冬瓜BhYAB4b基因的克隆、表达及功能研究

doi:10.16420/j.issn.0513-353x.2024-0720

摘要/Abstract

摘要：

YABBY基因家族是高等植物中一类特有的转录因子，在植物生长和发育过程中发挥着重要的调控作用。本研究中从冬瓜中克隆获得了1个YABBY基因，命名为BhYAB4b，并对该基因进行生物信息、表达模式、亚细胞定位及异源过表达拟南芥分析。BhYAB4b编码区长度为624 bp，编码蛋白分子量为23.469 kD，在N端含1个保守的C2C2型锌指结构域，C端含有1个YABBY保守结构域。系统发育分析表明BhYAB4b蛋白与西瓜、苦瓜的YAB4蛋白亲缘关系较近。qRT-PCR结果表明，BhYAB4b在冬瓜中的表达具有特异性。在冬瓜高代自交系B227中，BhYAB4b在根、茎、叶、雄花、雌花及果实中均有表达，但在果实中的表达显著高于其他组织，并且在授粉当天果实中的表达高于其他两个时期果实。亚细胞定位结果表明BhYAB4b蛋白定位于细胞核。BhYAB4b在拟南芥中异源过表达后，转基因拟南芥植株系表现出莲座叶变大且数量增多，果实的长和宽均增加，这表明BhYAB4b可能调控叶片和果实的发育。

关键词: 冬瓜, YABBY, 基因克隆, 表达分析, 亚细胞定位, 异源过表达

Abstract:

The YABBY gene family encodes a unique class of transcription factors in higher plants，which plays important regulatory roles in plant growth and development. In this study，a YABBY gene named BhYAB4b was cloned from wax gourd and this gene was subjected to bioinformatics analysis，expression pattern analysis，subcellular localization analysis，and heterologous overexpression in Arabidopsis analysis. The length of the coding sequence of BhYAB4b was 624 bp，and the predicted molecular weight of the encoded protein was 23.469 kD，containing a conserved structural domain of C2C2 zinc finger structure at the N-terminal end and a conserved structural domain of YABBY at the C-terminal end. Phylogenetic analysis showed that the BhYAB4b protein was closely related to the YAB4 proteins of watermelon and bitter melon，qRT-PCR results showed that BhYAB4b was expressed specifically in wax gourd. In the advanced-generation inbred line B227 of wax gourd，BhYAB4b was expressed in the roots，stems，leaves，male and female flowers，and fruits，while its expression in fruits was significantly higher than that in other tissues，and its expression in fruits was higher on the day of pollination than other two periods. Subcellular localization results showed that the BhYAB4b protein was localized in the nucleus of the cell. When the BhYAB4b gene was heterologous overexpression in Arabidopsis，the transgenic lines showed larger and more rosette leaves，along with enhanced fruit length and width，suggesting that the BhYAB4b gene may regulate the development of leaves and fruits.

Key words: wax gourd, YABBY, gene cloning, expression analysis, subcellular localization, heterologous overexpression

王宝琛, 罗陈, 闫晋强, 程晓欣, 莫仁连, 刘文睿, 张余洋, 江彪. 冬瓜BhYAB4b基因的克隆、表达及功能研究[J]. 园艺学报, 2025, 52(9): 2353-2362.

WANG Baochen, LUO Chen, YAN Jinqiang, CHENG Xiaoxin, MO Renlian, LIU Wenrui, ZHANG Yuyang, JIANG Biao. Cloning，Expression，and Functional Research of BhYAB4b in Wax Gourd[J]. Acta Horticulturae Sinica, 2025, 52(9): 2353-2362.

