Analysis Of The Function Of Transcription Factors GT And GATA On BnaFAD2 Gene Promoter In Brassica Napus L.

Fatty acid desaturase 2 gene(FAD2)is an important gene for controlling oleic acid content in rapeseed.And in this paper,the interaction between GATA family and and BnaFAD2.A5 and BnaFAD2.C5 gene promoters or GT family transcription factors and BnaFAD2.A5 and BnaFAD2.C5 gene promoters was studied to make sure the transcription regulation mechanism to provide the theoretical basis for high oleic acid breeding.The potential cis-elements were predicted by deletion of promoter fragments and bioinformatics analysis,and candidate transcription factors were screened from PlantTFDB transcription factor database.Real-time PCR(RT-qPCR)was used to detect the expression pattern of transcription factors to further screen candidate transcription factors by comparison with BnaFAD2.A5 and BnaFAD2.C5 gene expression patterns.Transcription factors were co-transformed with BnaFAD2.A5 and BnaFAD2.C5 genepromoters into yeast and verified interaction relationship between promoter and transcription factors by yeast one-hybrid.The transcription factors were transformed into Arabidopsis thaliana with BnaFAD2.A5 and BnaFAD2.C5 genepromoter sequence,and the reporter gene GFP was detected by western blot to analyze the effects of transcription factors on promoters.The main results are as follows:(1)The BnaFAD2.C5 promoter region of-1020~-319 bp mainly control gene expressing in the roots of Brassica napus L.;the promoter region of-1020~-581 bp mainly control gene expressing in the stem tissues;the promoter region of-581~-319 bp mainly control gene expressing in leaves;the promoter region of-1257~-1020 bp mainly control gene expressing in flowers;the promoter region of-319~-1 bp is a key region for controlling gene expression in seeds.By applying different concentrations of acetylsalicylic acid and jasmonic acid,it was found that two plant growth regulators promoted gene expression,indicating that there are acetyl salicylic acid and jasmonic acid response elements on BnaFAD2.C5 gene promoter.Informatics analysis found that the two response elements were located between-1150 and-1141 bp and-39 to-35 bp.(2)It was found that the BnaFAD2.A5 and BnaFAD2.C5 promoters have a homologous region of about 680 bp,which has a significant contribution to gene expression.The BnaFAD2.A5 and BnaFAD2.C5 promoters are highly homologous into four regions.Deletion analysis revealed that the-226 ~-314 bp of the BnA5.FAD2 promoter region and the-228~-312 bp region of the BnaFAD2.C5 promoter region are key promoter regions that regulate the expression of both in the seed.Through the prediction analysis of PlantCARE website,it was found that there are common regulatory elements GATA and GT in these two regions.By deleting two cis-acting elements of GATA and GT and mutating the two cis-acting elements of GATA and GT,it was found that they are regulating Gene expression plays an important role.(3)There 31 plant Brassica napus GATA transcription factor nucleic acid sequences and 6 Brassica napus GT transcription factor nucleic acid sequences being obtained by PlantTFDB.Analysis by real-time PCR showed that,in GATA family,the expression pattern of Bna010243 was similar with BnaFAD2.A5.The expression patterns of Bna006754,Bna015605,Bna026124 were similar with BnaFAD2.C5.In GT family,The expression pattern of Bna013749 was similar with BnaFAD2.A5,and Bna010915 was similar with BnaFAD2.C5.(4)The GATA family and GT family genes were cloned from the genome and cDNA genome sequences of Brassica napus L.oil,which were Bna006754(903 bp,301aa),Bna010243-1(594 bp,198aa),Bna010243-2(Bna010243-2,respectively).567 bp,189aa),Bna015605(963 bp,321aa),Bna026124-1(546 bp,182aa),Bna026124-2(582 bp,194aa),Bna010915(681 bp,227aa)and Bna013749(1 239 bp,413aa).The GATA family proteins all have a conserved single 18 residue zinc finger structure(CX2CX18CX2C).The GT family protein has three alpha-helices to form a Trihelix structure.(5)Analysis of the interaction between transcription factors and BnaFAD2.A5 and BnaFAD2.C5 promoters in yeast by yeast one-hybrid technique,and the result showed that the GATA family of transcription factors Bna010243-1,Bna026124-1,Bna026124-2 can be The BnaFAD2.A5 and BnaFAD2.C5 promoters interact;the GT family of transcription factors Bna010915 and Bna013749 were found to interact with the BnaFAD2.A5 and BnaFAD2.C5 promoters.(6)By analyzing the interaction between transcription factors and GATA and GT elements on the BnaFAD2.A5 and BnaFAD2.C5 promoters in the model plant Arabidopsis thaliana,the results showed that when the GATA family transcription factor and the promoter fragment containing GATA elements were co-transformed into Arabidopsis thaliana,the GFP protein content was significantly higher than that of no transcription factor.When the GATA element was knocked out,the GFP protein content did not change significantly.When the GT family transcription factor and the promoter fragment containing the GT element were co-transformed into Arabidopsis thaliana,the GFP protein content changed significantly.When the GT element was knocked out,the GFP protein content did not change significantly.The results showed that the GATA family transcription factors Bna010243-1,Bna026124-1,Bna026124-2 can interact with the GATA elements in the BnaFAD2.A5 and BnaFAD2.C5 gene promoters to enhance gene expression levels.The GT family transcription factor Bna010915 interacts with the GT elements in the BnaFAD2.A5 and BnaFAD2.C5 gene promoters to positively regulate gene expression;Bna013749 interacts with the GT elements in the BnaFAD2.A5 and BnaFAD2.C5 gene promoters to negatively regulate gene expression.