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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 896-907.doi: 10.3724/SP.J.1006.2022.14036

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯StMAPK4响应低温胁迫的功能解析

冯亚1,2(), 朱熙1,2, 罗红玉1,2, 李世贵1,2, 张宁1,2,*(), 司怀军1,2   

  1. 1甘肃农业大学生命科学技术学院, 甘肃兰州 730070
    2甘肃省干旱生境作物学省部共建国家重点实验室培育基地, 甘肃兰州 730070
  • 收稿日期:2021-02-26 接受日期:2021-06-16 出版日期:2022-04-12 网络出版日期:2021-07-25
  • 通讯作者: 张宁
  • 作者简介:E-mail: 2992919739@qq.com
  • 基金资助:
    国家自然科学基金项目(31960444);甘肃省干旱生境作物学重点实验室——省部共建国家重点实验室培育基地主任基金课题资助(GSCS-2019-Z03)

Functional analysis of StMAPK4 in response to low temperature stress in potato

FENG Ya1,2(), ZHU Xi1,2, LUO Hong-Yu1,2, LI Shi-Gui1,2, ZHANG Ning1,2,*(), SI Huai-Jun1,2   

  1. 1College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, Gansu, China
  • Received:2021-02-26 Accepted:2021-06-16 Published:2022-04-12 Published online:2021-07-25
  • Contact: ZHANG Ning
  • Supported by:
    National Natural Science Foundation of China(31960444);Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University(GSCS-2019-Z03)

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摘要：

马铃薯易受低温危害, 造成减产。MAPK基因广泛参与多种环境胁迫, 研究发现其参与低温调控。为探究马铃薯StMAPK4在响应低温胁迫过程中的功能, 本研究以马铃薯栽培品种‘Atlantic’为试验材料, 分析其在低温(4℃)胁迫下不同时间点在马铃薯根茎叶中的表达特性, 并对StMAPK4基因进行生物信息学分析及对其编码的蛋白进行亚细胞定位分析, 构建StMAPK4的过表达和RNAi干扰表达载体, 转化马铃薯获得转基因植株, 并分析了在4℃处理下非转基因(NT)、过表达和RNAi干扰表达转基因植株的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性、脯氨酸(Pro)和丙二醛(MDA)含量的变化。结果显示, StMAPK4蛋白的等电点为4.97, 属于酸性蛋白, 该蛋白定位于细胞核和细胞膜; 低温胁迫下, StMAPK4在根茎叶中的表达量显著升高; StMAPK4过表达植株的SOD、POD活性和脯氨酸含量较NT植株明显升高, 而MDA含量明显降低; StMAPK4干扰表达植株的SOD、POD活性和脯氨酸含量较NT植株明显降低, 而MDA含量明显升高; 通过表型观察发现, 非转基因和RNAi干扰表达植株的叶片萎蔫严重, 而过表达植株的叶片受影响较小。因此, 过表达StMAPK4基因可以增强马铃薯植株对低温胁迫的耐受性。

关键词: 马铃薯, StMAPK4, 低温, 亚细胞定位, 遗传转化

Abstract:

Potato is vulnerable to low temperatures resulting in reduced yield production. MAPK gene is widely involved in a variety of environmental stress, and it has been detected to be involved in low temperature regulation. In this present study, to explore StMAPK4 function in response to low temperature stress, potato cultivar ‘Atlantic’ as the experimental material, the expression characteristics of StMAPK4 gene were analyzed in potato root, stem, and leaf at the different time under low temperature (4℃) stress. StMAPK4 gene was analyzed using bioinformatics and its encoded protein subcellular localization was assayed. StMAPK4 overexpression and RNA interference expression vectors were constructed and obtained transgenic potato plants. The activities of superoxide dismutase (SOD) and peroxidase (POD), and the contents of proline (Pro) and malondialdehyde (MDA) in non-transgenic (NT), overexpressed and RNAi interfered transgenic plants were analyzed under 4℃. The results showed that the isoelectric point (pI) of StMAPK4 was 4.97 and it was acidic protein localized in the nucleus and cell membrane. The relative expression levels of StMAPK4 in roots, stems, and leaves significantly increased under low temperature stress. Compared with non-transgenic plants, the activities of SOD and POD and the content of proline in StMAPK4 overexpressed plants were significantly increased, while the content of MDA was significantly decreased. Compared with NT plants, the activities of SOD and POD, and the content of Pro in StMAPK4 overexpression plants were significantly decreased, while the content of MDA was significantly increased. Phenotypic observation revealed that the leaves of non-transgenic and RNAi interfered expression plants wilted seriously, while the leaves of overexpressed plants were less affected. In summary, the overexpression of StMAPK4 gene can enhance the tolerance of low temperature stress in potato plants.

