甜瓜钾离子通道基因MIRK的拟南芥转化及转基因植株的耐盐性研究
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摘要
土壤盐碱化是一个世界性的问题,培育新型的耐盐作物品系是改善这一状况的最有效途径。甜瓜(Cucumis melo L.)是我国重要的一种设施栽培作物,具有较强的耐盐性,这种特性使其有可能成为新型耐盐作物品系的培育的植物资源。
渗透凋节是植物耐盐的最基本特征之一,它具有两种方式:一是在细胞中选择性吸收和积累无机盐;二是在细胞中合成有机溶质。吸钾排钠降低植物地上部分盐分浓度是植物耐盐的重要机理之一。作为植物吸收转运大量钾的主要方式,K+通道可能与植物的耐盐性有关。
成功从甜瓜“春丽”中克隆KAT1类亚家族钾离子通道基因MIRK (Melon Inward Rectifying K+ channel, Gene Bank accession number: DQ116940),全长2506 bp,编码701个氨基酸残基的多肽。将MIRK转入爪蟾卵母细胞中表达,通过电生理分析发现Na+对MIRK有抑制作用(未发表),此现象暗示在甜瓜中MIRK在盐胁迫下可能扮演着重要的生理角色。
本研究构建了MIRK基因植物双元表达载体,利用农杆菌浸花法将其转入拟南芥(Arabidopsis thaliana L. Columbia ecotype)中。转化植株的Kan抗性筛选、转化植株叶片DNA的PCR检测和Southern-blotting结果显示MIRK已经整合到拟南芥基因组中,并且获得了可遗传的拟南芥转基因后代。
为了研究MIRK基因的功能及其与耐盐性的关系,以不同浓度的NaCl溶液(0、50、100、150、200 mmol/L)处理转基因拟南芥(T3),在0、4、8、12、16d后分别对其相对电导率、相对含水量、叶绿素含量、叶绿素荧光和钾钠含量比值K+/Na+进行测定。研究结果表明,随着NaCl浓度或处理时间的增加,转基因植株和阴性对照植株的生长状况均逐渐恶化,但转基因植株受影响较小;200 mmol/L NaC1胁迫16d后,转基因植株的相对电导率仅为对照植株的57.7%,相对含水量和Fv/Fm值高于对照植株,叶绿素含量为对照植株的1.2倍,钾钠含量比值K+/Na+约为对照植株的2.3倍;差异达显著水平。初步证明MIRK基因的转入在一定程度上提高了拟南芥的耐盐能力。
Soil salinization is a global problem; one method of utilizing saline-alkali soil is to cultivate salt tolerant plants. Melon (Cucumis melo L.), an important cultivation in china, has a strong salt tolerance. This character makes it to become a salt tolerant or desalination plant resource.
Infiltration wither section is an important mechanism of salt tolerant through absorbing and accumulating of inorganic salts or synthesizing of solute-sike in plant cells. Uptaking sodium potassium to reduce the concentration of salt on the ground part of plants is an important mechanism of salt tolerant in plants. As the main way of absorport and transformation of potassium, K + channel may be related to salt tolerance of plants.
In our previous work a K+ channel gene MIRK (melon inward rectifying K+ channel, Genbank accession number: DQ116940) was cloned from Chunli (Cucumis melo L.). The full length cDNA of MIRK is 2506 bp, encoding a peptide of 701 amino acids. Transferred MIRK into Xenopus oocytes, electrophysiological analysis showed that Na+ inhibited on the transport of MIRK (unpublished), this phenomenon demonstrate that MIRK probably play an important role under the conditions of salt stress in the melon plants.
We constructed a plant transformation vector of melon K+ channel Gene MIRK, and transferred it into Arabidopsis thaliana by Floral Dip method. Resistance testing, PCR results and Southern hybridization confirmed that MIRK has been integrated into the genome of Arabidopsis thaliana. Conversion efficiency of Floral Dip method was 4.3%. Transgenic plants provide a experimental system for further study.
In order to study the relationship between MIRK and salt tolerant in plants, watered transgenic plants(T3-generation) with different concentrations of NaCl solution (0、50、100、150、200 mmol/L) every day, and determined relative water content (Rwc), photochemical efficiency of PSⅡ(Fv/Fm), relative electronic conductivity (Rec)and chlorophyll content (Chla+b,Chla/b) after 0、4、8、12、16 days’salt stress. The phenotype of transgenic plants and negative control plants show that they were getting worse and worse while NaCl concentration and stress time increased gradually, but transgenic plants grown better than control plants; after 16 days’stress of 200 mmol/L NaCl, relative electronic conductivity of transgenic plants is 57.5 percent of the control plants, relative water content and the Fv/Fm value of transgenic plants were higher than those in control plants, chlorophyll content (Chla+b, Chla/b) of transgenic plants were 1.2 times of the control,,the ratio of potassium to sodium of transgenic plants were 2.3 times of the control; the differences between them reached a significant level. Those results indicated that melon K+ Channel Gene MIRK transformation could further improve salt tolerance of plant.
