【摘要】 叶绿素酶（Chlorophyllase,CLH）是酯酶的一种，广泛存在于叶绿体中，能将叶绿素水解成为羧酸的脱植基叶绿素和高级一价醇叶绿素（植醇）。以‘鸡心黄皮’为材料，研究根据已经报道的CLH基因的序列设计兼并引物，采用RACE方法，从黄皮果实中克隆得到了2个CLH家族基因，其全长cDNA序列分别为1324bp、1267bp，编码330个、320个氨基酸，分子重量为35.69kD、34.15kD，等电点为8.307、7.097。两个CLH基因编码的蛋白氨基酸组成主要是丙氨酸、甘氨酸、赖氨酸、亮氨酸等，其中两者丝氨酸、苏氨酸、色氨酸的含量差异比较大。通过系统发育分析发现基因编码的蛋白，黄皮CLH1与柠檬、温州蜜柑等植物具有较近的亲缘关系；CLH2与豇豆、刺田菁等植物具有较近的亲缘关系。对不同着色时期的黄皮果实进行表达分析发现：CLH1在绿果中的表达量较高，CLH2在褐色果实中的表达量比较高。对CLH蛋白相互作用的预测发现，CLH蛋白与NOL、CH1、NYE1、HCAR等蛋白相互作用。更多还原

【Abstract】 Chlorophyllase(CLH), a type of esterase widely found in chloroplasts, catalyzes the hydrolysis of chlorophyll into chlorophyllide and phytol, a long-chain monohydric alcohol derived from chlorophyll. In this study,’Chicken heart’ wampee was used as the experimental material. Based on previously reported CLH gene sequences,degenerate primers were designed, and the RACE method was used to clone two genes of the CLH family from the wampee fruit. The full-length cDNA sequences were 1324 bp and 1267 bp, encoding proteins of 330 and 320 amino acids, with molecular weights of 35.69 kD and 34.15 kD, and the isoelectric point of 8.307 and 7.097, respectively.The amino acid composition of the two proteins of CLH gene was mainly alanine, glycine, lysine, and leucine, among which the content of serine, threonine, and tryptophan differed significantly between the two proteins. Phylogenetic analysis revealed that the wampee proteins of CLH1 gene is closely related to those from lemon, Wenzhou tangerine and other citrus species, while CLH2 shows a close genetic relationship with cowpea, sesbania rostrata and other legumes. Expression analysis at different fruit coloration stages indicated that CLH1 was highly expressed in green fruits, whereas CLH2 exhibited higher expression in brown fruits. Protein–protein interaction predictions showed that CLH protein may interact with NOL, CH1, NYE1, HCAR and other proteins.更多还原