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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (12): 1773-1783.doi: 10.3724/SP.J.1006.2019.94040

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

Cloning and analysis of BoCDPK14 in self-incompatibility Brasscia olerace

Xiao-Jing BAI1,Xiao-Ping LIAN2,Yu-Kui WANG1,He-Cui ZHANG1,Qian-Ying LIU1,Tong- Hong ZUO1,Yi-Zhong ZHANG1,Qin-Qin XIE1,Deng-Ke HU1,Xue-Song REN2,Jing ZENG3,Shao-Lan LUO1,Min PU1,Li-Quan ZHU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400716, China
    2 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China
    3 School of Life Advanced Agriculture Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2019-03-15 Accepted:2019-06-12 Online:2019-12-12 Published:2019-06-12
  • 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(XDJK2017D073)

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Abstract:

Brasscia oleracea self-incompatibility (SI) is the reaction of rejection or inhibition of stigma to pollens from the same haplotype. Calcium-dependent protein kinase (CDPK) is an important component of plants involved in resistance to stress signals. In this study, we identified an up-regulated gene named BoCDPK14 based on the stigma transcriptome data in 0-60 min self-pollination. The gene was highly homologous to the calcium dependent protein kinase gene involved in stress signaling in Arabidopsis lyrata. BoCDPK14 gene contained an open reading frame (ORF) with the length of 1599 bp, encoding a protein with the length of 533 amino acid residues. It was a hydrophilic protein expressed in cytoplasm, and could be induced to express a 60.4 kD protein in the cytoplasm of E. coli Transetta (DE3). It was suggested that BoCDPK14 is an active cytoplasmic protein. The 2000 bp upstream of BoCDPK14 translation initial codon contained elements for responses to stress, hormone, and metabolic regulation. BoCDPK14 was expressed in stigma, pollen, flower bud, petal, and leaf, with lower expression level in stigma than in pollen. The results of qRT-PCR revealed that BoCDPK14 mRNA expression level after self- and cross-pollinations for 0 min to 60 min was consistent with that of RNA-seq data. The interaction between BoCDPK14 protein kinase domain and glutamate receptor channel protein BoGLR2.8d was identified by yeast two-hybrid, suggesting that BoCDPK14 might be a novel protein involved in SI reaction. These results suggest that BoCDPK14 may act as a Ca 2+ signal element to participate the process of response to pollen stimulation in the stigma of Brasscia oleracea, which provides a new insight into the further research and utilization of self-incompatibility of Brasscia oleracea.

Key words: Brasscia oleracea, BoCDPK14 gene, yeast two-hybrid, self-pollination, self-incompatibility

Table 1

Primers used in gene cloning and qRT-PCR"

引物名称
Primer		 引物序列
Primer sequence (5°-3°)		 用途
Use
GST
 F: GATCTGGTTCCGCGTGGATCCATGGGGAATTGCTGTGGAAC
R: GATGCGGCCGCTCGAGTCGACCTCTGCATCGCGATTATTAGAA		 基因的原核表达
Prokaryotic expression
QRP
 F: TCAAGAAAAGAGCACTCAGGG
R: GATGTGTACAGATATGGCTACGAAC		 荧光定量PCR引物
qRT-PCR
DActin
 F: GGCTGATGGTGAAGATATTCA
R: CAAGCACAATACCAGTAGTAC		 扩增内参基因
For the internal control
CDPK14-GFP
 F: AAGTCCGGAGCTAGCTCTAGAATGGGGAATTGCTGTGGAAC
R: GCCCTTGCTCACCATGGATCCCTCTGCATCGCGATTATTAGAA		 基因亚细胞定位
Subcellular location
CDPK14Δ-BD
 F: AGGACCTGCATATGGCCATGGATGGGGAATTGCTGTGGAAC
R: CCGCTGCAGGTCGACGGATCCTAACCATGGATGTTCAAGCACTT		 酵母表达
Yeast expression
GLR2.8d-AD
 F: GTACCAGATTACGCTCATATGAGCCCGACAAGTGAAATTAAAGTAG
R: ATGCCCACCCGGGTGGAATTCTTTAAGGAACACCCATGTGTTCTTG		 酵母表达
Yeast expression

Fig. 1

Expression pattern of BoCDPK14 in stigmas in response to self-pollination and cross-pollination SP: self-pollination; CP: cross-pollination."

Fig. 2

Amplification of BoCDPK14 gene from cDNA and gDNA of the stigma of Brassica oleracea"

Fig. 3

Gene structure of Brassica oleracea BoCDPK14 and its deduced amino acid sequence The underline show protein kinase II phosphorylation site; the dashed box show protein kinase C phosphorylation site; the oval show N-glycosylation site; the box show cAMP or cGMP-dependent protein kinase phosphorylation site; the double box show N-myristoylation site; the dot show the EF-hand calcium binding domain; double line show protein kinase ATP binding domain; stars show serine/threonine protein kinase active site."

Fig. 4

Core area of calcium ion binding (A) and predicted three-dimensional structure for BoCDPK14 protein (B) The figures at lower part in Fig. A are the serial number of the 12 amino acid residues of the core area of calcium ion binding."

Fig. 5

Phylogenetic tree of BoCDPK14 and other species CDPK14 amino acid sequence"

Table 2

cis-elements in the upstream regulation region of BoCDPK14 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
Abscisic acid responsiveness ABRE
Anaerobic induction ARE
DNA binding protein (ATBP-1) AT-rich element
Meristem expression CAT-box, CCGTCC-box
Ethylene-responsive element ERE
Gibberellin-responsive element GARE-motif
Endosperm expression GCN4_motif, Skn-1_motif
MYB binding site MRE
Zein metabolism regulation O2-site
Stress responsiveness TC-rich repeats, HSE
Light responsive ACE, Box 4, Box I, G-Box, GA-motif, GATA-motif, GTGGC-motif, I-box, 3-AF1 binding site

Fig. 6

Prokaryotic expression protein analysis of BoCDPK14 protein M: marker; 1: uninduced control; 2: BoCDPK14-GST fusion protein; 3: BoCDPK14-GST purified protein; 4: GST protein."

Fig. 7

Subcellular localization of BoCDPK14-GFP protein in protoplast cell GFP protein showed the green fluorescence under confocal laser scanning microscope."

Fig. 8

Expression pattern of BoCDPK14 in stigmas in response to self-pollination and cross-pollination of Brassica oleracea (A) and expression analysis of BoCDPK14 in different organs of Brassica oleracea (B) SP: self-pollination; CP: cross-pollination."

Fig. 9

Typical calcium-dependent protein kinase (CDPK) structure V: variable domain; J: junction domain; EF: EF-hand domain."

Fig. 10

Interaction of CDPK14Δ and GLR2.8d in yeast two-hybrid assay The first row indicates pGADT7-BoGLR2.8d×pGBDT7-BoCDPK14Δ; the second row indicates positive control (pGADT7-T×pGBDT7-p53); the third row indicates negative control (pGADT7-T×pGBDT7-Lam). DDO: SD/-Leu/-Trp; TDO: SD/-Leu/-Trp/-His; QDO/X/A: SD/-Leu/-Trp/-His/-Ade/X-α-gal/AbA."

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