芥菜BjuSPL10基因表达及调控抽薹开花的功能解析

doi:10.16420/j.issn.0513-353x.2024-0835

摘要/Abstract

摘要：

本研究中克隆了芥菜的BjuSPL10基因，qRT-PCR分析表明，该基因在芥菜不同部位的表达存在差异，在花中最高。亚细胞定位发现，BjuSPL10蛋白定位于细胞核中。进一步构建了BjuSPL10的过表达载体，遗传转化芥菜发现该基因能显著促进芥菜抽薹开花，同时提高开花整合子BjuSOC1的表达量。酵母单杂交和萤火虫素酶活性实验表明：芥菜BjuSPL10蛋白与BjuSOC1基因的启动子存在相互作用，并促进该基因的表达。

关键词: 芥菜, SPL10, 开花, 调控, BjuSOC1, 转录激活, 酵母单杂交, 萤火虫素酶活性

Abstract:

In this study，the BjuSPL10 gene was cloned from Brassica juncea. qRT-PCR analysis showed that the expression of BjuSPL10 gene was different in different parts of Brassica juncea，and the expression level was the highest in flowers. Subcellular localization revealed that BjuSPL10 protein was localized in the nucleus. Subsequently，the BjuSPL10 overexpression vector was constructed and transformed into Brassica juncea. The results showed that BjuSPL10 could significantly promote bolting and flowering in transgenic lines，and increase the expression level of flowering integrator BjuSOC1 gene. Furthermore，yeast one-hybrid and luciferase assays showed that the BjuSPL10 protein directly interacted with the promoter of BjuSOC1 gene，then enhanced its expression level.

Key words: Brassica juncea, SPL10, flowering, regulation, BjuSOC1, transcriptional activation, yeast one-hybrid, luciferase assay

赵一曼, 李景飞, 唐卓然, 葛家兴, 魏大勇, 王志敏, 邹家琪, 汤青林. 芥菜BjuSPL10基因表达及调控抽薹开花的功能解析[J]. 园艺学报, 2025, 52(5): 1341-1350.

ZHAO Yiman, LI Jingfei, TANG Zhuoran, GE Jiaxing, WEI Dayong, WANG Zhimin, ZOU Jiaqi, TANG Qinglin. Functional Identification and Expression Analysis of BjuSPL10 in Regulating Brassica juncea Bolting and Flowering[J]. Acta Horticulturae Sinica, 2025, 52(5): 1341-1350.

图/表 8

表1 本试验中所用引物

Table 1 Primers used in this study

用途 Purpose	基因名称 Gene name	引物序列（5′-3′） Sequences
基因克隆 Gene cloning	BjuSPL10	F：ATGGAATGCAACATGAGATC； R：TCATATAAAATGGGTAGGAG
实时荧光定量PCR qRT-PCR	qPCR-BjuSPL10	F：TGTGGGACTGGGAGCATTTG； R：ACAGGAGTGTGCAGGAAACC
实时荧光定量PCR qRT-PCR	BjuACTIN	F：GCTGACCGTATGAGCAAAGA； R：GTTGGAAAGTGCTGAGGGAT
亚细胞定位 Subcellular Localization	BjuSPL10-GFP	F：atacaccaaatcgactctagaATGGAATGCAACATGAGATCTCC； R：gcccttgctcaccatggtaccTATAAAATGGGTAGGAGGGAAA
过表达载体构建 Overexpressing vector	BjuSPL10-Bar	F：tttcatttggagaggagatctATGGAATGCAACATGAGATCTCC； R：caggacgtaacatggccatggTCATATAAAATGGGTAGGAGGGAAA
酵母单杂交 Yeast one-hybrid	BjuSPL10-AD	F：CCGGAATTCATGGAATGCAACATGAGATC； R：CGCGGATCCTCATATAAAATGGGTAGGAG
萤火虫素酶活性实验 LUC	BjuSPL10-SK	F：CGCGGATCCATGGAATGCAACATGAGATC； R：CCGGAATTCTCATATAAAATGGGTAGGAG

图1 芥菜BjuSPL10基因克隆产物

Fig. 1 PCR products electropherogram of BjuSPL10 in Brassica juncea

图2 芥菜（Bju）与拟南芥（At）SPL家族成员的系统发育进化树 Ⅰ ~ Ⅳ为亚家族，数据表示自展值

Fig. 2 Phylogenetic evolutionary tree of Brassica juncea and Arabidopsis thaliana SPL family members Ⅰ-Ⅳ are subfamilies，data indicate self-expansion values

图3 芥菜BjuSPL10在不同组织中的相对表达量（A）及其在烟草叶片中的亚细胞定位（B）使用Duncan’s法分析差异显著性，不同小写字母表示不同组织中的表达量差异显著（P < 0.05）

Fig. 3 Relative expression of BjuSPL10 gene in different tissues of Brassica juncea（A）and its subcellular localization in tobacco leaves（B） Significance of differences was analyzed using the Duncan’s method，and different letters indicate significant differences in expression in different tissues（P < 0.05）

图4 芥菜过表达BjuSPL10植株开花表型（A）及表达水平检测（B） WT：未转基因芥菜；OE-1和OE-2：芥菜BjuSPL10过表达株系。使用Duncan’s法分析，*、**和***分别代表P < 0.05、P < 0.01、P < 0.001水平差异显著，下同

Fig. 4 Flowering phenotype of the transgenic overexpressing BjuSPL10（A）and its expression level detection in Brassica juncea（B） WT：Wild type plant；OE-1，OE-2：The transgenic overexpressing BjuSPL10 lines. Significance of differences was analyzed using the Duncan’s method，*，** and *** respectively mean significant difference at the P < 0.05，P < 0.01 and P < 0.001. The same below

图5 芥菜BjuSOC1和BjuFT在过表达BjuSPL10转基因植株中的表达水平

Fig. 5 Expression levels of BjuSOC1 and BjuFT in BjuSPL10-overexpressing transgenic plants

图6 酵母单杂交鉴定 NC：阴性对照；PC：阳性对照

Fig. 6 Identification of the BjuSPL10 protein interacting with the BjuSOC1 promoter by yeast one-hybrid NC：Negative control；PC：Positive control

图7 萤火虫素酶活性实验鉴定BjuSPL10蛋白与BjuSOC1启动子相互作用

Fig. 7 Identification of the BjuSPL10 protein interacting with the BjuSOC1 promoter by luciferase assays

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