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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."

[1] 王汉中. 我国油菜产需形势分析及产业发展对策. 中国油料作物学报, 2007, 29:101-105.
Wang H Z. Analysis of my country’s rapeseed production and demand situation and industrial development countermeasures. Chin J Oil Crop Sci, 2007, 29:101-105 (in Chinese with English abstract).
[2] Fiil B K, Petersen K, Petersen M, Mundy J. Gene regulation by MAP kinase cascades. Curr Opin Plant Biol, 2009, 12:615-621.
doi: 10.1016/j.pbi.2009.07.017
[3] Xie Y F, Ding M L, Zhang B, Yang J, Pei T L, Ma P A, Dong J N. Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism. BMC Genomics, 2020, 21:630.
doi: 10.1186/s12864-020-07023-w
[4] 陆俊杏, 卢坤, 朱斌, 彭茜, 陆奇丰, 曲存民, 殷家明, 李加纳, 梁颖, 柴友荣. 芸薹属物种(B. napus, B. oleracea, B. rapa) MAPK1家族的克隆、进化和表达特征. 中国农业科学, 2013, 46:3478-3487.
Lu J X, Lu K, Zhu B, Peng Q, Lu Q F, Qu C M, Yin J M, Li J N, Liang Y, Chai Y R. Cloning, evolution and expression features of MAPK1 gene family from Brassica species (B. napus, B. oleracea, B. rapa). Sci Agric Sin, 2013, 46:3478-3487 (in Chinese with English abstract).
[5] Miao Y, Laun T M, Smykowski A, Zentgraf U. Arabidopsis MEKK1 can take a short cut: it can directly interact with senescence-related WRKY53 transcription factor on the protein level and can bind to its promoter. Plant Mol Biol, 2007, 65:63-76.
doi: 10.1007/s11103-007-9198-z
[6] Zhou C J, Cai Z H, Guo Y F, Gan S S. An Arabidopsis mitogen-activated protein kinase cascade, MKK9-MKK6, plays a role in leaf senescence. Plant Physiol, 2009, 150:167-177.
[7] Xing Y, Jia W, Zhang J. AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6 coupled signaling in Arabidopsis. Plant J, 2008, 54:440-451.
doi: 10.1111/j.1365-313X.2008.03433.x
[8] 王伟威, 林浩, 唐晓飞, 魏崃, 董兴月, 吴广锡, 刘丽君. 干旱胁迫下大豆相关基因的表达特性. 分子植物育种, 2014, 12:903-908.
Wang W W, Lin H, Tang X F, Wei L, Dong X Y, Wu G X, Liu L J. Expression characteristics of soybean-related genes under drought stress. Mol Plant Breed, 2014, 12:903-908 (in Chinese with English abstract).
[9] 潘月云, 朱寿松, 张银东, 陈银华. 木薯促分裂原激活蛋白激酶MeMAPK2基因的克隆和功能分析. 分子植物育种, 2019, 17:1112-1120.
Pan Y Y, Zhu S S, Zhang Y D, Chen Y H. Cloning and functional analysis of cassava mitogen-activated protein kinase MeMAPK2 gene. Mol Plant Breed, 2019, 17:1112-1120 (in Chinese with English abstract)
[10] Xia S T, Xiao L T, Bi D L, Zhu Z H. Arabidopsis replication factor C subunit 1 plays an important role in embryogenesis. Plant Physiol Mol Biol, 2007, 33:179-187.
[11] Xia S T, Cheng P, Jin-Kuib N I, Yan D Y, Xue D, Liange L. Mutation in Arabidopsis replication factor C subunit 3 compromises plant resistance to ultraviolet bombardment. J Hunan Agric Univ(Nat Sci), 2009, 35:606-610.
[12] Rosales-Munar A, Alvarez-Diaz D A, Laiton-Donato K, Jose A U. Efficient method for molecular characterization of the 5′ and 3′ ends of the dengue virus genome. Viruses, 2020, 12:72-87.
doi: 10.3390/v12010072
[13] 朱斌, 陆俊杏, 彭茜, 翁昌梅, 王淑文, 余浩, 李加纳, 卢坤, 梁颖. 甘蓝型油菜MAPK7基因家族及其启动子的克隆与表达分析. 作物学报, 2013, 39:789-805.
Zhu B, Lu J X, Peng Q, Weng C M, Wang S W, Yu H, Li J N, Lu K, Liang Y. Cloning and expression analysis of MAPK7 gene family and its promoter in Brassica napus. Acta Agron Sin, 2013, 39:789-805 (in Chinese with English abstract).
[14] 梁嘉扬. 番茄RBOH1在BR诱导光合效率中的作用及机械伤与不同光质对MAPK1/2的影响. 浙江大学硕士学位论文,浙江杭州, 2015.
