| Brassica oleracea var.acephala is cruciferous plants.And it also known as "Ye MuDan"because the appearance of its leaves are beautiful and colourful.Besides,it has strong cold hardiness and can survive at-10?.So it is very suitable for cultivating as landscape plants in northern region.But Brassica oleracea is a typical kind of cross-pollinated plants,and it always shows self-incompatibility.SI is a method for plant to regulate reproduction.The purpose is to prevent inbreeding and maintain genetic diversity.However,there is still little understanding on SI.But,it has been known that most signal teansduction factors of SI are phosphorylated protein.In this dissertation,we combined the methods of proteomics and molecular biology to discover the phosphorylated proteins involved SI.And we have discovered two candidate genes which haven't reported their relationship with SI.So they have provided clues to understanding SI.The main results obtained are as follows:1.In order to find the relevant phosphorylated proteins for SI,the self-incompatibility line(9#)and the self-compatibility line(14#)were selected as material,and then the stigmas of 9#were selfing and outcrossing pollination 0 min,15 min,60 min,respectively.The total protein was extracted,and then the differential proteins profile in phosphorylation level were selected through the method of 2D-DIGE after digesting by ?-phosphatase.Finally,11 spots were found and identified by mass spectrometry,two of these spots were positively correlated with SI,while nine of them were negatively correlated with SI.Among them,It has been reported that UGPase and MBP both involved in pollen development,and their functional loss can lead to genetic male sterility.It is the main reason why they were selected as candidate genes for further study.These results have provided clues and foundation for the research of SI signal transductional machenism.2.In order to analyse the expression pattern and functions,the full-length cDNA of BoUGPase and BoMBP were cloned firstly,and the sequence are respectively 1465 bp and 2797 bp.In addition,their ORFs respectively encode 476 and 931 amino acid polypeptide chains.Then it has been found that the expression of BoUGPase is higher in anther,while the expression of BoMBP is higher in style and ovary.The pattern of expression indicates that they may involve in SI.For further confirming their proteins' expressional patterns and they are phosphorylated proteins,the pHis-BoUGPase and pHis-BoMBP for Prokaryotic expression was constructed to identify the feasibility of BoUGPase and BoMBP expressing in lower organisms.The protein sequences were respectively located in 55 KDa and 100 KDa.3.For in vivo functional analysis of candidate gene,genetic transformation system of Brassica oleracea has been established from cotyledon and the best shoot initiation medium has been identified as MS0+6-BA 2mg/L.After that.the stigma specific promoter of BoARCl and BnSLR1 are transfermed into Arabidopsis,and the promoter of BnSLR1 can drive downstream genes highly expressed in the stigma and pistil,while the promoter of BoARC1 only can drive downstream genes expressed in the sepal.This indicated that the function of the BnSLR1 is more conversed in the whole cruciferous plants and it is more suitable for the further research.According to the above,they can be used for functional verification and analysis of the candidate genes... |
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