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作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1518-1531.doi: 10.3724/SP.J.1006.2023.24153

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

BnMAPK2 对甘蓝型油菜耐旱性的影响

袁大双1,2,3(), 张晓莉1,2, 朱冬鸣1,2, 杨友鸿1,2, 姚梦楠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
    3贵州省六盘水市六枝特区自然资源局, 贵州六盘水 553400
  • 收稿日期:2022-07-01 接受日期:2022-10-10 出版日期:2023-06-12 网络出版日期:2023-04-07
  • 通讯作者: *梁颖, E-mail: yliang@swu.edu.cn
  • 作者简介:E-mail: 1967548139@qq.com
  • 基金资助:
    国家自然科学基金项目(31872876)

Effects of BnMAPK2 on drought tolerance in Brassica napus

YUAN Da-Shuang1,2,3(), ZHANG Xiao-Li1,2, ZHU Dong-Ming1,2, YANG You-Hong1,2, YAO Meng-Nan1,2, 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
    3Natural Resources Bureau of Liuzhi Special Economic Zone, Liupanshui City, Guizhou Province, Liupanshui 553400, Guizhou, China
  • Received:2022-07-01 Accepted:2022-10-10 Published:2023-06-12 Published online:2023-04-07
  • Contact: *E-mail: yliang@swu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31872876)

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摘要：

促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)级联参与植物多种生物及非生物胁迫应答过程, BnMAPK2属于MAPK级联途径最下游C族基因。本研究成功获得BnMAPK2超量表达(OE-MAPK2)和RNA干扰表达(RNAi-MAPK2)转基因甘蓝型油菜, 在干旱条件下, OE-MAPK2植株耐旱性增加, RNAi-MAPK2植株耐旱性降低。相关生理指标试验结果证明, 在干旱胁迫下, BnMAPK2可减缓叶片脱水程度、促进植物体内脯氨酸积累、降低丙二醛含量, 在干旱后期增加POD活性。比较干旱相关基因(P5CSB、SCE1)、BnMAPK2互作干旱相关基因(STRS2、CRL1)以及STRS2依赖ABA信号途径相关基因(RD22、MYC、SnRK2), 在转基因植株和野生型植株中表达水平变化差异, 结果表明, BnMAPK2可正向调控P5CSB、SCE1、CRL1、RD22、MYC、SnRK2的表达; 负调控STRS2的表达, 并且STRS2依赖ABA信号途径相关基因在OE-MAPK2植株中的表达变化趋势和strs2突变体中一致。由此推测BnMAPK2可通过调控植株体内渗透能力、叶片含水量、细胞膜和蛋白质结构稳定性、清除自由基、降低膜脂过氧化来增加植株耐旱性; 还可通过与STRS2互作, 负调控STRS2基因的表达, 介导STRS2依赖ABA信号途径, 增加植株的耐旱性。本研究为进一步阐明BnMAPK2基因的抗逆作用机制奠定了基础。

关键词: 甘蓝型油菜, BnMAPK2, 耐旱性, qRT-PCR

Abstract:

The mitogen-activated protein kinase (MAPK) cascade is involved in various biotic and abiotic stress responses in plants. BnMAPK2 belongs to the most downstream C-family genes in the MAPK cascade pathway. In this study, BnMAPK2 overexpression (OE-MAPK2) and RNA interference expression (RNAi-MAPK2) transgenic Brassica napus were successfully obtained. Under drought conditions, the drought tolerance of OE-MAPK2 plants was increased, and the drought tolerance of RNAi-MAPK2 plants was decreased. The related physiological indicators showed that under drought stress BnMAPK2 could slow down the degree of leaf dehydration, promote the accumulation of proline in plants, reduce the content of malondialdehyde, and increase the activity of POD at the later stage of drought. We compared the differences in the relative expression levels between transgenic and wild-type plants in the drought-related genes (P5CSB, SCE1), BnMAPK2-interacting drought-related genes (STRS2, CRL1), and STRS2-dependent ABA signaling pathway-related genes (RD22, MYC, SnRK2). The results showed that BnMAPK2 positively regulated the relative expression levels of P5CSB, SCE1, CRL1, RD22, MYC, and SnRK2, negatively regulated the relative expression levels of STRS2, and the relative expression trends of genes related to STRS2-dependent ABA signaling pathway in OE-MAPK2 plants and strs2 mutants were consistent. Therefore, it was speculated that BnMAPK2 can increase plant drought tolerance by regulating the in vivo permeability, leaf water content, cell membrane and protein structure stability, scavenging free radicals, and reducing membrane lipid peroxidation, which can also negatively regulate STRS2 by interacting with STRS2. The relative expression of the genes mediated the STRS2-dependent ABA signaling pathway and increased the drought tolerance of plants. This study lays a foundation for further elucidating the anti-stress mechanism of BnMAPK2 gene.

Key words: Brassica napus, BnMAPK2, drought tolerance, qRT-PCR

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′)
OV-BnMAPK2 CACCATGTTATTGCATAACTTGTCTGAAGG GAGCTCAGAGTTAACAGTTTCTGGA
impk2-A ctGACGTCTATTCCCGGGGACAGAATGTC cgCCATGGTCTGAAGCAGATCAATGGCTAG
impk2-B ctGGATCCTATTCCCGGGGACAGAATGTC cgTCTAGATCTGAAGCAGATCAATGGCTAG
F35s3ND GGAAGTTCATTTCATTTGGAGAG GCTGCATAATTCTCGGGGCAGCA
Bar CGACATCCGCCGTGCCACCGA CAAATCTCGGTGACGGGCAGG
26S CACAATGATAGGAAGAGCCGAC CAAGGGAACGGGCTTGGCAGAATC
ACT7 TGGGTTTGCTGGTGACGAT TGCCTAGGACGACCAACAATACT
MAPK2-q GGGGACAGAATGTCTTAACCAGA GGGAGAGACTCTATGTATCTTTTG
P5CSB CAGAAGCCACAGACTGAACTTG AAACTGCTATCAGTCACCAGCA
PLC CTGATCGATGTTCAGAAGCAAG TCGAGGTGGAGACCGTTACTAT
SCE1 AGGCTTTTTCCACCCTAATGTCTATCCA ACCCTTTTCTTGTACTCAACTGCATCC
STRS2 GAAGCAAGATCATGAGTTCGTC CTGGTTCTGTAACTCTGAGGTT
SnRK ATATCGAGCGAGGTGAGAAGA AGCTGCGTATTCCATAACAATA
BnMYC2 CGACGATAACGCCTCTATGA CCTTCGTTTGTCCCTTCAAT
BnRD22 CGCAGCGGCTGGAGTAAAGA ACCGCGTAAACGCTCGTCAT

图1

转基因植株BnMAPK2和野生型植株表达量的鉴定 相同处理标以不同字母的柱值在 P < 0.05 水平差异显著。"

图2

干旱条件下BnMAPK2基因的表达量 数据表示为平均值±SD (n = 3)。"

图3

干旱胁迫下转基因植株和野生型植株叶片含水量变化和表型分析 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

图4

自然干旱条件下转基因植株和野生型植株脯氨酸和丙二醛的对比 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

图5

自然干旱条件下转基因植株和野生型植株抗氧化酶含量的对比 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

图6

干旱条件下转基因植株和野生型植株中干旱相关基因表达情况 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

图7

干旱条件下BnMAPK2互作基因表达情况 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

图8

ABA途径相关基因表达模式 数据表示为平均值±SD (n = 3)。*、**和***分别表示在0.05、0.01、0.001概率水平差异显著。显著性均指转基因植株与野生型植株之间比较。"

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