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Molecular cloning and functional analysis of lotus salt-induced NnDREB2C, NnPIP1-2 and NnPIP2-1 in Arabidopsis thaliana

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Abstract

Dehydration-responsive element bindings transcription factor (DREBs) and plasma membrane intrinsic proteins (PIPs) have been characterized multi-functions in plant growth and metabolism, as well as in the adaptation to various stresses. In this study, we cloned the full-length cDNA of NnDREB2C from a salt-tolerated lotus species with RT-PCR methods. Analysis of qRT-PCR demonstrated that NnDREB2C mRNA in the leaf dramatically increased after the treatments of NaCl, abscisic acid, low temperature and mannitol. Next, NnDREB2C was cloned into constitutive expression vector pSN1301, which in turn transformed into Arabidopsis thaliana to investigate its function in plants. NnDREB2C overexpression significantly elevated Arabidopsis tolerance against salt and drought stresses, showing higher survival rates, lower conductivity and more chlorophyll content than those of wild-type plants. Moreover, higher germination rates were observed in the NnDREB2C overproducing plants when subjected into the stresses of NaCl and mannitol. Furthermore, we investigate the potential down-stream genes regulated by NnDREB2C and observed a significant increase in expressions of several genes belonging to PIPs family, including PIP1-1, PIP1-2, PIP1-3, PIP1-4 and PIP1-5. Consistently, overexpressed NnPIP1-2 and NnPIP2-1 conferred Arabidopsis the tolerance to stresses. Taken together, we concluded that overexpression of NnDREB2C enhanced the tolerance of salt and drought stresses in plants, which might probably be derived from the increased expression of the genes belonging to PIPs family.

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Abbreviations

cDNA:

Complementary DNA

RT-PCR:

Reverse transcription polymerase chain reaction

qRT-PCR:

Quantitative real time polymerase chain reaction

DREB2C:

Dehydration responsive element-binding protein 2C

ABA:

Abscisic acid

PIPs:

Plasma membrane intrinsic proteins

LT:

Low temperature

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Acknowledgements

The authors thank Edanz Groop for their editorial assistance. This work was supported by Jiangsu Agriculture Science and Technology Innovation Fund (CX (18) 3066), and the modern agriculture of Yangzhou (YZ2017044).

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Authors and Affiliations

  1. School of Horticulture and Plant Protection, Yangzhou University, Jiangsu, People’s Republic of China

    Liu Ziyuan, Yang Jianjun, Liu Xian, Li Liangjun & Cheng Libao

  2. College of Guangling, Yangzhou University, Jiangsu, People’s Republic of China

    Li Shuyan

  3. Center for Multi-omics Research, Key Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, 475004, Kaifeng, China

    Wang Chunfei

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  1. Liu Ziyuan
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Contributions

CL and WC were responsible for most data analysis, and WC wrote the manuscript. LZ conducted the experimental work. The experiment was designed by LS and CL, who critically reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Cheng Libao or Li Shuyan.

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11033_2019_5156_MOESM1_ESM.jpg

Supplementary material 1 (JPEG 100 kb) Fig. S1 Alignment of the deduced amino acid sequence of NnDREB2C. DREB2C from Arabidopsis (NP_565929.1), Camalina (XP_010505763.1), Cucumber (XP_004138558.1) and Phoenix (XP_008799804.1) were collected for comparison with the DNAman software

Supplementary material 2 (DOCX 15 kb) Table S1: Isolation of NnPIPs of lotus and their deduced amino acids.

Supplementary material 3 (DOCX 20 kb) Table S2: The primer sequences for NnPIPs isolation and expression analysis

11033_2019_5156_MOESM4_ESM.doc

Supplementary material 4 (DOC 45 kb) Table S3: Primers for drought stress-related genes. Twenty-eight stresses-related genes from Arabidopsis were selected for expression analysis in NnDREB2C-transgenic and wild type plants. All these genes were derived from the NCBI database, and primers were designed according to the gene sequences with the Primer 5.0 software

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Ziyuan, L., Chunfei, W., Jianjun, Y. et al. Molecular cloning and functional analysis of lotus salt-induced NnDREB2C, NnPIP1-2 and NnPIP2-1 in Arabidopsis thaliana. Mol Biol Rep 47, 497–506 (2020). https://doi.org/10.1007/s11033-019-05156-0

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