酸化植物蛋白26 Heavy metal-associated isoprenylated plant protein 26	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
XP_004146297.1	COL5	CsGy1G019700.1	锌指蛋白CONSTANS-LIKE 5-like Zinc finger protein CONSTANS-LIKE 5-like	细胞质、细胞核、线粒体 Nucleus，cytoplasm，mitochondria	是 Yes
XP_004146881.1	DHNA2	CsaV3_4G004540.1	二氢蝶呤醛缩酶2 Dihydroneopterin aldolase 2	细胞质、细胞核 Cytoplasm，nucleus	是 Yes
XP_004150052.1	PERK1	CsaV3_1G024290.1	富含脯氨酸的受体样蛋白激酶PERK1 Proline-rich receptor-like protein kinase PERK1	细胞核 Nucleus	是 Yes
XP_004152924.1	DEAD32	CsaV3_3G005200.1	依赖型DEAD-box RNA解旋酶 DEAD-box ATP-dependent RNA helicase 32	细胞质、细胞核 Cytoplasm，nucleolus	是 Yes
XP_008439666.1	DNA-binding protein	Csa6G522140.1	DNA结合蛋白 DDB_G0278111 DNA-binding protein DDB_G0278111	细胞核、胞质溶胶 Nucleus，cytosol	是 Yes
XP_011658294.1	GLO1	CsaV3_6G051820.1	GLO1乙醇酸氧化酶 (S)-2-hydroxy-acid oxidase GLO1	细胞质、细胞核、线粒体 Cytoplasm，nucleus，mitochondria	是 Yes

图4 候选基因在黄瓜少蜡LCW和多蜡HCW材料中的差异表达（A）与启动子元件分析（B） A图红点代表在HCW和LCW中的表达量存在差异的基因。B图中的启动子元件为：光响应元件（AAAC、ACE、AE-box、Box-4、G-Box、GATA-motif、GT1-motif、I-box、GATT-motif、GA-motif、Gap-box、MRE），脱落酸响应（ABRE），厌氧诱导（ARE），生长素相关（AuxRE、AuxRR），MYB参与干旱诱导的结合位点元件（MBS），茉莉酸响应（CGTCA-motif），低温响应（LTR），玉米蛋白代谢调控（O2-site），栅栏叶肉细胞的分化（HD-Zip），分生组织相关（CAT-box），赤霉素响应（GARE-motif）。

Fig. 4 Differential expression（A）of candidate genes in different waxy cucumber materialsand promoter elements（B） The red dots in Figure A represent genes with different expression levels of‘ HCW and LCW. The promoter elements in Figure B is the light responsive（AAAC，ACE，AE-box，Box-4，G-Box，GATA-motif，GT1-motif，I-box，GATT-motif，GA-motif，Gap-box，MRE），the abscisic acid responsiveness（ABRE），the anaerobic induction（ARE），part of an auxin-responsive element（AuxRE，AuxRR），MYB binding sites involved in drought-inducibility（MBS），the MeJA-responsiveness（CGTCA-motif），low-temperature responsiveness（LTR）zein metabolism regulation（O2-site），differentiation of the palisade mesophyll cells（HD-Zip），cis-acting regulatory element related to meristem expression（CAT-box），gibberellin-responsive element（GARE-motif）.

图5 CsCOL5在黄瓜不同组织中表达量的实时荧光定量不同小写字母表示差异显著（P < 0.05），下同。

Fig. 5 Fluorescence quantification of CsCOL5 in different tissue sites Different lowercase letters indicate significant differences at 0.05 level. The same below.

图6 CRISPR/Cas9构建CsCOL5突变株

Fig. 6 CRISPR/Cas9 generation of CsCOL5 mutant

图7 黄瓜CRISPR/Cas9敲除株系（col5）及野生型（WT）中CsCOL5和CsCER1的表达量

Fig. 7 Expression level of CsCOL5 and CsCER1 in wild-type（WT）and CRISPR/Cas9 lines（col5）

图8 黄瓜col5敲除株系及其野生型（WT）果皮表型和蜡质含量

Fig. 8 Phenotype and wax content of cucumber epidermis in wild-type（WT）and CRISPR/Cas9 lines（col5） ** P < 0.01.