图/表 7

表1 本研究中所用的引物序列

Table 1 Primer sequences used for BhYAB4b gene in this study

引物用途Primer purpose	引物名称Primer name	引物序列（5′-3′）Primer sequence
基因克隆 Gene clone	Clone-BhYAB4b-F	ATGGTTTGGGGGGTGATCATTTACTTTTGT
基因克隆 Gene clone	Clone-BhYAB4b-R	TTATGTCAAAAATGAATGCCTTGGGAAACA
表达分析 Expression analysis	BhYAB4b-qRT-F	GCTCGAGCCTCCTTTCTGTT
表达分析 Expression analysis	BhYAB4b-qRT-R	GGTGTCACAGGAATCAAGCC
内参基因 Refenrence gene	BhUBQ-qRT-F	CCTAACTGGGAAGACGAT
	BhUBQ-qRT-R	CAAGACCAAGTGAAGGGT
	At-ACT-F	TAACAGGGAGAAGATGACTCAGATCA
	At-ACT-R	AAGATCAAGACGAAGGATAGCATGAG
亚细胞定位 Subcellular localization	BhYAB4b-pS1300-F	TACACCAAATCGACTCTAGTCTAGAAT GGTTTGGGGGGTGATCATTTACT
亚细胞定位 Subcellular localization	BhYAB4b-pS1300-R	CATGGTACCGGATCCACTAGTTGTCAA AAATGAATGCCTTGGGAAACA
拟南芥过表达 Overexpression in Arabidopsis	BhYAB4b-PCY-F	GAACACGGGGGACGAGCTCGGTACC ATGGTTTGGGGGGTGATCATTTACT
拟南芥过表达 Overexpression in Arabidopsis	BhYAB4b-PCY-R	GGTGGTGGTGGTGGTCGACGGATCCT TATGTCAAAAATGAATGCCTTGGGAAA

图1 BhYAB4b基因片段扩增

Fig. 1 Fragment amplification of BhYAB4b

图2 BhYAB4b蛋白亲水性分析（A）和二级结构预测（B）

Fig. 2 BhYAB4b protein hydrophilicity analysis（A）and prediction of secondary structure（B）

图3 冬瓜BhYAB4b蛋白与其他物种YAB4蛋白的系统发育树系统发育树由邻接法构建，bootstrap重复为1 000次

Fig. 3 Phylogenetic tree of wax gourd BhYAB4b protein and YAB4 proteins from other species The phylogenetic tree was constructed by the neighbor-joining method with 1 000 bootstrap replicates

图4 BhYAB4b蛋白的亚细胞定位

Fig. 4 Subcellular localization of BhYAB4b protein

图5 BhYAB4b在B227不同组织（A）及不同发育时期果实（B）中的表达水平不同字母表示差异显著（P < 0.05），下同

Fig. 5 Expression levels of BhYAB4b in different tissues（A）and fruits at different developmental stages（B）of B227 Different letters indicate significant differences（P < 0.05），the same below

图6 35S：BhYAB4b异源过表达拟南芥表型鉴定及分析 A ~ C：野生型（WT）和35S：BhYAB4b植株播种后30 d全株（A）、莲座叶（B）和果实表型（C）；D：BhYAB4b表达量；E ~ H：莲座叶及果实长度和宽度、果实长宽比比较。数值为平均值 ± 标准偏差（n = 3）

Fig. 6 Phenotypic observation of 35S：BhYAB4b overexpression Arabidopsis A-C：Phenotypes of whole plants（A），rosette leaves（B），and fruits（C）of wild-type（WT）and 35S：BhYAB4b plants at 30 days after sowing；D：Expression levels of BhYAB4b；E-H：Comparison of rosette leaf and fruit length and width，and fruit length-to-width ratio. Values represent mean ± standard deviation（n = 3）

