Mythimna Separata Transcriptome Sequencing And Cloning Of RNAi Mechanism Related Genes

Mythimna separata (lepidoptera, noctuidae) is a kind of omnivorous pest which harms nearly 100 families of more than 300 kinds of crops of economic importance. In recent years, mythimna separata is threatening the production of corn in North China and Northeast China and is leading to serious economic loss. Transcriptome or even genome sequencing projects have been reported for other families in lepidopteran insects such as diamondback moth, bombyx mori, monarch butterflies, as leading to the rich genetic information available in these families. But there are no such efforts reported for noctuidae family, as provided a new direction for this research.The sequence information and genome expression profiling of mythimna separata are very limited. Only the mitochondrial genome of mythimna separate has been sequenced, and only 46 expressed sequences have been included in NCBI database to date. Therefore, next-generation transcriptome sequencing was performed for mythimna separata in this study, and 12 families of genes associated with RNAi mechanism were verified by cloning and expression analysis, laying the basis for use of RNA interference technology to control mythimna separata.The results are as follows:1. The transcriptome sequence reads of mythimna separata has been assembled to 69238 unigenes after de novo assembly of the raw sequencing data.45,227 unigenes have been annotated and were highly homologous with the reported genes, getting the annotation frequency of 65.32%. Among them,42,064 (accounting for 93.01% of 45,227 unigenes) unigenes are annotated to the NR database; 13,083 (accounting for 28.93% of 45,227 unigenes) unigenes are annotated to the 25 COG gene cluster; 21,110 (accounting for 46.68% of 45,227 unigenes) unigenes are annotated to the 59 GO entries; 27,719 (accounting for 61.29% of 45,227 unigenes) unigenes are annotated to 258 KEGG metabolic pathways.2.998 unigenes from KEGG annotated metabolic pathway are related to RNA transport pathway.98 unigenes associated with RNAi mechanism from NR annotation and they are divided into 12 gene families. Used RT-PCR to detect the expression of 35 unigenes from these 98 unigenes, and verified the expression of 27 unigenes which are included in eight gene families such as Sid, Dicer,Argonaute, PIWI, Loquacious, SR-C, Juvenile hormone esterase and. juvenile hormone epoxide hydrolase, respectively. The qualification rate of RT-PCR is 77.14%, demonstrating the successful assembly of the transcriptome data for mythimna separata.3. Having been done successfully obtained a partial cDNA sequence of three genes:Sid (151 lbp), Dicer (2008bp), Argonaute (876bp), and also obtained a 5’end sequence of Dicer gene by RACE experiments and PCR amplification using combinated primers from different unigenes.4. Verified the expression of Sid、Dicer、Argonaute、PIWI with predicted size using northern hybridization experiment, and also successfully amplified the genes such as Sid、Argonaute、PIWI、Loquacious、Juvenile hormone esterase、juvenile hormone epoxide hydrolase from genomic DNA.