Cloning And Expression Analysis Of Anthocyanidin Biosynthesis Related Regulatory Genes In Viola×wittrockiana Gams

Violax-wittrockiana Gams.is an ornamental plant with typical spotted petal. We studied transcriptional factors related to anthocyanins biosynthesis in petal of yellow pansy with purple blotches. As we known, MYB, bHLH and WD40 repeat protein are dominate factors involved in anthocyanins biosynthesis regulation. We have already cloned cDNA sequences of structural genes VwCHS, VwCHI, VwF3H, VwF3’H, VwDFR, VwANS and regulated genes VwMYB8, VwMYB29. We’ve also researched expression patterns of these genes in previous studies. In this study, another two regulated genes have been cloned, VwMYB27 and VwbHLHl, The main results are as follows:(1) 29 sequences of MYB unigenes,14 sequences of bHLH unigenes and 77 sequences of WD40 were selected from unigenesfrom transcriptome data. Multiple alignments of these unigene sequences with transcriptional factors of Arabidopsis thaliana showed indenties between these genes.(2) Tubulin and 18S were validated and proved the most stable and appropriate reference genes for RT-qPCR of pansy. We analyzed expression profiles of VwMYB27, VwbHLH1 during different developmental stages of pansy petal. Both of their relative expression level peaked at stage V, which were consistent with the variation trend of anthocyanins accumulation in pansy. Expression level of VwMYB27 in stage V was higher in cynic area than acynic area, but VwbHLH1 expressed more in acynic areas. Thus VwMYB27 could improve the accumulation of pigments; and VwbHLHl coulde be a repressed transcriptional factor.(3) RACE was used to obtain 1517bp full length of VwMYB27 and 1125bp partial sequence of VwbHLH1.(4) VwMYB8, VwMYB27 GFP transient assay vectors were successfully constructed. Both of them did not result in any red color in acynic areas of pansy petals, which was similar to the negative control of vector expression. But red color spots occurred in rose petals with VwMYB8, VwMYB27 transient expression. Then sub-cellular location analysis of VwMYB27 showed it located in cell nucleus.(5) VwMYB27-Pcambia1301 expression vector were constructed, then transferred to tobacco by the Agrobacterium EHA105. And transgenic tobacco plant was expected to obtain to ensure the regulated function of VwMYB27 by observing the variation of petals color and anthocyanins accumulation.