Eexpression Analysis Of The CsLac Gene Family And CsLac29 In Camellia Sinensis L.in Response To Leafhopper Feeding

The tea tree（Camellia sinensis（L.）O.Kuntze）originated in China and is an important perennial cash crop in the country,while the small green leafhopper（Empoasca onukii Matsuda）is an important pest on tea trees,seriously affecting tea production and quality.The current control of leafhoppers relies mainly on chemical pesticides and the resulting pesticide residues in tea are a major problem.Therefore,it is important to explore the main insect resistance genes in tea trees and to investigate their role and mechanism in protecting tea trees,in order to further select insect-resistant tea tree varieties by molecular means.Laccase（LAC）is an important plant defence enzyme that plays an important role in the phytophagous insectinduced plant defence response.In this study,bioinformatics analysis,promoter cis-regulatory element analysis and q RT-PCR analysis of the tea tree laccase family were performed to analyse these genes under normal growth and small green leafhopper stress.The study focused on one laccase gene,Cs Lac29,and explored the mechanism of tea tree insect resistance defence mediated by feeding by the tea little green leafhopper.The main findings were as follows.1.A total of 38 members of the Cs Lac gene family were screened in the tea tree genome by bioinformatics analysis.The amino acid sequences of the tea tree laccase protein family were highly variable,with length variations ranging from 370-1146 amino acids.Tea tree laccase proteins are localised in the secretory pathway,they are extracellular proteins,and the structure of the exon and intron spacing is highly variable.The number of introns in this family of genes ranges from 4-7,and a phylogenetic tree constructed on the basis of the encoded products shows that the family members can be classified into a total of 6 clusters.2.Further analysis of Cs Lac family promoter cis-acting elements using the online software Plant CARE revealed the presence of several light-responsive elements and elements that respond to methyl jasmonate and salicylate hormone responses in the Cs Lac family promoter sequences.In addition,the functional elements of adversity response,including anaerobic induction response element,low temperature response element,defence and adversity response element and wound induction response element,suggest that Cs Lac genes are largely involved in defence responses in response to external environmental stimuli.3.The laccase family genes were subjected to q RT-PCR with different mature leaves of tea tree to detect the expression of laccase in tea tree leaves under normal growth and to measure their lignin content and m-phenylene triol staining.The results showed that the laccase family was mainly expressed in leaves,while the lignin content assay showed higher lignin content in stems than in leaves.The results of lignin content and laccase expression patterns in different tissues of tea trees were different,indicating that the laccase genes were not significantly expressed in normal growing tea tree tissues.To investigate the expression pattern of the Cs Lac family under the feeding of the tea leafhopper,it was detected that the Cs Lac family was all induced to different degrees by the feeding of the leafhopper,and the expression of all family members was highest on day 7 in leaves that had been fed on by the leafhopper,and varied between members at other times.4.The sequence analysis showed that the sequence of Cs Lac29 was identical to the sequence of the corresponding genome TEA028448 and the predicted coding region sequence in the Tea Tree Genome Database,and the length of Cs Lac29 open reading frame was 3335 bp.encoding a total of 1040 amino acid residues with an isochronous point of 9.42.The PGEX-Cs Lac29 expression vector was constructed to achieve the prokaryotic expression of Cs Lac29,and the results showed that the size of Cs Lac29 protein was basically consistent with the size of the predicted protein molecular weight（80.53 k Da）.5.The expression of Cs Lac29 in tea leaves at different time points after feeding by the tea little green leafhopper was examined by q RT-PCR in a small green leafhopper stress experiment on tea trees.The expression of Cs Lac29 in tea leaves was significantly increased by Cs Lac29 feeding,and the lignin content of tea leaves was significantly increased by Cs Lac29 feeding.The lignin staining by methanetriol also showed that the lignin staining area of the leaves fed on by the leafhopper was higher than that of the non-fed control.The lignin content of tea tree leaves under L.minor stress increased compared to the control,and laccase genes would be involved in the lignin synthesis pathway.It is therefore hypothesized that Cs Lac29 can play an important role in the defence of tea trees against the feeding damage caused by the tea leafhopper.