Abstract: 【Objective】The objective of this study is to clone Lycopene β-cyclase enzyme(LCYB) gene and its promoter from marigold(Tagetes erecta), to analyze their bioinformatics characters and predict the promoter function. This study provides a reference for the regulation of the metabolic mechanism of the carotenoids and the control of the lutein concentration in marigold.【Method】The cDNA sequence of TeLCYb gene was cloned using RT-PCR method. Bioinformatics tools were applied to analyze both the TeLCYb gene sequence and the characteristics of its encoded protein sequence. DNAMAN and online software BoxShade were used to make multiple sequence alignments between the TeLCYb amino acid sequence and their homologous sequences, and MEGA6.0 was used to constructed phylogenetic tree of homologous species. The promoter sequence was cloned according to its cDNA sequence using FPNI-PCR. Thereafter, two different 5′ UTR deletion mutants of the TeLCYb gene promoter were amplified by PCR, and then were inserted into the vector V152 to construct promoter-GUS fusion genes expression vectors, named pTeLCYb(-1969)::GUS and pTeLCYb(-1140)::GUS, respectively, and then were transformed into tobacco by agrobacterium tumefaciens-mediated transformation of tobacco, respectively. And the promoter activities were quantitatively estimated using GUS report gene.【Result】The TeLCYb was successfully cloned from marigold(Tagetes erecta). The bioinformatics analysis showed that its full length was 1 865 bp with an open reading frame (ORF) of 1 527 bp length, encoding 508 amino acids. Homology analysis showed that the deduced TeLCYb protein was highly homologous to other LCYb proteins from Chrysanthemum morifolium,Dendranthema lavandulifoliu and TeLCYb protein has typical protein special elements of LCYb protein. Phylogenetic analysis also indicated that TeLCYb was clustered into the branch with asteracea. A 1 806 bp promoter sequencewas obtained via FPNI-PCR. Bioinformatics analysis of promoter sequence revealed that this fragment contained some promoter core elements, such as TATA-box and CAAT-box, and 13 light responsive elements and 5 hormones responses elements. Meanwhile, MYB transcription factor binding elements and heat-resistant response elements, physiological control elements and cis-acting regulatory elements were found in this sequence. Functional characterization of promoters by constructing pTeLCYb (-1969)::GUSand pTeLCYb (-1140)::GUS plant expression vectors and transforming into tobacco respectively showed that both promoters were active in stems, petals, anthers and capitals. But there were significant differences of GUS enzyme activity among plant tissues, and blue spots found in anthers and capital were significantly stronger than that of petals. Compared with the expression level of pTeLCYb (-1969)::GUS, the shorter sequence of pTeLCYb (-1140) led to a significant increase in the activity of GUS in the transgenic leaves and root and sepals were also found GUS enzyme activity. 【Conclusion】 Different length of promoters can drive the expression of GUS gene, but there are differences in the sites and intensity where promoters functions, we hypothesize that a positive regulatory factor of light response elements maybe exist between 1 140 bp and 164 bp area of 5′ upstream to ATG, while hormone response elements and the heat stress response element may suppress or reduce the promoter function.

Key words: marigold(Tagetes erecta ), lycopene β-cyclase, promoter clone, analysis of promoter function