参考文献 31

[1]	Chen L, Zhang J, Li H, Niu J, Xue H, Liu B, Wang Q, Luo X, Zhang F, Zhao D, Cao S. 2017. Transcriptomic analysis reveals candidate genes for female sterility in pomegranate flowers. Frontiers in Plant Science,8:1430.
[2]	Clough S J, Bent A F. 1998. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal:for Cell and Molecular Biology,16:735-743.
[3]	Dong Hao, Li Yixing, Wang Tiankang, Song Shufeng, Qiu Mudan, Li Lei, Li Li. 2021. Functional study of OsYABBY4 gene regulating plant height in rice. Hybrid Rice, 36 (2):89-95. (in Chinese)
	董皓, 李懿星, 王天抗, 宋书锋, 邱牡丹, 李磊, 李莉. 2021. OsYABBY4基因调控水稻株高的功能研究. 杂交水稻, 36 (2):89-95.
[4]	Finet C, Floyd S K, Conway S J, Zhong B J, Scutt C P, Bowman J L. 2016. Evolution of the YABBY gene family in seed plants. Evolution & Development,18:116-126.
[5]	Han H Q, Liu Y, Jiang M M, Ge H Y, Chen H Y. 2015. Identification and expression analysis of YABBY family genes associated with fruit shape in tomato(Solanum lycopersicum L.). Genetics and Molecular Research, 14 (2):7079-7091. doi: 10.4238/2015.June.29.1 pmid: 26125918
[6]	He Changxia, Luo Chen, Yan Jinqiang, Liu Wenrui, Wang Min, Xie Dasen, Wu Zhimin, Jiang Biao. 2024. Screening and evaluation of reference genes in real-time fluorescent quantitative PCR of wax gourd. Acta Horticulturae Sinica, 51 (4):748-760. (in Chinese)
	何长霞, 罗陈, 闫晋强, 刘文睿, 王敏, 谢大森, 吴智明, 江彪. 2024. 冬瓜实时荧光定量PCR内参基因的筛选与评价. 园艺学报, 51 (4):748-760.
[7]	Huang Z J, Van Houten J, Gonzalez G, Xiao H, Knaap E V D. 2013. Genome-wide identification phylogeny and expression analysis of SUN OFP and YABBY gene family in tomato. Molecular Genetics and Genomics, 288 (3):111-129.
[8]	Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X:molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol,35:1547-1549.
[9]	Lee J Y, Baum S F, Oh S H, Jiang C Z, Chen J C, Bowman J L. 2005. Recruitment of CRABS CLAW to promote nectary development within the eudicot clade. Development, 132 (22):5021-5032. doi: 10.1242/dev.02067 pmid: 16236772
[10]	Li Hao. 2023. Functional study of PagYAB2 transcription factors in the secondary growth process of poplar trees[M. D. Dissertation]. Tai’an: Shandong Agricultural University. (in Chinese)
	李好. 2023. 杨树PagYAB2转录因子在次生生长过程的功能研究[硕士论文]. 泰安: 山东农业大学.
[11]	Letunic I, Doerks T, Bork P. 2011. SMART 7:recent updates to the protein domain annotation resource. Nucleic Acids Research,40:D302-D305.
[12]	Liu H L, Xu Y Y, Xu Z H, Chong K. 2007. A rice YABBY gene,OsYABBY4,preferentially expresses in developing vascular tissue. Development Genes and Evolution, 217 (9):629-637.
[13]	Liu X, Ning K, Che G, Yan S, Han L, Gu R, Li Z, Weng Y, Zhang X. 2018. CsSPL functions as an adaptor between HD-ZIP III and CsWUS transcription factors regulating anther and ovule development in Cucumis sativus(cucumber). The Plant Journal:for Cell and Molecular Biology, 94 (3):535-547.
[14]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆CT method. Methods,25:402-408.
[15]	Luo C, Yan J, Liu W, Xu Y, Sun P, Wang M, Xie D, Jiang B. 2022. Genetic mapping and genome-wide association study identify BhYAB4 as the candidate gene regulating seed shape in wax gourd(Benincasa hispida). Frontiers in Plant Science,13:961864.
[16]	Ma G L, Han Y Y, Li T T, Xia T, Gao L P. 2025. Phosphorylation modification reverses the transcriptional inhibitory activity of CsMYB4a in tea plants(Camellia sinensis). Horticultural Plant Journal, 11 (1):406-416.
[17]	Mei Yulin, Xu Jinjian, Jiang Mengyue, Yu Junhai, Zong Yu, Chen Wenrong, Liao Fanglei, Guo Weidong. 2023. Identification of F₁ hybrids and analysis of fruit shape difference between fingered citron and citron. Acta Horticulturae Sinica, 50 (7):1402-1418. (in Chinese)
