乌菜BcDET2的克隆及其抽薹开花调控功能

doi:10.3864/j.issn.0578-1752.2025.05.013

摘要/Abstract

摘要：

【目的】通过对乌菜（Brassica campestris L. ssp. chinensis var. rosularis Tsen）油菜素内酯（brassinosteroid，BR）合成途径中类固醇5α-还原酶基因BcDET2（DE-ETIOLATED 2）进行克隆和表达分析，遗传转化乌菜植株，分析其在抽薹开花调控中的作用，为乌菜遗传育种提供分子依据。【方法】基于大白菜（Brassica rapa L.）基因组DET2（BraA10g023600.3C）基因序列，通过同源克隆获得乌菜BcDET2。利用Expasy、SOPMA、SWISS-MODEL、TMHMM等在线工具对BcDET2进行生物信息学分析。运用荧光定量PCR（qRT-PCR）等方法分析BcDET2的表达模式。采用农杆菌介导法瞬时侵染烟草（Nicotiana tabacum）细胞，进行BcDET2的亚细胞定位；遗传转化乌菜分析BcDET2在乌菜抽薹开花中的调控功能。利用酵母双杂交技术筛选和鉴定BcDET2的互作蛋白，并运用qRT-PCR分析该互作蛋白基因对春化的响应，以及在BcDET2过表达植株中的表达水平。【结果】成功获得乌菜BcDET2的cDNA序列（825 bp），编码274个氨基酸。生物信息学分析结果显示，BcDET2蛋白质结构以α-螺旋为主，预测为弱亲水性、不稳定碱性蛋白；含有39个磷酸化位点和5个跨膜结构域，无信号肽，表明其可能为非分泌型膜蛋白。进化树分析表明，BcDET2与大白菜（B. rapa）和油菜（B. napus）的DET2具有较近的亲缘关系。亚细胞定位显示，BcDET2在细胞核和细胞膜上均有荧光信号。qRT-PCR等分析表明，BcDET2在春化15 d时表达量最高，表明其对春化过程具有响应性。功能研究发现，过表达BcDET2显著促进乌菜转基因植株的早花现象。酵母双杂交试验表明，BcDET2可与转录因子BcBHLH96互作，且该转录因子在春化后期具有较高的转录水平，并且在过表达BcDET2植株中表达水平显著升高。【结论】成功克隆了乌菜BcDET2，该基因可能通过春化途径参与乌菜抽薹开花的调控。

关键词: 乌菜, BcDET2, 基因克隆, 酵母双杂交, 抽薹开花

Abstract:

【Objective】 The steroid 5α-reductase gene BcDET2 (DE-ETIOLATED 2), involved in the brassinosteroid (BR) biosynthesis pathway, was cloned and analyzed in Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). The functions of BcDET2 in regulating bolting and flowering were investigated through genetic transformation, thereby providing a molecular basis for the genetic breeding of Wucai. 【Method】The BcDET2 was obtained via homologous cloning, based on the DET2 (BraA10g023600.3C) sequence from the Chinese cabbage (B. rapa) genome. Bioinformatics analyses were performed using online tools such as Expasy, SOPMA, SWISS-MODEL, and TMHMM. The expression pattern of BcDET2 was analyzed using quantitative real-time PCR (qRT-PCR). Subcellular localization of BcDET2 was determined through Agrobacterium-mediated transient transformation in tobacco (Nicotiana tabacum) cells. The role of the BcDET2 in regulating bolting and flowering was studied through genetic transformation of Wucai. Yeast two-hybrid (Y2H) assay was utilized to screen and identify BcDET2-interacting proteins, followed by qRT-PCR analysis to examine the response of BrbHLH96 to vernalization and its expression levels in BcDET2 transgenic plants. 【Result】The cDNA sequence of BcDET2 (825 bp), encoding 274 amino acids, was successfully obtained through homologous cloning. Bioinformatics analysis indicated that the BcDET2 protein predominantly consists of α-helices, is predicted to be a weakly hydrophilic, non-secretory membrane protein, and contains 39 phosphorylation sites and 5 transmembrane domains. Phylogenetic analysis revealed that BcDET2 shares a close evolutionary relationship with DET2 from Brassica rapa and Brassica napus. Subcellular localization showed fluorescent signals of BcDET2 in both the nucleus and cell membrane. qRT-PCR analysis indicated the highest expression level of BcDET2 at 15 days of vernalization, suggesting its responsiveness to the vernalization process. Functional assay demonstrated that overexpression of BcDET2 significantly promoted early flowering in transgenic Wucai plants. Y2H analysis revealed that BcDET2 interacts with the transcription factor BcBHLH96, which exhibited high transcription levels in the late vernalization stage and significantly increased expression in BcDET2 transgenic plants. 【Conclusion】In this study, the cDNA of BcDET2 from Wucai was cloned, and it is likely involved in the regulation of bolting and flowering in Wucai via the vernalization pathway.

