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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)

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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。"

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