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作物学报 ›› 2024, Vol. 50 ›› Issue (9): 2237-2247.doi: 10.3724/SP.J.1006.2024.34178

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过表达马铃薯StuPPO9基因对烟草抗旱能力的影响

刘波(), 池明* (), 曹梦琦, 唐达, 杨恒照, 张卫华, 薛聪   

  1. 天津农学院园艺园林学院, 天津 300384
  • 收稿日期:2023-11-01 接受日期:2024-05-21 出版日期:2024-09-12 网络出版日期:2024-05-28
  • 通讯作者: *池明, E-mail: chiming@tjau.edu.cn
  • 作者简介:E-mail: 17622576425@163.com
  • 基金资助:
    天津市教委科研计划项目(2018KJ192)

Impact of potato StuPPO9 gene overexpression on drought resistance in Nicotiana benthamiana

LIU Bo(), CHI Ming* (), CAO Meng-Qi, TANG Da, YANG Heng-Zhao, ZHANG Wei-Hua, XUE Cong   

  1. School of Horticulture and Landscape Architecture, Tianjin Agricultural University, Tianjin 300384, China
  • Received:2023-11-01 Accepted:2024-05-21 Published:2024-09-12 Published online:2024-05-28
  • Contact: *E-mail: chiming@tjau.edu.cn
  • Supported by:
    Tianjin Municipal Education Commission(2018KJ192)

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摘要：

为鉴定马铃薯多酚氧化酶基因StuPPO9在干旱胁迫响应中的功能, 本研究以利用农杆菌介导法获得的过表达StuPPO9基因的本氏烟草为材料, 通过盆栽试验模拟自然干旱胁迫, 对比转基因株系和空载转化(WT)烟草植株干旱胁迫0 d、3 d、6 d、9 d、12 d的生长形态, 测定叶绿素、丙二醛、脯氨酸含量和抗氧化酶活性等生理指标以及抗旱相关基因的表达情况。结果表明, 干旱处理后过表达株系叶片的相对含水量、叶绿素、脯氨酸含量以及SOD、POD活性均显著高于WT, 而丙二醛含量则显著低于WT。RT-qPCR分析发现, 活性氧(reactive oxygen species, ROS)清除系统基因SOD、POX, ABA生物合成基因NCED、RD29A, 脯氨酸生物合成基因P5CS以及其他应激反应基因LEA、ADC、SAMDC等的表达量明显升高。本研究表明, StuPPO9基因增强了烟草植株对干旱的耐受性, 为探明StuPPO9基因调控干旱胁迫响应的功能提供理论依据。

关键词: StuPPO9, 转基因烟草, 抗旱性, 功能鉴定

Abstract:

To investigate the role of the potato polyphenol oxidase gene StuPPO9 in response to drought stress, we utilized the overexpression of the StuPPO9 gene in Nicotiana benthamiana through Agrobacterium-mediated transformation as the experimental material. Pot experiments were conducted to simulate natural drought stress, and various physiological indicators including growth morphology, chlorophyll content, malondialdehyde and proline levels, antioxidant enzyme activity, and expression of related resistance genes were compared and measured in transgenic and wild-type tobacco plants under drought stress for 0, 3, 6, 9 and 12 days. The results revealed that following the drought treatment, the transgenic strains exhibited significantly higher relative water content, chlorophyll content, proline levels, and superoxide dismutase (SOD) and peroxidase (POD) activities compared to the wild-type plants. Conversely, the malondialdehyde content was significantly lower in the transgenic strains compared to the wild-type. RT-qPCR analysis demonstrated a significant upregulation of expression levels in ROS scavenging system genes (SOD, POX), ABA biosynthesis genes (NCED, RD29A), proline biosynthesis gene (P5CS), and other stress response genes (LEA, ADC, SAMDC). These findings suggest that the overexpression of the StuPPO9 gene enhances drought tolerance in tobacco plants, providing a theoretical foundation for further exploration of StuPPO9’s role in regulating drought stress response.

