Characteristics Analysis,Cloning And Function Research Of CaM/CML Gene From Triticum Aestivum

Calmodulin（CaM）and Calmodulin-like（CML）proteins are a primary family of plant-specific Ca²⁺ sensors.CaM/CML proteins have been identified in many plant species,such as the model plant Arabidopsis and rice.Ca M/CML can specifically bind to Ca2+ and transmit signals through the downstream binding proteins,which is widely involved in plant growth and development and response to various external stimuli.Based on the 232 wheat CaM/CML genes identified in the previous study,the bioinformatics method was used to analyze the gene characteristics.Then,the organ-specific expression pattern of 9 wheat CMLs and relative expression of m RNA under various treatments were observed by real-time PCR.And the subcellular localization of four CML proteins was determined.Finally,the overexpression vector of TaCML46-D was constructed and the function of the gene was preliminarily studied by transforming Fielder.The main results are as follows:（1）A total of 232 CaM/CML genes were identified in wheat,which were studied by bioinformatics analysis.These CaM/CML proteins ranged in length from78 to 1299 amino acids,with molecular weights ranging from 8.65 k Da to 139.62 k Da and the isoelectric points ranged from 3.70 to 9.63.Subcellular localization analysis indicated that all Ca M proteins are localized in the cytoplasmic;CML proteins are located in the cytoplasmic,extracellular,nuclear,periplasmic,chloroplast,outer Membrane,mitochondrial and plasma membrane.The 231 Ca M/CML genes are distributed on 21 chromosomes,while the gene TaCML70-U cannot be assigned to any chromosome.（2）A total of 9 CML genes of wheat including TaCML11,TaCML13,TaCML24,TaCML31,TaCML33,TaCML46,TaCML68,TaCML74 and TaCML75 were selected for tissue-specific detection.In addition to TaCML11 not expressed in seeds,TaCML13,TaCML24,TaCML31,TaCML33,TaCML46,TaCML68,TaCML74,and TaCML75 genes were expressed in the young roots,young shoot,and mature roots,stems,leaves,flag leaves,flowers,and seeds of wheat,but the expression abundance is different.The expression level of TaCML75 of the young shoot is 97.5 times that of the young root.（3）The response of the above nine TaCMLs to abiotic stress was examined.The expression of TaCML genes was induced or inhibited by Na Cl,PEG,cold and heat stress.The expression of different TaCML genes showed different responses to different abiotic stresses in different organs of wheat.Salt stress can induce the expression of TaCML31,TaCML33,TaCML46,TaCML74 and TaCML75 genes in wheat aerials,inhibit the expression of TaCML11,TaCML24 and TaCML68 genes,induce the expression of TaCML31,TaCML33 and TaCML75 genes in wheat roots,but inhibit TaCML13,TaCML24,TaCML46,Expression of TaCML68 and TaCML74 genes.Osmotic stress can induce the expression of the above-ground TaCML gene;the expression of TaCML11 and TaCML68 genes in wheat roots is inhibited.Cold stress can induce the expression of TaCML gene in the aerial part of wheat;inhibit the expression of TaCML24 and TaCML68 genes in wheat roots.Heat shock stress treatment can induce the expression of TaCML31 and TaCML74 genes in wheat aerial parts,inhibit the expression of TaCML11,TaCML13,TaCML24,TaCML33,TaCML46,TaCML68 and TaCML75 genes;induce wheat root TaCML11,TaCML24,TaCML31,TaCML33,TaCML46,TaCML68 and TaCML75 Gene expression,but inhibition of TaCML13 and TaCML74 gene expression.The expression of different TaCML genes in different tissues of wheat showed different responses to different abiotic stresses,suggesting that CML genes participate in different stress-tolerant signal transduction pathways in different tissues.Gene expression is affected by salt,infiltration,cold and heat shock stress and may play a role in the signal transduction process of wheat against stress.The TaCML74 in wheat shoot strongly responded to heat shock stress,the expression of which was 2622.0 times at 0 min at 30 min.The TaCML33 in wheat root strongly responded to cold stress.The TaCML75 in wheat root strongly responded to salt,osmotic and heat shock stress.The TaCML46 gene in wheat roots responded to osmotic,cold and heat shock stress,while the above-ground TaCML46 gene responded to salt,infiltration and cold stress.（4）By constructing a subcellular localization vector in which four calmodulin proteins TaCML11-A,TaCML31-A,TaCML33-A and TaCML46-D were fused with EGFP protein,four kinds of calcium-like tones were observed by agrobacterium infecting tobacco leaves.Proteins are expressed in the nucleus and cytoplasmic.（5）By constructing the TaCML46-D overexpression vectors and transforming the Fielder to obtain the overexpressing line OE1-OE11,and 7 overexpressing homozygous lines were obtained.The DNA and RNA levels of the overexpressing lines and the analysis of agronomic traits showed that the TaCML46-D gene overexpressing strain had premature ripening compared with the wild type,and the plant height and effective tiller number did not change regularly.