Expression Character Of BrF3H And Function Analysis Of Its Promoter In Turnip 'Tsuda'

Flavanone 3-hydroxylase (F3H) is a key enzyme during the pathway of anthocyanin biosynthesis in higher plants. An increase or decrease in the expression of F3H gene may lead to variety in flower colour. According to the former research, UV-A can specially induce F3H gene expression and anthocyanin biosynthesis in turnip'Tsuda'. For the study of expression character of the F3H gene induced by UV-A, turnip'Tsuda', which has a characteristic of the light-dependent anthocyanin biosynthesis, was used as materials. The gene cloning the analysis of gene copy numbers and gene expression character of BrF3H were investigated. To elucidate its regulatory pattern at the transcriptional level, the sequence of BrF3H promoter has been analyzed by bioinformatic tools. Moreover, the promoter activity in turnip 'Tsuda' and tobacco has been characterizatized. The overall results have been summarized as followings:1 The expression of BrF3H was induced by UV-AThe fragment of BrF3H gene was isolated from the turnip'Tsuda'. Blast analysis indicated that the degree of sequence homology is more than 92% with part of Brassica rapa subsp. pekinensis clone KBrS001M03. Southern blot analysis indicated that BrF3H belonged to a multi-gene family. Furthermore, Real-Time quantitative PCR analysis revealed that BrF3H gene was induced by UV-A. In the seedlings of 'Tsuda', the expression of BrF3H in the middle of the hypocotyl was the most obvious induced by UV-A, which indicated that there may exist light-specific induction regulatory pathway of anthocyanin biosynthesis.2 The functional dissection analysis of BrF3H promoter in turnip'Tsuda'The sequence of BrF3H promoter in turnip 'Tusda' contains CAAT box and TATA box and light-induced motifs such as AE-box, Box-Ⅰ, CATT-motif, G-Box, GT1-motif, LAMP-element, TCT-motif and chs-Unit 1. To identify the putative light-responsive region, four deletions from the 5'-end of BrF3H have been fused with GUS gene. pCAM-BrF3Hp1091, pCAM-BrF3Hp724, pCAM-BrF3Hp231 and pCAM-BrF3Hp158 have been constructed respectively and were transformed into Agrobacterium tumefaciens strain EHA105. Then the germinated seeds of turnip 'Tsuda' were transformed by vacuum infiltration. GUS histochemical stain revealed that the four BrF3H promoter fragments could propel the GUS gene expression in the seedlings of turnip 'Tsuda'. Furthermore, for the study of quantitative analysis of BrF3H promoter activity, the four BrF3H promoter fusions were transformed into tobacco plants by Agrobacterium-mediated genetic transformation technology. Real-time quantitative PCR analysis indicated that there were 10 lines with single copy,3 lines with 2 copies,1 line with 3 copies and 1 line with 4 copies-in the 15 transgenic tobacco lines. GUS histochemical stain showed that four BrF3H promoter fragments could propel GUS gene expression in the tobacco leaves and petioles. Then the BrF3H promoter fragment activity was detected by quantitative analysis of the GUS gene in transgenic tobacco lines with single copy insertion. The results revealed that the highest GUS activity was observed with BrF3Hp1091. BrF3Hp724 and BrF3Hp231 had relative activities of 69.2% and 35.2% compared with BrF3Hp1091, while the activity of promoter BrF3Hp158 was similar to BrF3Hp1091. GUS activity decreased when the region between-1091 and-232 was deleted. Further deletions from-231 to-159 led to the increase of promoter activity. According to these results, we could conclude that there may be some positive regulatory elements in the region between-1091 and-232 and some negative regulatory elements in the region between-231 and-159, and the minimal promoter region was-158～-1 which may be the core promoter of BrF3H gene.