Functional Analysis Of Red Swiss Chard Tyrosinase Gene And Its Promoter

Betalains, a group of water-soluble nitrogen pigments, confer to plants the beautiful and diverse color substituting anthocyanins in most of plants belonging to the order Caryophyllales. They are biosynthesized through three key enzymatic steps:hydroxylation of L-tyrosine to form L-DOPA, oxidation of L-DOPA to produce cyclo-DOPA and conversion of L-DOPA to betalamic acid, a common chromophore of betalains. Betalamic acid can condense with an amino acid or other amine group to spontaneously form betaxanthins, and can also spontaneously condense with cyclo-DOPA to form red betanidin. The PPO-type tyrosinase has been proposed involved in the first step (even the second step) for long time, but the molecular evidence is lacking. Our lab has cloned a PPO-type tyrosinase gene (BvcTYR) from leaves of red Swiss chard. As one part of works to analyze the function of BvcTYR, this work focus on the cloning and investigation of its promoter. In addition, the BvcTYR was co-expressed with DODs from diverse sources, transiently and stably in tobacco. The main results are listed as following:A 1670 bp 5’-flanking region of BvcTYR coding sequence was isolated from the leaves of red Swiss chard. The cloned fragment contains putative transcriptional start site (TSS), TATA-box, CAAT-box at its 3’end, and various cis-acting elements in other parts, and thus termed as BvcPPOP. BvcPPOP was introduced into the anthocyanins-producing plant Arabidopsis after fusing with GUS gene, mediated by Agrobacterium. GUS staining and quantitative analysis of T3 transgenic plants showed that BvcPPOP could direct GUS gene expression in vegetative organs with root- and petiole-preference, but not in reproductive organs including inflorescences shoot, inflorescences leaf, flower, pod and seed. This promoter was associated with developmental stages in its driving strength, but not in expression pattern. Its expression was also influenced by the abiotic stressors tested, positively by SA and MeJA but negatively by ABA, GA, NaCl and OH-. To search for the vegetative organ specific elements and obtain the core promoter, its four 5’-truncated fragments (F2-F5) were generated. Four versions varied in the driving strength, but not obviously in expression pattern, and even the shortest version (247 bp, F5) retained the root- and petiole-preference. For example, expression intensity of F3 began to decline sharply.Transient co-expression of BvcTYR and various biological origin DODs in Nicotiana benthamiana indicated that the BvcTYR+BvcDODA1 transformed leaves had the strongest betaxanthins characteristic fluorescence under blue light. Stable expression of BvcTYR/ BvcTYR+BvcDODA1 in common tobacco showed that leaf discs could catalyze tyrosine/tyramine to generate red/yellow substance with an absorption peak at about 470 nm. The red substance may be the Dopa-quinone.These results provide molecular evidences for the involvement of tyrosinase in the first step of betalain biosynthesis, and also a useful tool for the expression of target gene in vegetative organs.