图9 黄瓜col5敲除株系及其野生型（WT）果皮烷类含量

Fig. 9 Alkane content of cucumber epidermis in wild-type（WT）and CRISPR/Cas9 lines（col5） ** P < 0.01.

参考文献 33

[1]	Almada R, Cabrera N, Casaretto J A, Ruiz-Lara S, Villanueva E G. 2009. VvCO and VvCOL1,two CONSTANS homologous genes,are regulated during flower induction and dormancy in grapevine buds. Plant Cell Reports, 28 (8):1193-1203. doi: 10.1007/s00299-009-0720-4 pmid: 19495771
[2]	Bernard A, Domergue F, Pascal S, Jetter R, Renne C, Faure J D, Haslam R P, Napier J A, Lessire R, Joubès J. 2012. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a Very-Long-Chain alkane synthesis complex. The Plant Cell, 24 (7):3106-3118. doi: 10.1105/tpc.112.099796 pmid: 22773744
[3]	Cao D D, Lin Z Y, Huang L Y, Damaris R N, Li M, Yang P F. 2021. A CONSTANS-LIKE gene of Nelumbo nucifera could promote potato tuberization. Planta, 253 (3):1-11.
[4]	Chen G H, Wang J, Wang H, Wang C G, Tang X Y, Li J, Zhang L, Song J H, Hou J F, Yuan L Y. 2020. Genome-wide analysis of proline-rich extension-like receptor protein kinase(PERK)in Brassica rapa and its association with the pollen development. BMC Genomics, 21 (1):401-413.
[5]	Go Y S, Kim H, Kim H J, Suh M C. 2014. Arabidopsis cuticular wax biosynthesis is negatively regulated by the DEWAX gene encoding an AP2/ERF-type transcription factor. The Plant Cell, 26 (8):1666-1680.
[6]	Gong Si-yu, Yu Yue, Guo Dong-yue, Du Ya-lin, Hao Ning, Wu Tao. 2020. Cloning and expression analysis of CsCER4 gene related to wax synthesis in cucumber. Journal of Henan Agricultural Sciences, 49 (4):101-106. (in Chinese)
	宫思宇, 于越, 郭冬雪, 杜亚琳, 郝宁, 武涛. 2020. 黄瓜蜡质合成相关基因CsCER4的克隆与表达分析. 河南农业科学, 49 (4):101-106.
[7]	Greer S, Wen M, Bird D, Wu X, Samuels L, Kunst L, Jetter R. 2007. The cytochrome P 450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis. Plant Physiology, 145 (3):653-667.
[8]	Griffiths S, Dunford R P, Coupland G, Laurie D A. 2003. The evolution of CONSTANS-like gene families in barley,rice,and Arabidopsis. Plant Physiology, 131 (4):1855-1867. doi: 10.1104/pp.102.016188 pmid: 12692345
[9]	Hen-Avivi S, Lashbrooke J, Costa F, Aharoni A. 2014. Scratching the surface:genetic regulation of cuticle assembly in fleshy fruit. Journal of Experimental Botany, 65 (16):4653-4664. doi: 10.1093/jxb/eru225 pmid: 24916070
[10]	Hooker T S, Millar A A, Kunst L. 2002. Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiology, 129 (4):1568-1580. doi: 10.1104/pp.003707 pmid: 12177469
[11]	Kim D, Langmead B, Salzberg S. 2015. HISAT:a fast spliced aligner with low memory requirements. Nat Methods, 12 (4):357-360. doi: 10.1038/NMETH.3317
[12]	Kinmonth-Schultz H A, Tong X, Lee J, Song Y H, Ito S, Kim S H, Imaizumi T. 2016. Cool night-time temperatures induce the expression of CONSTANS and FLOWERING LOCUST to regulate flowering in Arabidopsis. New Phytologist, 211 (1):208-224. doi: 10.1111/nph.13883 pmid: 26856528
