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作物学报 ›› 2021, Vol. 47 ›› Issue (12): 2379-2393.doi: 10.3724/SP.J.1006.2021.04280

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

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

王珍1,2(), 张晓莉1,2(), 孟晓静1,2, 姚梦楠1,2, 缪文杰1,2, 袁大双1,2, 朱冬鸣1,2, 曲存民1,2, 卢坤1,2, 李加纳1,2,*(), 梁颖1,2,*()   

  1. 1西南大学农学与生物科技学院 / 油菜工程研究中心, 重庆 400715
    2西南大学现代农业科学研究院, 重庆 400715
  • 收稿日期:2020-12-21 接受日期:2021-04-14 出版日期:2021-12-12 网络出版日期:2021-06-09
  • 通讯作者: 李加纳,梁颖
  • 作者简介:王珍, E-mail: wangzhencq@swu.edu.cn;
    张晓莉, E-mail: zxl19930619@email.swu.edu.cn第一联系人：**同等贡献
  • 基金资助:
    国家自然科学基金项目(31872876);重庆市博士后科研特别资助(2010010006157688);中国博士后科学基金项目(2021M692683)

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 Published:2021-12-12 Published online:2021-06-09
  • Contact: LI Jia-Na,LIANG Ying
  • 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)

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摘要：

丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs)级联途径在植物的生长发育、分裂分化、细胞凋亡以及抗逆等多种生命过程中发挥着极其重要的作用。本研究从甘蓝型油菜中分离克隆了1个C族BnMAPK7基因的启动子, 序列长度为1612 bp, 命名为ProBnMAPK7。启动子分析工具PlantCARE预测结果表明, ProBnMAPK7含有大量响应光照、激素、防御和损伤等相关顺式作用元件, 包括ACE、MRE、ABRE、TGACG-motif和TC-rich repeats等。同时, 我们对拟南芥及甘蓝型油菜MAPK7基因的表达模式进行分析发现, MAPK7对生长发育以及生物和非生物胁迫应答过程具有重要调控意义。将ProBnMAPK7逐步分段连接至pCambia1305.1-GUS表达载体筛选启动子的核心区段, GUS组织化学染色分析显示该启动子的核心区段定位于-467~ -239 bp (ProBnMAPK7-rPE)。将核心区段3拷贝串联重复后整合至Y1H gold基因组, 并进行AbA背景测试, 结果显示AbA浓度为500 ng mL-1时, ProBnMAPK7-rPE×3在酵母细胞中的本底表达被完全抑制。利用酵母单杂交技术对BnMAPK7的上游调控因子进行文库筛选, 获得3个候选因子BnNAD1B (NADH dehydrogenase 1B)、BnERD6 (early response to dehydration 6)和BnPIG3 (quinone oxidoreductase PIG3-like)。表明BnNAD1B、BnERD6及BnPIG3蛋白可能通过结合ProBnMAPK7-rPE区段调控BnMAPK7的转录, 使得BnMAPK7参与光合作用以及逆境胁迫应答等生物学过程, 为进一步阐明甘蓝型油菜BnMAPK7基因的功能奠定了基础, 也为MAPKs级联的研究提供了新的思路。

关键词: 甘蓝型油菜, ProBnMAPK7, 启动子, 上游调控因子, 酵母单杂交

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

表1

本研究所用PCR引物"

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

表2

甘蓝型油菜ProBnMAPK7的主要顺式作用元件预测"

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

图1

AtMAPK7 (At2g18170)在拟南芥的不同组织器官(A)、不同生长时期(B)以及生物(C)和非生物(D)胁迫过程中的表达模式分析 横坐标表示不同的组织器官、发育时期或胁迫处理, 括号内的数字表示富集到的文库数量; 纵坐标表示AtMAPK7基因在均一化后的各个文库中的FPKM值。"

图2

BnMAPK7 (BnaA09g09520D)在甘蓝型油菜不同时期不同组织中的表达模式(A)和逆境响应模式(B)分析 Ro_24h、Ro_48h、Ro_72h分别表示种子萌发24 h、48 h、72 h的胚根; Hy_48h、Hy_72h分别表示种子萌发48 h、72h的下胚轴; Co_24h、Co_48h、Co_72h分别表示种子萌发24 h、48 h、72 h的子叶。Ro_s、Ro_s_f分别表示苗期的温室植株根系、大田植株根系; Le_s、Le_s_f分别表示苗期的温室植株叶片、大田植株叶片。Ro_b、St_b、LeY_b、LeO_b、Bu_b、IT_b分别表示蕾薹期的根系、茎、幼叶、成熟叶片、花蕾、主序顶端。Ro_i、St_i、LeY_i、LeO_i、Ao_i、Cal_i、Pe_i、Pi_i、Sta_i、At_i、Cap_i、IT_i分别表示初花期的根系、茎、幼叶、成熟叶片、花柄、萼片、花瓣、雌蕊、雄蕊、花药、花丝、主序顶端。Ro_f、St_f、LeY_f、LeO_f、Ao_f、Cal_f、Pe_f、Pi_f、Sta_f、At_f、Cap_f、IT_ f分别表示盛花期的根系、茎、幼叶、成熟叶片、花柄、萼片、花瓣、雌蕊、雄蕊、花药、花丝、主序顶端。St_24d表示青荚期花后24 d的茎; LeY_10d、LeY_24d、LeY_30d分别表示青荚期花后10 d、24 d、30 d的幼叶; LeO_10d、LeO_24d、LeO_30d分别表示青荚期花后10 d、24 d、30 d的成熟叶片; Se_10d、Se_24d、Se_30d分别表示青荚期花后10 d、24 d、30 d的种子; Em_21d、Em_30d分别表示青荚期花后21 d、30 d的种胚; SC_21d、SC_30d分别表示青荚期花后21 d、30 d的种皮; En_21d表示青荚期花后21 d的内种皮; Ep_30d表示青荚期花后30 d的外种皮; SP_10d、SP_24d、SP_30d表示青荚期花后10 d、24 d、30 d的荚果皮。St_50d表示成熟期花后50 d的茎; SC_43d、SC_46d分别表示成熟期花后43 d、46 d的种皮; Em_43d、Em_46d分别表示成熟期花后43 d、46 d的种胚; Ra_43d、Ra_46d分别表示成熟期花后43 d、46 d的胚芽; SP_43d、SP_46d分别表示成熟期花后43 d、46 d的荚果皮。"

图3

ProBnMAPK7不同长度片段示意图"

图4

瞬时表达筛选ProBnMAPK7核心区段的GUS组织化学染色"

图5

Y1H-Bait酵母菌株的毒性与自激活活性检测(A)以及Y1H文库筛选回转验证(B)"

表3

甘蓝型油菜ProBnMAPK7酵母单杂交文库筛选的上游调控因子注释信息"

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

附图1

甘蓝型油菜ProBnMAPK7的序列及其顺式作用元件"

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