欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (4): 840-850.doi: 10.3724/SP.J.1006.2022.14061

• 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BnMAPK2基因的克隆及功能分析

袁大双1,2(), 邓琬玉1,2, 王珍1,2, 彭茜1,2, 张晓莉1,2, 姚梦楠1,2, 缪文杰1,2, 朱冬鸣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:2021-04-15 接受日期:2021-06-16 出版日期:2022-04-12 网络出版日期:2021-07-24
  • 通讯作者: 梁颖
  • 作者简介:E-mail: 1967548139@qq.com
  • 基金资助:
    国家自然科学基金项目资助(31872876)

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 Published:2022-04-12 Published online:2021-07-24
  • Contact: LIANG Ying
  • Supported by:
    National Natural Science Foundation of China(31872876)

RichHTML

41

PDF

426

摘要：

从甘蓝型油菜中分离克隆了BnMAPK2 (BnaA01g21880D)基因, cDNA及其编码序列长度分别为1516 bp、1113 bp, 编码370个氨基酸。生物信息学分析表明, BnMAPK2蛋白分子量为42,497.0 kD, 等电点为6.36, 蛋白不稳定系数38.74, 为疏水性蛋白, 具有MAPKs蛋白特有的STKc_TEY_MAPK_plant (cd07858)保守结构域; 蛋白二级结构中α螺旋所占比例最大, 为44.05%, 无信号肽; 与拟南芥C族AtMAPK2的亲缘关系更近。核心元件预测结果显示, BnMAPK2-P含有响应水杨酸激素、热胁迫和光照等相关顺式作用元件, 包括TCA-element、HSE、AAAC-motif和MYB 结合位点等。实时荧光定量PCR (qRT-PCR)结果表明, BnMAPK2在甘蓝型油菜中的各个组织器官中均有表达, 受到茉莉酸甲酯、水杨酸、H2O2、损伤、高温和核盘菌的诱导。转基因异源表达BnMAPK2拟南芥株系的表型数据发现, 与野生型相比, 超量表达BnMAPK2使拟南芥植株的抽薹期提前, 株高、主花序有效长度和角果数显著增加, 由此推测BnMAPK2基因参与调节植物生长发育过程。本研究为深入探究BnMAPK2调控甘蓝型油菜生长发育过程的分子机制提供了参考资料和数据支撑。

关键词: 甘蓝型油菜, BnMAPK2, 表达模式, 过表达, 生长发育

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

表1

本研究所用引物"

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

图1

BnMAPK2基因全长cDNA扩增(A)和BnMAPK2基因启动子的克隆(B)"

附表1

启动子BnMAPK2-P序列分析"

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

图2

BnMAPK2核苷酸序列和推导的氨基酸序列"

图3

BnMAPK2跨膜结构(A)、磷酸化位点(B)和二级结构(C)预测 纵向从长到短的4种线段分别表示α螺旋、延伸链、β转角和随机卷曲。数字表示蛋白的氨基酸残基的计数。"

附图1

BnMAPK2蛋白亚细胞定位"

附图2

BnMAPK2蛋白的信号肽预测"

图4

BnMAPK2与其他MAPK2蛋白的系统发育树"

附图3

BnMAPK2基因的BLASTn分析"

图5

植物不同组织器官中的BnMAPK2基因的表达模式(A)和转基因拟南芥中BnMAPK2表达量检测(B) Ro、Hy、CO、St、Le、Bu、FI、SP、15DAF、30DAF、45DAF分别表示根、下胚轴、子叶、茎、真叶、蕾、花、荚果皮、开花15 d的种子、开花30 d的种子和开花45 d的种子。"

图6

不同胁迫条件下BnMAPK2基因的表达模式 MeJA: 茉莉酸甲酯; SA: 水杨酸。"

图7

BnMAPK2过表达拟南芥转基因植株的PCR鉴定 M: trans2K plus DNA marker; 1~37: 过表达拟南芥植株; 38~41: 野生型拟南芥; 41~42: 转化有重组质粒的农杆菌。"

图8

超量表达BnMAPK2对拟南芥苗期(A)、蕾薹期(A, G)、成熟期(B~D)和产量(E~F)的影响 WT和OE-MAPK2-25、OE-MAPK2-5、OE-MAPK2-9、OE-MAPK2-12、OE-MAPK2-13分别表示野生型拟南芥和5个拟南芥转基因株系。数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001水平差异显著。"

[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
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[4] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[5] 余慧芳, 张卫娜, 康益晨, 范艳玲, 杨昕宇, 石铭福, 张茹艳, 张俊莲, 秦舒浩. 马铃薯CrRLK1Ls基因家族的鉴定及响应晚疫病菌信号的表达分析[J]. 作物学报, 2022, 48(1): 249-258.
[6] 荐红举, 尚丽娜, 金中辉, 丁艺, 李燕, 王季春, 胡柏耿, Vadim Khassanov, 吕典秋. 马铃薯PIF家族成员鉴定及其对高温胁迫的响应分析[J]. 作物学报, 2022, 48(1): 86-98.
[7] 张明聪, 何松榆, 秦彬, 王孟雪, 金喜军, 任春元, 吴耀坤, 张玉先. 外源褪黑素对干旱胁迫下春大豆品种绥农26形态、光合生理及产量的影响[J]. 作物学报, 2021, 47(9): 1791-1805.
[8] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[9] 黄兴, 习金根, 陈涛, 覃旭, 谭施北, 陈河龙, 易克贤. 剑麻苯丙氨酸裂解酶基因的鉴定及表达分析[J]. 作物学报, 2021, 47(6): 1082-1089.
[10] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[11] 王慰亲, 唐启源, 陈元伟, 贾巍, 罗友谊, 王小卉, 郑华斌, 熊娇军. 水稻机械精量有序抛秧栽培的产量形成和生长发育特征研究[J]. 作物学报, 2021, 47(5): 942-951.
[12] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[13] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[14] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[15] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2