Transcriptome Sequencing Of Dastarcus Helophoroides And Analysis Of Important Functional Genes

Dastarcus helophoroides is known as the most valuable natural enemy insect againstmany large-body longhorned beetles. Under laboratory conditions, D. helophoroides can livefor more than8years with continued sexual reproduction. The molecular mechanism of itslong lifespan and reproduction makes it a unique resource for genomic research. During thepast two decades, its morphological and biological characteristics, artificial reproduction andthe controlling of longhorned beetles have been studied extensively. However, molecularbiology research about this parasitic beetle is scare, and genomic information for D.helophoroides is not currently available. Transcriptome data for this species are thereforeimportant resources needed to better understand the molecular mechanisms of D.helophoroides. In this study, we obtained the transcriptome information of D. helophoroidesusing Illumina HiSeq2000sequencing and the assembled unigenes were annotated. Theimportant functional genes related to development and reproduction were analyzed. Theexpression of Methuselah (mth) genes in different tissues, different stages and different alivetime adults were studied by using real-time quantitative PCR technology. The full-length oftwo mth genes were obtained by RACE PCR and Touchdown PCR technologies, and thenthey were aligned with mth genes from fruit fly and other species. The family analysis ofP450in D. helophoroides were also carried out. The main results are as follows:1. The transcriptome information of D. helophoroides was obtained. De novo sequencingof D. helophoroides transcriptome was carried out using Illumina HiSeq2000sequencing.A cDNA library was built using the total RNA of adult D. helophoroides. In total,27,543,746clean reads with accumulated length of2,478,937,140bp were obtained, which wereassembled into42,810unigenes and30,103unigenes (70.32%of the total) were matched theknown sequences in NCBI, among which26,828COG annotations were produced by14,280annotated unigenes,15,704were annotated as98,975GO terms, and22,102unigenes werelocated in258KEGG pathways.62.23%of the unigenes showed high homology withTribolium castaneum and30.43%of the unigenes showed low homology with the knownsequences in NCBI, which consisted our unique transcripts.2. The expression of mth genes were analyzed. In total,11mth genes were retrieved in the transcriptome of D. Helophoroides. Real-time quantitative PCR technology was used toanalyze the expression of seven mth genes in different tissues (head, chest, midgut, hindgut,testis, ovary, fatbody and residue), different stages (1~6instars’ larvae, pupae and newlyemerged adult) and different living time (adults lived for1,6,18,29,31,33,34and36months). The results showed that, mth genes were expressed in all of the samples and hadhigh tissue-specific. The expression of mth was the highest in testis, and followed by residueand ovary. Among different stages, the expression of mth was the highest in6instar larvae.The expression of mth in adults of different living time had no significant difference.3. The full-length of two mth genes were cloned. RACE PCR and touch-down PCRtechnologies were used to obtain the full-length of two mth genes (mth1，GenBank accessionno：KM588897、mth2，no：KM588898), which was counted2649bp and1947bp, and codingan amino-acid sequence of566aa and409aa, respectively. Both of them were belonged tothe B family of G protein coupled receptor, with the typical seven trans-membrane structure.The two sequences were then aligned and carried out phylogenetic tree analysis comparedwith the homologous mth sequences from fruit fly and other species. The results showed thatthe protein sequence of the two mth genes both showed the highest homology and the shortestgenetic distance with T. castaneum which also belonged to Coleoptera.4. The family of P450in D. helophoroides was analyzed.151P450related sequenceswere discovered in the transcriptome, which belonged to CYP4, CYP6CYP9, CYP18andCYP301-3414families, among which, the CYP6family was the bigest, counted33.11%ofthe total and followed by the CYP4family, counted21.19%. And these two families countedmore than half of the total P450genes.