[13]	Lewandowska M, Keyl A, Feussner I. 2020. Wax biosynthesis in response to danger:its regulation upon abiotic and biotic stress. New Phytologist, 227 (3):698-713. doi: 10.1111/nph.16571 pmid: 32242934
[14]	Li Cheng, Pan Jian, Lian Hongli, Wang Gang, He Huanle, Pan Junsong, Cai Run. 2018. Cloning and functional analysis of CsWIN1,a transcription factor regulated the wax synthesis in cucumber. Acta Horticulturae Sinica, 45 (2):359-370. (in Chinese) doi: 10.16420/j.issn.0513-353x.2017-0736
	李铖, 潘健, 连红莉, 王刚, 何欢乐, 潘俊松, 蔡润. 2018. 黄瓜蜡质合成调控基因CsWIN1的克隆与功能初步分析. 园艺学报, 45 (2):359-370. doi: 10.16420/j.issn.0513-353x.2017-0736
[15]	Li S Y, Zhao H Y, Marais D L D, Parsons E P, Wen X X, Xu X J, Bangarusamy D K, Wang G, Rowland O, Juenger T, Bressan R A, Jenks M A. 2012. Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status. Plant Physiol, 159 (3):930-944.
[16]	Li R J, Li L M, Liu X L, Kim J C, Jenks M A, Lü S. 2019. Diurnal regulation of plant epidermal wax synthesis through antagonistic roles of the transcription factors SPL9 and DEWAX. The Plant Cell, 31 (11):2711-2733.
[17]	Liu Bing-chen, Tang Yu-jin, Wei Rong, Wang Yue-jin, Zhang Chao-hong. 2017. Cloning and expression analysis of VvCOL5gene in the ovule development of grapevine. Journal of Northwest Forestry University, 31 (1):114-120. (in Chinese)
	刘炳臣, 唐玉瑾, 魏蓉, 王跃进, 张朝红. 2017. 葡萄VvCOL5基因克隆及在胚珠发育过程中的表达分析. 西北林学院学报, 32 (1):114-120.
[18]	Liu Xiao-feng, An Jing-bo, Zhang Li-xin, Wang Wen-jiao, Xu Chong, Ren Hua-zhong. 2014. Cloning and expression analysis of CsCER7,a relative gene may regulate wax synthesis in cucumber. Acta Horticulturae Sinica, 41 (4):661-671. (in Chinese)
	刘小凤, 安静波, 张立新, 王文娇, 徐冲, 任华中. 2014. 黄瓜调控蜡质合成相关基因CsCER7的克隆与表达分析. 园艺学报, 41 (4):661-671.
[19]	Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15 (12):1-21.
[20]	Luo B, Xue X Y, Hu W L. 2007. An ABC transporter gene of Arabidopsis thaliana,AtWBC11,is involved in cuticle development and prevention of organ fusion. Plant & Cell Physiology, 48 (12):1790-1802.
[21]	Ma Ai-ru. 2012. Cloning and analysis of CO gene and ASY1 gene in Chinese cabbage[M. D. Dissertation]. Baoding: Hebei Agricultural University. (in Chinese)
	马爱茹. 2012. 大白菜CO基因与ASY1基因的克隆及分析[硕士论文]. 保定: 河北农业大学.
[22]	Qiao P, Bourgault R, Mohammadi M, Gore M A, Molina I, Scanlon M J. 2020. A maize LIPID TRANSFER PROTEIN may bridge the gap between PHYTOCHROME-mediated light signaling and cuticle biosynthesis. Plant Signaling & Behavior, 15 (9):1790824.
[23]	Robson F, Costa M M R, Hepworth S R, Vizir I, Piñeiro M, Reeves P H, Putterill J, Coupland G. 2001. Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant Journal for Cell and Molecular Biology, 28 (6):619-631.
