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Expression Analysis And Promoter Methylation Of TaGAPC1 In Wheat

Posted on:2017-06-03Degree:MasterType:Thesis
Country:ChinaCandidate:Y FeiFull Text:PDF
GTID:2370330512951653Subject:Botany
Abstract/Summary:PDF Full Text Request
To analyze the functon of TaGAPC1 under abiotic stress and investigate the function of promoter methylation in this process,taking two wheat genotypes with contrasting drought resistance as materials,we cloned the CDS sequences and promoter squences of TaGAPC1 from two wheat genomes by homology-based cloning.Then rename the gene by predicting subcellular localization,analyzing the phylogeny and constructing the three-dimensional model of protein;Then detected the transcription level of TaGAPC1 when the different concentrations of 5-azaC used to treat with wheat seedlings,and tested the phenotype and photosynthetic rate changes in wheat seedlings under 5-azaC treatment;Subsequently,we detect the expression levels of gene under PEG,Na Cl,ABA and combined with 5-azaC treatment using q RT-PCR.Furthermore,the cis-acting elements related to stress response were analyzed by bioinformatics software;Finally,the methylation patterns were detected under PEG and Na Cl treatment by Bisulfite Sequencing(BSP)and Nested PCR.The results are as follows:1.The CDS sequence of TaGAPC1 in 1014 bp and promoter sequence in 972 bp were cloned from two wheat genomes,which showed a sequence similarity with 98%.The prediction of subcellular localization and phylogenetic analysis showed that this gene was 65.2% likelihood of cytoplastic,belonged to one clade of plant GAPCs in phylogenetic tree with At GAPC,and shared a similar three-dimensional structure with Os GAPC.Then the cis-acting elements related to stress response were found in this two promoters by bioinformatics software.Thus it was renamed of TaGAPC1,and submitted to Gen Bank.2.The different concentrations of 5-azaC used to treat with wheat seedlings to detected the transcription level of TaGAPC1 by q RT-PCR.The results showed that the transcription level of TaGAPC1 was changed most marked under 50?M concentrations of 5-azaC.Thus to treat wheat seedlings with 50?M 5-azaC and measured the phenotype and photosynthetic rate.The results showed that 50?M 5-azaC could promote the wheat plant height,root length,leaf area,biomass,leaf photosynthetic rate and transpiration rate.3.The expression patterns of TaGAPC1 were investigated under three abiotic stress,and observed the expression level under combined treatment with 5-azaC.The results showed that TaGAPC1 was strongly induced by this three abiotic stresses in drought-tolerant wheat Changwu134,but hardly induced in drought-sensitive wheat Zhengyin1.It was most strongly and rapidly induced by ABA treatment in both two wheat.In addition,the combined treatment with 5-azaC could promote the expression of TaGAPC1 under Na Cl stress in Changwu134 and PEG stress in Zhengyin1.4.The methylation patterns of promoter region were detected under PEG and Na Cl treatment by Bisulfite Sequencing(BSP)and Nested PCR.The results showed that it was hypermethylation in the upstream region of promoter and hypomethylation in the downstream of promoter.The total methylation level of promoter and CG context in percentage was decreased significantly under PEG stress in Changwu134,and CHG context was demethylated significantly by Na Cl stress.While all of the cytosine methylation were non-significantly impacted by PEG and Na Cl treatment in Zhengyin1.And the statistical results showed that the methylation frequency of cis-acting elements were decreased by stress in Changwu134 but non-significantly impacted by stress in Zhengyin1.
Keywords/Search Tags:Triticum aestivum, 5-azaC, GAPC, abiotic stress, promoter, methylation
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