Screening And Analysis Of LRE Binding Proteins For BrCHS1Promoter

Anthocyanins are important secondary metabolites of plants which can not only represent the major red, purple, violet and blue pigments in many flowers, fruits and vegetables, but also attract animal pollinators and seed dispersers, defend plants against a variety of abiotic and biotic stresses. Chalcone synthase (CHS) is one of the key enzymes in anthocyanin biosynthesis, the anthocyanin biosynthesis in Tsuda turnip (Brassica rapa L. subsp. rapa Tsuda) and expression of BrCHSl is specifically induced by UV-A light. In this study, we conducted bioinformatics analysis to find out the light responsive elements existing in BrCHSl promoter region. To analyze the ability of BrPAPl, BrPAP2, BrTT8, BrBZIP and BrHY5protein to interact with G-box-an improtant light responsive element in BrCHS promotor region, yeast One-hybrid method was employed. In addition, binding proteins of cis-acting element UNIT1are also isolated by yeast One-hybrid screening. The purpose of this study is to identify transcription factors which bind light responsive elements in BrCHSl promoter and lay the foundation for the study of molecular mechanism in UV-A specific induction of BrCHSl expression and anthocyanin biosynthesis. The main results obtained in this study are as follows:1:Sequence of1500bp from3’end of BrCHSl was analyzed using PlantCARE, results show that this promoter region contains conserved sequence in general eukaryotic promoters such as the CAAT box and TATA box. Besides, there are a lot of light responsive elements existing in BrCHSl promoter region such as AE-box, Box-I, CATT-motif, G-Box, GT1-motif, LAMP-element, TCT-motif and CHS-UNIT1.2:The bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast. BrPAP1, BrPAP2, BrTT8, BrBZIP and BrHY5gene sequences were clone to pGADT7-Rec expression vector and the recombinant expression vectors were introduced into bait yeast strain to determine binding ability of the proteins with G-box. Results show that BrTT8and BrBZIP have strong ability to bind to G-box while BrPAP1, BrPAP2and Br HY5can’t bind to G-box.3:Bait yeast strain was constructed by synthesizing oligonucleotides containing BrCHSl UNIT1cis-acting element sequence which consists of ACE, RRE and MRE and integrating it into the genome of yeast. The cDNA for turnip(Brassica rapa L. subsp. rapa Tsuda) was synthesized and transformed into bait yeast strain together whith pGADT7-Rec exression vector, one-hybrid cDNA library is simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination. Results show that the number of clones screened is2.73×106, the number of posive clones is68. CDNA inserts in posive clones are obtained byyeast clony PCR, sequencing and bioinformatics analysis results show that proteins isolated by yeast one-hybrid screening are mainly involved in signaling transduction and stress response.