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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (10): 2654-2664.doi: 10.3724/SP.J.1006.2023.24280

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

Functional identification of Saccharum spontaneum transcription factor SsWRKY1 to improve drought tolerance in sugarcane

SHEN Qing-Qing1(), WANG Tian-Ju2, WANG Jun-Gang3, ZHANG Shu-Zhen3, ZHAO Xue-Ting1, HE Li-Lian1(), LI Fu-Sheng1,4()   

  1. 1College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2College of Resources Environment and Chemistry, Chuxiong Normal University, Chuxiong 675000, Yunnan, China
    3Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China
    4Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2022-12-20 Accepted:2023-04-17 Online:2023-10-12 Published:2023-04-26
  • Contact: E-mail: lfs810@sina.com; E-mail: helilian905@sohu.com
  • Supported by:
    National Natural Science Foundation of China(31960451);National Key Research and Development Program of China(2018YFD1000503);Special Projects of the Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province(202105AG070007);Major Science and Technology Projects in Yunnan Province(202202AE090021);Scientific Research Fund Project of Department of Education Yunnan Province(2020Y203)

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Abstract:

SsWRKY1 is a member gene of the WRKY family in Saccharum spontaneum. The functional analysis provides valuable information for studying the molecular mechanism of SsWRKY1 involved in drought regulation. In this study, SsWRKY1 overexpression lines and RNAi interference expression lines were obtained by Agrobacterium-mediated transformation technology. The results showed that overexpression lines were significantly less damaged by drought stress than non-transgenic plants, while the interference expression lines showed severe drought stress damage with PEG to simulate drought stress. The content of proline in the overexpression lines increased significantly, the content of malondialdehyde decreased significantly, and the activities of antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase increased to varying degrees. The relative expression levels of MAPK Cascade signal transduction gene MAPK, ABA biosynthesis gene NCED, proline biosynthesis gene P5CS, ROS scavenging system gene SOD, POD, and CAT were significantly increased, indicating that SsWRKY1 overexpression could improve the ability of sugarcane to cope with drought stress. In conclusion, SsWRKY1 enhanced the drought resistance of sugarcane by activating the antioxidant system and regulating stress response genes and could be used as an important gene resource for genetic improvement of sugarcane resistance.

Key words: WRKY transcription factors, transgenic sugarcane, drought tolerance, functional identification

Fig. 1

Schematic representation of SsWRKY1 overexpression vector and RNAi interference expression vector A: SsWRKY1 overexpression vector. B: SsWRKY1 RNAi interference expression vector."

Table 1

Primers for stress responsive genes by qRT-PCR"

引物名称
Primer name		 引物序列
Primer sequences (5'-3')
SOD-F
SOD-R		 GTGAAGGCTGTTGCTGTGCTT
CGGTTCTCATCTTCTGGTGCT
POD-F
POD-R		 AATCGGAGAAGTCCTCGTCCCATAG
TTCTACCACAACAACCTCGCCAAG
CAT-F
CAT-R		 CATCCCACTCAACTACAGGCACATG
TCACACCACAAGTAGGCTTCCAATG
P5CS-F
P5CS-R		 TTTGGATTGGGTGCTGAGGTTGG
ATCCTTGTCACCGTTCACCACTTG
MAPK-F
MAPK-R		 GGTCTTCCTCTCCTCCGTCGTC
TCCTCCGCTCATCGTCTTCTTCC
NCED-F
NCED-R		 CTTCCACGGCACCTTCATCACTG
GGCTCCTCTGTTCCTCCTCCAAG

Fig. 2

Molecular detection of SsWRKY1 overexpression vector and RNAi interference expression vector A: restriction enzyme digestion identification of SsWRKY1 overexpression vector. B: bacterial solution detection of SsWRKY1 RNAi interference expression vector."

Fig. 3

Genetic transformation of sugarcane mediated by Agrobacterium-tumefaciens A: embryogenic callus. B: callus began to differentiate into seedlings. C: the first Basta resistance screening. D: the second Basta resistance screening. E: rooting of individual plant and seedling refining. F: transgenic plant in a pot."

Fig. 4

Molecular detection of SsWRKY1 transgenic sugarcane A: PCR detection of the target gene. M: DNA marker DL2000. +: positive contrast. -: negative contrast. 1-10: transgenic sugarcane. B: paper test detection of detection Bar gene. -: negative contrast. 1-5: transgenic sugarcane. C: the target gene was used by qRT-PCR detection in overexpression lines. WT: non-transformed sugarcane lines. OE4, OE5, and OE8 stands for different overexpression lines. D: the target gene was used by qRT-PCR detection in RNAi interference expression lines. WT: non-transformed sugarcane lines. R1, R2, and R5 stands for different RNAi interference expression lines. APRT gene expression is used as an internal control. The significant difference is evaluated by the Student's t-test. *: P < 0.05; **: P < 0.01."

Fig. 5

Drought resistance identification of SsWRKY1 overexpression lines A: phenotypic changes of overexpression lines under drought stress. WT: non-transformed sugarcane lines. OE4, OE5, and OE8 stands for different overexpression lines. The significant difference is evaluated by the Student's t-test. *: P < 0.05; **: P < 0.01."

Fig. 6

Drought resistance identification of SsWRKY1 interference expression lines A: phenotypic changes of interference expression lines under drought stress. WT: non-transformed sugarcane lines. R1, R2, and R5 stands for different RNAi interference expression lines. The significant difference is evaluated by the Student's t-test. *: P< 0.05; **: P < 0.01."

Fig. 7

Relative expression profile of drought stress response genes in SsWRKY1 overexpression lines WT: non-transformed sugarcane lines. OE4, OE5, and OE8 stands for different overexpression lines. The significant difference is evaluated by the Student's t-test. *: P < 0.05; **: P < 0.01."

Fig. 8

Relative expression profile of drought stress response genes in SsWRKY1 interference expression lines WT: non-transformed sugarcane lines. R1, R2, and R5 stands for different RNAi interference expression lines. The significant difference is evaluated by the Student's t-test. *: P < 0.05; **: P < 0.01."

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