Welcome to Acta Agronomica Sinica，

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)

RichHTML359

94

PDF

616

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."

[1] Gu T, Mazzurco M, Sulaman W, Matias D D, Goring D R . Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proc Natl Acad Sci USA, 1998,95:382-387.
[2] Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock J M . Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol, 2003,133:919-929.
[3] Stone S L, Anderson E M, Mullen R T, Goring D R . ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell, 2003,15:885-898.
[4] Nasrallah M E, Liu P, Nasrallah J B . Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata. Science, 2002,297:247-249.
[5] Nasrallah M, Liu P, Sherman-Broyles S, Boggs N, Nasrallah J . Natural variation in expression of self-incompatibility in Arabidopsis thaliana: implications for the evolution of selfing. Proc Natl Acad Sci USA, 2004,101:16070-16074.
[6] Nasrallah J B, Nasrallah M E . Robust self-incompatibility in the absence of a functional ARC1 gene in Arabidopsis thaliana. Plant Cell, 2014,26:3838-3841.
[7] Elleman C, Dickinson H G . Commonalities between pollen/ stigma and host/pathogen interactions: calcium accumulation during stigmatic penetration by Brassica oleracea pollen. Sexual Plant Reprod, 1999,12:94-202.
[8] Iwano M, Shiba H, Matoba K, Miwa T, Funato M, Entani T, Nakayama P, Shimosato H, Takaoka A, Isogai A . Actin dynamics in papilla cells of Brassica rapa during self and cross-pollination. Plant Physiol, 2007,144:72-81.
[9] Dearnaley J D W, Levina N N, Lew R R, Heath I B, Goring D R . Interrelationships between cytoplasmic Ca 2+ peaks, pollen hydration and plasma membrane conductances during compatible and incompatible pollinations of Brassica napus papillae. Plant Cell Physiol, 1997,38:985-999.
[10] Yang G, Shen S, Yang S, Komatsu S . OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced in response to cold and gibberellin. Plant Physiol Biochem, 2003,41:369-374.
[11] Yoon G M, Cho H S, Ha H J, Liu J R, Lee H S P . Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Mol Biol, 1999,39:991-1001.
[12] Ye S, Wang L, Xie W, Wan B, Li X, Lin Y . Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole lifespan and under phytohormone treatment conditions in rice (Oryza sativa L. ssp. indica). Plant Mol Biol, 2009,70:311-325.
[13] Chotikacharoensuk T, Arteca R N, Arteca J M . Use of differential display for the identification of touch-induced genes from an ethylene-insensitive Arabidopsis mutant and partial characterization of these genes. J Plant Physiol, 2006,163:1305-1320.
[14] Skelton N J, Kördel J, Akke M, Chazin W . Signal transduction versus buffering activity in Ca 2+-binding proteins . Nat Struct Biol, 1994,1:239-245.
[15] Ikura M . Calcium binding and conformational response in EF- hand proteins. Trends Biochem Sci, 1996,21:14-17.
[16] Kretsinger R H . EF-hands embrace. Nat Struct Biol, 1997,4:514-516.
[17] Hrabak E M, Chan C W, Gribskov M, Harper J F, Choi J H, Halford N, Kudla J, Luan S, Nimmo H G, Sussman M R . The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol, 2003,132:666-680.
[18] Rutschmann F, Stalder U, Piotrowski M, Oecking C, Schaller A . LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization. Plant Physiol, 2002,129:156-168.
