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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (10): 2458-2467.doi: 10.3724/SP.J.1006.2024.44017

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

Overexpression of wild soybean salt-alkali tolerance gene GsGSTU13 increases salt-alkaline tolerance in rice seedlings

LI Wan-Hong(), HU Bing-Shuang, SUN Xiao-Li, CAI Xiao-Xi*(), SUN Ming-Zhe*()   

  1. College of Agriculture, Heilongjiang Bayi Agriculture University / Crop Stress Molecular Biology Laboratory, Daqing 163316, Heilongjiang, China
  • Received:2024-01-28 Accepted:2024-06-20 Online:2024-10-12 Published:2024-07-11
  • Contact: *E-mail: sunmingzhe@byau.edu.cn;E-mail: 18746616279@163.com
  • Supported by:
    National Natural Science Foundation of China(U20A2025);National Natural Science Foundation of China(31971826);Natural Science Foundation of Heilongjiang Province(YQ2023C035)

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Abstract:

Glutathione S-transferases (GSTs) are a class of highly conserved enzymes that play crucial roles in plant responses to environmental stresses. Bioinformatic analysis has revealed that Glycine soja GsGSTU13, which positively regulates salt-alkaline tolerance, shares the highest sequence identity with the OsGSTU17 protein. To investigate the potential contribution of GsGSTU13 to rice salt-alkaline tolerance, we transformed GsGSTU13 into rice and obtained two homozygous transgenic lines with significantly elevated GST activity. Phenotypic assays showed that after treatment with 200 mmol L-1 NaHCO3, the accumulation of reactive oxygen species was significantly lower in GsGSTU13 transgenic lines compared to wild-type. Additionally, the survival rates, relative water contents, and the activities of superoxide dismutases, peroxidases, catalases, and GSTs were significantly higher in GsGSTU13 transgenic lines than in the wild-type. In summary, overexpression of GsGSTU13 in rice enhanced salt-alkaline tolerance by promoting ROS scavenging, which could facilitate the breeding of new rice cultivars with improved tolerance to salt-alkaline stress.

Key words: rice, wild soybean, salt-alkaline tolerance, glutathione S-transferases, GsGSTU13

Table 1

Primers used in the study"

引物名称
Primer name		 序列
Sequence (5°-3°)		 试验用途
Experimental purpose
pCAMBIA130035Su-GsGSTU13-F GGCTTAAUGGCTTCAAATCATGAAG 植物超量表达载体构建
Generation of plant expression construct
pCAMBIA130035Su-GsGSTU13-R GGTTTAAUCTACTTTTTAGCAGAAGCTTG
pCAMBIA35S-F ATAAGGAAGTTCATTTCATTTGGA 遗传转化抗性苗PCR检测
PCR identification of transgenic rice
GsGSTU13-PCR-R TCCTCCACTGCTTTCTCACG
GsGSTU13-RT-PCR-F TGCCTCGAGACCCTTATGAGA 遗传转化抗性苗RT-PCR检测
RT-PCR identification of transgenic rice
GsGSTU13-RT-PCR-R CCTCCTTGATCACAGGGTGATTG
OsGSTU17-qRT-F CTCCCAACTCCCAACTTCCC OsGSTU17基因表达Real-time PCR分析
Real-time PCR analysis of OsGSTU17 gene expression
OsGSTU17-qRT-R ACATGCCGAACACCTTCACT

Fig. 1

Phylogenetic analysis of GsGSTU13 and rice homologous GSTU proteins A: Phylogenetic tree of GsGSTU13 and homologous OsGSTU proteins (GsGSTU13 is marked by a red star, functionally reported OsGSTUs are marked by blue dots). B: Heatmap showing the similarity of GsGSTU13 and homologous OsGSTUs proteins."

Fig. 2

Multiple alignment of GsGSTU13 and homologous OsGSTU proteins"

Fig. 3

Generation of pCAMBIA130035Su-GsGSTU13 plant expression vector A: Representation of the pCAMBIA130035Su-GsGSTU13 construct; B: PCR product of the GsGSTU13 CDS region (M: DNA marker 2K); C: PCR identification of recombinant E. coli clones harboring the pCAMBIA130035Su-GsGSTU13 construct; M: DNA marker 2K; +: Positive plasmid control (pGEM-T-GSTU13); -: Negative H2O control; 1-4: GsGSTU13 colonies."

Fig. 4

Molecular identification of GsGSTU13 transgenic rice lines A: PCR identification of GsGSTU13 transgenic rice (M: DNA marker 2K Plus; +: positive plasmid control; -: negative H2O control; WT: wild type control; #1-#9: resistant seedlings transformed with GsGSTU13); B: RT-PCR identification of GsGSTU13 transgenic rice (WT: the wild type control; #1 and #2: GsGSTU13 transgenic lines; OsElf1-α is used as the reference gene). C: Detection of GST activity in GsGSTU13 transgenic lines; *: P < 0.05 by the Student’s t-test, n = 20. D: Relative expression of OsGSTU17 in GsGSTU13 transgenic lines. ns: non-significance, n = 3."

Fig. 5

Salt-alkaline tolerance of GsGSTU13 transgenic rice lines at the seedling stage A: Phenotype of WT and transgenic rice seedlings treated with NaHCO3; B: Survival rates of WT and transgenic rice seedlings; C: Relative water content of WT and transgenic rice seedlings. Significant differences were evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01, n = 20."

Fig. 6

ROS accumulation in the GsGSTU13 transgenic rice under salt-alkaline treatment A: NBT staining; B: DAB staining. Scale bars are 1 cm."

Fig. 7

Antioxidant enzyme activity of GsGSTU13 transgenic rice under salt-alkaline treatment A: Glutathione S-transferase (GST) activity; B: Superoxide dismutase (SOD) activity; B: Peroxidase (POD) activity; B: Catalase (CAT) activity. Significant differences were evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01, n = 20."

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