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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 561-569.doi: 10.3724/SP.J.1006.2023.22008

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Effects of GsERF6 overexpression on salt-alkaline tolerance in rice

CAI Xiao-Xi(), HU Bing-Shuang(), SHEN Yang, WANG Yan, CHEN Yue, SUN Ming-Zhe, JIA Bo-Wei, SUN Xiao-Li()   

  1. Crop Stress Molecular Biology Laboratory, College of Agriculture, Heilongjiang Bayi Agriculture University, Daqing 163319, Heilongjiang, China
  • Received:2022-01-28 Accepted:2022-06-07 Online:2022-06-21 Published:2022-06-21
  • Contact: SUN Xiao-Li E-mail:18746616279@163.com;alisa961102@gmail.com;sunxiaoli2016@byau.edu.cn
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(32101672);National Natural Science Foundation of China(31971826);National Natural Science Foundation of China(U20A2025);Special Funds from the Central Finance to Support the Development of Local Universities(202201005)

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Abstract:

Ethylene response factors (ERFs) are a family of plant specific transcription factors that play important roles in abiotic stress. Bioinformatic analysis revealed that GsERF6, a Glycine soja ERF transcription factor that positively regulated salt-alkaline tolerance, shared high sequence identity with homologous ERF proteins with one highly conserved AP2 domain in rice. To explore the effect of GsERF6 overexpression on the salt-alkaline tolerance in rice, we transformed GsERF6 into rice and obtained two homozygous transgenic lines via PCR and RT-PCR. Phenotypical and physiological assays indicated that, compared with the wild type rice under 200 mmol L-1 NaHCO3 treatment, the survival rate, relative water content, the activities of SOD, POD, and CAT, the soluble sugar and proline contents, were significantly increased, while ROS accumulation was significantly decreased in GsERF6 overexpression lines. The qRT-PCR showed that transcript levels of OsP5CS2 and OsLEA14 were significantly up-regulated in GsERF6 transgenic line after 40 mmol L-1 NaHCO3 treatment for 6 hours. In summary, GsERF6 overexpression in rice contributed to ROS scavenging, osmotic regulation, and activation of stress responsive genes, thus improving the salt-alkaline tolerance of transgenic rice.

Key words: rice, salt-alkaline tolerance, ethylene response factors, Glycine soja, GsERF6

Table 1

Primers used in this study"

引物名称
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
GsERF6-F/R GGCTTAAUATGGCTAACGCTGCTG GGTTTAAUTCACACAGCCACGAGCGGT
GsERF6-PCR-F/R ATAAGGAAGTTCATTTCATTTGGA TCTTCGGAACAGCGATTAGCAG
GsERF6-RT-F/R CTGGCCACTCCCCAAAACAAA GAAGGTTCCGAGCCAAACCC
OsElf1-α-F/R GCACGCTCTTCTTGCTTTCAC TCTTGTCAGGGTTGTAGCCGAC
OsP5CS2-F/R GTGGCTTGTGAAGGAGCTGT TTTGACATGCTTTCGTGCTC
OsLEA14-F/R TCGGGATGTCAGGCGATAA GCTTGTAGGTGCTGGTGTCCTT

Fig. 1

Phylogenetic analysis of GsERF6 and its homologous ERF proteins in rice “?” stands for ERF gene of rice; “Δ” stands for ERF gene of wild soybean."

Fig. 2

Multiple alignment of GsERF6 and its homologous ERF proteins in rice"

Fig. 3

Construction of the GsERF6 overexpression vector A: the GsERF6 overexpression vector; B: gene clone of GsERF6; M: DNA marker; 1-3: PCR product of GsERF6; C: PCR identification of recombinant E. coli clones harboring the pC35SU-GsERF6 con-struct; M: DNA marker; ?: negative H2O control; +: positive plas-mid control; 1: PCR products of the pC35SU-GsERF6 clones."

Fig. 4

Molecular detection of the GsERF6 transgenic lines A: PCR detection of resistant plants transformed with GsERF6; B: RT-PCR detection of GsERF6 transgenic rice; M: DNA marker; ?: negative H2O control; +: positive plasmid control; WT: wild type control; #1-#8: NeoR resistant seedlings."

Fig. 5

Salt-alkaline tolerance of GsERF6 transgenic rice A: phenotype of WT and transgenic rice seedlings treated with NaHCO3; B: the survival rates of WT and transgenic rice seedlings; C: the relative water content of WT and transgenic rice seedlings. The significant difference was evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 6

ROS accumulation and antioxidant enzyme activity in the GsERF6 transgenic rice under salt-alkaline treatment A: DAB staining; B: NBT staining; C: SOD activity; D: POD activity; E: CAT activity. The significant difference was evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 7

Content of osmolytes in the GsERF6 transgenic rice under salt-alkaline treatment A: soluble sugar content; B: proline content. The significant difference was evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 8

Relative expression patterns of salt-alkaline responsive genes in WT and GsERF6 transgenic lines A: the relative expression level of OsLEA14 genes; B: the relative expression level of OsP5CS2 genes. The significant difference was evaluated by the Student’s t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

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