Mechanism Analysis Of Ppd-D1b Promoter Of Photoperiod Gene In Wheat

Wheat(Triticum aestivum L.)is an important food crop.Photoperiod genes control the flowering time of wheat and determine the planting area and yield of wheat.Ppd-D1 gene is the most important photoperiodic gene in wheat.The dominant allele Ppd-D1 a is insensitive to photoperiodic response,while the recessive allele Ppd-D1 b is sensitive to photoperiodic response.Previous studies have shown that the photoperiod gene Ppd-D1 promoter is related to its photoperiod sensitivity,but the key regions,key elements and transcriptional regulation mechanism of the photoperiod response of the promoter are still unclear.Therefore,in this study,the promoter sequence of Ppd-D1 was isolated from the common wheat genome by PCR method,and the expression vector of Ppd-D1 b promoter fusing GUS reporter gene with different length fragments was constructed by combining the method of bioinformatics prediction and 5’ deletion.The expression of downstream reporter gene GUS driven by different lengths of promoters was analyzed by transforming Arabidopsis medium stable lines,and the key regions and cis-elements in the promoters that were sensitive to day length were identified.Further,candidate transcription factors binding to key elements were screened by yeast one hybridization assay,and the binding of transcription factors to key cis-elements in plants and their modes of action were identified by double luciferase assay.The main research results are as follows:1.The 709 bp and 2518 bp sequences of Ppd-D1 a and Ppd-D1 b promoters were cloned from wheat,respectively.Due to the large fragment deletion of Ppd-D1 a promoter,the PpdD1b promoter with relatively complete sequence was predicted and analyzed.The results showed that the Ppd-D1b promoter contained core promoter elements such as TATA-box.And G-Box and CHEBS(CCA1 HIKING EXPEDITION binding site)and other light response elements.On this basis,ten 5 ’-deficient expression vectors(D0～D9)of Ppd-D1b promoter were constructed and transformed into Arabidopsis thaliana for stable expression.Under short day(8h light /16 h dark),GUS reporter gene expression in transgenic Arabidopsis thaliana with full-length promoter(D0)showed a rhythm and reached the peak at 3h after dawn,which was the same as that of Ppd-D1b gene in wheat studied by previous studies,indicating that this promoter was a light-induced promoter.And it has the same activity in Arabidopsis allogeneic expression system.At 3h after dawn,GUS activity in transgenic Arabidopsis thaliana driven by each 5’ deletions promoter was compared.The results showed that GUS activity was significantly decreased due to the absence of CHEBS,suggesting that CHEBS may be the key element responsible for the sensitivity of Ppd-D1b gene promoter in response to day length.2.Wheat cDNA library was screened by yeast one hybridization(Y1H)experiment,and the transcription factor TaTCP20 was obtained.The binding of TaTCP20 to CHEBS element was verified by yeast one-to-one transformation.Dual-luciferase(Dual-Luc)assay results showed that Ta TCP20 could positively regulate Ppd-D1b gene expression by binding CHEBS.This study revealed that the transcription factor TaTCP20 positively regulates the expression of Ppd-D1b through specific binding with CHEBS,providing a theoretical basis for further exploration of the photoperiodic response mechanism of Ppd-D1 gene.