| Due to the climate change and environmental pollution,which are caused by environmental factors such as adverse stress(stress)including UV radiation,high and low temperature,salinity,drought,flood,heavy metals,diseases and pests,etc.,rapeseed production has been brought the serious negative impact.With the completion to the whole genome sequencing of rape,rapes' researches in functional genes for cultivating resistance of rapeseed varieties,have important theoretical and practical significance.On the basis of previous researches,we discover a gene associated with leaf trichome development and resistance,which may have a close contact with nsLTP family genes.We analyzed the gene in the part of function and mechanism and it will provide theoretical support for resistance rapeseed breeding work.Already researches have confirmed that the plant trichome is a natural barrier to resist outside stress environment.At the same time,trichome is a "biochemical factory" for secondary metabolites biosynthesis and secreting,which closely related with plant adverse resistance.Numerous studies have shown that the nsLTP family genes really have functions in plant trichome development,secondary metabolism and play an important role in the process of adverse stress.The purpose of this experiment is to find out the specific stage involved in trichome development,the specific effect on different sort of secondary metabolites in trichome,and the specific resistance of adverse stress.Following works were carried out in order to study related researches:1.Genetic analysis of basic functional characteristics in BraLTP2BraLTP2 gene and its homologous genes have been conducted evolutionary tree analysis,the structure of the conservative domains prediction,conservative site analysis,signal peptide prediction and promoter element analysis by the method of Bioinformatics.According to the results,BraLTP2 has 99% homology with BnaLTP1 genes in Brassica napus,and in Arabidopsis thaliana has 76% homology with AtLTP1.Protein characteristics analysis shows that it belongs to the nsLTP family proteins that are shared by conservative structure,which are eight cysteine residues and four disulfide bonds.Further research is carried by building a promoter analysis vector using genetic transformation system mediated by Agrobacterium.And then we can analyse spacial-temporal expression patterns of BraLTP2 gene in transgenic lines.We focus on the expression of the blade and its trichome.And histochemical results show that the BraLTP2 gene expressed in special spatio-temporal leaf trichome.In addition,by constructing BraLTP2::GFP fusion gene expression vector,we analyze BraLTP2 protein in subcellular localization.Microscopy observation results showed that the protein localization is in epidermal cells of extracellular location and edge of trichomes.2.Researches of BraLTP2 in trichome growth and development.In order to prove the BraLTP2 gene function in the process of trichome growth and development,we constructed the 35S::BraLTP2 overexpression vector,mediated by Agrobacterium via genetic transformation methods,over-expressed BraLTP2 in transgenic rapeseed plants and identified by the molecular biology methods.Based on the 6 lines of transgenic rapeseed plants to detect the relative expression,we selected two lines(35S::BraLTP2-3 and 35S::BraLTP2-4)to identify the phenotype.Through the optical microscope and scanning electron microscope,the transgenic plants leaf trichomes density are increased significantly,number of transgenic plants leaf trichomes in the same leaf area has increased by more than 20 times.However,in crop height,plant and trichome morphology,there are no difference compared with wild type.So we conclude that the gene specificly regulated the trichome growth and development process,it mainly reflected on the change of trichome density.3.Researches of BraLTP2 gene overexpression influence in the secondary metabolites of leaves.Based on LC-MS/MS platform with extensively targeted technology of metabonomics,we compared leaf differences in the composition and content of secondary metabolites in 35S::BraLTP2 overexpression transgenic lines and wild type.Results show that 43 kinds of secondary metabolites up-regulated in the 89 differences metabolites,which are divided into seven categories,including flavones,phenylpropanoids,phenolamines,alkaloids,terpenoids,organic acids,and others.We found that the secondary metabolites which are significantly increased related to plant resistance,such as flavones,phenylpropanoids,alkaloids,etc.The part of experimental study reveals that the over-expressed BraLTP2 gene can change the content of secondary metabolites in Brassica napus leaf,fuethermore,infer BraLTP2 can involve in the biosynthesis or accumulation of secondary metabolites.4.Pre-investigation on BraLTP2 gene function in the adverse stressCombined with the feature of the basic function of BraLTP2 gene,the research result in Brassica napus leaf trichome growth and development,and metabolism detected results,we infer that the BraLTP2 gene has the role in antioxidant function.We calculated the differences of seed germination rate and seedling growth traits such as crop height and root length in the transgenic and wild-type plants under different concentration of the MV solution stress.Datas show that the seed germination rate has the minor change under the strong oxidant treatment compared to wild type and BraLTP2 over-expression transgenic plants,however,the results of seedling growth traits did not reach the statistically significant level.After determined five kinds of major antioxidant enzymes activity,the results of 35S::BraLTP2 transgenic plant leaves,we find the activity of antioxidant enzymes(CAT,APX,POD and SOD)were significantly improved,and it is the further proof to support the antioxidant function of BraLTP2 gene. |
PDF Full Text Request