Transcriptome Analysis And Identification Of Chitin Metabolism Pathway And Resistance Gene In Cnaphalocrocis Medinalis Guenee

The rice leaf roller,Cnaphalocrocis medinalis?C.medinalis?is a kind of significant rice pests.Due to its migrate character,it is difficult to control and break out in the middle and lower reaches of the Yangtze river area every year.At present,the control of the rice leaf folder mainly depend on chemical insecticide and influence environment and human life seriously.What's more,the pest resistance to insecticide greatly increased.Chitin as a linear polymer mainly exist in the cell walls of fungi,insect cuticle and peritrophic membrane,which plays a crucial role in insect against outside microorganism.Developing the target of pesticides through destroy insect chitin metabolism pathway will benefit to lay the foundation for controlling the rice leaf folder.So far,only few reports about chitin metabolism and insect resistance in the rice leaf folder.Due to the whole genome sequencing of the rice leaf folder has not been completed,the transcriptome sequencing become a new method in identifying new gene and approaching gene function.Especially in the absence of genomic information,available in transcriptional level of specific tissues,organs and individual genetic information,greatly promote the insect molecular biology research.In our study,the transcriptome sequencing of four instars larvae obtains a large number of reads,then gained Unigene by sequence assembly.Identify chitin metabolism related enzymes and metabolic pathway,screen drug resistance related genes on the basis of gene annotation,consequently laid the foundation for pesticides for the development of new targets and the research on drug metabolism.Fully studies the transcriptome data and identify functional SSR in order to obtain molecular markers for genetic research.The results are as follows:1.A total of 29,367,797 reads were obtained by using high-throughput sequencing technology Illumina HiSeq^TM2000 for the rice leaf folder,four instars larvae of the transcriptome sequencing and gained the corresponding number of Contig and Transcript according to Trinity software assembly,3,381,765 and 106,407 respectively.The use of paired end joining and gap-filling technology obtain 63,174 Unigene joining together,the bases for an average of 753 bp.In total,63,174 Unigene were submitted to NCBI database BLASTx and the threshold of E value was setted10^-5,among them annotations to the COG,GO,KEGG,Swiss-prot,Nr,respectively8,669,18,176,10,043,24,246,31,810.In total,32,035 Unigenes were annotated successful.2.Sixteen chitin metabolism related enzymes were identified according to BLASTx analysis,setting the threshold E value for 10^-5,including CHSA,CHSB,PGM,UNAP,NAGK,CHT1,CHT2,CHT3,CHT5,CHT7,Hex,CDA1,CDA2,CDA4,CDA5 and CDA6,the corresponding sequence had submitted to Gene Bank.The chitin metabolism pathway was obtained by KEGG pathway from transcriptome sequencing and referenced related reference.Three major genes were verified and tested in midgut,intugument,larvae and adult tissue by qRT-PCR,including CmCHS1,CmCDA1 and CmCHT1.The result showed that CmCHS1 has a high expression level in integument and adult expression more than larvae;CmCHT1has a high expression level in integument and larvae expression more than adult;CmCDA has a high a high expression level in midgut and adult expression level more than larvae.3.In total,378 insect resistance related genes were identified by analysis of BLASTx for C.medinalis,setting the threshold E value for 10^-5.It mainly can be divided into 15 categories : cytochrome P450,carboxylesterase,glutathione transferase,?-aminobutyric acid sodium,acetylcholine receptors,ion channels,acetylcholinesterase,water channel protein,catalase,calcium ion channels,chloride ion channels,glutathione peroxidase high methoxyl puling,superoxide dismutase and serine protease inhibitors.4.Take advantage of the data of the transcriptome sequencing and excavate the SSR molecular markers of C.medinalis.In total,10,394 SSR marker were obtained,5050 single nucleotide motifs,1,826 dinucleotide motifs,3,408 trinucleotide motifs,101 tetranucleotide motifs,6 pentanucleotide motifs and 3 hexanucleitide motifs.As described above,this study through C.medinalis four instar transcriptome sequencing and identified 16 kinds of chitin metabolism related enzymes,analysised chitin metabolic pathway,identified 378 insect resistance related genes and mined10,394 SSR molecular markers.All of these results will develop a foundation for molecular level study and pest control.