Identification And Transcriptional Regulation Of Cis Acting Elements Of The 5'UTR Intron And Promoter Of Cotton FAD2-1 Gene

Object:In Gossypium,the?12-desaturase enzyme gene FAD2-1 is highly expressed and seed-specific,and it encodes the key enzyme that catalyzes the conversion of oleic acid to linoleic acid in the developing seed.The expression level of FAD2-1 determines the nutritional properties and oxidative stability of cottonseed oil.It is essential for understanding the mechanism of FAD2-1 expression to identify and analyze upstream regulatory regions of FAD2-1,the promoter and 5'UTR intron.To reveal the regulatory mechanism of FAD2-1 gene and explore the physiological significance of the formation of cottonseed fatty acid composition,the important cis-regulatory elements in 5'UTR intron and promoter region would therefore be identified.The spatial and temporal expression patterns of the promoter and 5'UTR intron and their effects on gene expression regulation,and elucidate the synergistic effects between the 5'UTR intron and promoter and their utility value in molecular plant breeding should be cleared.It would also provide scientific basis for improving cottonseed oil quality.Method:1.In this study,the 5'UTR sequence of GhFAD2-1 was cloned by 5'RACE technology.And then,5'UTR intron of GhFAD2-1 was cloned in combination with the cotton genome sequence.Cis elements were analyzed by PLACE and other bioinformatics software.2.the promoter sequence of GhFAD2-1 was cloned by bioinformatics technology based on cotton genome database.Cis elements of promoter were also analyzed by PLACE and other bioinformatics software.3.The expression vector with full length or partial deletion of GhFAD2-1 5'sequence,driving GUS gene expression were constructed by Gateway technology.Transgenic Arabidopsis plants were obtained by Agrobacterium transformation.4.The GUS gene expression of transgenic plants was analyzed by qRT-PCR.GUS histochemistry analysis of transgenic plants in different tissues was also carried out.Results:1.the GhFAD2-1 5'UTR sequence 77bp was obtained from Gossypium hirsutum using 5'RACE technique.The result showed that the transcription start site is T based on a combination of 5'UTR and genome sequence analysis.GhFAD2-1 5'UTR contained a full-length 1103bp intron sequence in the A genome of cotton.The full-length sequence of GhFAD2-1 5'UTR intron is 1111bp in D genome.The cleavage sites of 5'UTR intron were AA-GG,CA-GC,respectively.Cis element analysis showed that the intron includes some typical function and optical response related elements and elements related to hormone regulation or stress response.2.The full-length GhFAD2-1 5'promoter of cotton A and D genome is 1349bp and 1279bp,respectively,and there are 238 base differences between them.The analysis of functional components showed that the A genome and the D genome sequence also had seed specific expression elements,as well as response elements related to hormone and stress factors.3.The full-length and deletion sequences of GhFAD2-1 5'sequences were amplified by PCR technology.The following expression vectors were successfully constructed by Gateway technology:pFAD2-1^2467bp::GUS,pFAD2-1^2219bp::GUS,pFAD2-1^2165bp::GUS,pFAD2-1^1642bp::GUS,pFAD2-1^1348bp::GUS,pFAD2-1^1279bp::GUS,pFAD2-1^2163bp::GUS,pFAD2-1^1799bp::GUS,pFAD2-1^1428bp::GUS,pFAD2-1^1119bp::GUS and pFAD2-1^1111bp::GUS.4.The results of real-time fluorescent quantitative PCR of Arabidopsis transgenic plants with deletion promoters revealed that GUS gene expression was driven during Arabidopsis seed and ovule development but not in other tissues,indicating that the promoter is Seed dominant expression promoter.At the same time,we also confirmed that the GhFAD2-1 5'UTR intron has promoter activity.5.The result of GUS gene expression indicates that-300 element?TGHAAARK?elements play an important role in regulating the expression of genes.a certain number of E-Box?CANNTG?,SEF4MOTIFGM7S?RTTTTTR?,SEF3MOTIFGM?AACCCA?,SEF1MOTIF?ATATTTAWW?,and SEF1MOTIF?ATATTTAWW?,were proportional to the level of gene expression.Conclusion:The 5'UTR intron of GhFAD2-1 also plays a transcriptional enhancer function,which contains regulatory elements such as-300 element?TGHAAARK?,an important impact on the transcriptional level of the regulated genes.In this study,the promoter and 5'UTR intron sequences of FAD2-1 and their functional components were analyzed.The results will help to understand the spatio-temporal expression and regulation of FAD2-1.It provides a scientific basis for cultivating high quality cottonseed oil with high oleic acid content.At the same time,it is also expected to provide efficient promoters and efficient enhancer elements for plant genetic transformation.