Screening Of Citrus Laccase Genes Related To Cold Stress And Functional Characterization Of CsLAC18 In Cold Resistance

Abiotic stress seriously affected the yield,quality and cultivation range of cultivated citrus,among which low temperature was one of the important reasons.Genetic engineering is an effective method to create cold-resistant citrus germplasm,and it is an important basis and prerequisite to analyze the mechanism of cold response and identify key cold-resistant genes by using cold-resistant resources.Laccase(LAC)is a copper-based glycoprotein oxidase involved in lignin synthesis and stress response in higher plants.At present,the role of laccase in cold resistance of citrus is not clear.In this study,the expression of laccase gene family under low temperature stress was analyzed.On this basis,function identification and mechanism analysis of CsLAC18 were carried out.The main results are as follows:1.Sweet orange seedlings were treated with low temperature stress(4℃)to analyze the changes of laccase activity and lignin content in sweet orange leaves.The results showed that the activity of laccase was up-regulated by low temperature,and the lignin content increased accordingly.The expression patterns of laccase gene family members at low temperature were studied by real-time quantitative PCR.The results showed that most laccase genes of sweet orange responded to low temperature stress,but showed different expression patterns,among which CsLAC18 was the most highly up-regulated.CsLAC18 is 1743 bp long and encodes 580 amino acids.Subcellular localization analysis indicated that the gene was located on the cell membrane.2.In order to verify the cold resistance of CsLAC18,overexpressed tobacco and gene silencing immature orange were obtained by transgenic method.Tobacco plants with overexpression of CsLAC18 showed higher tolerance under low temperature stress.Compared with wild-type tobacco overexpressed plants,it has low conductivity,MDA and ros content,high photosynthetic efficiency,survival rate,antioxidant level and laccase activity.On the contrary,CsLAC18 silencing line has higher sensitivity to low temperature stress.It can be seen that CsLAC18 may play a positive regulatory role in the response of citrus to low temperature.3.The promoter sequence of CsLAC18 was cloned,and the analysis of promoter elements showed that CsLAC 18 promoter contained several transcription factor binding elements.Multiple possible regulatory genes upstream of CsLAC18,such as Cs4g02630,were obtained by yeast single hetero sieve library technique.