渗透凋节是植物耐盐的最基本特征之一,它具有两种方式:一是在细胞中选择性吸收和积累无机盐;二是在细胞中合成有机溶质。吸钾排钠降低植物地上部分盐分浓度是植物耐盐的重要机理之一。作为植物吸收转运大量钾的主要方式,K+通道可能与植物的耐盐性有关。
成功从甜瓜“春丽”中克隆KAT1类亚家族钾离子通道基因MIRK (Melon Inward Rectifying K+ channel, Gene Bank accession number: DQ116940),全长2506 bp,编码701个氨基酸残基的多肽。将MIRK转入爪蟾卵母细胞中表达,通过电生理分析发现Na+对MIRK有抑制作用(未发表),此现象暗示在甜瓜中MIRK在盐胁迫下可能扮演着重要的生理角色。
本研究构建了MIRK基因植物双元表达载体,利用农杆菌浸花法将其转入拟南芥(Arabidopsis thaliana L. Columbia ecotype)中。转化植株的Kan抗性筛选、转化植株叶片DNA的PCR检测和Southern-blotting结果显示MIRK已经整合到拟南芥基因组中,并且获得了可遗传的拟南芥转基因后代。
为了研究MIRK基因的功能及其与耐盐性的关系,以不同浓度的NaCl溶液(0、50、100、150、200 mmol/L)处理转基因拟南芥(T3),在0、4、8、12、16d后分别对其相对电导率、相对含水量、叶绿素含量、叶绿素荧光和钾钠含量比值K+/Na+进行测定。研究结果表明,随着NaCl浓度或处理时间的增加,转基因植株和阴性对照植株的生长状况均逐渐恶化,但转基因植株受影响较小;200 mmol/L NaC1胁迫16d后,转基因植株的相对电导率仅为对照植株的57.7%,相对含水量和Fv/Fm值高于对照植株,叶绿素含量为对照植株的1.2倍,钾钠含量比值K+/Na+约为对照植株的2.3倍;差异达显著水平。初步证明MIRK基因的转入在一定程度上提高了拟南芥的耐盐能力。
Soil salinization is a global problem; one method of utilizing saline-alkali soil is to cultivate salt tolerant plants. Melon (Cucumis melo L.), an important cultivation in china, has a strong salt tolerance. This character makes it to become a salt tolerant or desalination plant resource.
Infiltration wither section is an important mechanism of salt tolerant through absorbing and accumulating of inorganic salts or synthesizing of solute-sike in plant cells. Uptaking sodium potassium to reduce the concentration of salt on the ground part of plants is an important mechanism of salt tolerant in plants. As the main way of absorport and transformation of potassium, K + channel may be related to salt tolerance of plants.
In our previous work a K+ channel gene MIRK (melon inward rectifying K+ channel, Genbank accession number: DQ116940) was cloned from Chunli (Cucumis melo L.). The full length cDNA of MIRK is 2506 bp, encoding a peptide of 701 amino acids. Transferred MIRK into Xenopus oocytes, electrophysiological analysis showed that Na+ inhibited on the transport of MIRK (unpublished), this phenomenon demonstrate that MIRK probably play an important role under the conditions of salt stress in the melon plants.
We constructed a plant transformation vector of melon K+ channel Gene MIRK, and transferred it into Arabidopsis thaliana by Floral Dip method. Resistance testing, PCR results and Southern hybridization confirmed that MIRK has been integrated into the genome of Arabidopsis thaliana. Conversion efficiency of Floral Dip method was 4.3%. Transgenic plants provide a experimental system for further study.
In order to study the relationship between MIRK and salt tolerant in plants, watered transgenic plants(T3-generation) with different concentrations of NaCl solution (0、50、100、150、200 mmol/L) every day, and determined relative water content (Rwc), photochemical efficiency of PSⅡ(Fv/Fm), relative electronic conductivity (Rec)and chlorophyll content (Chla+b,Chla/b) after 0、4、8、12、16 days’salt stress. The phenotype of transgenic plants and negative control plants show that they were getting worse and worse while NaCl concentration and stress time increased gradually, but transgenic plants grown better than control plants; after 16 days’stress of 200 mmol/L NaCl, relative electronic conductivity of transgenic plants is 57.5 percent of the control plants, relative water content and the Fv/Fm value of transgenic plants were higher than those in control plants, chlorophyll content (Chla+b, Chla/b) of transgenic plants were 1.2 times of the control,,the ratio of potassium to sodium of transgenic plants were 2.3 times of the control; the differences between them reached a significant level. Those results indicated that melon K+ Channel Gene MIRK transformation could further improve salt tolerance of plant.
引文
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