甘蓝锌指蛋白转录因子BoC2H2的克隆、定位与表达分析
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摘要
C2H2型锌指蛋白是植物中最重要的转录调节子之一,主要调控植物的生长发育和胁迫应答反应。本研究通过分析自交不亲和甘蓝未授粉,自花授粉15、30和60min,异花授粉15、30和60min柱头转录组数据,筛选到一个自花授粉诱导上调表达的C2H2型锌指蛋白基因,命名为BoC2H2。BoC2H2开放阅读框756bp,编码251个氨基酸残基,蛋白分子量为26.7kDa,等电点为4.62,是一种亲水性蛋白,不含信号肽和跨膜域,含有C2H2型锌指蛋白家族高度保守的ZnF_C2H2结构域。BoC2H2起始密码子上游2000 bp的核苷酸序列中含有光响应、昼夜节律、茉莉酸响应、防御和应激反应等多种顺式作用元件。通过原生质体亚细胞定位和瞬时浸染烟草,发现BoC2H2蛋白在细胞核和细胞质中表达。BoC2H2在下胚轴、叶片和花中均有表达,柱头中的表达量随发育时间而变化,开花当天后表达量降低。荧光定量PCR结果显示, BoC2H2在自花授粉和异花授粉0～60 min的表达量变化趋势与转录组分析结果基本一致。综上所述,甘蓝BoC2H2属于C2H2型锌指蛋白家族,可能参与了柱头响应花粉刺激的分子过程,这有利于揭示BoC2H2在甘蓝自交不亲和过程中的作用机制,为研究C2H2型转录因子在甘蓝自交不亲和过程中的调控机制提供依据。
C2H2-type zinc finger protein family is one of the most important transcriptional regulator in plants, which it mainly involved in regulation plant growth and stress response. In this study, we screened and compared non-pollinated, self-or cross-pollinated 15, 30, and 60 min pistil transcriptome data, and isolated a gene with specifically up-regulated expression induced by self-pollination, named as BoC2H2. Molecular cloning indicated that BoC2H2 is a single exon gene, encoding a 251 amino acids protein. The protein molecular weight is 26.7 kDa and theoretical isoelectric point is 4.62. BoC2H2 contains a highly conserved ZnF_C2H2 domain. Physicochemical property analysis found BoC2H2 is a hydrophilic protein, not contains signal peptide and transmembrane domain. The 2000 bp upstream of BoC2H2 translation start codon contains light response, circadian rhythm, jasmonic acid response, defense and stress response cis acting elements and so on. The sub-cellular location of BoC2H2 in nuclear and cytoplasm were verified by transforming to Arabidopsis protoplast and tobacco. RT-PCR analysis indicated that BoC2H2 expressed in hypocotyls, leaves and flowers. The expression level of BoC2H2 in pistil changed with development stages and specifically decreased after flowering day. qRT-PCR analysis revealed that BoC2H2 mRNA expression level after self-and cross-pollination 0 min to 60 min were similar with RNA-seq data. In conclusion, BoC2H2 belongs toC2H2 type of zinc finger protein family and may involve in pistil-pollen stimulating molecular processes. Our founding is helpful to reveal the mechanism of BoC2H2 in Brassica oleracea self-incompatibility response and provide a clue for studying the function ofC2H2 type of transcription factors in B. oleracea self-incompatibility response.
C2H2-type zinc finger protein family is one of the most important transcriptional regulator in plants, which it mainly involved in regulation plant growth and stress response. In this study, we screened and compared non-pollinated, self-or cross-pollinated 15, 30, and 60 min pistil transcriptome data, and isolated a gene with specifically up-regulated expression induced by self-pollination, named as BoC2H2. Molecular cloning indicated that BoC2H2 is a single exon gene, encoding a 251 amino acids protein. The protein molecular weight is 26.7 kDa and theoretical isoelectric point is 4.62. BoC2H2 contains a highly conserved ZnF_C2H2 domain. Physicochemical property analysis found BoC2H2 is a hydrophilic protein, not contains signal peptide and transmembrane domain. The 2000 bp upstream of BoC2H2 translation start codon contains light response, circadian rhythm, jasmonic acid response, defense and stress response cis acting elements and so on. The sub-cellular location of BoC2H2 in nuclear and cytoplasm were verified by transforming to Arabidopsis protoplast and tobacco. RT-PCR analysis indicated that BoC2H2 expressed in hypocotyls, leaves and flowers. The expression level of BoC2H2 in pistil changed with development stages and specifically decreased after flowering day. qRT-PCR analysis revealed that BoC2H2 mRNA expression level after self-and cross-pollination 0 min to 60 min were similar with RNA-seq data. In conclusion, BoC2H2 belongs toC2H2 type of zinc finger protein family and may involve in pistil-pollen stimulating molecular processes. Our founding is helpful to reveal the mechanism of BoC2H2 in Brassica oleracea self-incompatibility response and provide a clue for studying the function ofC2H2 type of transcription factors in B. oleracea self-incompatibility response.
