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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 840-850.doi: 10.3724/SP.J.1006.2022.14061

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

Cloning and functional analysis of BnMAPK2 gene in Brassica napus

YUAN Da-Shuang1,2(), DENG Wan-Yu1,2, WANG Zhen1,2, PENG Qian1,2, ZHANG Xiao-Li1,2, YAO Meng-Nan1,2, MIAO Wen-Jie1,2, ZHU Dong-Ming1,2, LI Jia-Na1,2, LIANG Ying1,2,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University / Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
    2Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
  • Received:2021-04-15 Accepted:2021-06-16 Online:2022-04-12 Published:2021-07-24
  • Contact: LIANG Ying E-mail:1967548139@qq.com;yliang@swu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31872876)

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Abstract:

The mitogen-activated protein kinase (MAPK) cascade is involved in plant growth and development and it is in response to a variety of biotic and abiotic stresses. In this study, a BnMAPK2 (BnaC01g28210D) gene was isolated and cloned from Brassica napus. The cDNA and its coding sequence were 1516 bp and 1113 bp in length, respectively, encoding 371 amino acids. Bioinformatics analysis revealed that the molecular weight of BnMAPK2 protein was 42,497.0 kD, the isoelectric point was 6.36, protein instability coefficient was 38.74, it was a hydrophobic protein, and it had STKc_TEY_MAPK_ plant (cd07858) conserved structure domain unique to MAPKs protein, protein secondary level. The alpha helix accounted for the largest proportion of 44.05% in the secondary structure of protein, and there was no signal peptide, which was more closely related to the C group AtMAPK2 of Arabidopsis. The core element prediction indicated that BnMAPK2-P contained related cis-acting elements in response to salicylic acid hormone, heat stress, and light, including TCA-element, HSE, AAAC-motif, and MYB binding sites. Real-time quantitative PCR (qRT-PCR) demonstrated that BnMAPK2 was expressed in various tissues and organs in Brassica napus, which was induced by methyl jasmonate, salicylic acid, H2O2, injury, high temperature, and Sclerotinia sclerotiorum. The phenotypic data of transgenic Arabidopsis lines expressing BnMAPK2 heterologously showed that compared with the wild type, the overexpression of BnMAPK2 made the bolting period of Arabidopsis plants earlier, and significantly increased plant height, the effective length of main inflorescence, and the number of siliques. We speculated that BnMAPK2 gene was involved in the regulation of plant growth and development. This study provides reference materials and data support for in-depth exploration of the molecular mechanism of BnMAPK2 regulating the growth and development in Brassica napus.

Key words: Brassica napus, BnMAPK2, expression pattern, overexpression, growth and development

Table 1

Primers used in this study"

引物名称
Primer name		 上游引物
Forward sequence (5′-3′)		 下游引物
Reverse sequence (5′-3′)
MPK2OF ATGGCGACTCCGGTTGATCC TCAGAGCTCAGAGTTAACAGTTTCTGGATG
MPK2-31 GATGCTCGTTCTTGACCCTTCCA CATGCGCGCTAAACCAAAGTCAC
RACE 5 CGACTGGAGCACGAGGACACTGA GGACACTGACATGGACTGAAGGAGTA
RACE 3 GCTGTCAACGATACGCTACGTAACG CGCTACGTAACGGCATGACAGTG
PMPK2 CTCCGCGGCTTGGCAATCTAAAGATAA TCTTCCTTCAGACAAGTTATGCAATAACAT
MAPK2-Q GATCGATGCGTTGAGGACAC CCGATGGAGAATGTTGGCTG
UBC21 CCTCTGCAGCCTCCTCAAGT CATATCTCCCCTGTCTTGAAATGC
ACT7 TGGGTTTGCTGGTGACGAT TGCCTAGGACGACCAACAATACT
OE-MAPK2 CGGAATTCATGGCGACTCCGGTTGATCC CGGGATCCTCAGAGCTCAGAGTTAACAGTTTCTG
F35S GGAAGTTCATTTCATTTGGAGAG GTTCACGGTGCCCTCC
eGFP ATGGTGAGCAAGGGCGAGGAG GGACTTGTACAGCTCGTCCATGCC
Hyg TTCCATGTGCAAACAAAGAGAG GAAGTTGTTAACTGTGTCGACC
PMPK2 CTCCGCGGCTTGGCAATCTAAAGATAAATAGCAAGGAGGC TCTTCCTTCAGACAAGTTATGCAATAACAT

Fig. 1

PCR products of full-length cDNA of BnMAPK2 gene (A) and cloning of BnMAPK2 gene promoter (B)"

Table S1

Analysis of promoter sequences of BnMAPK2-P genes"

元件名称 Element name 功能 Function
TATA-box, CAAT-box Core promoter element
TCA-element Cis-acting element involved in salicylic acid responsiveness
Skn-1_motif Cis-acting regulatory element required for endosperm expression
AAAC-motif, Box I, GT1-motif, Box4, BoxⅠ Light responsive element
Box-W1 Fungal elicitor responsive element
LTR Cis-acting element involved in low-temperature responsiveness
HSE Cis-acting element involved in heat stress responsiveness
MBS MYB binding site

Fig. 2

Nucleotide sequences of BnMAPK2 and deduced amino acid sequences"

Fig. 3

Transmembrane structure (A), phosphorylation site (B), and secondary structure (C) of BnMAPK2 Four kinds of line segments from long to short in longitudinal direction represent α-helix, extended chain, β-turn, and random curl, respectively. The number represents the number of amino acid residues in the protein."

Fig. S1

Prediction of subcellulal localization of BnMAPK2 protein"

Fig. S2

Prediction of signal peptide of BnMAPK2 protein"

Fig. 4

Phylogenetic relationship of BnMAPK2 and MAPK2 from others plants"

Fig. S3

BLASTn analysis of BnMAPK2 gene"

Fig. 5

Relative expression patterns of BnMAPK2 gene in different tissue and organ (A) and detection of BnMAPK2 expression level in transgenic Arabidopsis thaliana (B) Ro, Hy, CO, St, Le, Bu, FI, SP, 15DAF, 30DAF, and 45DAF represent roots, hypocotyls, cotyledons, stems, true leaves, buds, flowers, pods, 15-day seeds, 30-day old seeds, and 45 days old seeds."

Fig. 6

Relative expression patterns of BnMAPK2 gene in response to different abiotic stresses MeJA: methyl jasmonate; SA: salicylic acid."

Fig. 7

PCR identification of transgenic Arabidopsis plants overexpressing BnMAPK2 M: trans2K plus DNA marker; 1-37: overexpression of Arabidopsis plants; 38-41: wild type in Arabidopsis thaliana; 41-42: Agrobacterium tumefaciens with recombinant plasmid."

Fig. 8

Effects of overexpression of BnMAPK2 on Arabidopsis seedling (A), budding (A, G), maturity (B-D), and yield (E-F) WT, OE-MAPK2-25, OE-MAPK2-5, OE-MAPK2-9, OE-MAPK2-12, and OE-MAPK2-13 represent wild-type Arabidopsis and five Arabidopsis transgenic line, respectively. Value indicates mean ± SD (n = 3). *, **, and *** indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

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