红海榄脱水素基因的克隆及转化研究
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摘要
红树林生长于热带、亚热带陆海交汇的海湾河口潮间带,长期生长在高盐、高渗透压下的海水环境中,已进化出一套有别于陆生植物或淡水生植物的耐盐机制,属于真正的盐生植物。红海榄(Rhizophora stylosa)是这类植物中比较典型的一种,它属于红树科(Rhizophoraceae)红树属(Rhizophora),耐盐优势明显,是研究植物耐盐机制的理想材料。为了克隆红海榄的耐盐相关基因,本实验室在前期试验中构建了红海榄cDNA文库,然后将红海榄幼苗分别进行淡栽和盐栽处理,通过基因芯片分析在其根部差异表达基因,找到了一些有明显差异表达的cDNA片段。本文采用RT-PCR和cDNA末端快速扩增(RACE)技术,克隆了其中一个差异表达片段的全长cDNA序列,该序列包括146 bp的5’端非编码区,43bp的3’端非编码区和一个编码241个氨基酸的开放阅读框。该cDNA编码的蛋白具有植物脱水素蛋白的特征性结构,属于SKn类脱水素蛋白,我们将之命名为RsDHN1,与其它植物的SKn类脱水素蛋白具有43-55%的氨基酸序列同源性,序列存在较大差异。半定量RT-PCR分析表明,盐胁迫对RsDHNl的表达有明显上调作用。
为了深入研究这些基因的功能,我们构建了红海榄耐盐相关基因RsDHNl和RsGLP基因的植物表达载体并进行植物转化,通过抗生素抗性筛选,得到了RsDHNl基因的烟草转化植株和RsGLP基因的水稻转化植株。两基因转化植株的耐盐性状将在后续试验中鉴定。在RsGLP护基因的GFP融合表达转基因植株的荧光细胞定位分析中,发现RsGL基因在细胞中分布在细胞壁位置。这些研究结果为鉴定红树耐盐基因和理解红树耐盐机理做了初步探讨,并为红树耐盐基因在培育耐盐作物品种的研究打下了良好的基础。
Mangroves grow in estuarine or intertidal zone of tropical and subtropical area. Due to growth in marine environment with high-salt and long-term endurance of high osmotic pressure, mangroves have evolved a set of salt-tolerant mechanism, which differs from that of terrestrial plants or other aquatic plants.Mangroves are referred as true halophytes.
Rhizophora stylosa is one species of mangrove and belongs to Rhizophoraceae, Rhizophora. Rhizophora stylosa is a good target to study mechanism of plant salt-tolerance. In order to clone salt-tolerant-related genes of Rhizophora stylosa, a cDNA library has been constructed. Furthermore, Rhizophora stylosa were grown in fresh water or in saline respectively, differentially expressed genes at its roots under two different growth conditions were analysed by macroarray, some salt-tolerant related ESTs (expression sequence tag) were identified.
Full-length sequence of a EST was identified by RT-PCR and RACE technology (rapid amplification of cDNA ends). It contained a 146bp 5'-UTR (untranslation region), a 43-bp 3'-UTR and an open reading frame encoding a protein of 241 amino acid. The deduced protein showed typical domain of plant dehydrin proteins contained conserved S and K segments, belonged to SKn class of plant dehydrin and was termed as RsDHNl.RsDHNl shared 43-55% homology with the SKn dehydrins of other plant species. Semi-quantative RT-PCR analysis showed that salt treatment significantly upregulated the expression of RsDHNl.
Finally, RsDHNl expression vector was constructed and successfully transformed it into tobacco (Nicotiana tabacum). Additionally, an expression vector containing another salt-tolerant related gene, germin like proteins (RsGLP), was also constructed and successfully transformed it into tobacco and rice (Oryza sativa). RsGLP-GFP fusion expression shows that the protein locates in cell wall. The trangenic plants would be investigated in the late study. These results provide a preliminary explore to identify the salt-tolerance-related genes from Rhizophora stylosa for understanding the mechanism of mangrove, which lays a good foundation for characterization of mangrove salt-related genes and breeding salt-tolerant crops.
为了深入研究这些基因的功能,我们构建了红海榄耐盐相关基因RsDHNl和RsGLP基因的植物表达载体并进行植物转化,通过抗生素抗性筛选,得到了RsDHNl基因的烟草转化植株和RsGLP基因的水稻转化植株。两基因转化植株的耐盐性状将在后续试验中鉴定。在RsGLP护基因的GFP融合表达转基因植株的荧光细胞定位分析中,发现RsGL基因在细胞中分布在细胞壁位置。这些研究结果为鉴定红树耐盐基因和理解红树耐盐机理做了初步探讨,并为红树耐盐基因在培育耐盐作物品种的研究打下了良好的基础。
Mangroves grow in estuarine or intertidal zone of tropical and subtropical area. Due to growth in marine environment with high-salt and long-term endurance of high osmotic pressure, mangroves have evolved a set of salt-tolerant mechanism, which differs from that of terrestrial plants or other aquatic plants.Mangroves are referred as true halophytes.
Rhizophora stylosa is one species of mangrove and belongs to Rhizophoraceae, Rhizophora. Rhizophora stylosa is a good target to study mechanism of plant salt-tolerance. In order to clone salt-tolerant-related genes of Rhizophora stylosa, a cDNA library has been constructed. Furthermore, Rhizophora stylosa were grown in fresh water or in saline respectively, differentially expressed genes at its roots under two different growth conditions were analysed by macroarray, some salt-tolerant related ESTs (expression sequence tag) were identified.
Full-length sequence of a EST was identified by RT-PCR and RACE technology (rapid amplification of cDNA ends). It contained a 146bp 5'-UTR (untranslation region), a 43-bp 3'-UTR and an open reading frame encoding a protein of 241 amino acid. The deduced protein showed typical domain of plant dehydrin proteins contained conserved S and K segments, belonged to SKn class of plant dehydrin and was termed as RsDHNl.RsDHNl shared 43-55% homology with the SKn dehydrins of other plant species. Semi-quantative RT-PCR analysis showed that salt treatment significantly upregulated the expression of RsDHNl.
Finally, RsDHNl expression vector was constructed and successfully transformed it into tobacco (Nicotiana tabacum). Additionally, an expression vector containing another salt-tolerant related gene, germin like proteins (RsGLP), was also constructed and successfully transformed it into tobacco and rice (Oryza sativa). RsGLP-GFP fusion expression shows that the protein locates in cell wall. The trangenic plants would be investigated in the late study. These results provide a preliminary explore to identify the salt-tolerance-related genes from Rhizophora stylosa for understanding the mechanism of mangrove, which lays a good foundation for characterization of mangrove salt-related genes and breeding salt-tolerant crops.
引文
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