Functional Analysis Of Arabidopsis Thaliana AtMYBL And Brassica Napus BnMYBL In Flowering Time Regulation

MYB transcription factors play an important role in plant growth and development,organ morphogenesis and so on.MYB family is one of the largest families of plant transcription factors and has a variety of biological functions.In this paper,one MYB gene,AtMYBL was isolated and cloned from Arabidopsis,and the expression pattern and function of AtMYBL were studied.At the same time,the homologous gene of AtMYBL in Brassica napus,BnMYBL was cloned to preliminary study the expression pattern and function too.The main findings as follows:1.Isolation,identification and sequence analysis of AtMYBLOne gene encoding MYB family transcription factor,AtMYBL was isolated and cloned from Arabidopsis.The total genomic sequenc of AtMYBL was 1351 bp,and the CDS sequence was 711 bp,which could encode 236 amino acids.So far,the biological function of AtMYBL is unclear.2.Subcellular localization of AtMYBLThe 35S:GFP-AtMYBL transgenic Arabidopsis was obtained.Then the fluorescence in roots of transgenic seedlings was observed.The results showed that AtMYBL was localized in the nucleus.3.Detection of transcriptional activity of AtMYBLMYB family is a kind of transcription factors.In order to verify whether AtMYBL is a transcriptional activator or a transcriptional inhibitor,yeast cells are used to detect AtMYBL self-activation activity.The results showed that AtMYBL had no yeast self-activation activity and might be a transcriptional inhibitor.4.AtMYBL expression pattern analysisThe expression patterns of AtMYBL in roots,stems,leaves and flowers of Arabidopsis were analyzed.The results showed that the expression of AtMYBL was higher in flowers and kernels.AtMYBL promoter sequence was cloned and AtMYBLp:GUS was constructed and transformed into Arabidopsis thaliana.Gus activity analysis showed that AtMYBL promoter was expressed in cotyledons of transgenic Arabidopsis thaliana seedlings,but not in the roots of transgenic Arabidopsis thaliana seedlings.In the flowers,rosette leaves and kernels of seedlings,there was strong gus signal.5.Phenotypic Analysis of AtMYBL transgenic Arabidopsis thalianaThe over-expression vector pMD-AtMYBL was constructed and transformed into Arabidopsis thaliana so that the transgenic Arabidopsis thaliana with high expression of AtMYBL was obtained.At the same time,the seed of MYBL mutant was identified,and myb6 was identified as a homozygous mutant.The flowering time of over-expressed AtMYBL transgenic Arabidopsis thaliana in WT,myb6 was analyzed statistically.The results showed that the flowering time of over-expressed transgenic Arabidopsis thaliana was earlier than that of wild-type Arabidopsis thaliana,while the flowering time of myb6 was delayed.And the number of rosette leaves increased.6.Analysis of flowering Regulation Gene expression in AtMYBL transgenic Arabidopsis thalianaThe flowering genes in myb6 and and over-expression AtMYBL transgenic Arabidopsis thaliana were detected.In myb6,FLC was up-regulated,LFY,SOC1 and AP1 were down-regulated.FLC was down-regulated,LFY,SOC1 and AP1 were up-regulated in over-expressed lines.7.AtMYBL can interact with FLC PromoterYeast single hybrid assay showed that AtMYBL could interact with FLC promoter.8.BnMYBL expression pattern analysisThe transgenic plant of Arabidopsis thaliana was obtained by constructing the vector 1300-BnMYBLP and transforming it into Arabidopsis thaliana.Gus Activity analysis showed that AtMYBL promoter was expressed in cotyledons of transgenic Arabidopsis thaliana seedlings,but not in roots of transgenic Arabidopsis thaliana seedlings.In the flowers,rosette leaves and kernels of seedlings,there was strong Gus signal.9.Over-expression of AtMYBL in Arabidopsis thalianaThe overexpressing vector of BnMYBL was constructed and transformed to obtain the overexpressed transgenic plants L17 and L18 of BnMYBL.In the overexpressing lines L17 and L18,the gene BnFLC decreased relative to the wild type,while the expression levels of the BnLCY,BnSOC1 and BnAP were up-regulated and the related flowering phenotype was in a further experiment.