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4-香豆酸辅酶A连接酶(4-coumarate-CoA ligase,4CL)是木质素合成途径的关键酶。该研究以‘红玉石籽’(Punica granatum cv.Hongyushizi)石榴为试验材料,采用RACE和RT-PCR方法获得4CL同源基因Pg4CL。Pg4CL基因cDNA全长2 121bp,编码544个氨基酸;序列比对和功能域分析发现,Pg4CL包含有2个保守域SSGTTGLPKGV和GEICIRG;系统进化树分析显示,Pg4CL与其他物种起源相同,而与赤桉Ec4CL亲缘关系最近。荧光定量PCR分析表明,Pg4CL基因在叶、果皮、种皮和茎等组织中均有表达,其中果皮和叶片中表达量较高,种皮中表达量最低;Pg4CL基因在6个石榴品种的种皮中均有表达,其中在‘突尼斯软籽’中表达较高,‘会理软籽’表达量最低;Pg4CL基因在‘红玉石籽’籽粒的不同时期均有表达,在15~60d相对表达量逐步上升,并在60d时达到最高;75d以后表达量急剧下降,仅维持较低水平表达。
4-coumarate-CoA ligase (4CL) is a key enzyme in the lignin biosynthesis pathway. In this study, Punica granatum cv Hongongushizi was used as experimental material, 4CL homologous gene Pg4CL was obtained by RACE and RT-PCR methods. Sequence analysis and functional analysis showed that Pg4CL contained two conserved domains, SSGTTGLPKGV and GEICIRG. Phylogenetic tree analysis showed that Pg4CL shared the same origin as other species but not with Eucalyptus Ec4CL kinship recently. Quantitative real-time PCR analysis showed that the Pg4CL gene was expressed in the leaves, pericarp, seed coat and stems, among which, the expression level of Pg4CL gene was the highest in the pericarp and the leaves and the lowest in the seed coat. The Pg4CL gene was expressed in 6 pomegranate species The expression of Pg4CL gene was the highest in ’Tunisian Soft Seeds’ and ’Huili Soft Seeds’ was the lowest. The expression of Pg4CL gene was expressed in different stages of ’Redstone’ seed and was relatively expressed in 15-60 days Gradually increased and reached the highest level at 60 days. After 75 days, the expression decreased sharply and maintained at a relatively low level.