Studies On Molecular Characterization And Function Analysis For Inverse Resistance Gene TaCaM1 And TaMAPK3 In Wheat

In order to adapt to the changeable external environment,plants have evolved a set of molecular mechanisms to resist the abiotic stresses,by using biological processes associated with signal perception,signaling transduction,and downstream transcriptional regulation and response.Among them,calcium signal transduction pathway and mitogen activated protein kinase(MAPK)cascade play a crucial role in plant response to external stresses.In this study,the molecular characteristics and stress tolerance of wheat resistant genes TaCaM1 and TaMAPK3 were studied.The main research results are as follows:1.The nucleotide sequence and coded amino acid sequence of wheat calcineurin gene TaCaM1 were analyzed and predicted using bioinformatics methods,such as those for the phylogenetic analysis,homologous amino acid sequence alignment,transmembrane helical domain analysis,tertiary structure prediction,and subcellular localization prediction.The nucleotide sequence of TaCaM1 is 759 bp,encoding 149 amino acids that does not contain transmembrane helical domain.The predicted subcellular localization of TaCaM1 was mainly onto the cytoplasm,with a small rate onto nucleus.2.Analysis on the expression of TaCaM1 under different stress conditions suggested that the transcripts of TaCaM1 were shown to be decreased under low P stress and to be a slight increased under stresses of low N,NaCl,and PEG.3.Using DNA recombination technology,the positive and antisense binary expression vectors integrated with TaCaM1 and those fused TaCaM1 and TaMAPK3 for subcellular localization detection were constructed.Transgenic tobacco and wheat lines with overexpression of TaCaM1 were obtained by agrobacterium-mediated transgenic transformation approach.The subcellular localization of TaCaM1 and TaMAPK3 was detected by observing green fluorescence signals in transgenic tobacco lines.Results indicated that TaCaM1 was located in the cytoplasm and nucleus,whereas TaMAPK3 was located in the nucleus.4.Based on soil culture and hydroponic methods,the functions of sense and antisense TaCaM1 transgenic tobacco lines in mediating plant low P stress tolerance were investigated.Under low P stress condition,compared with wild type tobacco,the sense tobacco plants displayed phenotypes of weak,short root,small leaf area,low chlorophyll content,and reduced fresh and dry weights.In contrast,the antisense tobacco plants were shown to be flourish,long root length,enlarged leaf area,high chlorophyll content,and increased fresh and dry weights;Under the stresses of low nitrogen,salt and drought,the sense tobacco plants grew more bloom than those of the wild type,with long root,large leaf area,high chlorophyll content and significant big fresh and dry weight.These results indicated that TaCaM1 mediates plant adaptations to stresses of low phosphorus,low nitrogen,salt and drought.5.Combined with the previous work of the research group,the stomatal characteristics of TaMAPK3 transgenic tobacco lines were observed and analyzed under different stress conditions.The results showed that under NaCl stress treatment,the TaMAPK3 transgenic tobacco promoted stomatal closure by increasing the ABA content.6.Using high-throughput sequencing technology,the transcriptome of TaMAPK3 transgenic tobacco and wild-type tobacco treated by 48 h of low P stress was analyzed,and the GO function enrichment and KEGG metabolic pathway enrichment of the two differentially expressed genes were explored.The results showed that the GO functions of the differential genes were mainly concentrated on the response to injury and the regulation of various metabolic enzymes.The KEGG metabolic pathways are mainly concentrated on isoquinoline alkaloid biosynthesis,distyrene,diarylheptanoic acid and gingerol biosynthesis,amino acid biosynthesis,tyrosine metabolism and pyruvate metabolism.These results suggestsed that TaMAPK3 regulates the corresponding metabolic processes and responds to low P stress by enrichment of related stress genes.7.The interaction between TaMAPK3 and TaWRKY12 was screened by yeast double hybridization and bimolecule fluorescence complementary technique.This suggests that TaMAPK3 may exert stress-defensive function by regulating downstream WRKY transcription factor genes.