Prokaryotic Expression, Preparation Polyclonal Antibody and Protein Expression of BoMAPK4 of Ornamental Kale(Brassica oleracea var. acephala)

doi:10.7525/j.issn.1673-5102.2022.04.008

Abstract

Abstract:

The mitogen-activated protein kinase 4（MAPK4）cDNA were isolated from the stigma of ornamental kale（Brassica oleracea var. acephala）self-incompatibility line（S_13-bS_13-b ）by RT-PCR. The obtained BoMAPK4 cDNA contained a 1 122 bp open reading frame in length， encoded 373 amino acids， and had a serine/threonine domain， no signal peptide and transmembrane structure. The deduced amino acid sequence of BoMAPK4 shared 99.7%， 99.5% and 95.4% identity with Brassica napus BnMAPK4， Brassica rapa BrMAPK4， Arabidopsis thaliana AtMAPK4， respectively. The ORF of BoMAPK4 was constructed into the prokaryotic expression vector pET-14b and transformed into Escherichia coli BL21（DE3）for expression. SDS-PAGE showed that BoMAPK4 protein was specifically expressed at a molecular weight of 45 kDa. The recombinant BoMAPK4 was obtained by affinity chromatography， and the polyclonal antibody against BoMAPK4 was prepared by immunizing mice. The total proteins of sepals， petals， anthers， stigmas， styles and ovaries of ornamental kale were extracted， and protein expression was detected by Western blot. The results showed that BoMAPK4 was less expressed in petals， anthers and ovary， more expressed in sepals and styles， and the highest expressed in stigmas. The study lays the foundation for exploring the biological functions of BoMAPK4.

Key words: ornamental kale, stigma, the mitogen-activated protein kinase 4, polyclonal antibody, western blot

CLC Number:

S635

Heming LI, Hongtao QIN, Deqiang WEI, Xu LI, Xingguo LAN. Prokaryotic Expression, Preparation Polyclonal Antibody and Protein Expression of BoMAPK4 of Ornamental Kale(Brassica oleracea var. acephala)[J]. Bulletin of Botanical Research, 2022, 42(4): 584-591.

