Study On The Biological Functional Characterization Of Lycopene Epsilon-cyclase Gene In Tobacco

The existence of highly homologous copies is a thorny problem in process of functional research of the gene in polyploid plants.The functional variation of different homologous copies and whether they obtain new function have long been a research hot spot.In this paper,we will combine gene editing technology,bioinformatic,metabolomic,plant physiology and other technologies to analyze tobacco lycopene epsilon-cyclase gene members Nt?-LCY1 and Nt?-LCY2 in allotetraploid tobacco K326(Nicotiana tabacum)features.At the same time,the biological functions of Nt?-LCY1 and Nt?-LCY2 genes under high light stress will be studied.The main results are as follows:(1)Bioinformatics analysis showed that allotetraploid tobacco Nt?-LCY1 and Nt?-LCY2 were derived from the ancestral Nicotiana sylvestris and Nicotiana tomentosiformis.The homology of Nt?-LCY1 and Nt?-LCY2 genes was up to 87.95%in coding sequence.Besides,both Nt?-LCY1 and Nt?-LCY2 were closely related to solanaceae plants such as tomato,potato and so on.There were significant differences in terms of structure,ligand binding sites and enzyme active sites between Nt?-LCY1 and Nt?-LCY2 proteins.Nt?-LCY1 and Nt?-LCY2 genes also differed in promoter cisacting element,The common ground was that both Nt?-LCY1 and Nt?-LCY2 genes contained several light-responsive elements.(2)Tissue expression pattern analysis showed that Nt?-LCY1 and Nt?-LCY2 genes were expressed in root,stem,leaf,flower,young fruit.Besides,it was obviously that they had the highest expression level in leaves,and Nt?-LCY2 gene had the higher expression level in leaf tissue compared with Nt?-LCY1 gene.(3)The homozygous mutants of Nt?-LCY1 and Nt?-LCY2 genes were successfully created by gene editing technology.In order to avoid off-target effect of gene editing,phenotypic statistics were carried out in two parallel lines in this study.The phenotypic statistics showed that the mutations of Nt?-LCY1 and Nt?-LCY2 genes could promote the growth and development of tobacco leaves,but the latter had more significant effects.(4)Metabolism analysis showed that the mutations of Nt?-LCY1 and Nt?-LCY2 genes could increase the contents of chlorophyll and carotenoids in tobacco leaves to varying degrees.(5)Nt?-LCY1 and Nt?-LCY2 genes had different response patterns.The expression of Nt?-LCY2 gene was inhibited in nt?-lcy1-1 under normal light;but it was induced in nt?-lcy1-1 under high light for 2 hours.The expression of Nt?-LCY1 gene in nt?-lcy2-1was inhibited under normal light and high light for 2 hours.(6)Compared with nt?-lcy1-1,the mutation of Nt?-LCY2 gene had a more significant effect on the expression of upstream and downstream related genes in the carotenoid synthesis pathway under normal light or high light stress.(7)Mutations in both Nt?-LCY1 and Nt?-LCY2 genes led to the improvement of photosynthetic efficiency and photoprotective ability of tobacco plants,and the order of their photosynthetic capacity and photoprotective capacity was nt?-lcy2-1>nt?-lcy1-1>WT.(8)Mutations of Nt?-LCY1 and Nt?-LCY2 genes could reduce the accumulation of reactive oxygen species to varying degrees,and the mutation of Nt?-LCY2 gene was more conducive to improve the antioxidant capacity of tobacco plants.In summary,the expression patterns and biological functions of the Nt?-LCY gene members Nt?-LCY1 and Nt?-LCY2 showed some characteristics.The mutation of Nt?-LCY2 gene was more conducive to increase the content of chlorophyll and various carotenoid components,and enhanced the stress resistance of tobacco plants under high light stress.This study will help to analyze the genetic and functional characteristics of homologous copies of ?-LCY genes in polyploid plants,and provide references for genetic improvement of polyploid crops.