Sequencing Of The Transcriptome And Cloning And Characterization Of The CcLys Gene In A. Chinensis

Coridius chinensis is an important insect used for both medicine and food in China,and belongs to Dinidoridae,Hemiptera.It has the functions of regulating qi,relieving pain,and promoting telotism.Lysozyme is a glycoside hydrolase,also known as muramidase,and is widely distributed in nature with many roles such as antibacteria,antiinflammation,antivirus,and digestion.In this study,the full-length transcriptome from C.chinensis was sequenced with the third generation sequencing technique.Based on this data,the cDNA of CcLys in C.chinensis was cloned and characterized,and expression patterns of this gene also analyzed.CcLys was expressed in Pichia pastoris,and the antibacterial activity of the expressed CcLys was detected.This study will lay a foundation for further clarifying the function of CcLys,developing new antimicrobial agents,and deeply excavating the C.chinensis resource.1.Sequencing and analysis of full-length transcriptome from C.chinensisThe full-length transcriptome of C.chinensis was sequenced by Pac Bio Sequel platform and55,021 transript isoforms were obtained,of which 41,258 transcripts belonged to known genes.Transcripts from C.chinensis showed a similarity of 79.77% with those from Halyomorpha halys.The 10,456 transcripts(19.00% of total)were categorized into 55 functional groups in the GO(gene ontology)database;14,680 transcripts(26.68% of total)were categorized into 25 functional groups in the COG(cluster of orthologous groups of proteins)database;and 27,752 transcripts(50.44% of total)were annotated to 44 metabolic pathways in the KEGG(Kyoto encyclopedia of genes and genomes)database.The 36,126 coding sequences(CDSs)and 19,353 simple repeated sequences(SSRs)were predicted from the transcripts,respectively.2.Cloning and characterization of CcLys from C.chinensisThe CcLys cDNA is 883 bp in length,containing a 669 bp open reading frame(ORF)that encodes 222 amino acids.CcLys contains a signal peptide composed of 18 amino acids at N-terminus,and the mature CcLys forms a three-demensional structure consisted of 7 ?-helices and 8 ?-pleated sheets.Homology and cluster analyses showed CcLys possesses the closest relationship with that of Halyomorpha haly.Gene expression analyses indicated that CcLys is expressed throughout the developmental stages of C.chinensis with the highest expression level in the 3rd instar numph and is expressed in the adult tissues tested with the highest expression level in the fat body.The m RNA epression level of this gene reached the maximum 12 h postinduction by bacteria.3.Secretory expression of CcLys and detection of antibacterial activity of this lysozymeAccording to the codon preference in P.pastoris,the nucleotide sequence of mmature CcLys protein was optimized and then synthesized,with cleavage sites of Xho I and Xba I at both ends of this sequence.After double enzyme digestion,the optimized sequence was cloned into the expression vector p PICZ?A to construct the recombinant expression vector p PICZ?A-CcLys.CcLys was expressed in P.pastoris GS115 and the expressed Cclys protein was secreted to medium.The antibacterial activity of the expressed product was tested and the results showed that the lysozyme CcLys has antibacterial activity against Bacillus subtilis.