关键词: 欧李；番茄红素&beta, &ndash, 环化酶；基因克隆；基因表达；功能鉴定

Abstract:

The full length of cDNA 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 cDNA sequence of ChLCYb was 1 798 bp in length，
containing a 1 515 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.

Key words: Cerasus humilis；lycopene &beta, -cyclase；gene cloning；gene expression；functional identification