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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (11): 1650-1660.doi: 10.3724/SP.J.1006.2018.01650

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-11-12 Published:2018-07-30
  • Contact: Li-Quan ZHU E-mail:zhuliquan@swu.edu.cn
  • 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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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

Table 1

Primers used for gene cloning and analysis by real-time PCR"

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

Fig. 1

Expression pattern of BoC2H2 in stigma in response to self-pollination and cross-pollination"

Fig. 2

Amplification of BoC2H2gene from gDNA and cDNA of the stigma of Brassica oleracea M: DL2000; gDNA: products of BoC2H2 gene gDNA; cDNA: products of BoC2H2 gene cDNA."

Fig. 3

Nucleotide sequence of BoC2H2 and its amino acid sequence"

Fig. 4

Phylogenetic tree of BoC2H2 and C2H2 amino acid sequence in other species"

Table 2

cis-elements in the upstream regulation region of BoC2H2 gene"

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

Fig. 5

Subcellular location of BoC2H2-1300-GFP in epidermal cells of tobacco A: subcellular location of 1300-GFP vector; B: subcellular location of BoC2H2-1300-GFP; the arrows indicate the location of nucleus."

Fig. 6

Subcellular location of BoC2H2-1300-GFP in epidermal cells of Arabidopsis thaliana A: subcellular location of 1300-GFP vector; B: subcellular location ofBoC2H2-1300-GFP; the arrows indicate the location of nucleus."

Fig. 7

Expression analysis of BoC2H2 in different organs of Brassica oleracea"

Fig. 8

PCR analysis of transgenic plants1, 2, and 3 bands were BoC2H2-GUS and pCAMBIA 1391 transgenic plants, respectively. WT was wild-type Arabidopsis thaliana."

Fig. 9

GUS staining analysisa-b: seedlings at different developmental stages; c-f: leaves at different developmental stages; g-k: flowers at different developmental stages; l-m: pods at different developmental stages."

Fig. 10

Expression pattern of BoC2H2 in stigmas in response to self-pollination and cross-pollination"

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