Genome-wide Identification And Characterization Of Laccase Gene Family And Resistance Study In Citrus Sinensis

Citrus is the first fruit tree with the highest planting area in China and the second highest yield.It has extremely high economic value.Laccase is a copper ion-containing glycoprotein oxidase,which plays an important role in the lignin synthesis and stress resistance of higher plants,and has broad application prospects.At present,the study of the function of laccase in citrus has not been reported,the gene expression and regulation mechanism of laccase in citrus is not clear.In this paper,we introduced the results of bioinformatics analysis of the laccase gene family of Citrus sinensis first;Furthermore,we cloned two promoters of genes with stress resistance.Moreover,the results of qRT-PCR based on the analysis of promoter cis-regulatory elements were described,which is expression characteristics of some members of the family genes in different tissues and organs,and expression characteristics under stress and hormone treatments under different stress conditions;Finally,the change analysis of the laccase activity of citrus under low temperature stress was described.The main results were as follows:1.Bioinformatics analysis indicate that there are 27 members of the Laccase gene in citrus genome,their physicochemical properties are significantly different from those of the encoded products.CsLACs clustered on chromosomes 6,7,and 8,and were not distributed on chromosomes 4,5,and 9.The phylogenetic tree constructed from the encoded products indicates that family members can be divided into 6 clusters.The amino acid sequences of the 27 members were similar,the structural of exons-introns are differents.The number of introns in this family is between 2 and 5.The promoter analysis showed that the promoter region of the CsLAC gene contains a large number of cis-acting elements that imply the promoters of this family may respond to external stimi such as light,temperature,moisture,hormones,microorganisms,and specate that the CsLAC gene may be involved various biological functions,such as citras morphological development,adaptation to biological and abiotic stress,etc.2.The promoters of CsLACl and CsLAC3 with specific functions were cloned by PCR.The promoter sequence of CsLAC1 was 1478 bp and that of CsLAC3 was 1534 bp.3.The qRT-PCR technique was used to analyze the expression level of CsLAC gene in different tissues of Volusia orange under different stress treatments.The results showed that the CsLAC gene has tissue-specific expression characteristics.Although gene expression was detected in roots,stems,leaves,and calli,most of the genes were highly expressed in citrus roots.The results also show that the CsLAC gene can be induced by a variety of hormones and abiotic stresses,which is consistent with the cis-acting elements of the promoter.For example,CsLAC1-01 and CsLAC1-02 were significantly up-regulated in light,hypothermia,ABA treatment,and SA treatment.CsLAC3 was significantly and highly expressed by low temperature and MeJA induction.CsLAC12 was significantly up-regulated by PEG treatment.CsLAC9,CsLAC10,and CsLAC19 were significantly up-regulated by high temperature induction.4.Citrus sinensis and Poncirus trifoliata seedlings was performed at 40C to analyze the changes of soluble protein content,laccase activity and REC of the two citrus materials.The results suggest that the laccase activity of Citrus sinensis and Poncirus trifoliata could be significantly up-regulated by low temperature induction.It was also found that the Poncirus trifoliata laccase activity and soluble protein content were consistently significantly higher than Citrus sinensis at all stages.The REC of Citrus sinensis was significantly higher than that of Poncirus trifoliata,indicating that the cold resistance of different citrus varieties was positively correlated with laccase activity and soluble protein content,and negatively correlated with conductivity.The results of this dissertation can further reveal the expression characteristics of laccase gene in citrus,especially its role in response to stress,and lay the foundation for improving the stress resistance of citrus using laccase gene.