日本结缕草ZjZFN1基因对拟南芥的转化及其耐旱性分析

doi:10.11686/cyxb2018675

摘要/Abstract

摘要： 锌指蛋白在植物干旱胁迫中起重要作用,尽管已经在各种植物中克隆并鉴定了编码这些蛋白质的基因,但是它们在日本结缕草中的功能和潜在的转录机制还不清楚。通过花序侵染法将ZjZFN1基因及其启动子转化拟南芥,并通过草铵膦/潮霉素抗性筛选和PCR鉴定,获得转基因株系。ZjZFN1的启动子能够驱动GUS基因的表达,在甘露醇处理下的GUS基因表达水平显著高于对照;在拟南芥中过表达ZjZFN1降低了种子发芽率,减弱了植物对干旱胁迫的适应性和植物在干旱胁迫下的生长状况;干旱处理后,转基因植株中的丙二醛含量高于野生型植株,而脯氨酸含量显著低于野生型植株;实时荧光定量分析表明对过表达ZjZFN1的拟南芥进行干旱处理后,POD、SOD、P5CS、LEA的表达水平降低,而APX的表达水平升高。ZjZFN1基因的过表达可减弱植物的耐旱性,为进一步开发利用该基因奠定基础。

关键词: 日本结缕草, ZjZFN1转录因子, 转基因拟南芥, 抗旱性, 抗性基因表达

Abstract: Zinc finger proteins play an important role in drought stress. Although genes encoding these proteins have been cloned and identified in various plants, their functions and potential transcriptional mechanisms in Zoysia japonica are also not well understood. In the present study, the ZjZFN1 gene and its promoter were transformed into Arabidopsis thaliana by the inflorescence infection method, and then the transgenic A. thaliana was screened using glufosinate or hygromycin resistance screening and PCR identification. It was found that the promoter of ZjZFN1 can drive the expression of the GUS reporter gene system, and that the expression level of GUS genes under mannitol treatment was significantly higher than that of control plants. The results demonstrated that overexpression of ZjZFN1 in Arabidopsis reduced seed germination rate, and weakened plant adaptability to drought stress and plant growth status under drought stress. After drought treatment, malondialdehyde content was higher in plants overexpressing ZjZFN1 than that in the wild type plants, while the proline content in the transgenic plants was significantly lower than that in the wild type plants. Real-time fluorescence quantitative analysis showed that the over-expression of ZjZFN1 in Arabidopsis decreased the expression levels of peroxidase, superoxide dismutase, and pyrroline-5-carboxylate synthase enzymes and late embryogenesis abundant proteins, but increased the expression level of ascorbate peroxidase compared with control under drought treatment. This study indicates that overexpression of ZjZFN1 gene causes drought sensitivity in Arabidopsis, and provides a basis for further utilization of this gene.

Key words: Zoysia japonica, ZjZFN1 transcription factor, transgenic Arabidopsis, drought tolerance, resistant gene expression

姜红岩, 滕珂, 檀鹏辉, 尹淑霞. 日本结缕草ZjZFN1基因对拟南芥的转化及其耐旱性分析[J]. 草业学报, 2019, 28(4): 129-138.

JIANG Hong-yan, TENG Ke, TAN Peng-hui, YIN Shu-xia. Heterologous expression of a novel Zoysia japonica C₂H₂ zinc finger protein gene, ZjZFN1, caused drought sensitivity in Arabidopsis[J]. Acta Prataculturae Sinica, 2019, 28(4): 129-138.

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