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

摘要/Abstract

摘要： 【目的】ZmAN14是玉米A20/AN1型锌指蛋白基因家族成员，该家族在水稻中广泛参与非生物胁迫应答。通过分析玉米旱21（H21）及转ZmAN14烟草在非生物胁迫下的表达情况，为玉米ZmAN14及其家族的功能和分子机制的全面解析提供新信息。【方法】通过对ZmAN14进行序列分析，发现其属于玉米A20/AN1型锌指蛋白基因家族。以玉米自交系H21为研究材料，在三叶期时对其进行非生物胁迫和ABA诱导处理，0、1、3、6、12和24 h时取整株样品，同时在玉米不同生长期取根、茎、叶、胚芽鞘、雌蕊、雄蕊、花丝、苞叶等不同组织样品，利用实时荧光定量PCR方法对ZmAN14的组织表达谱和非生物胁迫及ABA诱导的表达谱进行分析，并结合启动子区顺式作用元件分布情况进行对比分析。将ZmAN14编码序列克隆至GFP表达载体pMDC85，利用亚细胞定位技术验证ZmAN14在胞内的定位情况。将ZmAN14克隆至GAL4 DNA结合结构域载体，并转化酵母，将其涂布在缺陷培养基上观察酵母生长情况，分析其有无转录激活活性。将ZmAN14编码区与植物表达载体p1300-221连接构建超表达载体，并将其转入烟草，选择ZmAN14 mRNA 表达量高的T₂纯合体株系进行盐、干旱胁迫和ABA诱导，观察其对非生物胁迫的应答。【结果】序列分析表明，ZmAN14开放阅读框（ORF）为516 bp，编码一个171个氨基酸的蛋白，预测分子量为18.3 kD，等电点是8.28。ZmAN14具有A20和AN1结构域，属于玉米A20/AN1型锌指蛋白基因家族。实时定量荧光PCR结果表明ZmAN14在叶中大量表达，受NaCl、干旱胁迫和ABA诱导上调。ZmAN14与GFP融合蛋白定位于细胞质和细胞核中，定位结果与ZNF216 和ZmAN13类似，而ZmAN14本身并没有核定位信号，暗示ZmAN14与其他蛋白结合进入细胞核中发挥其生物学功能。ZmAN14在酵母中表达时不能在酵母缺陷培养基（SD/-Leu-His medium）上生长，因此，未发现其具有转录激活活性。将ZmAN14在烟草中过表达会增加烟草苗期对盐和干旱的抗性。【结论】ZmAN14属于玉米A20/AN1型锌指蛋白基因家族。ZmAN14主要在叶中表达，受非生物胁迫和ABA诱导，在烟草中过表达可以提高烟草苗期对盐和干旱的抗性。ZmAN14可能通过与其他蛋白的互作行使功能。相比较于其他的A20/AN1型锌指蛋白，在转基因烟草中，ZmAN14也参与非生物胁迫应答。ZmAN14与ZmAN13具有高同源性，但在盐和干旱胁迫时，二者却具有向反方向调控的功能。

关键词: 玉米, 锌指蛋白, ZmAN14, 非生物胁迫, 转基因烟草

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

宣宁，柳絮，张华，陈高，刘国霞，边斐，姚方印. 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[J]. 中国农业科学, 2015, 48(5): 841-850.

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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