Cloning And Expression Analysis Of Four R2R3-MYB Encoding Genes From Caragana Intermedia

Abiotic stresses are major limiting factors for crop production worldwide.including drought,salinity,high temperature,low temperature,oxidative stress and so on.Application of transcription factors to improve plant resistance has become a hot area of research nowadays.MYB transcription factor constitutes one of the largest transcription families in plant,while R2R3-MYB is the most abundant MYB proteins.R2R3-MYBs play crucial roles in the primary and secondary metabolism of plant,grovwth development,and responses to biotic and abiotic stress.Caragana intermedia is one of bushes that can hold water,protect against wind and sand,hence it is one of the main shrubs in alpine sandland.In this study,four R2R3-MYBs were cloned by PCR technique from C.intermedia,named CiMYB 102.CiMYB31,CiMYB60 and CiMYB185,and their expression patterns and promoter cis-elements are analyzed.The main results are as follows:1.Four R2R3-MYB are chosen from drought transcriptome database,which cDNA and gDNA were cloned.The ORF length of CiMYB102,CiMYB31,CiMYB60 and CiAMY-B185 is 1107 bp,969 bp,1038 bp and 906 bp,and encoding 369,323.346 and 302 amino acids respectively.The gDNA length of CiAMYB102.CiMYB31.CiMYB60 and CiMYB185 is 1396 bp.1724 bp.1485 bp and 1592 bp respectively.CiMYB102,CiMYB31 and CiMYB60 have two introns and three exons,while CiMYB185 has one intron and two exons.2.The expression pattern of four R2R3-MYBs under various stresses was analyzed by qRT-PCR.CiMYB31 and CiMYB185 were mainly induced by cold stress.CiMYB102 was induced by dehydration,NaCl.ABA and drought.The expression of CiMYB60 was reduced by dehydration.NaCl and UV-B treatments.These results showed that four R2R3-MYBs might play a role in response to abiotic stress of C.intermedia.3.The promoter sequences of four R2R3-MYBs were cloned by genome walking.The promoter length of CiMYB102,CiMYB31,CiMYB60 and CiMYB185 is 1359 bp.902 bp.1848 bp and 908 bp,respectively.Analysis predicts that the promoter contains hormone related,light responsive,tissue specific and some abiotic stress responsive cis-elements.4.The expression pattern of CiMYB102.CiMYB60 and CiMYB31 in different tissues varies.but with the lowest level in root.CiMYB102 expresses highly in stem.The expression of CiMYB31 reaches the highest in leaves.While the expression of CiMYB60 in leaf and stem is relatively high.5.Binary expression vectors p35s::CiMYB102-GFP.p35s::CiMYB31-GFP and p35s::CiMYB60-GFP wre constructed and transformed into wild-type A.thaliana.Homozygous transgenic Arabidopsis thaliana lines were obtained.