Key words: Wucai, BcDET2, gene cloning, yeast two-hybrid, bolting and flowering

郑雅琴, 刘雪晴, 吴思文, 唐小燕, 杨丹妮, 汪永康, Ahmad Aftab, Khna Afrsyab, 汪承刚, 陈国户. 乌菜BcDET2的克隆及其抽薹开花调控功能[J]. 中国农业科学, 2025, 58(5): 991-1003.

ZHENG YaQin, LIU XueQing, WU SiWen, TANG XiaoYan, YANG DanNi, WANG YongKang, AHMAD Aftab, KHAN Afrsyab, WANG ChengGang, CHEN GuoHu. Cloning and Expression of BcDET2 Gene and Functional of Its Regulatory Effect on Bolting and Flowering in Wucai (Brassica campestris L.)[J]. Scientia Agricultura Sinica, 2025, 58(5): 991-1003.

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引物 <BOLD>P</BOLD>rimer	序列 Sequences (5′-3′)	用途 Application
BcDET2-F	ATGGAGATGGTGACGAGTTTT	基因克隆 Gene cloning
BcDET2-R	CTAGAACACAAAGGGAATCAGAG	基因克隆 Gene cloning
1300-BcDET2-F	ggagaggacagggtacccgggATGGAGATGGTGACGAGTTTTGT	载体构建 Vector construction
1300-BcDET2-R	ggtactagtgtcgactctagaGAACACAAAGGGAATCAGAGCC	载体构建 Vector construction
35S-BcDET2-F	TTCATTTTGGAGAGAACACGGGGGAC	转基因植株鉴定 Identification of transgenic plants
35S-BcDET2-R	TCGGCCGTCGAGCTCCACGAGG
HTP-F	AAATCCGCGTGCACGAGGT
HTP-R	TCGTTATGTTTATCGGCACTTTGCA
BcDET2-qF	AAAACTTCACGCTAGGACTACCC	实时荧光定量PCR qRT-PCR
BcDET2-qR	ATGGTGATACAGATTCCCAACG
BcFLC1-F	CACGGCACAGAGACCTCTCG
BcFLC1-R	AGAAGGTGACTTGTCTGCTACTGTTT
BcSOC1-F	CGAGCAAGAAAGACTCAACTGTTTA
BcSOC1-R	AGATCCCCACTTTTCAGTGAGC
Actin-F	TGGGTTTGCTGGTGACGAT
Actin-R	TGCCTAGGACGACCAACAATACT

序号 Number	基因ID Gene ID	基因名称 Gene name	拟南芥同源ID Arabidopsis gene ID	E值 E value
1	BraA08g004120.3C	SRP54	AT1G48900	0.0
2	BraA05g014300.3C	CYP5	AT2G29960	2e-129
3	BraA01g013480.3C	PGRL1A	AT4G22890	0.0
4	BraA07g036580.3C	bHLH96	AT1G72210	7e-167
5	BraA06g043480.3C	ADH2	AT5G43940	0.0
6	BraA08g022960.3C	ATFP6	AT4G38580	2e-108
7	BraA03g015610.3C	PABN1	AT5G51120	2e-88
8	BraA03g028470.3C	ATHM2	AT4G03520	7e-88