Cloning And Functional Analysis Of The Gene Encoding Lycopene β-Cyclase From Gentiana Lutea

The naturalβ-carotene is very important to the human health, especially the cis stereoisomer ofβ-carotene which is lacking in the synthetic chemicals. The artificial synthetic chemicals are pure homogeneous materials, but a large proportion of cis-trans isomer of carotenes were included in the natural products. They cooperate and take on certain biological functions in human or animal body. Cis isomers of carotenoids appear to be more bioavailable than the all-trans forms. It has been proven that 9-cis stereoisomer ofβ-carotene was powerful antioxidant and applied in preventing cardiovascular disease.β-carotene is a precursor to synthesize vitamin A(retinol) in animal and human body. It is a best way to supply vitamin A. One molecule ofβ-carotene can be converted into two molecular of vitamin A, butα-carotene andβ-cryptoxanthin with oneβ-ring can only produce one molecule of vitamin A. Thus, how to increase the content ofβ-carotene in crops by carotenoid genetic engineering in order to get enough naturalβ-carotene in daily diet has been a studying focus. We cloned and identified lycopeneβ-cyclase 2(LYCB2) cDNA from Gentiana lutea,which is key regulatory enzyme in carotenoid biosynthetic pathway. The functional analysis of G. lutea LYCB2 cDNA is not only helpful to the understanding of biosynthesis and accumulation mechanism ofβ-carotene, but also provide target gene for genetic engeering which can directionally control the expression of target gene to adjust metabolic approach in order to make corps accumulate some valuable metabolites.In this thesis, lycopeneβ-cyclase 2(LYCB2) cDNA was cloned from flower petal cDNA library of G. lutea. The LYCB2 cDNA informatuion was deposited in NCBI (National Center for Biotechnology Information) GenBank (Accession number: EF062505). The full-length of nucleotide sequence of G. lutea LYCB2 cDNA consisted of 2165 bp with a 1592 bp open reading frame (ORF), a 177 bp 5' terminal and a 515 bp 3' terminal non-coding regions, respectively. There has the significant polyadenylation signal (poly(A) signal) in this cDNA. The deduced amino acid sequences of G. lutea LYCB2 cDNA was a polypeptide of 508 amino acid residues with molecular weight of 57169.20 D. The isoelectric point of G. lutea LYCB2 was 8.64. The deduced amino acid sequences of G. lutea LYCB2 cDNA has higher identites with known functional LYCB sequences. The function of G. lutea LYCB2 cDNA was established by complementation in engineering Escherichia coli. The results indicated G. lutea LYCB2 can convert lycopene intoβ-carotene.