Identification And Function Analysis Of TILLING Mutants Of TaCKX4 And TaNAC1 Genes In Wheat

Cytokinin(CTK),a class of adenine derivatives,is one of the most important plant hormones involved in the regulation of plant growth and development.At present,cytokinin has been confirmed to have many important physiological functions by research,such as promoting plant cell division and differentiation,delaying senescence,increasing grain yield,regulating abiotic stress,etc.It can be irreversibly degraded by cytokinin oxidase/dehydrogenase(CKX)and has strong specificity.As one of the three major crops in the world,wheat is the main food source in China.The study of wheat CKX genes is of great significance for cultivating wheat varieties with high yield,high quality and strong adaptability,optimizing wheat germplasm resources and national food security.This study was based on the Jimai 22 TILLING mutant library which constructed in the early stage of the laboratory,and the effective mutation sites of TaCKX4 and TaNAC1 genes were investigated and identified in its whole exon capture sequencing database.Several physiological indexes and CKX-related gene expression levels were analyzed for some of the mutant lines which been successfully identified.The physiological functions and traits of each mutant line were preliminarily analyzed and compared,and the regulatory relationship between the TaCKX4 and TaNAC1 genes was summarized.At the same time,this study was also based on the HRM mutant screening system constructed and optimized in the early stage of the laboratory,with TaCKX1 as the target gene for screening applications.The main experimental results are as follows:1.Detection of mutation sites of TaCKX4 and TaNAC1 genes in Jimai 22 TILLING mutant lineIn order to study the function of wheat TaCKX4 and TaNAC1 genes,the corresponding mutant lines were searched in the whole exon capture sequencing database of Jimai 22 TILLING mutant population constructed by our laboratory,and obtained 6TaCKX4 mutant lines and 8 TaNAC1 mutant lines.Through further analysis and detection,4 TaCKX4 mutant lines and 6 TaNAC1 mutant lines were successfully identified,all of which had single base mutation sites of G→A and C→T.It laid the foundation for further analysis of the influence of wheat TaCKX4 and TaNAC1 gene mutations on wheat growth and the study of wheat CKX gene function.2.Physiological function analysis and gene expression analysis of TaCKX4 and TaNAC1 mutantsSome of the mutant lines(JM58,JM73,JM105,JM108,JM112,and JM286)that were successfully identified in the early stage of the experiment were selected for the analysis of physiological indicators and gene expression levels,and compared with the wild-type JM22.Wheat seedlings of different mutant lines were treated with 0,2,4 and 6mmol/L zinc sulfate solution,respectively,and measured the root growth,plant height,chlorophyll content,root cell viability found that:the effects of different concentrations of zinc sulfate on the root growth and plant height of wheat tended to be promoted by high concentration and inhibited by low concentration,and high-concentration zinc sulfate(6 mmol/L)had obvious effects on JM112 and JM286.The chlorophyll content decreased gradually with the increase of zinc sulfate solution concentration,and the chlorophyll content may increase under the treatment of 2 mmol/L zinc sulfate solution,it was also found that the basic chlorophyll content of mutant lines JM73 and JM108 was higher than that of other lines when treated with zinc sulfate free solution.Low concentration zinc sulfate solution(0,2 mmol/L)had little effect on the viability of wheat root cells,and JM58,JM73 and JM108 had better root cell viability than other lines.Wheat seedlings of different mutant lines were treated with 0%,20%,30%and40%PEG for about a week,and after measuring its maximum photosynthetic efficiency(F_V/F_m),photochemical quenching coefficient(q_p),and actual photosynthetic efficiency of PSⅡ(Y(Ⅱ)),it was found that:F_V/F_m,q_p,Y(Ⅱ)had the most significant decline under severe PEG drought stress(40%),the general trend was that the fluorescence parameters decreased with the increase of PEG drought stress concentration.The fluorescence kinetic parameters of JM22 and JM286 did not decrease significantly,and the drought resistance performance was good.Severe drought stress(40%PEG)was used to treat wheat seedlings of different mutant lines,after analyzing the changes in the expression of TaNAC1 and TaCKX4 genes at 0,0.5,1,2,and 12 h,it was found that:within 12 hours of severe drought stress,the expression of TaNAC1 gene in all wheat lines was slightly down-regulated and then returned to normal level,while the expression levels of TaCKX4 and TaCKX5 genes were significantly up-regulated.Through the analysis of the above physiological indexes and gene expression,it was preliminarily found that wild-type JM22,mutant JM73 and JM105 had better drought resistance,which could be used as improved wheat germplasm resources to further explore their potential.3.Preliminary application of HRM screening mutant platformBased on the HRM mutant screening platform established and optimized in the early stage of the laboratory,with TaCKX1 related to wheat yield traits as the target gene,and 8 randomly selected Jimai 22 TILLING mutant lines were screened.The G→A and A→G single-base mutations were successfully screened in the 7A019 and 7A106 mutant lines respectively.The screening application of the HRM mutant screening platform to the TaCKX1 gene verified the operation of the platform and provided technical support for the subsequent screening of other genes in the TILLING mutant library.