玉米质膜内在蛋白ZmPIP2;6响应渗透、盐和 干旱胁迫的功能鉴定

doi:10.3864/j.issn.0578-1752.2020.03.001

摘要/Abstract

摘要：

【目的】质膜内在蛋白（plasma membrane intrinsic proteins,PIPs）广泛存在于植物细胞的膜系统上,在植物体内水分运输和水分平衡的过程中至关重要。对ZmPIP2;6在植物水分胁迫耐性中的功能进行探究,为玉米培育抗旱耐盐新品种提供优秀基因资源。【方法】分析并比对ZmPIP2;6与其他物种中报道参与水分胁迫的PIPs的氨基酸序列,构建ZmPIP2;6-GFP载体并通过PEG介导转化玉米原生质体,对ZmPIP2;6进行亚细胞定位。采集玉米的不同组织样品,包括根、茎、叶、未成熟雄穗、未成熟雌穗、胚和胚乳;对玉米进行PEG或NaCl处理,在处理的不同时间点采集玉米的根和叶样品。提取总RNA并通过qRT-PCR调查ZmPIP2;6在玉米不同组织以及在水分胁迫下的表达模式。构建ZmPIP2;6超表达载体,发展并鉴定ZmPIP2;6超表达拟南芥材料,观察转基因植株对渗透、盐及干旱胁迫的耐性生理表型,并测量其根长、叶片水分散失率等性状。检测在干旱或盐胁迫条件下,拟南芥胁迫信号通路上的相关基因在ZmPIP2;6超表达植株中的表达。【结果】氨基酸序列分析比对结果显示ZmPIP2;6具有PIP蛋白的典型结构与并且其他物种的PIP蛋白具有很高的同源性。转化玉米原生质体试验结果显示ZmPIP2;6蛋白定位在细胞质膜。qRT-PCR结果显示ZmPIP2;6在玉米未成熟雄穗中表达量最高,并且在玉米受到渗透和盐胁迫后根和叶中的ZmPIP2;6表达受到显著诱导。在MS固体培养基上进行渗透胁迫处理和盐胁迫处理以及进一步的土培试验中进行干旱胁迫处理,ZmPIP2;6超表达拟南芥植株相对野生型都显示出更强的胁迫耐性。在干旱或盐胁迫条件下,拟南芥胁迫信号通路上的相关基因在ZmPIP2;6超表达植株中的表达受到不同程度的影响。【结论】玉米内在质膜蛋白基因ZmPIP2;6在渗透或盐胁迫下表达上调,在拟南芥中超表达ZmPIP2;6会增强植株对渗透、盐和干旱胁迫的耐性,并且在盐或干旱胁迫条件下会影响拟南芥中胁迫相关基因的表达。ZmPIP2;6可能参与植物水分胁迫响应过程。

关键词: 玉米, 质膜内在蛋白, 渗透胁迫, 干旱胁迫, 盐胁迫

Abstract:

【Objective】Plasma membrane Intrinsic Proteins (PIPs) exist widely in the membrane system of plant cells, which are essential to water transport and water balance in plant. The aim of this study is to explore the function of ZmPIP2;6 in plant water stress tolerance, and provide potential gene resources for new varieties of stress tolerance maize breeding. 【Method】Amino acid sequences of ZmPIP2;6 was analyzed and compared with other PIPs that involved in water stress tolerance. To verify the subcellular localization of ZmPIP2;6, ZmPIP2;6-GFP infusion vector was constructed and assessed using maize protoplasts isolated from leaves of maize seedlings. Tissues from root, stem, mature leaf, immature tassel, immature ear, endosperm and embryo of maize were isolated. Samples from root and leaf of maize were collected at different time after PEG or NaCl treatment. Total RNA were extracted, and expression pattern of ZmPIP2;6 in different tissues or under water stress condition was investigated by qRT-PCR. Transgenic Arabidopsis plants that overexpressed ZmPIP2;6 were generated and identified. The phenotype of ZmPIP2;6 overexpression transgenic Arabidopsis that tolerated to osmotic, salt or drought stress were monitored and primary root length and leaf water loss rate were measured. A number of stress responsive genes in ZmPIP2;6 overexpression Arabidopsis were detected under drought or salt condition.【Result】Analysis and comparison of the amino acid sequences showed that ZmPIP2;6 shared the same conserved structural domains and had a high degree of sequence similarity with other PIPs. The subcellular localization was assessed using maize protoplasts indicated ZmPIP2;6 was located on the plasma membrane. qRT-PCR result showed that ZmPIP2;6 was highly expressed in tassel. Treatment with PEG or NaCl resulted in induced expression of ZmPIP2;6 in root and leaf of maize. Overexpression of ZmPIP2;6 in transgenic Arabidopsis showed enhanced osmotic and salt stress tolerance in MS media plate and improved drought stress tolerance in soil condition compared to wild type. Expressions of related genes in the stress signaling pathway were changed in ZmPIP2;6 overexpression Arabidopsis under drought or salt condition. 【Conclusion】 Expression of ZmPIP2;6 was up-regulated under osmotic or salt stress condition. Overexpression of ZmPIP2;6 in Arabidopsis enhanced osmotic, salt and drought stress tolerance. A number of stress responsive genes in ZmPIP2;6 overexpression Arabidopsis were affected under salt or drought stress condition. These results indicated that ZmPIP2;6 might be involved in plant water stress responsive pathway.

Key words: maize (Zea mays L.), plasma membrane intrinsic proteins, osmotic stress, drought stress, salt stress

周练,熊雨涵,洪祥德,周京,刘朝显,王久光,王国强,蔡一林. 玉米质膜内在蛋白ZmPIP2;6响应渗透、盐和干旱胁迫的功能鉴定[J]. 中国农业科学, 2020, 53(3): 461-473.

ZHOU Lian,XIONG YuHan,HONG XiangDe,ZHOU Jing,LIU ChaoXian,WANG JiuGuang,WANG GuoQiang,CAI YiLin. Functional Characterization of a Maize Plasma Membrane Intrinsic Protein ZmPIP2;6 Responses to Osmotic, Salt and Drought Stress[J]. Scientia Agricultura Sinica, 2020, 53(3): 461-473.

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