Cloning And Expression Analysis Of BnF3H And BnDFR Genes And Related MYB Transcription Factors In Brassica Napus L.

Anthocyanins are water-soluble pigments widely found in plants and play an important role in plant stress resistance and prevention of chronic diseases in humans.At present,the biosynthesis process of anthocyanins is well studied in model plants,and its process is mainly regulated by enzymes and transcriptional regulators(MYB,bHLH and WD40 proteins)encoded by various structural genes.As an important economic crop,rapeseed not only has edible value,but also has ornamental value.At present,there have been some reports on the anthocyanin synthesis pathway in other plants,but little research has been done on the anthocyanin synthesis genes and related transcription factors in rapeseed.Therefore,this study selected Brassica napus L.as the research object.Cloning and expression analysis of the genes BnF3 H and BnDFR and their related MYB transcription factors in the anthocyanin synthesis pathway by molecular cloning,subcellular localization,real-time fluorescence quantification,yeast one-hybridizationand and other molecular biology techniques.It provides a theoretical basis for studying the regulation mechanism of related genes and transcription factors in the anthocyanin anabolic pathway of Brassica napus L.in the future.The main findings are as follows:1.The CDS sequences of RFBnF3H1,RFBnF3H2,RFBnDFR genes,RFBnMYB114 and RFBnMYB90 transcription factors were cloned from Brassica napus L.,and the obtained sequences were analyzed by bioinformatics.2.The subcellular localization indicated that the localization was different in the cells.The RFBnF3H1,RFBnF3H2 and RFBnDFR genes in the Brassica napus L.were distributed in the nucleus and cell membrane,while the transcription factors RFBnMYB114 and RFBnMYB90 were mainly distributed in the nucleus.3.The expression patterns of RFBnF3H1,RFBnF3H2,RFBnDFR,RFBnMYB114 and RFBnMYB90 in different tissues of Brassica napus L.were analyzed by real-time quantitative PCR.The results showed that RFBnF3H1,RFBnF3H2 and RFBnDFR and related RFBnMYB114 and RFBnMYB90 transcription factors were expressed in roots,stems,leaves,flowers,fruit pods 27 days after flowering and 35 days after flowering,but the expression level was tissue Specificity.The expression level of RFBnF3H1 gene in fruit pods at 27 days after safflower flowering was significantly higher than that in other tissues.The expression of RFBnF3H2 gene was higher in roots,stems and leaves,but it was lower in fruit pods 27 days after flowering,but increased in fruit pods 35 days after flowering.The expression of the RFBnDFR gene in roots was significantly higher than in other tissues.Analyzing the expression pattern of the relevant MYB transcription factor,We found that the expression level of RFBnMYB114 transcription factor in roots was significantly higher than that of other tissues,which showed the same trend as the expression of RFBnDFR gene,and there was an increasing trend in the expression of fruit pods in fruit pods on the 27 th day after flowering and 35 days after flowering;The expression of RFBnMYB90 transcription factor was higher in leaves,flowers and flowering 35 days after fruiting,especially in fruit pods 35 days after flowering,and in fruit pods 27 days after flowering and 35 days after flowering,it also shows an increasing trend.4.The corresponding promoters of RFBnF3H1,RFBnF3H2 and RFBnDFR genes were cloned,and the binding of RFBnMYB114 and RFBnMYB90 to the promoters of RFBnF3H1,RFBnF3H2 and RFBnDFR genes in Brassica napus L.was preliminarily verified by yeast one-hybrid technique.5.A preliminary analysis of the cis-acting elements on the gene promoter was performed using Plant Care software.The results indicate that the promoter contains important cis-acting elements such as the hormone response-related cis-regulator ABRE,the anaerobic-inducing response element ARE,the photoresponsive element Box4,the defense-related response element TC-rich repeats,and the low-temperature response element LTR.