引文
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[3]Jiang L,Pan L J.Identification and expression of C2H2 transcription factor genes in Carica papaya under abiotic and biotic stresses.Mol Biol Rep,2012,39:7105-7115
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[6]黄骥,王建飞,张红生.植物C2H2型锌指蛋白的结构与功能.遗传,2004,26:414-418Huang J,Wang J F,Zhang H S.Structure and function of plant C2H2 zinc finger protein.Hereditas,2004,26:414-418(in Chinese with English abstract)
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[14]Agarwal P,Arora R,Ray S,Singh A K,Singh V P,Takatsuji H,Kapoor S,Tyagi A K.Genome-wide identification of C2H2zinc-finger gene family in rice and their phylogeny and expression analysis.Plant Mol Biol,2007,65:467-485
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[18]Yang B,Jiang Y Q,Rahman M H,Deyholos M K,Kav N N.Identification and expression analysis of WRKY transcription factor genes in canola(Brassica napus L.)in response to fungal pathogens and hormone treatments.BMC Plant Biol,2009,9:68
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[22]Mito T,Seki M,Shinozaki K,Takagi M O,Matsui K.Generation of chimeric repressors that confer salt tolerance in Arabidopsis and rice.Plant Biotechnol J,2011,9:736-746
[23]Zhang H,Liu Y P,Wen F,Yao D M,Wang L,Guo J,Ni L,Zhang A,Tan M P,Jiang M Y.A novel rice C2H2-type zinc finger protein,ZFP36,is a key player involved in abscisic acid-induced antioxidant defence and oxidative stress tolerance in rice.J Exp Bot,2014,65:5795-5809
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[25]Qureshi M K,Sujeeth N,Gechev T S,Hille J.The zinc finger protein ZAT11 modulates paraquat-induced programmed cell death in Arabidopsis thaliana.Acta Physiol Plant,2013,35:1863-1871
[26]Khatun K,Nath U K,Robin A H K,Park J I,Lee D J,Kim M B,Kim C K,Lim K B,Nou I S,Chung M Y.Genome-wide analysis and expression profiling of zinc finger homeodomain(ZHD)family genes reveal likely roles in organ development and stress responses in tomato.BMC Genomics,2017,18:695
[27]Wang W L,Wu P,Li Y,Hou X L.Genome-wide analysis and expression patterns of ZF-HD transcription factors under different developmental tissues and abiotic stresses in Chinese cabbage.Mol Genet Genomics,2016,291:1451-1464
[28]柳琼,黄毅,陶章生,黄顺谋,王新发,黄邦全,刘贵华.甘蓝型油菜TF III A型锌指蛋白基因鉴别和表达模式.中国油料作物学报,2014,36:562-571Liu Q,Huang Y,Tao Z S,Huang S M,Wang X F,Huang B Q,Liu G H.Identification and expression pattern of TF III A zinc finger protein gene in Brassica napus L.Chin J Oil Crop Sci,2014,36:562-571(in Chinese with English abstract)
[29]Bowman J L,Sakai H,Jack T,Weigel D,Mayer U,Meyerowitz EM.SUPERMAN,a regulator of floral homeotic genes in Arabidopsis.Development,1992,114:599-615
[30]Kobayashi A,Sakamoto A,Kubo K,Rybka Z,Kanno Y,Takatsuji H.Seven zinc-finger transcription factors are expressed sequentially during the development of anthers in petunia.Plant J,1998,13:571-576
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