Key words: potato, StMAPK4, low temperature, subcellular localization, genetic transformation

表1

amiR-StMAPK4的PCR扩增引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
I GATATACCCTGTGTAAATCGCACTCTCTCTTTTGTATTCC
II GAGTGCGATTTACACAGGGTATATCAAAGAGAATCAATGA
III GAGTACGATTTACACTGGGTATTTCACAGGTCGTGATATG
IV GAAATACCCAGTGTAAATCGTACTCTACATATATATTCCT
A CTGCAAGGCGATTAAGTTGGGTAAC
B GCGGATAACAATTTCACACAGGAAACAG

表2

PCR和qRT-PCR的特异性引物"

引物名称
Primer name		 引物编号
Primer ID		 引物序列
Primer sequence (5'-3')
PCR primers for StMAPK4 正向Forward CGGGGGACGAGCTCGGTACCATGGATGCTGAAAACATTGAAAAT
反向Reverse CCATGTCGACTCTAGACTTGGTTGTATCGGGATCAAACT
PCR primers for Ef1a 正向Forward CAGCCAATCCCATCAAGACG
反向Reverse ATCATTCCGGAGCACTCGAT
qRT-PCR primers for StMAPK4 正向Forward ACGCTCTTCACAGGCCCTCACAGAAG
反向Reverse TGGAACTTGAGGCAGCTGCTTGACGT

图1

马铃薯StMAPK4蛋白与其他物种同源蛋白的氨基酸序列比对"

图2

马铃薯StMAPK4基因与其他11个物种的同源基因发育树"

表3

StMAPK4启动子区顺式作用元件分析"

位点名称
Site name		 序列
Sequence		 功能
Function
ABRE ACGTG 脱落酸反应的顺式作用元件
cis-acting element involved in abscisic acid response
ARE AAACCA 厌氧诱导反应的顺式作用调节元件
cis-acting regulatory element essential for the anaerobic induction
CAAT-box CAAAT, CCAAT 启动子和增强子区域顺式作用元件
Common cis-acting element in promoter and enhancer regions
G-Box CACGTT, TAAACGTG 参与光响应的顺式作用调节元件
cis-acting regulatory element involved in light response
LTR CCGAAA 参与低温反应的顺式作用元件
cis-acting element involved in low-temperature response
MBS CAACTG 参与干旱诱导的MYB结合位点
MYB binding site involved in drought-inducibility
P-box CCTTTTG 赤霉素响应元件
Gibberellin-responsive element
TC-rich repeats CACGTT 参与防御和应激反应的顺式作用元件
cis-acting element involved in defense and stress response

图3

StMAPK4蛋白的二级结构 蓝色表示α-螺旋(Hh); 红色代表延伸链(Ee); 绿色表示β转角(Tt); 紫色表示无规则卷曲(Cc)。"

图4

StMAPK4蛋白的三级结构及结构域"

图5

StMAPK4的蛋白质互作网络"

图6

StMAPK4的扩增产物条带及amiR-MAPK4前体片段扩增 A: StMAPK4的扩增产物条带; B: 以pRS300质粒为模板, 扩增的a (引物A和IV)、b (引物Ⅲ和II)和c (引物I和B)小片段; C: 以a、b和c小片段混合物为模板扩增的d片段(引物A和B); M: DL 2000 marker。"

图7

pCE-StMAPK4 质粒(A)及pBI121-miR-StMAPK4重组质粒双酶切(B) M: DL 2000 marker."

图8

pCEGFP-StMAPK4的亚细胞定位 pCEGFP: 空白对照; pCEGFP-StMAPK4: pCEGFP-StMAPK4融合蛋白; A: 在暗视野中的pCEGFP荧光信号; B: 叶绿体的自身荧光; C: 亮视野下的细胞形态; D: 叠加场。"

图9

qRT-PCR分析低温处理下StMAPK4基因的组织特异性表达 *和**分别表示在0.05和0.01水平差异显著, n = 9。"

图10

转基因植株的qRT-PCR检测 OE-1, 2, 3, 4, 5, 6: 过表达植株; RNAi-1, 2, 3, 4, 5, 6: 干扰表达植株; NT: 非转基因植株; *和**分别表示在0.05和0.01水平差异显著, n = 9。"

图11

低温胁迫下马铃薯转基因植株表型分析 NT: 非转基因植物; OE: 过表达转基因马铃薯植株; RNAi: 干扰表达转基因马铃薯植株。"

图12

低温胁迫下转基因植株各生理指标含量的测定 NT: 非转基因植株; OE-2, 3, 6: pCE1300-StMAPK4转化植株; RNAi-1, 2, 4: pBI121-amiR-StMAPK4转化植株; 不同小写字母表示在0.05水平差异显著。"

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