Transcriptome Anaylsis And Identification Of Stress-Responsive Genes In Citrus Whitefly Dialeurodes Citri (Ashmead)

Citrus whitefly Dialeurodes citri (Ashmead) is a worldwide-distributed insect pest, and it has caused great damage to citrus-producing areas. However, there is little researches conducted on this species.So far, no effective bioassay method has been extablished for this insect pest. Meanwhile, no molecular information of D. citri is available. Therefore, the need to monitor the insecticide resistance of citrus whitefly and investigate its molecular mechanisms is very urgent. Although the genomic information isabsent, we can get some tissues, organs, and genetic information of individuals at the transcriptome level with the next generation sequencing technology. Application of thehigh-throughputsequencing technology in the field of entomologicalresearchhas greatly promoted its progress.Here, our research focuses on the establishment the bioassay methods againstcitrus whitefly, screening of resistance related genes and mining microsatellite loci. Furthermore, the high-throughput sequencing technology was employed to obtain moremolecular of citrus whitefly. On the basis of gene annotation and parsing, the screening and identification of major stress resistance genes will lay the foundation for the development of novel drug targets and the understanding of the molecular mechanisms of the insecticide metabolism. The main findings are as follows:1. Improved leaf dip bioassay method was established to measure the susceptibility to seven different types of insecticide in citrus whitefly collected from field in Chongqing. The ranking of the toxicity is avermectins> fipronil> imidacloprid> spinosad> cypermethrin> chlorpyrifos> carbosulfan.2. The citrus whitefly transcriptome sequencing generated43075transcripts with total length of23238374bp and the average length is539bp. Using paired-end joining and gap-filling, the transcripts were further assembled into36766Unigene with the total length of18282349bp. To annotate these Unigenes, we searched against the non-redundant (Nr) NCBI protein database using BLASTX with a cutoff E-value of10-5. A total of16460distinct sequences returned a blast result. Besides the Nr database, there were11458Unigenes successfully annotated in Nt,14441in Swiss-Prot,7507in KEGG,4850in GO, and4850in COG. Taken together,17788Unigenes were annotated across these databasestotally. The result of GO classification showed that4850Unigenes were mapped to48functional groups. These functional groups were classified into three categories:namely "cellular component","biological process" and "molecular function".2978(61.40%) Unigenes were in "molecular function",1184(24.41%) in "biological processes", and688(14.19%) in "cellular components".5732sequences had a COG annotation and were mapped to25functional groups. Among the COG categories, most Unigenes (19.96%) were mapped to the "General function prediction". The second was "translation, ribosomal structure and biogenesis"(10.33%), the third was "post-translational modification, protein turnover, chaperones"(8.43%). The results of the annotations of GO and COG showed that most citrus whitefly genes bear with basic life activities related functions, biological control, metabolism, etc.3. By BLAST in Nr,117Unigenes related to detoxification were identified. They are mainly detoxification enzymes including Glutathione S-transferases (GSTs), carboxylesterase (CarEs) and cytochrome P450. There are15glutathione S-transferases belonging to sigma, delta, epsilon and omega families. Furhtermore, the49carboxylesterases belong to clade A, clade D and clade E family, respectively. In addition,53cytochrome P450belonging to CYP4, CYP3and CYP2family were identified. Meanwhile,67Unigenes coding hot-shock proteins were identified from the transcriptome of citrus whitefly. They encoded18sHsps,4Hsp60,36Hsp70,8Hsp90and1Hsp100. Mining and the subsequent identification of genes will lay the foundation for the functional researchof these resistance related genes.4. Bioinformatics methods and related softwares were used to identify and assess of the quality and quantity of EST-SSR Loci for D. citri.In summary,150EST-SSR loci were identified from the transcriptome of D. citri. There were30di-nucleotide,64tri-nucleotide,27tetra-nucleotide,23penta-nucleotide and5hexa-nucleotide SSR, followed by20%of the total number of SSR,42.67%,18%,15.33%and3.33%respectively. Addtionally,79SSR were confirmed so that can be further used.The results will lay a solid foundation for the population genetic structure studies of citrus whitefly.