[24]	Wang Shi-feng, Guo Ye, Li Mei-lan, Hou Lei-ping, Wang Wen-jiao. 2019. Expression patterns and biological information analysis of waxes synthesis-related gene CsCER10 in cucumber. Journal of Shanxi Agricultural Sciences, 47 (5):719-722,729. (in Chinese)
	王世峰, 郭烨, 李梅兰, 侯雷平, 王文娇. 2019. 黄瓜蜡质合成基因CsCER10的表达模式及生物学信息分析. 山西农业科学, 47 (5):719-722,729.
[25]	Wang W J, Zhang Y, Xu C, Ren J J, Liu X F, Black K, Gai X S, Wang Q, Ren H Z. 2015. Cucumber ECERIFERUM1(CsCER1),which influences the cuticle properties and drought tolerance of cucumber,plays a key role in VLC alkanes biosynthesis. Plant Molecular Biology, 87 (3):219-233.
[26]	Wei Xiaoyu, Wang Yuejin. 2022. Correlation between anatomical structure and resistance to powdery mildew in Chinese wild Vitis species. Acta Horticulturae Sinica, 49 (6):1200-1212. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0367
	魏晓羽, 王跃进. 2022. 中国野生葡萄果皮解剖结构与白粉病抗性的相关性研究. 园艺学报, 49 (6):1200-1212. doi: 10.16420/j.issn.0513-353x.2021-0367
[27]	Wu Hui-jie, Kang Bao-shan, Peng Bin, Liu Li-feng, Gu Qin-sheng. 2020. Construction of yeast two-hybrid cDNA library in response to MNSV infection in melon. China Vegetables,(9):36-40. (in Chinese)
	吴会杰, 康保珊, 彭斌, 刘丽锋, 古勤生. 2020. 甜瓜响应甜瓜坏死斑点病毒侵染的cDNA文库构建. 中国蔬菜,(9):36-40.
[28]	Yang H B, Zhu Z F, Zhang M F, Li X, Xu R W, Zhu F, Xu J, Deng X X, Cheng Y J. 2022. QTL analysis reveals reduction of fruit water loss by NAC 042 through regulation of cuticular wax synthesis in citrus fruit. Horticultural Plant Journal, 8 (6):737-746.
[29]	Yang Y, Cai C, Wang Y, Wang Y, Ju H, Chen X. 2022. Cucumber glossy fruit 1(CsGLF1)encodes the zinc finger protein 6 that regulates fruit glossiness by enhancing cuticular wax biosynthesis. Horticulture Research, 10 (1):uhac237.
[30]	Yeats T H, Rose J K C. 2013. The formation and function of plant cuticles. Plant Physiology, 163 (1):5-20. doi: 10.1104/pp.113.222737 pmid: 23893170
[31]	Yu Qinhan, Li Junduo, Cui Ying, Wang Jiahui, Zheng Qiaoling, Xu Weirong. 2023. Screening and identification of interacting protein of VaMYB4a from Vitis amurensis. Acta Horticulturae Sinica, 50 (3):508-522. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-1219
	俞沁含, 李俊铎, 崔莹, 王佳慧, 郑巧玲, 徐伟荣. 2023. 山葡萄转录因子VaMYB4a互作蛋白的筛选与鉴定. 园艺学报, 50 (3):508-522. doi: 10.16420/j.issn.0513-353x.2021-1219
[32]	Zhang Jun-xia, Li Ding-li, Wang Ran, Liu Lian-cheng, Yuan Yong-bing, Ma Chun-hui. 2014. Cloning and prokaryotic expression analysis of CONSTANS gene in Pyrus pyrifolia Nakai. Journal of Plant Genetic Resources, 15 (4):824-830. (in Chinese)
	张军霞, 李鼎立, 王然, 刘成连, 原永兵, 马春晖. 2014. 砂梨CONSTANS基因的克隆及原核表达分析. 植物遗传资源学报, 15 (4):824-830. doi: 10.13430/j.cnki.jpgr.2014.04.021
[33]	Zhang R, Ding J, Liu C X, Cai C P, Zhou B L, Zhang T Z, Guo W Z. 2015. Molecular evolution and phylogenetic analysis of eight COL superfamily genes in Group I related to photoperiodic regulation of flowering time in wild and domesticated cotton(Gossypium)Species. PLoS ONE, 10 (2):e0118669.