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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (12): 2379-2393.doi: 10.3724/SP.J.1006.2021.04280


Identification of upstream regulators for mitogen-activated protein kinase 7 gene (BnMAPK7) in rapeseed (Brassica napus L.)

WANG Zhen1,2(), ZHANG Xiao-Li1,2(), MENG Xiao-Jing1,2, YAO Meng-Nan1,2, MIU Wen-Jie1,2, YUAN Da-Shuang1,2, ZHU Dong-Ming1,2, QU Cun-Min1,2, LU Kun1,2, 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:2020-12-21 Accepted:2021-04-14 Online:2021-12-12 Published:2021-06-09
  • Contact: LI Jia-Na,LIANG Ying E-mail:wangzhencq@swu.edu.cn;zxl19930619@email.swu.edu.cn;ljn1950@swu.edu.cn;yliang@swu.edu.cn
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31872876);Chongqing Special Funding in Postdoctoral Scientific Research Project(2010010006157688);China Postdoctoral Science Foundation Project(2021M692683)


Mitogen-activated protein kinases (MAPKs) cascade plays a key role in plant growth and development, division, differentiation, apoptosis, and stress resistance. In this study, a 1612 bp promoter of C group BnMAPK7 gene, designated ProBnMAPK7, was cloned from Brassica napus. Promoter structure prediction by PlantCARE revealed that ProBnMAPK7 contained a lot of ACE, MRE, ABRE, TGACG-motif, and TC-rich repeats cis-acting elements, which involved in light, hormones, defense, and wounding responsiveness. At the same time, we analyzed the expression patterns of MAPK7 genes in Arabidopsis and B. napus, and found that MAPK7 played an important regulatory role in growth and development process and responding to biotic and abiotic stresses. Different lengths of ProBnMAPK7 were gradually ligated to the pCambia1305.1-GUS expression vector to identify the core fragment. GUS histochemical staining analysis showed that the core fragment of ProBnMAPK7 was located in the -467 to -239 bp (ProBnMAPK7-rPE) region. Three copies of the promoter core fragment were integrated into the genome of Y1H gold to test the AbA background. The data demonstrated that the expression background of ProBnMAPK7-rPE in yeast cells was completely inhibited by 500 ng mL-1 AbA. Using yeast one-hybrid, we screened the library of the upstream regulatory factors of BnMAPK7, and obtained three candidates, including BnNAD1B (NADH dehydrogenase 1B), BnERD6 (early response to dehydration 6), and BnPIG3 (quinone oxidoreductase PIG3-like). Taken together, these results suggested that BnNAD1B, BnERD6, and BnPIG3 might bind to ProBnMAPK7-rPE to regulate the transcription of BnMAPK7, to further involve in photosynthesis and responding to stresses. This study lays a foundation for further elucidating the function of BnMAPK7 in rapeseed, and provides a new perspective for research into MAPKs cascade.

Key words: Brassica napus, ProBnMAPK7, promoter, upstream regulators, yeast one-hybrid

Table 1

PCR primers used in this study"

Primer name
Primer sequence (5°-3°)
ProBnMAPK7-PA (1612 bp)全长启动子表达载体的构建
Construction of promoter expression vector of full-length ProBnMAPK7-PA (1612 bp)
ProBnMAPK7-lPB (539 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-lPB (539 bp)
ProBnMAPK7-rPB (1073 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-rPB (1073 bp)
ProBnMAPK7-lPC (212 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-lPC (212 bp)
ProBnMAPK7-rPC (861 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-rPC (861 bp)
ProBnMAPK7-lPD (623 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-lPD (623 bp)
ProBnMAPK7-rPD (238 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-rPD (238 bp)
ProBnMAPK7-lPE (394 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-lPE (394 bp)
Primer name
Primer sequences (5°-3°)
ProBnMAPK7-rPE (229 bp)启动子表达载体的构建
Construction of promoter expression vector of ProBnMAPK7-rPE (229 bp)
Insertion detection of promoter expression
Detection of GUS gene in promoter expression vector
Detection of Hyg gene in promoter expression vector
Detection of expression vector of pAbAi- ProBnMAPK7-rPE×3
Detection of ProBnMAPK7-rPE×3 integration in to Y1Hgold chromosome
Detection of candidate upstream regulators of ProBnMAPK7-rPE
Detection of Prey yeast cells from library screening

Table 2

Key cis-acting elements of ProBnMAPK7 in Brassica napus"

Element name
Sequence (5°-3°)
ABRE ACGTGGC 响应ABA Abscisic acid responsiveness
ACE CTAACGTATT 响应光照 Light responsiveness
ARE TGGTTT 厌氧诱导 Anaerobic induction
Box 4 ATTAAT 光响应的保守DNA模块 A conserved DNA module involved in light responsiveness
CGTCA-motif CGTCA 响应MeJA MeJA responsiveness
G-box ACACGTGGC 响应光照 Light responsiveness
GARE-motif TCTGTTG 响应GA Gibberellin responsiveness
MBS CGGTCA 干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
MRE AACCTAA 响应光照的MYB结合位点 MYB binding site involved in light responsiveness
TC-rich repeats GTTTTCTTAC 响应防御和胁迫 Defense and stress responsiveness
TCT-motif TCTTAC 响应光照 Light responsiveness
TGA-element AACGAC 响应IAA Auxin responsiveness
TGACG-motif TGACG 响应MeJA MeJA responsiveness
WUN-motif TCATTACGAA 响应损伤 Wound responsiveness

Fig. 1

Relative expression patterns of AtMAPK7 (At2g18170) in different tissues (A), at different stages (B), biotic (C), and abiotic (D) stresses in Arabidopsis The abscissa represents different tissues, developmental stages, or stress treatments, the number in parentheses represents the number of enriched libraries; the ordinate represents the FPKM value of AtMAPK7 gene in each normalization library."

