| Our former research showed that the accumulation of anthocyanidin in Brassica rapa'Tsuda' is induced by UV-A radiation,while blue light does not.We suggested that there must be a unique signal transduction pathway for UV-A among heterogeneous light by an new light receptor.Recently,we found that cyclophilins are overexpressed after UV-A radiation.Cyclophilins are highly conservative supper-family protein,which are called as rotamase cyclophilin(ROC)for possessing peptidyl-prolyl cis-trans isomerase activity.Plant CyPs play significant roles in plant biology,light-damage defense and stress responses.In this study,we try to get clones of these candidate in Brassica rapa 'Tsuda'.,and make expression analysis for each one.we harvest many details of these genes through biological methods for studying whether the over-expression of CyPs affect the synthesis of anthocyanin and stress responses.Besides,it might provide certain help for further research of the CyPs' fuctions.The main results of this study are as follows:(1)Clone and Bioinformatic analysis of BrROC1-1 and BrROC1-2Two overexpressed genes were cloned from Brassica rapa 'Tsuda'.Bioinformatic methods reveal that they are CyPs with conserved domains.These two genes are homologous gene named BrROC1-1 and BrROC1-2.The full-length of BrROC1-1 is 743bp,including a 218bp 3'untranslated region,The full-length of BrROC1-2 is 822bp,including a 253bp 3'untranslated region.all of they encoded 172 amino acids.Southem blot showed that BrROC have many copes in the Brassica rapa 'Tsuda' genome.The phylogeny reconstruction analyses on these genes reveal that each sequence therein has high degree of identity in the predicted protein sequence with Arabidopsis CyP1(ROCS)genes.(2)The expression characteristics of BrROC1-1 and BrROC1-2Expression of BrROC1-1 and BrROC1-2 in different tissues:The results reveal that the expression of BrROC1-1 was higher in 'Tsuda' flower buds,less in the leaves,while the other organizations are not different significantly.BrROCl-2 is mainly expressed in petals,buds,but very little in leaves.It suggest that ROCs play an important role in flower development.Expression of BrROC1-1 and BrROC1-2 in different light treatment:the expression of BrROC1-1,BrROC1-2 in taproot white epidermis increase with UVA irradiation time,and reach a peak at 24 hours and 3 hours respectively.LiV-A treat the 4.5 day seedlings which growing in the dark,BrROC1-1 expression is increasing with UV-A irradiation time only in the hypocotyl section,while no changes in the other part;in the blue light treatment,expression of BrROC1-1 increased only in cotyledons;Under the red and red/farred light treatment,expression of BrROC1-1 increase slightly in cotyledon,but decrease slightly in other patrs.In all parts of seedlings,the expression of BrROC1-2 is induced by UV-A and blue light.Under the red and red/farred light treatment,the expression of BrROC1-2 only decreased in the lower part of hypocotyl,while increase significantly in other parts.These results suggest that the expression of BrROC1-1 and BrROC1-2 were induced by light,including UV-A.blue light,red light and red/Tarred light.Expression of BrROC1-1 and BrROC1-2 in different stress treatment:in 8 day seedlings,cold,hot,dry,PEG,mannitol,CsCL,ABA and NaCl treatment improved expression of BrROC1-1 and BrROC1-2 significantly,It suggest that BrROC1-1 and BrROC1-2 involved in these stress response.(3)The transfomation of BrROC1-1 and BrROC1-2 in ArabidopsisWith Gateway clonging system,BrROC1-1 and BrROC1-2 were constructed into over expression vector pH7WG2D,of which the selective gene is hygomycin when transforming.Both of them were then transformed into Arabidopsis flowers by agrobacterium-mediated method.We got plenty of transgenic candidates by hygomycin selection system. |
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