Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (5): 991-1003.doi: 10.3864/j.issn.0578-1752.2025.05.013

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression of BcDET2 Gene and Functional of Its Regulatory Effect on Bolting and Flowering in Wucai (Brassica campestris L.)

ZHENG YaQin1(), LIU XueQing1, WU SiWen1,2, TANG XiaoYan1, YANG DanNi1, WANG YongKang1, AHMAD Aftab1, KHAN Afrsyab1, WANG ChengGang1,2, CHEN GuoHu1,2()   

  1. 1 College of Horticulture, Anhui Agricultural University/Anhui Provincial Engineering Center of Horticultural Crop Breeding, Hefei 230036
    2 Anhui Provincial Wanjiang Vegetable Industrial Technology Institute, Maanshan 238200, Anhui
  • Received:2024-07-13 Accepted:2024-08-16 Online:2025-03-07 Published:2025-03-07
  • Contact: CHEN GuoHu

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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

Table 1

The primers used in this study"

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
1300-BcDET2-F ggagaggacagggtacccgggATGGAGATGGTGACGAGTTTTGT 载体构建
Vector construction
1300-BcDET2-R ggtactagtgtcgactctagaGAACACAAAGGGAATCAGAGCC
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

Fig. 1

Cloning and analysis of BcDET2 gene A: cDNA and protein sequences of BcDET2; B: Protein hydrophobicity prediction of BcDET2; C: Multiple sequence alignment, * Glutamate is required for 5α-reductase; D: Phylogenetic tree analyses of DET2 proteins. AlDET2: Arabidopsis lyrata, Araly1|488450; AtDET2: A. thaliana, AT5G16010; CasDET2: Camelina sativa, Csa20g023550.1; CruDET2: Capsella rubella, XP_023636418.1; BcDET2: B. campestris, BcDET2; BnaDET2: B. napus, CAF2345619.1; BoDET2: B. oleracea, BolC09g057210.2J; BrDET2: B. rapa, BraA10g023600.3C; EsDET2: Eutrema salsugineum, XP_006400130.1; RsDET2: Raphanus sativus, Rsa10025279"

Fig. 2

Analysis of BcDET2 gene expression pattern A: Relative expression of BcDET2 gene in various tissues of Wucai; B: Relative expression of BcDET2 gene at 0, 15, and 30 days of vernalization; C-D: Transcription level and protein abundance of BcDET2 gene at 0, 15, and 30 days of vernalization. Different letters indicate significant differences at P<0.05"

Fig. 3

BcDET2 protein structure and bioinformatics analyses A: Tertiary structure modeling; B: Potential phosphorylation sites; C: Potential N-glycosylation sites; D: Transmembrane domain prediction; E: Signal peptide analysis"

Fig. 4

Subcellular localization of BcDET2 A: Vector construction; B: Fluorescence microscopy observation"

Fig. 5

Analysis of BcDET2 gene genetic transformation into Wucai A-B: PCR detection of BcDET2 (A) and HPTⅡ (B) in transgenic plants; M: Marker1000; N: Negative control (WT); P: Positive control (p1300-BcDET2); 0: (ddH2O); 1-2: Transgenic plants; C: Relative expression levels of BcDET2 gene in transgenic plant; D: Phenotype of BcDET2 gene transgenic Wucai; E-F: Analysis of flowering days and rosette leaf number at the initial bolting of OE#2 transgenic plants. G-H: Relative expression levels of BcSOC1 and BcFLC1 genes in OE#2 transgenic plant. *: Significant differences at P<0.05, ***: Significant differences at P<0.001. The same as below"

Fig. 6

BcBHLH96 interaction with BcDET2 and its expression analyses A-B: Yeast library screening using BcDET2 and transfection verification; +: Positive control (pGADT7-largeT+pGBKT7-p53); -: Negative control (pGADT7-largeT+pGBKT7-laminC); C: Yeast two-hybrid verification of the interaction between BcBHLH96 and BcDET2; D: Expression of BcBHLH96 gene in response to vernalization; E: Expression of BcBHLH96 gene in the BcDET2 transgenic Wucai plants. ns: No significant difference"

Table 2

Screening of candidate interacting proteins of BcDET2 from yeast cDNA library"

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
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