Fig. 2

Relative expression patterns of BnMAPK7 (BnaA09g09520D) in different tissues at different stages (A) and stresses responses (B) in Brassica napus Ro_24h, Ro_48h, and Ro_72h represent the root at 24, 48, and 72 hours after seed germination, respectively; Hy_48h and Hy_72h represent the hypocotyl at 48 hours and 72 hours after seed germination, respectively; Co_24h, Co_48h, and Co_72h represent the cotyledon at 24, 48, and 72 hours after seed germination, respectively. Ro_s, Ro_s_f represent the root from seedlings planted in greenhouse and field at the seedling stage, respectively; Le_s, Le_s_f represent the leaf from seedlings planted in greenhouse and field at the seedling stage, respectively. Ro_b, St_b, LeY_b, LeO_b, Bu_b, and IT_b represent the root, stems, young leaf, mature leaf, bud, and the tip of main inflorescence at the bolting stage, respectively. Ro_i, St_i, LeY_i, LeO_i, Ao_i, Cal_i, Pe_i, Pi_i, Sta_i, At_i, Cap_i, and IT_i represent the root, stem, young leaf, mature leaf, anthocallus, calyx, petal, pistil, stamen, anther, capillament, and the tip of main inflorescence at the initial bloom stage, respectively. Ro_f, St_f, LeY_f, LeO_f, Ao_f, Cal_f, Pe_f, Pi_f, Sta_f, At_f, and Cap_f, IT_f represent the root, stem, young leaf, mature leaf, anthocallus, calyx, petal, pistil, stamen, anther, capillament, and the tip of main inflorescence at the full bloom stage, respectively. St_24d represents the stem at 24 days after flowering at the green pod stage; LeY_10d, LeY_24d, and LeY_30d represent the young leaf at 10, 24, and 30 days after flowering at the green pod stage, respectively; LeO_10d, LeO_24d, and LeO_30d represent the mature leaf at 10, 24, and 30 days after flowering at the green pod stage, respectively; Se_10d, Se_24d, and Se_30d represent the seed at 10, 24, and 30 days after flowering at the green pod stage, respectively; Em_21d and Em_30d represent the embryo at 21 days and 30 days after flowering at the green pod stage, respectively; SC_21d and SC_30d represent the seed coat at 21 days and 30 days after flowering at the green pod stage, respectively; En_21d represents the endotesta at 21 days after flowering at the green pod stage; Ep_30d represents the episperm at 30 days after flowering at the green pod stage; SP_10d, SP_24d, and SP_30d represent the silique pericarp at 10, 21, and 30 days after flowering at the green pod stage, respectively. St_50d represents the stem at 50 days after flowering at the mature stage; SC_43d and SC_46d represent the seed coat at 43 days and 46 days after flowering at the mature stage, respectively; Em_43d and Em_46d represent the embryo at 43 days and 46 days after flowering at the mature stage, respectively; Ra_43d and Ra_46d represent the radicle at 43 days and 46 days after flowering at the mature stage, respectively; SP_43d and SP_46d represent the silique pericarp at 43 days and 46 days after flowering at the mature stage, respectively."

Fig. 3

Schematic diagram of different lengths of ProBnMAPK7 regions"

Fig. 4

GUS histochemical staining of transient expression screening the core fragment of ProBnMAPK7"

Fig. 5

Toxicity and autoactivation test of Y1H-Bait in yeast cells (A) and retransformation validation in Y1H screening assays (B)"

Table 3

Annotation of upstream regulators of ProBnMAPK7 in Y1H library screening assays in Brassica napus"

Gene name
Gene ID (B. napus/A. thaliana)
Gene description
Gene function
BnNAD1B chrUn_random
2 编码从3个前体NAD1A、NAD1B和NAD1C反式剪接的线粒体NAD(P)H脱氢酶的亚基。
Encodes subunit of mitochondrial NAD(P)H dehydrogenase that is trans-spliced from three precursors, NAD1A, NAD1B, and NAD1C.
Involved in cellular respiration, oxidation-reduction process, photorespiration.
BnERD6 BnaC08g42900D
2 编码假定的蔗糖转运蛋白, 其基因表达受到脱水和冷诱导。
Encodes a putative sucrose transporter whose gene expression is induced by dehydration and cold.
参与碳水化合物的跨膜转运, 响应ABA和几丁质, 响应冷、盐、缺水胁迫。
Involved in carbohydrate transmembrane transport, response to abscisic acid, response to chitin, response to cold, response to salt stress, response to water deprivation.
BnPIG3 BnaA01g11430D
1 氧化还原酶, 锌结合脱氢酶家族蛋白。
Oxidoreductase, zinc-binding dehydrogenase family protein.
Involved in oxidation-reduction process

Fig. S1

Sequence of ProBnMAPK7 and its cis-acting elements in Brassica napus"

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