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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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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