玉米海藻糖含量测定及其合成酶基因的克隆与序列分析
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摘要
玉米是我国主要粮食作物之一,同时也是需水较多、对水分胁迫比较敏感的作物。干旱对玉米等农作物所造成的损失在所有非生物胁迫中居首位。我国每年约有60%的玉米面积受到干旱胁迫,因旱灾而减产达15%-20%。因此,研究玉米耐旱机理,弄清玉米对干旱的适应和调节机制,开发玉米耐旱新材料,是减少干旱对玉米生产影响的有效措施。
耐旱植物卷柏在干旱条件下,体内大量合成的海藻糖具有保护细胞内活性物质的功能,以降低逆境对细胞造成的伤害;在其他高等植物如拟南芥、水稻等植物中,也发现海藻糖的存在并与干旱存在相关性。目前已有研究者将活性较高的外源海藻糖相关合成酶基因转入水稻、拟南芥、烟草等植物中,使植物耐旱抗逆性得到提高。
本试验采用高效液相色谱法测定了玉米、小麦和卷柏叶片中的海藻糖,其中卷柏体内海藻糖的含量达10.31mg/g干重;而在玉米和小麦中未能检测出海藻糖。本试验以水稻TPS的cDNA序列为探针,对玉米EST数据库进行同源检索,通过DNASTAR软件的Seqman程序对同源性序列进行拼接组装,根据拼接序列设计特异引物,采用RT-PCR方法对序列进行验证:然后用cDNA末端快速扩增技术扩增序列的5'端,最后得到了一条长3055bp的cDNA全序列,命名为ZmTPS,GenBank登录号为EU659122。序列分析表明,该序列具有完整的末端结构,并含有一个2691bp的完整阅读框,编码897个氨基酸。通过比对分析,发现ZmTPS对应的氨基酸序列与拟南芥、水稻TPS氨基酸序列的相似性分别达52.26%和41.56%;ZmTPS具有TPS保守功能域和糖基转移酶模体,以及与合成海藻糖底物相结合的保守氨基酸如:Asp130、His154、Arg300等。在卷柏和拟南芥TPS氨基酸序列中存在突出的N端结构,这部分序列起到抑制TPS活性的功能(Van Dijck et al,2002),而玉米ZmTPS中也存在类似的N端结构;另外在玉米等海藻糖含量较低的植物中,其TPS序列均缺失了部分重要氨基酸,如Gly-Gly-Leu保守模体和一些与底物相结合的位点。这些因素可能影响了玉米体内TPS的活性,使得其海藻糖含量很低。
Maize is the second crops in China.Maize is a special variety that needs a lot of water and is sensitive to the water stress.In China,about 60%of maize planting area are harmed by drought,and annual losses are appraised at 15%-20%,just inferior to the losses caused by plant diseases and insect pests.So,studying the mechanism of maize in tolerant to drought and developing new variety to resist drought,is a effective way to reducing the harm of drought to maize production.
A lot of trehalose was produced in Selaginella lepidophylla on the drought condition.The stress-protective properties of trehalose in vitro on proteins,membranes and intact cells have been amply documented.Trehalose was discoveried in other plants such as Arabidopsis thaliana,Oryza sativa et al,and had some correlation to drought.Some scientises have used the TPS/TPP gene transgenie into Arabidopsis thaliana,Oryza sativa and tobacco to improve the drought-resistant ability of plants.
The trehalose amount was quantified by high performance liquid chromatography(HPLC).the contents of trehalose was10.31mg/g dry weight in Selaginella lepidophylla.But there was no visible trehalose in maize and wheat.In this study,I used the tps cDNA of Oryza sativa as the querying probe,and highly homologous EST sequences were blasted in dbEST database of maize at Genbank. The downloaded EST sequences were aligned and assembled into contig by Seqman program of DNAstar software.To validate the correction of the splicing sequence,the specific primers were designed and used to amplify the complementary sequences of the assembled contig by RT-PCR,According to the sequencing contig,open reading frames(ORF) were analysed by ORF Finder program at NCBI website.The full length cDNA sequence of ZmTPS gene was obtained by using rapid amplificationof cDNA ends(RACE) technology.The results showed that the cDNA of ZmTPS gene was 3055 bp,encoding897 amino acid residues,and deduced nucleic acid an amino acid sequence possessed high identity with higher plants.Alignment showed that the putative amino acid sequences had 52.26%and 41.56%similarity with tps genes in Arabidopsis thaliana and Oryza sativa,and had a conservative region of TPS.ZmTPS gene might have the function of TPS.We found that the sequences of ZmTPS had a some amino acid at N termination as similar as Arabidopsis thaliana and Selaginella lepidophylla,these amino acid might inhibit the activity of TPS(Van Dijck et al,2002).Some plants had a little of trehalose,because of these TPS sequence lost some important amino acid,such as GGL motif and some amino acid which link to substrate.These factors that degraded the activity of TPS caused little trehalose in plant.
耐旱植物卷柏在干旱条件下,体内大量合成的海藻糖具有保护细胞内活性物质的功能,以降低逆境对细胞造成的伤害;在其他高等植物如拟南芥、水稻等植物中,也发现海藻糖的存在并与干旱存在相关性。目前已有研究者将活性较高的外源海藻糖相关合成酶基因转入水稻、拟南芥、烟草等植物中,使植物耐旱抗逆性得到提高。
本试验采用高效液相色谱法测定了玉米、小麦和卷柏叶片中的海藻糖,其中卷柏体内海藻糖的含量达10.31mg/g干重;而在玉米和小麦中未能检测出海藻糖。本试验以水稻TPS的cDNA序列为探针,对玉米EST数据库进行同源检索,通过DNASTAR软件的Seqman程序对同源性序列进行拼接组装,根据拼接序列设计特异引物,采用RT-PCR方法对序列进行验证:然后用cDNA末端快速扩增技术扩增序列的5'端,最后得到了一条长3055bp的cDNA全序列,命名为ZmTPS,GenBank登录号为EU659122。序列分析表明,该序列具有完整的末端结构,并含有一个2691bp的完整阅读框,编码897个氨基酸。通过比对分析,发现ZmTPS对应的氨基酸序列与拟南芥、水稻TPS氨基酸序列的相似性分别达52.26%和41.56%;ZmTPS具有TPS保守功能域和糖基转移酶模体,以及与合成海藻糖底物相结合的保守氨基酸如:Asp130、His154、Arg300等。在卷柏和拟南芥TPS氨基酸序列中存在突出的N端结构,这部分序列起到抑制TPS活性的功能(Van Dijck et al,2002),而玉米ZmTPS中也存在类似的N端结构;另外在玉米等海藻糖含量较低的植物中,其TPS序列均缺失了部分重要氨基酸,如Gly-Gly-Leu保守模体和一些与底物相结合的位点。这些因素可能影响了玉米体内TPS的活性,使得其海藻糖含量很低。
Maize is the second crops in China.Maize is a special variety that needs a lot of water and is sensitive to the water stress.In China,about 60%of maize planting area are harmed by drought,and annual losses are appraised at 15%-20%,just inferior to the losses caused by plant diseases and insect pests.So,studying the mechanism of maize in tolerant to drought and developing new variety to resist drought,is a effective way to reducing the harm of drought to maize production.
A lot of trehalose was produced in Selaginella lepidophylla on the drought condition.The stress-protective properties of trehalose in vitro on proteins,membranes and intact cells have been amply documented.Trehalose was discoveried in other plants such as Arabidopsis thaliana,Oryza sativa et al,and had some correlation to drought.Some scientises have used the TPS/TPP gene transgenie into Arabidopsis thaliana,Oryza sativa and tobacco to improve the drought-resistant ability of plants.
The trehalose amount was quantified by high performance liquid chromatography(HPLC).the contents of trehalose was10.31mg/g dry weight in Selaginella lepidophylla.But there was no visible trehalose in maize and wheat.In this study,I used the tps cDNA of Oryza sativa as the querying probe,and highly homologous EST sequences were blasted in dbEST database of maize at Genbank. The downloaded EST sequences were aligned and assembled into contig by Seqman program of DNAstar software.To validate the correction of the splicing sequence,the specific primers were designed and used to amplify the complementary sequences of the assembled contig by RT-PCR,According to the sequencing contig,open reading frames(ORF) were analysed by ORF Finder program at NCBI website.The full length cDNA sequence of ZmTPS gene was obtained by using rapid amplificationof cDNA ends(RACE) technology.The results showed that the cDNA of ZmTPS gene was 3055 bp,encoding897 amino acid residues,and deduced nucleic acid an amino acid sequence possessed high identity with higher plants.Alignment showed that the putative amino acid sequences had 52.26%and 41.56%similarity with tps genes in Arabidopsis thaliana and Oryza sativa,and had a conservative region of TPS.ZmTPS gene might have the function of TPS.We found that the sequences of ZmTPS had a some amino acid at N termination as similar as Arabidopsis thaliana and Selaginella lepidophylla,these amino acid might inhibit the activity of TPS(Van Dijck et al,2002).Some plants had a little of trehalose,because of these TPS sequence lost some important amino acid,such as GGL motif and some amino acid which link to substrate.These factors that degraded the activity of TPS caused little trehalose in plant.
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