Caspase-1Gene Expression Of Dastarcus Helophoroides

The adults of Dastarcus helophoroides Fairmaire (Coleoptera: Bothrideridae) could livemore than12years, which is a suitable insect to study genes related in aging process.Members of the caspase genes family play a central role in programmed cell death (PCD), andthey also play an important function in the development and cell decline of the organism.Real-time quantitative PCR is one of the advanced technologies to study gene expression,which need suitable reference genes for analyzing expression accurately. In this study, we hada preliminary analysis of caspases families from D. helophoroides, cloned a full length ofcaspase-1, selected the reference genes, and then, we detected the characteristics of caspase-1gene expression in D. helophoroides during growth and the aging. The main results are asfollows:1. There are9caspase genes in D. helophoroides transcriptome dataset. We determinedthe caspase-1, caspase-2, caspase-4, caspase-5and caspase-8by homology comparisonwhereas the other4genes need to be amplified to complete open reading frame (ORF).2. In this article, We determined the expression stability of10candidate housekeepinggenes was determined in D. helophoroides by qRT-PCR.10housekeeping genes includedRPS, SDHA, Histone, UBC, PGK, EF-1α, β-Actin, GAPDH, L13and α-Tubulin. GeNorm,NormFinder and BestKeeper analyses showed that the RPS or α-Tubulin was a better choiceas a reference gene for expression analysis of adult D. helophoroides differing in ages,whereas the combination of RPS，GAPDH and α-Tubulin was the most suitable. GAPDH wasa better choice in different tissues, wheras GAPDH and EF-1α were the best combination.Histone or RPS was a better choice in life stages of D. helophoroides, whereas Histone andRPS were the best combination.3. A1,201bp full-length caspase-1cDNA from D. helophoroides contained a996bpORF that encodes332amino acids was cloned by RT-PCR and RACE. The amino acids’molecular weight was32.83kDa, and the isoelectric point was8.67which include QACQGpentapeptide active-site motif and four substrate binding sites LSHG.4. qRT-PCR (real-time quantitative PCR) results demonstrated that the expression ofcaspase-1was highly tissue-specific in adult, the transcript appeared a low level in the head,chest, midgut, hindgut, fatbody, spermary and adipose, but a much higher level in ovary.Besides, Dahel caspase-1was expressed in all life stages of D. helophoroides, however its mRNA level rose gradually from the1st instar to5th instar, and then reduced in6th instar.qRT-PCR analysis among different age groups showed that the expression level of the Dahelcaspase-1increased with age. The caspase-1enzyme had a highly activity in the5th instar andpupal stage, whereas the activity increased with age. The result showed that the Dahelcaspase-1expression and its enzyme activity changing are related to cell death anddevelopment, and it also affected the adult’s age progress.