Identification And Expression Pattern Analysis Of TaDEAD-box Gene Family In Response To Low Temperature In Winter Wheat

Wheat(Triticum aestivum L.)is one of the important crops widely planted all over the world.In China,the varieties of wheat can be divided into winter wheat and spring wheat.The cultivation of winter wheat is not common in Heilongjiang,due to the low temperature in the high-cold area preventing it from overwintering and growing normally.New winter wheat varieties Dongnong Dongmai 1(Dn1)and Dongnong Dongmai 2(Dn2)are strong cold-resistant(tolerant to low temperature of-30℃)varieties which are independently developed and cultivated by Northeast Agricultural University and they are important materials for studying cold resistance of plants.DEAD box RNA helicase is a key enzyme in RNA metabolism pathway,which is involved in almost all RNA metabolism activities,including transcription,translation,m RNA precursor splicing,ribosomal biogenesis,m RNA nuclear cytoplasmic export and RNA nonsense decay.Because of its diverse functions in RNA metabolism,dead box RNA helicase widely regulates plant growth and development and stress resistance.The study of TaDEAD-box helicase involved in wheat response to abiotic stress has not been reported yet.In this study,we used bioinformatics methods to screen,identify and analyze the TaDEAD-box gene family in wheat.According to the low-temperature transcriptome database obtained in the previous laboratory,seven significantly differentially expressed TaDEAD-box genes were screened out.The expression levels of seven TaDEAD-box genes in tillering node and leaf of three winter wheat varieties(two strongly and one weakly cold resistant)with exogenous ABA treatment at different sampling temperatures(5℃,0℃,-10℃ and-25℃)were analyzed.The Ta RH7 gene with significant differential expression at low temperature was selected for cloning and the expression vector was constructed to transform Arabidopsis thaliana,which laid the foundation for the functional study of the wheat TaDEAD-box genes.The main findings are as follows:(1)Using 60 rice Os DEAD-box genes as references,134 wheat TaDEAD-box genes were screened and identified.The analysis of physical and chemical properties showed that the amino acid number of the wheat TaDEAD-box family protein was between 408-1276,and the average number of amino acids was 633.The highest isoelectric point was 10.17 and the lowest was 4.99.Subcellular localization analysis revealed that 78 DEAD-box proteins were located in the nucleus,19 DEAD-box proteins were located in the extracellular matrix,9 DEAD-box proteins were located in the plasma membrane,21 DEAD-box proteins were located in the cytoplasm,6DEAD-box proteins were located in the mitochondria,and 1 DEAD-box proteins was located in the chloroplast.The sequence uniformity of the TaDEAD-box gene family screened with the Os DEAD-box gene family in rice as the reference was higher than that of the Arabidopsis At DEAD-box gene family as the reference screen;Interspecific phylogenetic tree analysis showed that wheat and rice had closer affinity for the same kind of dead box gene;interspecific collinearity analysis showed that there were more collinearity lines between wheat and rice DEAD-box gene family genes than wheat and Arabidopsis thaliana.The above results indicated that the relationship between the TaDEAD-box gene family in winter wheat and the Os DEAD-box gene family in rice was higher than that of the At DEAD-box gene family in Arabidopsis.The results of intraspecific collinearity analysis showed that there was a collinearity crossover between chromosome 4 and chromosome 5,and between chromosome 2 and chromosome 6,which indicated that there were heterotopic mutations in TaDEAD-box genes in wheat.Further analysis showed that the heterotopic mutation sites were relatively conservative,and the distributions of heterotopic mutation sites on non-homologous chromosomes were similar,which may play a role in promoting the evolution of TaDEAD-box gene family in wheat.(2)Analysis of TaDEAD-box gene expression in different organs(tillering nodes and leaves)showed that the expression levels of Ta RH7,Ta RH30,Ta RH20,Ta RH10-2 and Ta RH5 in the leaves of the three wheat varieties were higher than those in the tiller nodes.Among them,the expression of Ta RH10-2 in Dn1 and Dn2 leaves increased by about 55 folds and 127 folds,respectively,compared with that at 5°C.(3)The results of TaDEAD-box gene expression analysis of different wheat varieties showed that most of Dn1 gene expression peaks were concentrated at-10℃,while most Dn2 gene expression peaked at-25℃.There were four TaDEAD-box genes(Ta RH7,Ta RH10-1,Ta RH10-2and Ta RH35)in the Dn1 tillering node.The expression of Ta RH7,Ta RH10-1,Ta RH10-2 and Ta RH35 peaked at-10℃.Among them,the expression of Ta RH35 changed most significantly at-10℃,and there were four TaDEAD-box genes(Ta RH30,Ta RH20,Ta RH10-2 and Ta RH5)in leaves of which the expression peaked at-10°C,and the expression of Ta RH10-2 changed most significantly at-10°C.There were five TaDEAD-box genes(Ta RH7,Ta RH30,Ta RH20,Ta RH10-2and Ta RH5)in the Dn2 tillering node.The expression levels peaked at-25℃.Among them,the expression level of Ta RH7 changed most significantly at-25℃,and there were six TaDEAD-box genes(Ta RH7,Ta RH30,Ta RH20,Ta RH10-1,Ta RH10-2 and Ta RH5)in leaves of which the expression peaked at-25°C,and the expression of Ta RH10-2 changed most significantly at-25°C.(4)ABA increased the expression of five TaDEAD-box genes(Ta RH7,Ta RH30,Ta RH20,Ta RH10-2 and Ta RH5)in Dn1 tillering nodes and four TaDEAD-box genes(Ta RH30,Ta RH20,Ta RH35 and Ta RH5)in Dn2 tillering nodes,but limited the overexpression of five TaDEAD-box genes(Ta RH7,Ta RH30,Ta RH20,Ta RH10-2,and Ta RH5)in Dn1 and Dn2 leaves.The expression level of TaDEAD-boxs of J22 at 5℃ was basically the same as that of Dn1 and Dn2,but as the temperature decreased,the overall expression level of TaDEAD-boxs in J22 was lower than those in Dn1 and Dn2(except the expression level of Ta RH35 at-25℃ which was higher than Dn1 and Dn2).