Key words: StuPPO9, transgenic tobacco, drought tolerance, functional identification

表1

试验所用引物序列"

基因名称
Gene name		 GenBank ID 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (3°-5°)
NtSOD XM_016657042.1 CTTATGTGTAGTCGCTGGTTGA AAGAGTTGCGCTCAAACGAT
NtApxL NM_001324874.1 AAGGCTCTCCTCTCTGATCC CTCTTCCTCCTATCGCAAGC
NtADC1 AF127239 TGCGTAGACGCTACTGTTTC GAGTTTGTAGGAGGTACGCC
NtSAMDC NM_001325698.3 AGTAGTGTCTTGAGGGCTGT AAATCCGAACGACACAGCTT
NtPOX2 AB178953 GACGAGTTGTTAACAGAGCCA AGAAGAACTCCTCCATCGCA
NtP5CS1 HM854026 AGACCTTAGTGGGCTTGAGA GTTAGGCTTCAACCTGCCTT
NtERD10C AB049337 TGGGGTTTGAAGGGTAATACAG AAGGTGAAAACCAAACCAACTG
NtLEA5 AF053076 GCTGCATCATCAGCTAGTGT CTCAGCAGCGTCAATCTCTT
NtNCED1 KM605435 AGCCCAAGATATCAATGCCAAG AGGAGCTTGGAGTGAGGAATTA
NtRD29A NM_001325924.1 TCATGGTGGACCCTATGCTAT CTGCAAAACTGGCCTATGTG
NtActin XM_016579509.1 GTCTCTTTGGATGCCTCTGC CATCCTATCAGCAATGCCCG
StuPPO9 XM_006347021.2 GGACCCGACGTTACCAAATG TGATGGAAGCTGGAAGTCGA
STPO XM_006347021.2 ATGTTCATGAATACATCTCAAAC CTAATCCTCAAGCACAATC

图1

StuPPO9过表达载体PCR检测 M: Trans2K Plus DNA marker; STPO: 马铃薯StuPPO9基因扩增产物。"

图2

StuPPO9过表达烟草阳性植株PCR鉴定 M: Trans2K Plus DNA marker; +: 阳性质粒pCambia2301- StuPPO9对照; -: 空载转化烟草; 5、7、9、11、15、18: 转基因烟草阳性株系Line-5、Line-7、Line-9、Line-11、Line-15、Line-18。"

图3

StuPPO9过表达烟草阳性植株株系StuPPO9基因表达量 图中小写字母表示在0.05概率水平差异显著。"

图4

干旱胁迫下StuPPO9转基因烟草地上部表型观察"

图5

干旱胁迫下StuPPO9转基因烟草根表型观察"

表2

干旱胁迫下StuPPO9转基因烟草生长指标变化"

株系
Line		 株高Height (cm) 茎粗Stem diameter (mm) 叶面积Leaf area (cm2)
对照组CK 干旱Drought 对照组CK 干旱Drought 对照组CK 干旱Drought
野生型WT 15.07±0.318 b 10.27±0.273 d 1.60±0.024 b 1.18±0.035 d 24.28±0.274 b 13.67±0.418 e
Line-5 15.60±0.306 b 13.47±0.203 c 1.67±0.021 ab 1.35±0.021 c 25.75±0.279 a 16.80±0.393 d
Line-9 16.40±0.173 a 14.00±0.252 c 1.75±0.031 a 1.42±0.029 c 26.41±0.188 a 18.77±0.256 c

表3

干旱胁迫下StuPPO9转基因烟草叶片/根系相对含水量"

株系
Line		 叶片Leaf 根系Root
对照组CK 干旱Drought 对照组CK 干旱Drought
野生型WT 92.25±0.584 a 85.06±0.208 c 94.36±0.220 b 87.24±0.347 d
Line-5 92.76±0.201 a 89.66±0.236 b 95.18±0.045 a 91.19±0.073 c
Line-9 93.15±0.075 a 90.12±0.602 b 95.27±0.074 a 91.34±0.044 c

图6

干旱胁迫下StuPPO9转基因烟草叶绿素含量变化 对于干旱胁迫处理下各处理间的差异显著性以不同小写字母(P < 0.05)标记。"

图7

干旱胁迫下StuPPO9转基因烟草叶片丙二醛(A)和脯氨酸(B)含量的变化 对于干旱胁迫处理下各处理间的差异显著性以不同小写字母(P < 0.05)标记。"

图8

干旱胁迫下StuPPO9转基因烟草叶片超氧化物歧化酶(A)、过氧化物酶(B)活性的变化 对于干旱胁迫处理下各处理间的差异显著性以不同小写字母(P < 0.05)标记。"

图9

干旱胁迫下转基因植株ROS和ABA相关基因表达量 对于干旱胁迫处理前后各处理间的差异显著性以不同小写字母(P < 0.05)标记。"

图10

干旱胁迫下转基因植株抗旱相关基因表达量 对于干旱胁迫处理前后各处理间的差异显著性以不同小写字母(P < 0.05)标记。"

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