Study On Expression Of Anthocyanin Biosynthesis Key Genes And Differential Protein From Brunfelsia Acuminata Flowers

Brunfelsia acuminata flowers’ s color change from dark purple to white with high ornamental value.Because of this rapid color change,their common name is yesterday-today-tomorrow.Brunfelsia acuminata flowers at different stages were used to study the expression of differential proteins and anthocyanin biosynthesis key genes by two-dimensional electrophoresis technique and real-time quantitative PCR,and the physiological index of membrane lipid peroxidation were also determined in Brunfelsia acuminata petals during color change.Results showed as follows:1.Expression Analysis of CHS,CHI,F3’5’H gene in Brunfelsia acuminataThe content of anthocyanin was measured.The gene expression levels of CHS,CHI and F3’5’H from Brunfelsia acuminata petals at different stages(day 0 to day 5),stems and leaves were analyzed by real-time quantitative PCR.The results showed that anthocyanin content decreased gradually in the petals,stems and leaves are also present in small amounts.The CHS,CHI and F3’5’H genes expressed in different tissues.In different days of flower opening,the transcriptional level is changed.The highest expression of CHS gene occurred at the first day of flower opening,and then decreased.The expression of petals at 3-5 day was almost not detected.Transcription of F3’5’H was the highest at day 0,while transcription of CHI was low expression at this stage.CHI was roughly equivalently expressed in all stages,but its expression at day 1 was somewhat higher level than that at day 0.The expression of CHI gene expression was the highest in the sample of new leafs,and lowest in old leaves.In contrast,F3’5’H expression was highest in day 0.The transcript levels of the gene quickly decreased at day 2.The expression of F3’5’H gene was almost not detected in stems and old leaves.Meanwhile,the anthocyanin content was correlated with CHS gene and F3’5’Hgene.2.The differential expression of protein in different stage of Brunfelsia acuminata petalsDifferent range of pH strips and different sample amount were used to optimize two-dimensional electrophoresis conditions of Brunfelsia acuminata petals.The result showed that 2-DE map is the best under the condition of the IPG strip with 24 cm and pH value from 4.0 to7.0,and the sample volume of 1.5 mg.The proteome profile of petals(day 1,day 3,day 5)were mapped by two-dimensional gel electrophoresis and differentially abundant proteins from distinct color change were subjected to MALDI-TOF/TOF-MS analysis.Among the total protein spots(70 spots)reproducibly detected on each gel,60 spots representing 53 proteins were identified to be differentially regulated,among of which proteins were up-regulated and the 20 proteins down-regulated,These proteins were annotated in various biological function such as photosynthesis and energy metabolism,secondary metabolism,protein stability,amino acid and nitrogen metabolism and defense,of which sugar metabolism and energy metabolism and respiration pathway occupied 19%,and lignin metabolic pathways anthocyanin biosynthesis 9%.Signaling and photosynthesis-related protein 7%,stress resistance and aging-related protein 36%,floral and terpene synthesis pathway 7%,cytoskeletal and chaperone 13%,protein Unclassified 9%.Coenzyme A coffee oxygen methyl transferase enzyme,salicylic acid carboxyl methyltransferase enzyme glutamine synthetase,ubiquitin-related modifier protein SUMO-1,etc.,and their high expression may be associated with changes in flower color.Protemoic techniques were demonstrated a reliable and robust tool to expand our understanding of differentially expressed proteins associated with color change of Brunfelsia acuminata petals.Functional analysis of these proteins further revealed biochemical and physi-ological basis of the petals during the color change would provide basic data for exploring mechanism of the color change of Brunfelsia acuminata flower.3.The physiological index of membrane lipid peroxidation during color change of Brunfelsia acuminata petalsA particularly interesting feature of Brunfelsia acuminata petals is their striking color change from dark purple through light purple and ultimately to pure white as petal coloration continued to develop.The MDA content,SOD,CAT,POD and PPO activity in Brunfelsia acuminata petals at different stages were detected.The results showed that the color change is associated with an decline in content of anthocyanin.The anthocyanin content were significantly negatively correlated with POD activity and positively correlated with MDA content where membrane lipid peroxidation may play an important role in this change.POD activity increased with the development of petal,showing their positive correlation,while PPO activity decreased,and was correlated with the activity of SOD activity which was highest in flower bud and significantly decreased at 1 day petal,and kept stable from day 3 to day 5.petal.However,CAT activity jumped during the color change.