Different Gene Expression Induced By Short-wave Length Light Qualities In Anthocyanin Biosynthesis Of Brassica Rapa ’Tsuda’

Short-wave length light, such as blue and ultraviolet light, plays an important role in plants. Blue light affects are known to affect the morphology of plants, including phototropism, chloroplast migration, stomatal opening and anthocyanin accumulation. Short-wave length (high-energy) UV light can physically damage DNA, RNA and proteins, while UV at low fluence rates were found to induce morphological changes, flavonoid production and the expression of defense-related genes. However, details of UV signaling pathways control anthocyanin synthesis are still unknown.The Brassica rapa’Tsuda’which anthocyanin biosynthesis is light-dependent was used as materials in this research. The investigations were carried out for various patterns of anthocyanin accumulation by different short-wave length light qualities; the cloning and expression of CHS family genes in response to different short-wave length light qualities; the cloning of anthocyanin biosynthesis related R2R3MYB factors and expression pattern of other regulatory genes in response to different short-wave length light qualities; the interactions of BrPAP1and BrTT8with Unitl element of BrCHS1promoter and the analysis of transcriptome in response to different short-wave length light qualities. The main results were obtained as below.1Different anthocyanin accumulation pattern of Brassica rapa’Tsuda’in response to different short-wave length light qualities.The effects of irradiating blue, UV-A, UV-B and a combination of the lights on anthocyanin accumulation at different hypocotyl positions were investigated in the seedlings of turnip ’Tsuda’. The location of anthocyanin accumulation varied depending on different light spectra. Stronger accumulation of anthocyanin was induced (1) at the upper hypocotyl positions by blue light;(2) mainly at the upper position, but also at the middle position by UV-B light; and (3) at the middle to lower position by UV-A light. There were synergistic effects between blue and UV-B, while such effects were not observed for the other light combinations. UV-A and blue+UV-B can induce anthocyanin biosynthesis in the swollen hypocotyls of turnip’Tsuda’.2Cloning and expression of CHS family genes in response to different short-wave length light qualities.Southern blot result showed that there were six copies of BrCHS genes in the genome of turnip’Tsuda’. The full length of BrCHS5and BrCHS6were amplified by RT-PCR. Among the six chalcone synthase (CHS) genes identified in this turnip, BrCHS1,4and5exhibited light-dependent expression patterns, while the other three showed no apparent light responses. The expression of BrCHS1,4and5were increased particularly by UV-A and blue+UV-B irradiation at the middle to lower hypocotyl positions, in accordance with anthocyanin accumulation patterns. BrCHS5increased in a linear manner as irradiation time and light intensity increased. The highest induction of gene expression was observed for BrCHS4upon blue+UV-B co-irradiation. In contrast, CHS expression was induced only slightly at higher hypocotyl positions by blue light. Meanwhile, the expression patterns of CHS and DFR gene induced by light were the same as anthocyanin accumulation in the swollen hypocotyls of turnip’Tsuda’.3Cloning of anthocyanin-related R2R3-type MYB transcription factor genes and expression analysis of regulatory genes in anthocyanin pathway in response to different short-wave length light qualities.Six R2R3-type MYB transcription factor genes were isolated by PCR, and the result of Real-time PCR showed that:PAP1was up regulated by light both in hypcotyls of seedling and swollen hypocotyls of turnip’Tsuda’, and the expression pattern was the same as anthocyanin accumulation. Meanwhile, the MYB4, MYB12and MYB111genes exhibited differential expression patterns at different hypocotyl positions; these patterns were unique for different light spectra. BrTT8genes also induced by light, but had a lower expression. Which indicated that it might be a co-factor participating in anthocyanin biosynthesis.4Two key factors controlling anthocyanin synthesis:interaction of BrPAP1and BrTT8with Unit1element of BrCHSl promoter.BrCHS4and BrCHS5promoters were isolated by PCR, and the CHS-Unit1motif which consisted of the light-responsive element ACE、RRE and MRE in the BrCHS1,4,5promoters was predicted by bio informatics method. Yeast one-hybrid assay showed that BrPAP1, BrTT8and BrHY5had high binding activities to the CHS-Unit1motif of the BrCHSl promoter.5Transcriptome induced by different short-wave length light qualities.Transcriptome changes of6hours’different short-wave length light qualities treatment with Brassica rapa’Tsuda’were analyzed by the RNA-seq.50703Unigene with average length of1286.44bp were obtained. Among them,847were specifically induced by UV-A, included anthocyanin synthesis genes CHS, F3H and DFR, and also contained MYB, bHLH, zinc finger and other signal transduction factors. These genes were involved in response to stress, sulfur amino acid biosynthetic process, response to chemical stimulus and pigment biosynthetic process respectively. One conservative element was predicted from promoter of genes in UV-A-specific cluster by MEME program, but further analysis is needed to confirm the functions of it.These results demonstrate that there is a distinct UV-A response, blue+UV-B synergistic response and blue/UV-A light response for anthocyanin biosynthesis in turnip. UV-A light- dependent anthocyanin biosynthesis appeared to be regulated in a manner that is distinct from that mediated by cryptochromes and UV-B photoreceptors.