| Histone acetylation plays an important role in epigenetic regulation and was mediated by antagonistic activities of two types of enzymes including histone acetyltransferases (HATs) and histone deacetylases (HDACs). As the catalytic subunit of the six-subunit Elongator complex of RNA polymerase II (RNAP-II), Elp3 is a representative of highly conserved GNAT family HATs, and possesses acetylation activity against histone H3 and H4 in vitro and vivo. The red flour beetle, Tribolium castaneum (Coleoptera:Tenebrionidae), is a serious global pest of agriculture grain, and shows the most efficient systemic RNA interference response in insects. In this paper, the Elp3 gene was cloned, and it’s expression profiles in different developmental stages of T. castanuem were analyzed. Furthermore, the function of Elp3 gene in T. castanuem was studied using RNAi (RNA interference) technology. The results will assist in understanding the role of histone acetylation in the development of insects and mechanisms of insect adaption to stress.A full length T. castaneum Elp3-like cDNA (TcElp3) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). Sequence analysis showed that the 1776 bp ORF of TcElp3 encoded 591 amino acid residues, and predicted a protein with molecular mass of 67.74 kDa and an isoelctric point (pI) of 8.66. The highly conserved GNAT-type HAT domain (Pfam accession no. PF00583) was located in the C-terminus within positions 493-539 aa. Another conserved domain belonging to the Radical SAM (S-adenosylmethionine) superfamily (Pfam accession no. PF04055) was located in the N-terminus within amino acid positions 98-346. An amino acid sequence alignment shows that TcElp3 shares the greatest amino acid identity with the Elp3 in Dendroctonus ponderosae (81.36%) and is 76.48%,75.59%,75.30%,74.62% and 74.54% identical in pairwise comparisons with the Elp3 amino acid sequences in Plutella xylostella, Drosophila melanogaster, Acyrthosiphon pisum and Nilaparvata lugens, respectively. Phylogenetic analysis reveals that TcElp3 protein is grouped with Elp homologue in D. ponderosae.To gain understanding into the developmental roles of TcElp3 inT. castaneum, the mRNA levels of TcElp3 were analyzed using qRT-PCR at various developmental stages of T. castaneuminsects, including 1 and 3-day old eggs,1,5 and 20-day old larvae,1 and 5-day old pupae,1 and 7-day old female adults,1 and 7-day old male adults. The developmental expression pattern revealed that TcElp3 was expressed at all developmental stages tested, the mRNA level of TcElp3 is the lowest in 3-day old eggs and highest in 5-day old larvae, while there is no significant difference among pupal and adult stages.RNAi was employed to investigate the putative function of TcElp3. The results showed that when the mRNA expression levels of TcElp3 were suppressed in late larval stage, all injected larvae with dsTcElp3 underwent normal larval-larval and larval-pupal molts, however, the pupal development was delayed, and 98.33% insects died during pupal stage. While the rest pupae could develop into adult, the adult exhibited abnormal folding of hind wings, and died the next day after eclosion. On the 6th day after dsRNA injection, insects in dsRNA injected group and wild-type larvae with no injection (WT group) as well as buffer-injected larvae (IB group) were treated with hydrogen peroxide, and the results showed that at 48 h after treatments, the mortality of dsRNA injected larvae was as high as 84.72%, while insects in WT group and IB group exhibited 25% and 26.39% mortality, respectively. Further study showed that two catalase genes, TcCatl and TcCat2, were significantly suppressed by 49.65% and 85.17%, respectively, when compared with the control insects. These results suggested that TcElp3 plays an important role in the development and adaption to oxidative stress in T. castanuem. |
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