菜薹BrWRKY57的特性及其对BrPPH1和BrNCED3的调控

doi:10.16420/j.issn.0513-353x.2020-0432

摘要/Abstract

摘要：

从菜薹叶片中分离获得1个WRKY转录因子,命名为BrWRKY57。氨基酸序列比对及进化树分析发现BrWRKY57与拟南芥AtWRKY57同源性较高,含有1个WRKY保守结构域,且同属于WRKY转录因子家族Ⅱc亚族。实时荧光定量PCR分析表明,BrWRKY57随着菜薹叶片衰老表达水平升高,外源脱落酸（abscisic acid,ABA）处理显著诱导其表达。亚细胞定位和转录活性分析表明,BrWRKY57定位于细胞核,且在酵母和烟草体内都具有转录激活活性。双荧光素酶瞬时表达试验显示,BrWRKY57可以激活叶绿素降解相关基因BrPPH1和ABA合成相关基因BrNCED3的启动子活性。以上研究结果表明,BrWRKY57参与菜薹叶片衰老过程可能与影响叶绿素降解和ABA合成相关基因的表达有关。

关键词: 菜薹, 叶片衰老, 脱落酸, WRKY转录因子, 转录调控

Abstract:

In this study,a WRKY transcription factor termed BrWRKY57,was obtained from Chinese flowering cabbage. Amino acid sequence alignment and phylogenetic analysis revealed that BrWRKY57 contained one WRKY conserved domain,exhibited the highest homology with Arabidopsis thalianaAtWRKY57,and belonged to sub-group IIc. Real-time quantitative PCR analysis demonstrated that BrWRKY57 was up-regulated during leaf senescence of Chinese flowering cabbage,and its expression was significantly enhanced by abscisic acid（ABA）treatment. Subcellular localization and transcriptional activity analysis suggested that BrWRKY57 was a nuclear protein and had transcriptional activation activity. Moreover,dual-luciferase transient expression assay revealed that BrWRKY57 could activate the promoter activities of chlorophyll catabolic gene BrPPH1and ABA biosynthetic gene BrNCED3. These results indicate that BrWRKY57 might be associated with Chinese flowering cabbage leaf senescence via effecting the genes expression of chlorophyll degradation and ABA synthesis.

Key words: Chinese flowering cabbage, leaf senescence, abscisic acid, WRKY transcription factor, transcriptional regulation

中图分类号:

S634.5

曾泽湘, 肖显梅, 谭小丽, 范中奇, 陈建业. 菜薹BrWRKY57的特性及其对BrPPH1和BrNCED3的调控[J]. 园艺学报, 2021, 48(3): 518-530.

ZENG Zexiang, XIAO Xianmei, TAN Xiaoli, FAN Zhongqi, CHEN Jianye. Characteristics of the Transcription Factor BrWRKY57 and Its Regulation on BrPPH1 and BrNCED3[J]. Acta Horticulturae Sinica, 2021, 48(3): 518-530.

图/表 10

表1 引物序列

Table 1 Primer sequences

引物用途 Primer assay	引物名称 Primer name	引物序列（5′-3′） Primer sequence	限制性内切酶 Enzyme
全长扩增Full- length amplification	BrWRKY57	F:ATGAACGATCCTAAAGATCCCGAT; R:TCAAGGATTGCGCATGGTTTGAG
实时荧光定量PCR Real-time quantitative PCR	BrAct1 BrWRKY57 BrPPH BrNCED3	F:CGCTTAACCCGAAAGCTAAC;R:TACGCCCACTAGCGTAAAG F:ACGGAGGAGAAGAAGAAGGC;R:ACTCTCTTCTTCACCGTGCA F:ATCGAGTATGGGCGGTTGAT;R:CCTGCAATGTACACTGGCTC F:GTCCTCTGCGAGATTCGGCT;R:GGAGACTTTAGGCCACGGCT
亚细胞定位 Subcellular localization	BrWRKY57	F:ATCTAGAGCAGTCGACGGTACCATGAACGATCCTAAAGATCCCGAT R:CTCCTCGCCCTTGCTCACCATAGGATTGCGCATGGTTTGAGGTAC	KpnⅠ BamHⅠ
酵母体内转录活性分析Transcriptional activity in yeast cells	BrWRKY57-pGBKT7	F:CATGGAGGCCGAATTCATGAACGATCCTAAAGATCCCGAT R:GCCGCTGCAGGTCGACGTCAAGGATTGCGCATGGTTTGAG	EcoR Ⅰ SalⅠ
双荧光素酶瞬时表达 Dual-luciferase transient expression assay	62SK-BD-BrWRKY57 62SK-BrWRKY57 0800-BrPPH1 pro 0800-BrNCED3 pro	F:CGCCGTCTAGAACTAGTGGATCCATGAACGATCCTAAAGATCCCGAT R:TCGATAAGCTTGATATCGAATTCTCAAGGATTGCGCATGGTTTGAG F:GGCCGCTCTAGAACTAGTGGATCCATGAACGATCCTAAAGATCCCGAT R:ATCGATAAGCTTGATATCGAATTCTCAAGGATTGCGCATGGTTTGAGG F:TATAGGGCGAATTGGGTACCCTTTAGAGCATACTGAGGCTG R:TTGGCGTCTTCCATGGTACACAAATCAATCCTGTAAGGAAC F:TATAGGGCGAATTGGGTACCCTTAGAGACCAAGTCATATGATGTA R:TTGGCGTCTTCCATGGTTTAAATATGATTAGTCAGTATTGGTGC	BamHⅠ EcoRⅠ BamHⅠ EcoRⅠ KpnⅠ NcoⅠ KpnⅠ NcoⅠ

图1 ABA处理加速采后菜薹叶片外观衰老和Fv/Fm荧光成像

Fig. 1 ABA treatment accelerates appearance senescence in Chinese flowering cabbage and chlorophyll fluorescence imaging（Fv/Fm）

图2 叶绿素含量和Fv/Fm值降低

Fig. 2 The decrease in chlorophyll content and Fv/Fm ratio *P < 0.05;** P < 0.01

图3 菜薹BrWRKY57与拟南芥（At）和番茄（Sl）的WRKY氨基酸序列比对

Fig. 3 The sequences alignment of BrWRKY57 with WRKYs in Arabidopsis（At）and tomato（Sl）

图4 BrWRKY57（“●”）与拟南芥（At）和番茄（Sl）WRKY蛋白的进化树分析

Fig. 4 The phylogenetic tree of BrWRKY57（“●”indicated）with Arabidopsis（At）and tomato（Sl）WRKY proteins

图5 对照组（H2O）和ABA处理组的采后菜薹叶片衰老过程中BrWRKY57、BrPPH1和BrNCED3的表达

Fig. 5 Expression of BrWRKY57,BrPPH1 and BrNCED3 in control（H2O）and ABA-treated cabbages leaves during senescence *P < 0.05,** P < 0.01.

图6 BrWRKY57在烟草叶片的亚细胞定位

Fig. 6 Subcellular localization of BrWRKY6 in tobacco leaves

图7 BrWRKY57在酵母体内的转录活性分析

Fig. 7 Transcriptional activity assay of BrWRKY57 in yeast cells

图8 BrWRKY57在烟草体内的转录活性分析

Fig. 8 Transcriptional activity assay of BrWRKY57 in tobacco leaves **P < 0.01.

图9 BrWRKY57激活BrPPH1和BrNCED3基因的启动子活性

Fig. 9 BrWRKY57 activates the promoter activities of BrPPH1 and BrNCED3 **P < 0.01.

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