Figures/Tables 8

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References 32

1	KELKAR N， GUPTA S， DICKENS M，et al.Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3［J］.Molecular and Cellular Biology，2000，20（3）：1030-1043.
2	KAZUYA ICHIMURA M G， SHINOZAKI K， TENA G，et al.Mitogen-activated protein kinase cascades in plants：a new nomenclature［J］.Trends in Plant Science，2002，7（7）：301-308.
3	RODRIGUEZ M C S， PETERSEN M， MUNDY J.Mitogen-activated protein kinase signaling in plants［J］.Annual Review of Plant Biology，2010，61：621-649.
4	SINHA A K， JAGGI M， RAGHURAM B，et al.Mitogen-activated protein kinase signaling in plants under abiotic stress［J］.Plant Signaling & Behavior，2011，6（2）：196-203.
5	ZHANG M M， SU J B， ZHANG Y，et al.Conveying endogenous and exogenous signals：MAPK cascades in plant growth and defense［J］.Current Opinion in Plant Biology，2018，45：1-10.
6	ZHANG S Q， KLESSIG D F.MAPK cascades in plant defense signaling［J］.Trends in Plant Science，2001，6（11）：520-527.
7	CRISTINA M S， PETERSEN M， MUNDY J.Mitogen-activated protein kinase signaling in plants［J］.Annual Review of Plant Biology，2010，61：621-649.
8	PITZSCHKE A， SCHIKORA A， HIRT H.MAPK cascade signalling networks in plant defence［J］.Current Opinion in Plant Biology，2009，12（4）：421-426.
9	LAMPARD G R， MACALISTER C A， BERGMANN D C. Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS［J］.Science，2008，322（5904）：1113-1116.
10	BECK M， KOMIS G， ZIEMANN A，et al.Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana ［J］.New Phytologist，2011，189（4）：1069-1083.
11	ZENG Q N， CHEN J G， ELLIS B E. AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis ［J］.The Plant Journal，2011，67（5）：895-906.
12	GUAN Y F， MENG X Z， KHANNA R，et al.Phosphorylation of a WRKY transcription factor by MAPKs is required for pollen development and function in Arabidopsis ［J］.PLoS Genetics，2014，10（5）：e1004384.
13	ZHAO F， ZHENG Y F， ZENG T，et al.Phosphorylation of SPOROCYTELESS/NOZZLE by the MPK3/6 kinase is required for anther development［J］.Plant Physiology，2017，173（4）：2265-2277.
14	JAMSHED M， SANKARANARAYANAN S， ABHINANDAN K，et al.Stigma receptivity is controlled by functionally redundant MAPK pathway components in Arabidopsis ［J］.Molecular Plant，2020，13（11）：1582-1593.
15	BECK M， KOMIS G， MÜLLER J，et al. Arabidopsis homologs of nucleus-and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essential for microtubule organization［J］.The Plant Cell，2010，22（3）：755-771.
16	KOSETSU K， MATSUNAGA S， NAKAGAMI H，et al.The MAP kinase MPK4 is required for cytokinesis in Arabidopsis thaliana ［J］.The Plant Cell，2010，22（11）：3778-3790.
17	POPESCU S C， POPESCU G V， BACHAN S，et al.MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays［J］.Genes & Development，2009，23：80-92.
18	XU J， ZHANG S Q.Mitogen-activated protein kinase cascades in signaling plant growth and development［J］.Trends in Plant Science，2015，20（1）：56-64.
19	ZHANG W W， SONG J F， YUE S Q，et al. MhMAPK4 from Malus hupehensis Rehd.decreases cell death in tobacco roots by controlling Cd²⁺ uptake［J］.Ecotoxicology and Environmental Safety，2019，168：230-240.
20	DORY M， HATZIMASOURA E， KÁLLAI B M，et al.Coevolving MAPK and PID phosphosites indicate an ancient environmental control of PIN auxin transporters in land plants［J］.FEBS Letters，2018，592（1）：89-102.
21	LI S N， WANG W Y， GAO J L，et al. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis ［J］.The Plant Cell，2016，28（11）：2866-2883.
22	HETTENHAUSEN C， BALDWIN I T， WU J Q.Silencing MPK4 in Nicotiana attenuata enhances photosynthesis and seed production but compromises abscisic acid-induced stomatal closure and guard cell-mediated resistance to Pseudomonas syringae pv tomato DC3000［J］.Plant Physiology，2012，158（2）：759-776.
23	LAN X G， YANG J， ABHINANDAN K，et al.Flavonoids and ROS play opposing roles in mediating pollination in ornamental kale（Brassica oleracea var. acephala）［J］.Molecular Plant，2017，10（10）：1361-1364．
24	乌木提·巴合提别克，唐玉倩，王淑婷，等.暴马桑黄倍半萜合酶基因SbTps1的克隆及表达分析［J］.森林工程，2021，37（4）：33-39.
	WUMUTI B， TANG Y Q， WANG S T，et al.Cloning and function identification of the sesquiterpenes synthase gene SbTps1 in Sanghuangporus baumii ［J］.Forest Engineering，2021，37（4）：33-39，46.
25	李素，陈芳，殷缘，等.山新杨PdPapGH12基因克隆及其响应胁迫的组织表达［J］.森林工程，2021，37（4）：11-21.
	LI S， CHEN F， YIN Y，et al.Cloning of PdPapGH12 gene of Shanxin poplar（Populus davidiana × P.alba var.pyramidlis） and its tissue expression in response to stress［J］.Forest Engineering，2021，37（4）：11-21.
26	李阳，施亚坤，高士科，等.羽衣甘蓝类蛋白质二硫键异构酶PDIL1-2的基因克隆及蛋白表达分析［J］.植物研究，2020，40（2）：293-300.
	LI Y， SHI Y K， GAO S K，et al.Gene cloning and expression analysis of PDIL1-2 of ornamental kale（Brassica oleracea var. acephala）［J］.Bulletin of Botanical Research，2020，40（2）：293-300.
27	LI X， ABHINANDAN K， ZHANG T，et al.Temporal regulation of two cytosolic phosphoglucomutases during stigma development in ornamental kale（Brassica oleracea var. acephala）［J］.Plant Signaling & Behavior，2018，13（5）：e1467698.
28	JONAK C， ÖKRÉSZ L， BÖGRE L，et al.Complexity，cross talk and integration of plant MAP kinase signalling［J］.Current Opinion in Plant Biology，2002，5（5）：415-424.
29	ZHANG M M， SU J B， ZHANG Y，et al.Conveying endogenous and exogenous signals：MAPK cascades in plant growth and defense［J］.Current Opinion in Plant Biology，2018，45：1-10.
30	SINGH P，ARA H， TAYYEBA S，et al.Development of efficient protocol for rice transformation overexpressing MAP kinase and their effect on root phenotypic traits［J］.Protoplasma，2019，256（4）：997-1011.
31	ZHANG M M， WU H J， SU J B，et al.Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis ［J］.The Plant Journal，2017，92（6）：1005-1019.
32	XU H Y， ZHANG C， LI Z C，et al.The MAPK kinase kinase GmMEKK1 regulates cell death and defense responses［J］.Plant Physiology，2018，178（2）：907-922.

引物名称 Primer name	引物序列 Primer sequence（5′→3′）	作用 Fuction
MAPK4-RT-For	GCTAAGCTATGTCGGCGGA	cDNA片段扩增 cDNA fragment amplification
MAPK4-RT-Rev	ATAACTCTCCCTTACTGAGG	cDNA片段扩增 cDNA fragment amplification
MAPK4-pET-For	GACGGTACCATGTCGGCGGAGAACTGTTT	编码区片段扩增 Coding region fragment amplification
MAPK4-pET-Rev	GACGGATCCTTACTGAGGATTGAACTTGA	编码区片段扩增 Coding region fragment amplification

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[5]	WANG Si-Yuan, WU Hai-Yan, CAI Zheng-Wang, SU Xue, HOU Qin-Zheng, SUN Kun. Characteristics of Stigma in Six Clematis Species and Its Adaptation to Pollination [J]. Bulletin of Botanical Research, 2017, 37(3): 334-339.
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[9]	ZHAO Tong-Xin;LIU Lin-De;ZHANG Li;WANG Yan-Jie;JIA Xing-Jun. Bionomics of Pollen and Stigma of Celastrus orbiculatus* Thunb. [J]. Bulletin of Botanical Research, 2014, 34(3): 309-316.
[10]	GUO Zheng-Hong;WEI Lan-Zhen;WANG Quan-Xi. Antibody Preparation and Protein Expression of NdhK in Nostoc flagelliforme* [J]. Bulletin of Botanical Research, 2012, 32(2): 204-207.
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[13]	Wu Li-hong, Wang Sui-yi. A NEW SPECIES OF DICRANOSTIGMA FROM HENAN AND ITS KARYOTYPE [J]. Bulletin of Botanical Research, 1997, 17(1): 42-46.