Liang J Y. The Role of Tomato RBOH1 in BR-induced Photosynthetic Efficiency and the Effect of Mechanical Injury and Different Light Quality on MAPK1/2. MS Thesis of Zhejiang University, Hangzhou, Zhejiang,China, 2015 (in Chinese with English abstract).
[15] 陆俊杏, 陆奇丰, 张凯, 柴友荣, 李加纳, 钱伟, 吕俊, 卢坤, 梁颖. 甘蓝型油菜MAPK1在损伤和病原菌胁迫下的表达模式分析. 中国农业科学, 2013, 46:4388-4396.
Lu J X, Lu Q F, Zhang K, Chai Y R, Li J N, Qian W, Lyu J, Lu K, Liang Y. Analysis of the expression pattern of Brassica napus MAPK1 under injury and pathogen stress. Sci Agric Sin, 2013, 46:4388-4396 (in Chinese with English abstract).
[16] 潘月云, 朱寿松, 张银东, 陈银华. 木薯促分裂原激活蛋白激酶MeMAPK2基因的克隆和功能分析. 分子植物育种, 2019, 17:1112-1120.
Pan Y Y, Zhu S S, Zhang Y D, Chen Y H. Cloning and functional analysis of cassava mitogen-activated protein kinase MeMAPK2 gene. Mol Plant Breed, 2019, 17:1112-1120 (in Chinese with English abstract).
[17] 李云洲. 外源水杨酸诱导RNAi与MAPK3级联信号抗番前黄化曲叶病毒研究. 西北农林科技大学博士学位论文,陕西杨凌, 2017.
Li Y Z. Exogenous Salicylic acid Induces RNAi and MAPK3 Cascade Signal to Resist the Pre-yellowing Leaf Curl Virus. PhD Dissertation of Northwest A&F University, Yangling, Shaanxi,China, 2017 (in Chinese with English abstract).
[18] Ding T P, Ding Y L. Stories of salicylic acid: a plant defense hormone. Trends Plant Sci, 2020, 25:549-565.
doi: 10.1016/j.tplants.2020.01.004
[19] Lim G H, Liu H, Yu K, Liu R, Kachroo P. The plant cuticle regulates apoplastic transport of salicylic acid during systemic acquired resistance. Sci Adv, 2020, 6: veaaz0478.
[20] Winston G W. Physiochemical basis for free radical formation in cells: production and defenses. Plant Biol, 1990, 12:57-86.
[21] Mehdy M C. Active oxygen species in plant defense against pathogens. Plant Physiol, 1994, 105:467-472.
pmid: 12232215
[22] Wu G S, Shortt B J, Lawrence E B, Leon J, Shah D M. Activation of host defense mechanisms by elevatedproduction of H2O2 in transgenic plants. Plant Physiol, 1997, 115:427-435.
[23] Seger R, Wexler S. The MAPK Signaling Cascades. Encycl Cell Biol, 2016, 3:122-127.
[24] Banerjee G, Singh D, Sinha A K. Plant cell cycle regulators: Mitogen-activated protein kinase, a new regulating switch? Plant Sci, 2020, 301:110-660.
[25] 霍强, 杨鸿, 陈志友. 基于QTL定位和全基因组关联分析筛选甘蓝型油菜株高和一次有效分枝高度的候选基因. 作物学报, 2020, 46:214-227.
doi: 10.3724/SP.J.1006.2020.94067
Huo Q, Yang H, Chen Z Y. Candidate genes screening for plant height and the first branch height based on QTL mapping and genome- wide association study in rapessed (Brassica napus L.). Acta Agron Sin, 2020, 46:214-227 (in Chinese with English abstract).
[26] Folter S D, Busscher J, Colombo L, Losa A, Angenent G C. Transcript profiling of transcription factor genes during silique development in Arabidopsis. Plant Mol Biol, 2004, 56:351-366.
doi: 10.1007/s11103-004-3473-z
[27] Millar A A. The Arabidopsis GAMYB-like genes MYB33 and MYB65 are microRNA-regulated genes that redundantly facilitate anther development. Plant Cell, 2005, 17:705-721.
doi: 10.1105/tpc.104.027920
[28] Browse M J. MYB108 acts together with MYB24 to regulate jasmonate-mediated stamen maturation in Arabidopsis. Plant Physiol, 2009, 149:851-862.
doi: 10.1104/pp.108.132597
[29] Zheng B C, Cui C, Zhang J F, Li H J, Chai L, Jiang J, Jiang L C. Correlation analysis of yield per plant and agronomic traits in breeding lines in Brassica napus L. J Plant Genet Resour, 2019, 20:113-121.
[30] Zhao W, Zhang L, Chao H, Wang H, Li M. Genome-wide identification of silique-related traits based on high-density genetic linkage map in Brassica napus. Mol Breed, 2019, 39:86.
doi: 10.1007/s11032-019-0988-1
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