茄子Ⅳ型查尔酮异构酶基因SmCHI2的克隆与分析

Cloning and analysis of typeⅣchalcone isomerase gene SmCHI2 in eggplant

【目的】探究茄子花青素合成通路中关键酶SmCHI2的蛋白特性和表达特性,为完善茄子花青素生物合成途径和品质育种提供一定的理论基础。【方法】通过同源克隆获得茄子SmCHI2基因全长序列,利用在线分析工具对其蛋白理化性质进行预测,利用ClustalX 1.7、GeneDoc 2.7.0和Mega 6.0软件进行蛋白序列比对和系统进化树分析,利用qRT-PCR分析SmCHI2基因的表达模式。【结果】SmCHI2基因编码区全长633 bp,编码210个氨基酸。蛋白理化性质分析显示SmCHI2蛋白分子量为23.98 kDa,理论等电点为4.81,且为不稳定的亲水性蛋白。二级和三级结构预测显示SmCHI2蛋白结构主要包括α-螺旋、随机卷曲、延伸链和β-转角。多序列比对表明SmCHI2与LcCHI(枸杞AIC33515.1)、LrCHI(黑枸杞AQZ26680.1)、CaCHI2(辣椒XP_016573438.1)和StCHI3(马铃薯XP_006365329.1)等Ⅳ型CHI蛋白具有相同的活性和识别位点,且在系统进化树中属于同一分支。亚细胞定位结果显示,SmCHI2蛋白在细胞核、细胞膜和细胞质中均有分布。表达分析显示,SmCHI2基因主要在紫色组织中表达,且在白色果皮中表达量显著低于紫色果皮。【结论】茄子中Ⅳ型CHI蛋白SmCHI2正调控茄子花青素的生物合成。

【Objective】The study aimed to explore the protein characteristics and expression characteristics of SmCHI2,a key enzyme in eggplant anthocyanin synthesis pathway and provide a theoretical basis for improving eggplant anthocyanin biosynthesis pathway and quality breeding in the future.【Method】The full-length sequence of SmCHI2 gene was obtained from eggplant by homologous cloning.The physical and chemical properties of SmCHI2 protein were predicted by online analysis tools,and protein sequence alignment and phylogenetic tree analysis were performed by ClustalX 1.7,GeneDoc 2.7.0 and Mega 6.0 software.And the expression pattern of SmCHI2 gene was analyzed by qRT-PCR.【Result】The full-length coding sequence of SmCHI2 was 633 bp,encoding 210 amino acids.The physicochemical properties analysis showed that the molecular weight of SmCHI2 protein was 23.98 kDa and the theoretical isoelectric point was 4.81.SmCHI2 protein was an unstable and hydrophilic protein.The secondary and tertiary structure prediction showed that SmCHI2 protein mainly consisted ofα-helix,random coil,extended strand andβ-turn.Multiple alignment showed that SmCHI2 had the same activity and recognition sites with typeⅣCHI protein,including LcCHI(Lycium chinense AIC33515.1),LrCHI(Lycium ruthenicum AQZ26680.1),CaCHI2(Capsicum annuum XP_016573438.1)and StCHI3(Solanum tuberosum XP_006365329.1),and they were classified into the same branch in phylogenetic tree.The result of subcellular localization showed that SmCHI2 protein was distributed in nucleus,cytomembrane and cytoplasm.Expression analysis showed that SmCHI2 gene was mainly expressed in purple tissues,and the expression level in white peel was significantly lower than that in purple peel.【Conclusion】TypeⅣCHI protein SmCHI2 positively regulates the anthocyanin biosynthesis of eggplant.