| Based on the heat stress-responsive transcriptome analysis of filling stage flag leaf tissue from wheat variety(Triticum aestivuml L.C S),we screened 2,429 candidate genes about heat stress response.Then based on the results of transcriptome analysis,we selected two genes(TaG1 and TaG12)which strongly induced by high temperature,and one gene(TaG16)which negatived by high temperature to depth study.In order to explore the response mechanism of wheat under high temperature stress from the genes angle,and get excellent heat stress related genetic resources,to provide guidance and lay the foundation for improved wheat varieties resistant traits,we studied these three genes by analysis of the genomic and cDNA and amino acid sequence,analysis of tissue expression and high temperature stress responses,plant expression vector construction and genetic transformation.Specific findings are as follows:1.In order to obtain a large amount of heat stress-related candidate genes resources,we constructed a heat stress-responsive transcriptome sequencing library of filling stage flag leaf tissue from wheat.Sequence analysis showed that the percentage of clean tags of control and treatment groups of the total sequences were respectively 96.74%and 96.63%,and the number of the detected genes in control and treated samples has been basically saturated,folly compliant transcriptome sequencing analysis needs.High temperature stress-response genes analysis showed that 1058 genes were upregulated by heat stress and 1371 genes were inhibited by high temperature of the detected 93,003 genes.It provided a number of excellent genetic resources to study the mechanism of heat resistance in wheat.2.In order to explore the response meehanism of wheat under high temperature stress from the genes angle,we selected two genes(TaG1 and TaG12)which strongly induced by high temperature,and one gene(TaG16)which negatived by high temperature to depth study.RT-PCR were used to clone TaG1,TaG12 and TaG16,respectively.FQ-PCR assay were performed to examine tissue expression of TaG1,TaG12 and TaG16 and expressing pattern during high temperature.Sequencing and structural analysis showed that TaG1,TaG12 and TaG16 were 1071 bp,1297 bp and 1756 bp in full length genome,and encoding 139,323 and 360 amino acids,respectively.Homology analysis showed that the deduced amino acid sequence of TaG1 shared 100%,89%and 83%identity with photosystem Ⅱ 10 kD polypeptide from Equisetum,Brachypodium and Rice.the deduced amino acid sequence of TaG12 shared 66%and 60%identity with unknown protein from Rice and Aegilops.the deduced amino acid sequence of TaG16 shared 83%and 99%identity with caffeic acid-O-methyl-transferase protein from Ryegrass and Aegilops.Tissue expression analysis showed that TaGI,TaG12 and TaG16 were constitutively expressed in wheat tissues including roots,husk,stems,leaves and seeds.High temperature stress response analysis showed that TaG1 and TaG12 was up-regulated expression pattern,and TaG16 was inhibited by high temperatures.3.In order to research the function of these three heat candidate genes,we constructed plant expression vectors,transformed into Arabidopsis thaliana by Agrobacterium mediated gene transfer technology and identified phenotype of seed germination at high temperature(45℃).The results showed that three plant expression vectors pCAMBIA 3301::TaG1,PCAMBIA 3301::TaG12 and pCAMBIA 3301::TaG16 were constructed successfully.RT-PCR and sequencing confirmed that the foreign gene was integrated into the genome of Arabidopsis,and have got 15,20 and 23 transgenic plants of TaG1,TaG12 and TaG16,respectively.Transgenic plants seeds germination rate at heat stress analysis showed that gene transfer TaG1 Arabidopsis seed germination of wild-type Arabidopsis thaliana "Col-o" no significant difference in the rate of seed germination,instead,transgenic of TaG12 and TaG16 germination rates were higher than "Col-o" by 29.1%and 23.9%,respectively. |
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