玉米锌指蛋白基因ZmAN14过表达转基因烟草对非生物胁迫的响应

doi:10.3864/j.issn.0578-1752.2015.05.02

Abstract

Abstract: 【Objective】 ZmAN14 is a member of the maize A20/AN1 zinc finger protein gene family. This gene family of rice is involved in the response to abiotic stress. The expression of ZmAN14 in the maize inbred line H21 and transgenic ZmAN14 tobacco under abiotic stress was analyzed. The results will provide novel information for the comprehensive analysis of the functional and molecular mechanisms of maize ZmAN14 and the entire gene family. 【Method】 Sequence analysis of ZmAN14 was performed to confirm that this gene is a member of the maize A20/AN1 zinc finger protein gene family. The experiment was conducted by using maize inbred line H2. At the three-leaf stage, the seedlings were treated with multiple abiotic stress or induced by ABA. After treatment for 0, 1, 3, 6, 12 and 24 h, the whole plants were harvested. In the meantime, the roots, stems, leaves, coleptiles, pistil, stamen, silks and bract leaves were harvested from different growing periods of maize. Real-time quantitative fluorescence PCR was used to analyze the expression profile in different tissues and abiotic stress response of ZmAN14 and its ABA-induced expression profile. The cis-acting element of the promoter area was cloned to perform the comparative analysis. The ZmAN14 coding sequence was cloned onto the GFP expressing vector pMDC85. A subcellular localization method was used to verify the localization of the ZmAN14 protein in the cell. The ZmAN14 coding sequence was cloned onto the GAL4 DNA-binding domain vector. This vector was used to transform yeast. The transformed yeast was spotted onto the defect medium, and the resulted transcriptional activation activity was analyzed. The ZmAN14 coding region was ligated to the plant expression vector p1300-221 to create an overexpression vector. The overexpression vector was transformed into tobacco. T2 homozygous transgenic lines with high ZmAN14 mRNA expression level were used to conduct salt-, drought- and ABA induced experiments, and its response to abiotic stress was observed. 【Result】Sequence analysis showed that the ORF was 516 bp in length, encoding a polypeptide of 171 amino acid residues with an estimated molecular mass of 18.3 kD and a pI of 8.28. The ZmAN14 gene, which contains the A20 and AN1 domains, belongs to the maize A20/AN1 zinc finger protein gene family. Results of the real-time quantitative fluorescence PCR showed that ZmAN14 was abundantly expressed in leaves, and its expression increased under NaCl-, drought-, and ABA-induced stresses. The ZmAN14-GFP fusion proteins were localized in the cytoplasm and nucleus, which was similar to the localization of ZNF216 and ZmAN13, but the ZmAN14 gene itself did not produce a nuclear localization signal. Therefore, ZmAN14 performed its biological function by entering the nucleus together with other proteins. The ZmAN14 transgenic yeast was not grown on the SD/-Leu-His defect medium. No transcriptional activation activity was found when ZmAN14 was expressed in yeast. The over-expression of ZmAN14 in tobacco increased plant resistance to salt and drought at seedling stage. 【Conclusion】The ZmAN14 gene belongs to the maize A20/AN1 zinc finger protein gene family. The ZmAN14 was abundantly expressed in leaves and the increase of its expression level was induced by abiotic stress and ABA. The overexpression of ZmAN14 in tobacco increased plant resistance to salt and drought stresses under abiotic stress and ABA induction at the seedling stage. ZmAN14 might function via its interaction with other proteins. Compared with other A20/AN1 zinc finger proteins, ZmAN14 participated in the response to abiotic stress. ZmAN14 and ZmAN13 showed high homology, but these genes had different control mechanisms (in the opposite directions) in terms of response to salt and drought stresses.

Key words: maize, Zinc finger protein, ZmAN14, abiotic stress, transgenic tobacco

XUAN Ning, LIU Xu, ZHANG Hua, CHEN Gao, LIU Guo-xia, BIAN Fei, YAO Fang-yin. The Response of a Putative Maize Zinc-Finger Protein Gene ZmAN14 in Transgenic Tabacco to Abiotic Stress[J].Scientia Agricultura Sinica, 2015, 48(5): 841-850.

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The Response of a Putative Maize Zinc-Finger Protein Gene ZmAN14 in Transgenic Tabacco to Abiotic Stress

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