Cloning And Expression Identification Of TYDC Gene In Viola×wittrikiana Gams.

Viola×wittrockiana is an important biennial ornamental plant of viola in violaceae,which has large petals and abundant colors. It is also an ideal material for the formation of flower blotch and color researches due to the stable heredity and large areas of blotches. Our previous researches found the expression of VwTYDC gene in Viola×wittrockiana showed a very significant difference in the cyanic blotches regions and acyanic areas , through the De novo transcriptome analysis of RNA, suggesting that the VwTYDC gene may be a potential candidate gene which lead to the formation of the blotches. Therefore VwTYDC gene became a new focus on the molecular mechanism of the colors in plants.In this study, 'Mengdie', the yellow pansy with purple blotches in petals, was used as research material to clone the tyrosine decarboxylase (TYDC) gene and to analysis the gene expression. That could verify the expression of VwTYDC and the correlation between its product, tyramine, and the blotches , thus provided a new research field for the formation and regulation patterns of color spots. The main results are as follows:(1) Real-time PCR detection of the VwTYDC gene revealed that different expression levels were in different flowering phase and different organs of Viola×wittrockiana. The gene was not expressed in root, but the expression level in petal was significantly higher than that of stems and leaves. Furthermore, the expression quantity of the gene in cyanic blotches was higher than that of acyanic areas.(2) By the methods of RT-PCR and RACE, the VwTYDC gene was cloned and the cDNA full-length nucleotides were 1634 bp of VwTYDC 1 and 1575 bp of VwTYDC 2, and the ORF Finder revealed that the two homologous genes contained the same 1485 bp open read frame encoding 494 amino acids.(3) The bioinformatics analysis of amino acid sequences were conducted for the full-length of VwTYDC, and the result indicated that the gene belonged to aspartate amino transferase pyridoxal phosphate dependent superfamily (AAT_I superfamily). The multiple comparisons and cluster analysis of amino acid sequences turned out that the amino acids encoded by VwTYDC of Viola×wittrockiana could reach to 82.43% consistency compared with the same genes of other plants, and the genetic relationship was close to Jatropha curcas and Populus euphratica.(4) The tyramine contents in petals of Viola×wittrockiana were then determinated by HPLC and the results showed that tyramine content in cyanic blotches were higher than that in acyanic areas, which was proportional to the expression level of VwTYDC gene.(5) The significant spectrum red-shift and absorption value increase of anthocyanin were found by UV spectrophotometry after adding tyramine which could be catalyzed by the VwTYDC gene, which showed that tyramine may act as copigments to affect the color pattern of pansy.(6) Exogenous application of tyramine and tyrosine could effectively induce the biosynthesis and accumulation of tobacco anthocyanin.(7) Plant expression vector VwTYDC-pCAMBIA1301 was construted, mediated and transferred to tobacco by the Agrobacterium. Consequently, resistant transgenic tobacco plants were screened successfully.These results showed that different concentration of tyramine was caused by the different expression level of VwTYDC gene between the cyanic blotches and the acyanic areas.Moreover, tyramine could promote the anthocyanin biosynthesis and may act as copigments to affect the color of Viola×wittrockiana to promote the formation of spots.