粉蕉MbACO2基因克隆及其表达分析

【目的】克隆粉蕉1-氨基环丙烷-1-羧酸（ACC）氧化酶（ACO）基因（MbACO2），并进行表达分析，为研究ACO基因家族在香蕉果实发育成熟过程及逆境胁迫过程中的功能作用提供参考依据，也为改良香蕉采后贮藏提供潜在的基因资源。【方法】利用RT-PCR从粉蕉果实克隆MbACO2基因，运用生物信息学软件分析其编码蛋白的理化性质、亲疏水性、保守结构域及二、三级结构。利用农杆菌介导的瞬时转化法进行亚细胞定位，并利用实时荧光定量PCR检测MbACO2基因在果实发育成熟过程及高盐、干旱和低温胁迫处理下的表达情况。【结果】克隆获得的MbACO2基因开放阅读框（ORF）长度为918 bp，编码305个氨基酸残基，其蛋白分子量为34.583 kD，理论等电点（pI）为5.43，属于亲水性蛋白，具有典型的植物ACO蛋白家族的结构特征，含有DIOX_N和2OG-FeⅡ_Oxy 2个保守结构域。MbACO2氨基酸序列与同属的香蕉（Musa acuminata AAA group）ACO氨基酸序列（XP_010908825.1）相似性最高，为98.04%，表明其具有高度的保守性。MbACO2蛋白在细胞核和细胞质中均有表达。随着粉蕉果实发育成熟，MbACO2基因表达量整体呈升高趋势，极显著高于开花后0 d（对照）（P< 0.01，下同），尤其是在果实采后6 d，其相对表达量达最高值。高盐、干旱和低温胁迫处理下，MbACO2基因的相对表达量较对照（未胁迫处理）显著（P< 0.05）或极显著提高。【结论】MbACO2基因属于ACO基因家族成员，含有该家族的典型结构域，在粉蕉果实发育成熟过程及逆境胁迫的应答中发挥正向调控作用，高盐、干旱和低温胁迫处理均能诱导其上调表达。

Cloning and expression analysis of MbACO2 gene in Musa ABB group,cv Pisang Awak,FJ

【Objective】To clone 1-aminocyclopropane-1-carboxylic acid(ACC) oxidase(ACO) gene(MbACO2) from Musa ABB group, cv Pisang Awak, FJ, and analyze its bioinformatics and expression, so as to provide reference for studying the function of ACO gene family in banana fruit ripening and stress process, and to provide potential gene resources for improving banana postharvest storage.【Method】Gene MbACO2 was cloned from Musa ABB group, cv Pisang Awak, FJ by RT-PCR.The physicochemical properties, hydrophilicity/hydrophobicity, conserved domains and secondary/tertiary structures of the encoded protein were predicted by bioinformatics analysis.Subcellular localization was conducted using Agrobacterium-mediated transient transformation.The expression level of MbACO2 gene in fruit development and stress response(high-salt, drought and low temperature) was detected by real-time quantitative PCR.【Result】The open reading frame(ORF) length of MbACO2 was 918 bp, encoding 305 amino acid residues.The molecular weight of the protein was 34.583 kD and the theoretical isoelectric point(pI) was 5.43.The protein encoded by MbACO2 gene belonged to hydrophilic protein, which had typical plant ACO structural characteristics and contained two conservative domainsDIOX_N and 2OG-FeⅡ_Oxy.The amino acid sequence of MbACO2 was similar to that of ACO amino acid sequence(XP_010908825.1) in banana(Musa acuminata AAA group) and the similarity reached 98.04%, indicating that the amino acid sequence of MbACO2 protein was highly conserved.The subcellular localization results showed that MbACO2 protein was expressed in both nucleus and cytoplasm.The expression of MbACO2 gene increased with the development of fruit ripening, and was extremely higher than 0 d after flowering(control) (P< 0.01, the same below).The expression level of MbACO2 was the highest 6 d after harvest.Under high-salt, drought and low temperature, the relative expression of MbACO2 gene was significantly up-regulated(P< 0.05) or extremely up-regulated compared with control(no stress treatment).【Conclusion】MbACO2 gene belongs to ACO gene family, it contains the typical domains of the family, and it playsa positive regulation role in banana fruit ripening and stress resistance of Musa ABB group, cv Pisang Awak, FJ.High-salt, drought and low temperature can induce its upregulation of expression.