Plant Cell Wall Degrading Enzymes In The Gut Of Eucryptorrhynchus Scrobiculatus And Eucryptorrhynchus Brandti

Eucryptorrhynchus scrobiculatus and E.brandti are both wood-boring pests of afforestation tree species Ailanthus altissima,and have caused the death of a large number of Ailanthus altissima.Plant cell wall degrading enzymes are the key to feeding on A.altissima and digesting lignocellulose for two weevils.In this paper,a series of studies were carried out on the endogenous plant cell wall degrading enzymes of two weevils and plant cell wall degrading enzymes of their gut microbiota.Through 16 S r RNA sequencing,the diversity of gut microbiota of two weevils was analyzed,the dominant population was determined,and the role of the dominant population was analyzed;through transcriptome sequencing of the midgut,plant cell wall degrading enzyme genes of two weevil were identified,and the phylogenetic analysis and expression profile analysis of plant cell wall degrading enzyme genes of E.scrobiculatus were carried out;14 plant cell wall degrading enzyme genes of two weevils were expressed in Escherichia coli.The main research results are as follows:1.The composition of the gut microbiota of two weevils was clarified.16 samples of two weevil species and soil were annotated to 39 phyla,61 classes,135 orders,272 families,and 556 genera of bacteria.At the phylum level,Proteobacteria and Firmicutes are the dominant phyla in the gut microbial of two weevils.At the genus level,the dominant genera in the gut of E.scrobiculatus larvae are Lactococcus,Wolbachia,Enterobacter and Bacillus;The dominant genera in the gut of E.scrobiculatus adult are Wolbachia,Chelonobacter and Lactococcus;The dominant genera in the midgut of E.scrobiculatus adult are Wolbachia,Nardonella,Enterococcus and Lactococcus;The dominant genera in the midgut of E.brandti adult are Wolbachia,Cyanobacteria and Enterobacter;The dominant genera in soil are Pseudomonas,Flavobacterium and Stenotrophomonas.2.The best reference genes for different tissues of E.scrobiculatus were clarified.18 candidate reference genes were identified from midgut transcriptome of E.scrobiculatus,and the expression levels of these reference genes were estimated by quantitative real-time fluorescent quantitative PCR(RT-q PCR).Four algorithms(ge Norm,Norm Finder,Best Keeper,and delta Ct)were used to analyze the differences of expression levels,and Ref Finder was used for comprehensive analysis.The expression levels of RPL13,RPS3 and RPL36 were the most stable in different tissues,which were suitable for use as reference genes in different tissues of E.scrobiculatus.In addition,the expression profile of the target gene GH45(glycoside hydrolase family 45)confirmed the reliability of the selected candidate reference genes.This study provided a set of appropriate reference genes for gene expression studies in different tissues of E.scrobiculatus.This study will be helpful to the further study of transcriptomics and functional genes of E.scrobiculatus.3.The midgut transcriptome sequencing analysis of E.scrobiculatus and E.brandti was completed.We identified 25 putative PCWDEs(GH48,GH45,GH9,GH28,CE8,and PL4)from the midgut transcriptome of E.scrobiculatus,including 11 cellulases and 14 pectinases.And we identified23 putative PCWDEs(GH48,GH45,GH9,GH28,CE8,and PL4)from the midgut transcriptome of E.brandti,including 8 cellulases and 15 pectinases.Through the phylogenetic tree constructed by maximum likelihood algorithm,the evolutionary relationship between PWCDEs genes of E.scrobiculatus and PWCDEs genes of other insects was studied,and the function of PCWDE gene of E.scrobiculatus was inferred.The expression patterns of 17 enzymes in midgut transcriptome of E.scrobiculatus were analyzed by RT-q PCR.The relative expression levels of 11 genes in the midgut and four genes in the proboscis were significantly higher than the relative expression levels in other tissues.Proboscis and midgut are related to the digestion of insects,and high expression levels indirectly indicate that these genes are related to digestion.4.Partial PCWDEs of E.scrobiculatus and E.brandtia were successfully expressed by prokaryotic expression.The full lengths of 14 PCWDEs genes of E.scrobiculatus and E.brandti were obtained by RACE PCR.6 PCWDEs of E.scrobiculatus and 8 PCWDEs of E.brandti were heterologously expressed by prokaryotic expression system to obtain recombinant proteins,and their activities were tested.This paper focuses on plant cell wall degrading enzymes in the gut of E.scrobiculatus and E.brandti.The plant cell wall degrading enzymes derived from gut microbiota and two endogenous weevils were studied separately,and their potential functions were analyzed.The results lay a foundation for further exploring the digestive process of two weevils and the differentiation of feeding sites.