欧李番茄红素β–环化酶基因ChLCYb的分离与功能鉴定  

Molecular Cloning and Functional Characterization of a Lycopene β-cyclase Gene ChLCYb in Cerasus humilis

作　　者：张建成[1] 高利敏 王鹏飞[1] 杨淑一 郑斌[1] 王裕添 杜俊杰[1] 

机构地区：[1]山西农业大学园艺学院,山西太谷030801

出　　处：《园艺学报》2017年第11期2075-2088,共14页Acta Horticulturae Sinica

基　　金：山西省重点研发计划项目(201703D221028-4);高等学校博士学科点专项科研基金项目(20101403120006);山西省自然科学基金项目(2012011032-4);山西省科技重大专项计划项目(20121101010)

摘　　要：利用RACE技术和RT-PCR相结合,从欧李[Cerasus humilis(Bge)Sok]果实中克隆获得长度为1798 bp的番茄红素β–环化酶(lycopeneβ-cyclase)基因ChLCYb的cDNA全长序列,开放读码框为1515 bp,编码503个氨基酸。序列分析发现,ChLCYb具有植物LCYb保守区(Plant LCYb conserved region)、FAD/NAD(P)结合区(Dinucleotide-binding signature)、"Cyclase motif 1"、"Cyclase motif 2"和"lycopeneβ-cyclase motif"等典型结构特征,在N–端存在1~84个氨基酸残基组成的转运肽信号序列,在85~106、209~227、373~391和460~480氨基酸区域包含4个跨膜结构域。荧光定量PCR结果表明,ChLCYb在叶中表达量最高,其次是幼果,在根中最低;在欧李果实发育过程中,果皮中ChLCYb表达量高于果肉,果皮中ChLCYb表达量先升高,在花后90 d左右表达量最高,然后呈缓慢下降趋势,而果肉中ChLCYb表达量相对平稳。ChLCYb表达模式与果皮和果肉中β–胡萝卜素含量的积累呈显著正相关(r=0.824和r=0.712,P<0.05)。利用大肠杆菌工程菌体系诱导表达了ChLCYb蛋白,并证实其可催化工程菌株中番茄红素向β–胡萝卜素转化,其转化效率达71.22%。在番茄中超量表达ChLCYb促进果实中番茄红素向β–胡萝卜素的转化和积累,转基因株系L-11号果实中的β–胡萝卜素含量高达692.18μg·g^(-1) DW,是非转基因对照的4.42倍。The full length of c DNA sequence of lycopene β-cyclase（LCYb）gene named ChLCYb was cloned from Cerasus humilis（Bge）Sok. using Reverse Transcription Polymerase Chain Reaction（RT-PCR）combined with RACE techniques. The c DNA sequence of ChLCYb was 1798 bp in length,containing a 1515 bp open reading frame（ORF）which encoded a protein of 503 amino acids. Sequence analysis indicated that ChLCYb contain typical plant LCYb conserved region,dinucleotide-binding signature,Cyclase motif 1,Cyclase motif 2 and lycopene β-cyclase motif. The ChLCYb protein has a signal transit peptide consists of 84 amino acid residues in the N-terminal region and four predicted transmembrane domains in the sites of 85–106,209–227,373–391 and 460–480 amino acids. Quantitative real-time PCR results showed that the expression of ChLCYb was the highest in the leaf,followed by in the fruitlets,and the lowest in the root. The expression of ChLCYb in the peel was markedly higher than that in the pulp during the fruit development. The expression of ChLCYb in the peel increased at first,which peaked on 90 days after bloom,and then started to drop slowly,while that in the pulp keep a relatively stable situation. There was a significantly positive correlation between ChLCYb transcript expression and β-carotene contents in peel and pulp（r = 0.824,r = 0.712,P 0.05）. The heterogenous expression in E. coli system produced the fused ChLCYb protein and confirmed that ChLCYb could catalyze the conversion of lycopene into β-carotene in E. coli engineered to produce lycopene,whose conversion efficiency reaches 71.22%. Overexpression of ChLCYb gene resulted in a virtually complete conversion and accumulation of lycopene into β-carotene in tomato fruit. The amount of β-carotene in the fruit of transgenic plant L-11 was 692.18 μg · g^（-1） DW,which was 4.42 times that of the non-transgenic control.

关 键 词：欧李 番茄红素β–环化酶 基因克隆 基因表达 功能鉴定 

分 类 号：S662.5[农业科学—果树学]