[19] Iwano M, Ito K, Fujii S, Kakita M, Asano-Shimosato H, Igarashi M, Kaothien-Nakayama P, Entani T, Kanatani A, Takehisa M . Calcium signalling mediates self-incompatibility response in the Brassicaceae. Nature Plants, 2015,1:15128.
[20] 许俊强, 孙梓健, 宋明, 汤青林, 王志敏, 王小佳 . 甘蓝花粉管钙感应蛋白CaM与SRK相互作用研究. 园艺学报, 2013,40:2429-2440.
Xu J Q, Sun Z J, Song M, Tang Q L, Wang Z M, Wang X J . Studies on the interactions between the pollen tube. calmodulin (CaM) and SRK from Brassica oleracea var. capitata. Acta Hortic Sin, 2013,40:2429-2440 (in Chinese with English abstract).
[21] Sheen J . Ca 2+-dependent protein kinases and stress signal transduction in plants . Science, 1996,274:1900-1902.
[22] Botella J R, Arteca J M, Somodevilla M, Arteca R N . Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata). Plant Mol Biol, 1996,30:1129-1137.
[23] 岳尧 . 基于转录组数据的甘蓝型油菜自交不亲和相关基因的挖掘. 华中农业大学硕士学位论文, 湖北武汉, 2016.
Yue Y . The Mining of Brassica napus Self-incompatibility Related Gene Based on RNA-Seq. MS Thesis of Huazhong Agricultural University, Wuhan, Hubei, China, 2016 (in Chinese with English abstract).
[24] Messerli M A, Creton R, Jaffe L F, Robinson K R . Periodic increases in elongation rate precede increases in cytosolic Ca 2+ during pollen tube growth . Dev Biol, 2000,222:84-98.
[25] Hamel L P, Sheen J, Seguin A . Ancient signals: comparative genomics of green plant CDPKs. Trends Plant Sci, 2014,19:79-89.
[26] Kunz C, Chang A, Faure J D, Clarke A E, Polya G M, Anderson M A . Phosphorylation of style S-RNases by Ca 2+ dependent protein kinases from pollen tubes . Sexual Plant Reprod, 1996,9:25-34.
[27] Kim S A, Kwak J, Jae S K, Wang M H, Nam H . Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant Cell Physiol, 2001,42:74-84.
[28] Dubos C, Huggins D, Grant G H, Knight M R, Campbell M M . A role for glycine in the gating of plant NMDA-like receptors. Plant J, 2003,35:800-810.
[29] Kang S, Kim H B, Lee H, Choi J Y, Heu S, Oh C J, Kwon S I, An C S . Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca 2+ influx and delays fungal infection. Mol Cells , 2006,21:418-427.
[30] Qi Z, Stephens N R, Spalding E P . Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiol, 2006,142:963-971.
[31] Cho D, Kim S A, Murata Y, Lee S, Jae S K, Nam H G, Kwak J M . De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca 2+-programmed stomatal closure . Plant J, 2009,58:437-449.
[32] Vincill E D, Bieck A M, Spalding E P . Ca 2+ conduction by an amino acid-gated ion channel related to glutamate receptors . Plant Physiol, 2012,159:40-46.
[33] Michard E, Lima P T, Borges F, Silva A C, Portes M T, Carvalho J E, Gilliham M, Liu L H, Obermeyer G, Feijó J A . Glutamate receptor-like genes form Ca 2+ channels in pollen tubes and are regulated by pistil D-serine . Science, 2011,332:434-437.
[34] 何明洁, 孙伊辰, 程晓园, 时冬雪, 李迪秦, 陈益银, 冯永坤, 刘璐, 范腾飞, 杨超, 曹凤秋, 刘来华 . 植物谷氨酸受体的研究进展. 植物学报, 2016,51:827-840.
He M J, Sun Y C, Cheng X Y, Shi D X, Li D Q, Chen Y Y, Feng Y K, Liu L, Fan T F, Yang C, Cao F Q, Liu L H . Current research advances on glutamate receptors (GLRs) in Plants. Chin Bull Bot, 2016,51:827-840 (in Chinese with English abstract).
[35] Jiang J, Jiang J, Qiu L, Miao Y, Yao L, Cao J . Identification of gene expression profile during fertilization in Brassica campestris subsp. chinensis. Genome, 2012,56:39-48.
[1] WANG Yan-Hua, JIAN Hong-Jiu, QIU Xiao, LI Jia-Na. Regulatory mechanism of the seed coat color gene BrTT1 in Brassica rapa L. [J]. Acta Agronomica Sinica, 2020, 46(11): 1678-1689.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd