甘蔗ATP结合蛋白基因克隆及其干旱胁迫下的表达特性分析
Cloning of ATP binding protein gene in sugarcane and its expression characteristics under drought stress
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摘要: 目的克隆甘蔗ATP结合蛋白基因,分析其基本生物学信息及在干旱胁迫下的表达特性,为ATP结合蛋白的功能研究提供理论依据.方法从乙烯诱导甘蔗差异表达转录组和水分胁迫下甘蔗cDNA文库中获取ATP结合蛋白基因序列,设计其引物进行RT-PCR扩增,利用生物信息学软件进行序列分析及其蛋白结构和功能预测,并采用实时荧光定量PCR(qPCR)检测干旱胁迫处理下的甘蔗ATP结合蛋白基因的表达情况.结果克隆获得的甘蔗ATP结合蛋白基因全长2145bp,编码714个氨基酸,其蛋白分子质量为79.430kD,理论等电点(pI)为9.33,不稳定系数为44.38;甘蔗ATP结合蛋白与玉米ATP结合蛋白的核苷酸序列及其推导氨基酸系列同源性最高,均达93%.甘蔗ATP结合蛋白主要由无规则卷曲(42.02%)、α螺旋(25.63%)和延伸链(22.27%)结构组成,具有蛋白激酶催化结构域、ATP结合位点、核苷酸结合位点(NBS)、糖基化位点、激酶磷酸化结合位点及核定位信号等;甘蔗ATP结合蛋白可能主要定位在细胞核、叶绿体、线粒体和过氧化物酶体中,起中间代谢调控作用,或参与转录调控、免疫应答、胁迫应答及信号转导等生物反应.qPCR检测结果显示,在干旱胁迫7d后,甘蔗ATP结合蛋白基因的表达量明显下调,约为对照的30%,恢复正常供水(复水)后,表达量回升.结论甘蔗ATP结合蛋白基因表达受干旱胁迫诱导,可能参与甘蔗对干旱逆境胁迫响应的代谢调控.Abstract: Objective The present experiment was conducted to clone ATP binding protein gene in sugarcane,analyze its bioinformatics and expression under drought stress,and provide reference for function research in ATP binding protein gene.MethodSequence of ATP binding protein gene was obtained from differential expression transcriptome induced by ethylene and sugarcane cDNA library under water stress.Its primers were amplified by RT-PCR,sequence analysis and protein structure and function prediction were carried out by bioinformatics software.Real-time fluorescence quantitative analysis was used to detect expression of sugarcane ATP binding protein gene under drought stress.ResultSugarcane ATP binding protein gene was cloned.It included 2145 bp and encoded 714 amino acids.Its relative protein molecular weight was 79.430 kD,isoelectric point(pI) 9.33 and instability index 44.38.The homology in nucleotide sequence and deduced amino acid sequence between sugarcane ATP binding protein gene and maize ATP binding protein gene was the highest (93 %).Sugarcane ATP binding protein gene mainly consisted of random coil (42.02%)、alpha helix (25.63 %) and extended strand (22.27%).Sugarcane ATP binding protein gene consisted of protein kinase catalytic domain,ATP binding site,nucleotide binding site (NBS),glycosylation site,kinase phosphorylation binding site and nuclear localization signal.The results of subcellular localization and protein function classification prediction showed that sugarcane ATP binding protein might be mainly located in nucleus,chloroplast,mitochondria and peroxidase,and played a role of intermediary metabolism.It might involved in biological reactions such as transcription regulation,immune response,stress response and signal transduction.The results of real-time qPCR analysis showed that,7 d after drought stress,the expression of sugarcane ATP binding protein gene down regulated to 30% ofthat in control,and after returning to normal water supply,the expression was on the rise.conclusionSugarcane ATP binding protein gene is induced by drought stress and may be involved in metabolic process of sugarcane responsive to drought adversity stress.