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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1650-1660.doi: 10.3724/SP.J.1006.2018.01650

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝锌指蛋白转录因子BoC2H2的克隆、定位与表达分析

罗绍兰1,廉小平2,蒲敏1,白晓璟1,王玉奎1,曾静4,施松梅3,张贺翠1,朱利泉1,*()   

  1. 1 西南大学农学与生物科技学院, 重庆 400700
    2 西南大学园艺园林学院, 重庆 400700
    3 西南大学资源与环境科学学院, 重庆 400700
    4 长江师范学院生命科学与技术学院, 重庆 408100
  • 收稿日期:2018-02-12 接受日期:2018-07-20 出版日期:2018-11-12 网络出版日期:2018-07-30
  • 通讯作者: 朱利泉
  • 基金资助:
    本研究由国家自然科学基金项目(31572127);西南大学基本科研业务费专项资金项目(XDJK2017E073)

Molecular Cloning, Location and Expression Analysis of Brasscia oleracea Zinc Finger Protein Transcription Factor BoC2H2

Shao-Lan LUO1,Xiao-Ping LIAN2,Min PU1,Xiao-Jing BAI1,Yu-Kui WANG1,Jing ZENG4,Song-Mei SHI3,He-Cui ZHANG1,Li-Quan ZHU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400700, China
    2 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400700, China
    3 College of Resources and Environmental Sciences, Southwest University, Chongqing 400700, China
    4 College of Life Science and Biotechnology, Yangtze Normal University, Chongqing 400718, China
  • Received:2018-02-12 Accepted:2018-07-20 Published:2018-11-12 Published online:2018-07-30
  • Contact: Li-Quan ZHU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31572127);the Fundamental Research Fund of Southwest University(XDJK2017E073)

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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60 min, 异花授粉15、30和60 min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756 bp, 编码251个氨基酸残基, 蛋白分子量为26.7 kDa, 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。

关键词: 甘蓝, C2H2锌指蛋白, BoC2H2基因, 自交不亲和, 定位, 表达分析

Abstract:

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 to C2H2 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 of C2H2 type of transcription factors in B. oleracea self-incompatibility response.

Key words: Brassica oleracea, C2H2-type zinc finger protein, BoC2H2, self-incompatibility, location, expression analysis

表1

基因克隆及其荧光定量 PCR 分析所用引物"

引物名称
Primer		 引物序列
Primer sequence (5°-3°)		 引物说明
Primer annotation
1300-GFP-F GAGAACACGGGGGACTCTAGAATGAGTGATCCCGAGAAAACAAAAG 基因的亚细胞定位
1300-GFP-R GCCCTTGCTCACCATGAGCTCCTCGGCTTTGTCCTCTTTTGC Subcellular localization
35S-R CCGATCTAGTAACATAGATGACACCG 通用引物 Universal primer
Gus-F CAAGCTTGGCTGCAGGTCGACTTCGACTCAGCGTGTTATG 基因的启动子活性分析
Gus-R GGTGGACTCCTCTTAGAATTCGATTTTGACTTTGTTGAGAG Promoter activity analysis
1391-F GAACTGATCGTTAAAACTGC 通用引物
1391-R TGGTCTTCTGAGACTGTATC Universal primer
RT-PCR-F GTGAACACTGAGGAAAGAATTAATG 荧光定量PCR引物
RT-PCR-R CAACCTCTTGTTCCATGTTGTC Primers for real-time PCR
Actin3-F GAGTAGAAAATGGCTGATGGTGAAG 扩增内参基因
Actin3-R TCATCTTCTCACGGTTAGCCTTTG For the internal control

图1

柱头内BoC2H2响应自花和异花授粉后表达模式0: 未授粉柱头。0: unpollinated stigma."

图2

甘蓝BoC2H2基因gDNA和cDNA PCR序列扩增 M: DL2000; gDNA: BoC2H2基因gDNA全长扩增条带; cDNA: BoC2H2基因cDNA全长扩增条带。"

图3

BoC2H2核苷酸序列及对应的氨基酸序列实线框表示ZnF_C2H2结构域。The box was ZnF_C2H2 domain."

图4

BoC2H2与其他物种C2H2氨基酸序列的系统进化树"

"

相关功能预测Associated putative function 启动子顺式作用元件 cis-elements in the promoter region
Light responsive element CATT-motif, GAG-motif, Gap-box
Circadian control Circadian
Auxin-responsive element TGA-element
Gibberellin-responsive GARE-motif
Zein metabolism regulation O2-site
Meristem expression CAT-box
Endosperm expression GCN4_motif, Skn-1_motif
Defense and stress responsiveness TC-rich repeats
Elicitor-responsive element EIRE, ELI-box3
Essential for the anaerobic induction ARE
Promoter and enhancer regions CAAT-box
MeJA-responsiveness CGTCA-motif, TGACG-motif
Anoxic specific inducibility GC-motif
Low-temperature responsiveness LTR
MYB binding site involved in drought-inducibility MYB MBS
Core promoter element around -30 of transcription start TATA-box
Salicylic acid responsiveness TCA-element

图5

BoC2H2-1300-GFP融合蛋白在烟草表皮细胞中的亚细胞定位 A: 1300-GFP载体的亚细胞定位(对照); B: BoC2H2-1300-GFP的亚细胞定位; 箭头所指为细胞核位置。"

图6

BoC2H2-1300-GFP融合蛋白在拟南芥原生质体中的亚细胞定位 A: 1300-GFP载体的亚细胞定位(对照); B: BoC2H2-1300-GFP的亚细胞定位; 箭头所指为细胞核位置。"

图7

甘蓝BoC2H2基因在不同组织中的表达分析"

图8

转基因植株PCR检测1, 2条带为BoC2H2-GUS转基因植株, 3条带为pCAMBIA 1391转基因植株, WT为野生型拟南芥。"

图9

GUS染色分析a~b: 幼苗不同发育时期; c~f: 叶子不同发育时期; g~k: 花的不同发育时期; l~m: 果荚不同发育时期。"

图10

甘蓝柱头内BoC2H2响应自花和异花授粉后表达模式"

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