甘蓝型油菜丝裂原活化蛋白激酶7基因(BnMAPK7)上游调控因子的鉴定

doi:10.3724/SP.J.1006.2021.04280

Abstract

Abstract:

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

WANG Zhen, ZHANG Xiao-Li, MENG Xiao-Jing, YAO Meng-Nan, MIU Wen-Jie, YUAN Da-Shuang, ZHU Dong-Ming, QU Cun-Min, LU Kun, LI Jia-Na, LIANG Ying. Identification of upstream regulators for mitogen-activated protein kinase 7 gene (BnMAPK7) in rapeseed (Brassica napus L.)[J].Acta Agronomica Sinica, 2021, 47(12): 2379-2393.

Figures/Tables 9

Table 1

PCR primers used in this study"

引物名称 Primer name		引物序列 Primer sequence (5°-3°)	用途 Usage
ProBnMAPK7-PA		F: GACGTCGACAATATAATGTCTAATGAGTGAACCAAAC R: GACCCATGGGCTTTCTTGTCCCTAAACTCAACC	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		F: GACGTCGACAATATAATGTCTAATGAGTGAACCAAAC R: GACCCATGGTATAAAAAACATGTTTATCTTTTAGGTTG	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		F: GACGTCGACGTTTGGATGAGAGAGTATTATGGATCCCGG R: GACCCATGGGCTTTCTTGTCCCTAAACTCAACC	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		F: GACGTCGACGTTTGGATGAGAGAGTATTATGGATCCCGG R: GACCCATGGAATATGACACGTGGCTTACG	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		F: GACGTCGACCCACTGTACCCACGTGCTTAGCCGGTTGC R: GACCCATGGGCTTTCTTGTCCCTAAACTCAACC	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		F: GACGTCGACCCACTGTACCCACGTGCTTAGCCGGTTGC R: GACCCATGGCCAGATTGTAATTACAGAAGCTG	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		F: GACGTCGACGTATGTCGAAATCTTCTTTTCTTTGC R: GACCCATGGGCTTTCTTGTCCCTAAACTCAACC	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		F: GACGTCGACCCACTGTACCCACGTGCTTAGCCGGTTGC R: GACCCATGGTTTCTTACTTCTTTTCTTTTTTCAGAA	ProBnMAPK7-lPE (394 bp)启动子表达载体的构建 Construction of promoter expression vector of ProBnMAPK7-lPE (394 bp)
引物名称 Primer name	引物序列 Primer sequences (5°-3°)			用途 Usage
ProBnMAPK7-rPE	F: GACGTCGACGGCTTCTTCATCAAACATAACGTATTGCTTC R: GACCCATGGCCAGATTGTAATTACAGAAGCTG			ProBnMAPK7-rPE (229 bp)启动子表达载体的构建 Construction of promoter expression vector of ProBnMAPK7-rPE (229 bp)
p1305.1	F: GATTCATTAATGCAGCTGGCACGACAGG R: GTTGATCGGGTACAGACTAGTTCGTCGG			启动子表达载体的插入检测 Insertion detection of promoter expression vector
GUS	F: CAACTCGCTGCGTGATGGCATG R: GATAGGAGTGTCCTCATGTTTGCC			启动子表达载体的GUS基因检测 Detection of GUS gene in promoter expression vector
Hyg	F: GAACTCACCGCGACGTCTGT R: GGCGTCGGTTTCCACTATCG			启动子表达载体的Hyg基因检测 Detection of Hyg gene in promoter expression vector
pAbAi	F: CTTCCTTCTGTTCGGAGATTACCGAATC R: TATACATACAGAGCACATGCCTCG			pAbAi-ProBnMAPK7-rPE×3表达载体的检测 Detection of expression vector of pAbAi- ProBnMAPK7-rPE×3
Bait	F: GGCGGATAATGCCTTTAGCGGCTTAACTG R: CCCGGAATGCAGTGAAGGAAAAGCACCG			ProBnMAPK7-rPE×3整合到Y1Hgold染色体的检测 Detection of ProBnMAPK7-rPE×3 integration in to Y1Hgold chromosome
Y1H	F: CTATCTATTCGATGATGAAGATACCCC R: GCACAGTTGAAGTGAACTTGCGGGGTTTTTC			ProBnMAPK7-rPE的候选上游调控因子检测 Detection of candidate upstream regulators of ProBnMAPK7-rPE
AD	F: CTATTCGATGATGAAGATACCCCACCAAACCC R: AGTGAACTTGCGGGGTTTTTCAGTATCTACGAT			文库筛选Prey酵母菌落的检测 Detection of Prey yeast cells from library screening

Table 1

Table 2

Fig. 1

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. 2

Fig. 3

Fig. 4

Fig. 5

Table 3

Fig. S1

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元件名称 Element name	序列 Sequence (5°-3°)	功能 Function
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

基因名称 Gene name	基因编号(甘蓝型油菜/拟南芥) Gene ID (B. napus/A. thaliana)	出现次数 Frequency	基因注解 Gene description	基因功能 Gene function
BnNAD1B	chrUn_random ATMG01120	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.	参与细胞呼吸、氧化还原过程、光呼吸。 Involved in cellular respiration, oxidation-reduction process, photorespiration.
BnERD6	BnaC08g42900D BnaA09g48640D AT1G08930	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 AT4G21580	1	氧化还原酶, 锌结合脱氢酶家族蛋白。 Oxidoreductase, zinc-binding dehydrogenase family protein.	参与氧化还原过程。 Involved in oxidation-reduction process

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

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