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【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2145 bp,编码714个氨基酸,其蛋白分子质量为79.430 kD,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7 d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。
【Objective】 The objective of this study was to clone the ATP binding protein gene of sugarcane and analyze its basic biological information and expression characteristics under drought stress to provide a theoretical basis for the study of the function of ATP-binding protein. 【Method】 ATP-binding protein sequences were obtained from ethylene-induced sugarcane differential expression transcriptome and sugarcane cDNA library under water stress. The primers were designed for RT-PCR amplification. Bioinformatics software was used to analyze the sequence and protein structure and function The expression of ATP binding protein (ATP-binding protein) gene in sugarcane under drought stress was detected by real-time fluorescence quantitative PCR (qPCR). 【Result】 The cloned ATP-binding protein gene was 2145 bp in length and encoded 714 amino acids with a molecular mass of 79.430 kD, a theoretical isoelectric point (pI) of 9.33 and an instability coefficient of 44.38. The ATP binding protein of sugarcane The nucleotide sequence of maize ATP-binding protein and its deduced amino acid sequence have the highest homology of 93%. The sugarcane ATP-binding protein mainly consists of random coil (42.02%), α-helix (25.63%) and extended chain (22.27%) and has protein kinase catalytic domain, ATP binding site and nucleotide binding site ), Glycosylation sites, kinase phosphorylation sites and nuclear localization signals, etc .; sugarcane ATP-binding protein may be mainly located in the nucleus, chloroplast, mitochondria and peroxisomes, play an intermediate metabolic regulation, or involved in transcription Regulation, immune response, stress response and signal transduction. The results of qPCR showed that the expression of ATP-binding protein (ATP-binding protein) gene was significantly down-regulated after 30 days of drought stress, which was about 30% of that of the control. After resuming normal water supply (rehydration), the expression level rose. 【Conclusion】 Sugarcane ATP binding protein gene expression is induced by drought stress, which may be involved in the metabolic regulation of sugarcane response to drought stress.