	梅煜琳, 徐劲剑, 蒋梦玥, 尉俊海, 宗宇, 陈文荣, 廖芳蕾, 郭卫东. 2023. 香橼与佛手F₁杂种鉴定及果形差异分析. 园艺学报, 50 (7):1402-1418.
[18]	Romanova M A, Maksimova A I, Pawlowski K, Voitsekhovskaja O V. 2021. YABBY genes in the development and evolution of land plants. International Journal of Molecular Sciences, 22 (8):4139.
[19]	Sun L, Wei Y Q, Wu K H, Yan J Y, Xu J N, Wu Y R, Li G X, Xu J M, Harberd N P, Ding Z J, Zheng S J. 2021. Restriction of iron loading into developing seeds by a YABBY transcription factor safeguards successful reproduction in Arabidopsis. Molecular Plant, 14 (10):1624-1639. doi: 10.1016/j.molp.2021.06.005 pmid: 34116221
[20]	Sun Li. 2021. The mechanism and biological function of transcription factor INO regulating iron loading in Arabidopsis seeds[Ph. D. Dissertation]. Hangzhou: Zhejiang University. (in Chinese)
	孙鹂. 2021. 转录因子INO调控拟南芥种子铁装载的机制及其生物学功能[博士论文]. 杭州: 浙江大学.
[21]	Vijayan A, Tofanelli R, Strauss S, Cerrone L, Wolny A, Strohmeier J, Kreshuk A, Hamprecht F A, Smith R S, Schneitz K. 2021. A digital 3D reference atlas reveals cellular growth patterns shaping the Arabidopsis ovule. ELife,10:e63262.
[22]	Villanueva J M, Broadhvest J, Hauser B A, Meister R J, Schneitz K, Gasser C S. 1999. INNER NO OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules. Genes & Development, 13 (23):3160-3169.
[23]	Xie Dasen, Jiang Biao, Liu Wenrui, Xue Shudan, Zhang Baige. 2020. Research progress on the diversification breeding of high-quality and disease-resistant wax gourd. Guangdong Agricultural Sciences, 47 (11):50-59. (in Chinese)
	谢大森, 江彪, 刘文睿, 薛舒丹, 张白鸽. 2020. 优质、抗病冬瓜多样化育种研究进展. 广东农业科学, 47 (11):50-59.
[24]	Yamada T, Ito M, Kato M. 2004. YABBY2-homologue expression in lateral organs of Amborella trichopoda(Amborellaceae). International Journal of Plant Sciences, 165 (6):917-924.
[25]	Yamada T, Yokota S, Hirayama Y, Imaichi R, Kato M, Gasser C S. 2011. Ancestral expression patterns and evolutionary diversification of YABBY genes in angiosperms. The Plant Journal:for Cell and Molecular Biology, 67 (1):26-36.
[26]	Yang C, Ma Y, Li J. 2016. The rice YABBY4 gene regulates plant growth and development through modulating the gibberellin pathway. Journal of Experimental Botany, 67 (18):5545-5556.
[27]	Yang H, Shi G, Li X, Hu D, Cui Y, Hou J, Yu D, Huang F. 2019. Overexpression of a soybean YABBY gene,GmFILa,causes leaf curling in Arabidopsis thaliana. BMC Plant Biol, 19 (1):234. doi: 10.1186/s12870-019-1810-2 pmid: 31159746
[28]	Yang Z, Peng Z, Wei S, Liao M, Yu Y, Jang Z. 2015. Pistillody mutant reveals key insights into stamen and pistil development in wheat(Triticum aestivum L.). BMC Genomics, 16 (1):211.
[29]	Yu Tingting, Shen Shurong, Xu Yiling. 2024. Identification and expression analysis of the strawberry YABBY gene family. Chinese Journal of Biotechnology, 40 (1):104-121. (in Chinese) doi: 10.13345/j.cjb.230118 pmid: 38258635
	余婷婷, 沈淑容, 许以灵. 2024. 草莓YABBY基因家族的鉴定及表达分析. 生物工程学报, 40 (1):104-121. pmid: 38258635
[30]	Zhang S L, Wang L, Sun X M, Li Y D, Yao J, Nocker S, Wang X P. 2019. Genome-wide analysis of the YABBY gene family in grapevine and functional characterization of VvYABBY4. Frontiers in Plant Science,10:1207.
[31]	Zhang Zhanping, Sun Jiaying, Lu Jiaxin, Kong Lingyang, Xu Jiao, Ma Wei, Liu Xiubo. 2023. Current status of research on the characteristics and functions of plant-specific YABBY gene. Zhejiang Agricultural Sciences, 64 (6):1545-1552. (in Chinese)
	张占平, 孙嘉莹, 陆佳欣, 孔令阳, 徐姣, 马伟, 刘秀波. 2023. 植物特有的YABBY基因特征与功能研究现状. 浙江农业科学, 64 (6):1545-1552. doi: 10.16178/j.issn.0528-9017.20220911