Identification Of Laccase Genes From Poncirus Trifoliata Under Salt Stress And Regulation By Exogenous Methyl Jasmonate

Trifoliate orange(Poncirus trifoliata L.)is one of the commonly used rootstocks in citrus,has strong cold tolerance and disease resistance,but the salt tolerance of citrus is relatively weak,limiting the geographical range of citrus production,so new cultivars with better salt tolerance are urgently needed in production.Laccase is a copper containing polyphenol oxidase whose main function is to participate in the process of lignin monomer polymerization together with peroxidase,which is important for plant growth and resistance to abiotic stresses.A previous study found that sweet orange laccase gene was significantly expressed in response to multiple stresses and its promoter element contained numerous methyl jasmonate responsive elements,but the role of trifoliate orange laccase gene,methyl jasmonate(MeJA),in response to salt stress was temporarily unclear.Therefore,in this study,we intended to mine and clone the key laccase gene of trifoliate orange in response to salt stress,and to initially explore its salt tolerance function and the alleviating effect of MeJA on the stress of trifoliate orange by transgenic means.The main results are as follows:A total of 20 PtrLACs were identified,which were distributed on 5 chromosomes and unassigned contigs.The PtrLACs were predicted to localize to the cell membrane and the nucleus.The PtrLACs have 6-14 exons and 9-11 domain.Phylogenetic analysis showed that the PtrLACs were divided into 7 groups.Synteny analysis revealed 19 paired collinearity relationships between P.trifoliata and Arabidopsis thaliana.The PtrLACs contained 24 kinds of cis-acting elements,among which anaerobic induction elements,drought response elements and methyl jasmonate responsive elements were dominant.The expression profiles revealed that 16 PtrLACs were significantly upregulated under salt stress,with PtrLAC9 showing the highest fold change.It was speculated that PtrLACs was involved in the plant response to salt stress.PtrLAC9 was cloned,whose full-length cDNA was 1704 bp,encoding 568 amino acids.Overexpression of PtrLAC9 transgenic sweet orange calli and Poncirus VIGS lines were obtained by Agrobacterium mediated transgenic technology and subjected to positive and negative bidirectional salt tolerance analysis.The results showed that overexpression of the transgene in sweet orange calli significantly improved salt tolerance and interference in the salinity tolerance of PtrLAC9 aurantium significantly decreased.It can be seen that the PtrLAC9 gene is indeed involved in a regulatory grid of salt stress responses in plants and plays an important role in regulating salt tolerance in plants.The leaf surface of trifoliate orange was subjected to salt stress by 400 μM of MeJA,the determination of physiological indexes at multiple time points was performed,and the effect of MeJA on the stress of citrus aurantium was investigated according to the temporal variation trend of each physiological indicator.The results showed that the application of MeJA under salt stress significantly increased the chlorophyll content,osmoregulatory substances content,antioxidant enzymes activity,laccase activity and lignin content and significantly decreased the lipid peroxidation index of citrus aurantium leaf membranes.Overall,exogenous MeJA alleviated the damage caused by salt stress on trifoliate orange plants to some extent,according to the different treatment durations the alleviating effect of exogenous MeJA on trifoliate orange stress under salt stress can be seen,and with the increasing treatment time,the alleviating effect of exogenous MeJA on trifoliate orange stress gradually increased.