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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (9): 2237-2247.doi: 10.3724/SP.J.1006.2024.34178

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-09-12 Published:2024-05-28
  • Contact: *E-mail: chiming@tjau.edu.cn
  • Supported by:
    Tianjin Municipal Education Commission(2018KJ192)

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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

Table 1

Primer sequences used in the study"

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

Fig. 1

PCR detection of StuPPO9 overexpression vector M: Trans2K Plus DNA marker; STPO: potato StuPPO9 gene amplification product."

Fig. 2

PCR identification of StuPPO9-overexpressing tobacco plants M: Trans2K Plus DNA marker; +: positive plasmid pCambia2301-StuPPO9 control; -: empty conversion tobacco; 5, 7, 9, 11, 15, 18: positive transgenic tobacco lines Line-5, Line-7, Line-9, Line-11, Line-15, and Line-18."

Fig. 3

StuPPO9 overexpression of tobacco positive plant strain StuPPO9 gene expression The lowercase letters indicate significant difference at the 0.05 probability level."

Fig. 4

Aboveground phenotypes of StuPPO9 transgenic tobacco under drought stress"

Fig. 5

Root phenotypes of StuPPO9 transgenic tobacco under drought stress"

Table 2

Changes in growth indicators of StuPPO9 transgenic tobacco under drought stress"

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

Table 3

Relative moisture content of StuPPO9 transgenic tobacco shoot/root under drought stress (%)"

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

Fig. 6

Changes in chlorophyll content in StuPPO9 transgenic tobacco under drought stress Significance of differences between treatments under drought stress treatment marked with different lowercase letters (P < 0.05)."

Fig. 7

Changes in malondialdehyde (A) and proline (B) content in StuPPO9 transgenic tobacco leaves under drought stress Significance of differences between treatments under drought stress treatment marked with different lowercase letters (P < 0.05)."

Fig. 8

Changes in superoxide dismutase (A) and peroxidase (B) activities in StuPPO9 transgenic tobacco leaves under drought stress Significance of differences between treatments under drought stress treatment marked with different lowercase letters (P < 0.05)."

Fig. 9

Expression of ROS and ABA-related genes in transgenic plants under drought stress Significance of differences between treatments under drought stress treatment marked with different lowercase letters (P < 0.05)."

Fig. 10

Expression of drought-resistant genes in transgenic plants under drought stress Significance of differences between treatments under drought stress treatment marked with different lowercase letters (P < 0.05)."

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