Prokaryotic Expression Of Wheat Lycopnene Cyclase (TaLCYB) And Preparation Of Its Polyclonal Antibody

Carotenoids are one of the most prevalent natural products in nature. As carotenoids’physiological functions have been revealed in recent years, they have been widely used,especially in nutrition, health care and disease prevention. With the emergence of golden rice,genetic engineering is regarded as an effective tool to improve carotenoid contents in thestaple food and other economic crops. However, when analysing carotenoids synthetic genessuch as in golden rice, it was found that the expression in gene level and protein level werenot consistent, especially in seeds and fruits. This phenomenon can bring about difficulty toanalyse positive generation’phenotype. Therefore, it is necessary to analyse the gene leveland protein level at the same time in transgenic lines.Lycopene β-cyclase (LCYB) is a key synthase in carotenoid biosynthesis pathway ofplant, of which is an important precursor of vitamin A. The research in golden wheat showedthat lycopene β-cyclase was a key enzyme for accumulating β-carotene. So it is ofsignificance to prepare the antibodies and to analyse the relative quantity of LCYB incarotenoids synthetic pathways. In the present study research, TaLCYB was cloned intopET32a(+) to construct the prokaryotic expression vector. The vector was then transformedinto E.coli BL21(DE3) competent cells. The relative molecular weight of about72.25kDrecombinant protein was obtained after adding IPTG at37℃; Then the recombinant proteinwas used as antigen to immune the rabbit, the polyclonal antibody thus produced was used forWestern blot test. Our results showed that the polyclonal can be used to analyse theexpression of LCYB at protein level in transgenic wheat.