Clone And Expression Of Methuselah-like Genes In Dastarcus Helophoroides

Dastarcus helophoroides(Fairmaire)(Coleoptera: Bothrideridae), which has a relatively longer lifespan compared to other insects(survive more than 8 years under the condition of artificial breeding in the laboratory), is one of the most effective natural enemy of many trunkborer-insects such as large-body longhorned beetles, buprestid beetles and snout beetles.methuselah(mth) which associated with lifespan, stress resistance and reproduction in Drosophila melanogaster is a kind of important receptor genes, but mth is not present in nondrosophiline insects. A number of methuselah-likes(mth-likes,mthls) have been identified in nondrosophiline insects, but it is still unknown whether they are present in D.helophoroides. The aim of this study was to explore the presence and expression patterns of mth-likes in D. helophoroides. mth-likes was cloned using the method of rapid amplification of cDNA ends(RACE), the structure of Mth-like receptors was analyzed by bioinformatic methods and the expression profiles were analyzed by real-time qPCR on the foundation of selection and identification of mth-like genes. The main results are as follow:Six clones of mth-like genes with full length sequences were obtained using the method of 5’- and 3’-rapid amplification of cDNA ends(RACE) based on the six mth-like fragments obtained from the D. Helophoroides transcriptome database. The six nucleotide sequences were named as mth-like1, mth-like2-1, mth-like2-2, mth-like2-3, mth-like5-1 and mth-like5-2,and the corresponding proteins were therefore designated as Mth-like1, Mth-like2-1,Mth-like2-2, Mth-like2-3, Mth-like5-1 and Mth-like5-2, respectively.The results of on line prediction and multiple sequence alignment showed that all these putative Mth-likes have conserved seven transmembrane domains. The results of phylogenetic analysis showed that Mth-like1, Mth-like2-1, Mth-like2-2, Mth-like5-1 and Mth-like5-2 were more closely related to Tribolium castaneum Mth-likes, while Mth-like2-3 was closely related to Hymenoptera insects.The results of RT-qPCR showed that six mth-likes were expressed throughout all the developmental stages and in all the examined tissues, but they displayed different expression patterns and appeared to be specific.The expression of six mth-likes were all regularly changed in the process of aging with levels recovered to the normal level(mth-like2-2) or elevated to a higher level(mth-like1, mth-like2-3, mth-like5-1 and mth-like5-2) exceptmth-like2-1(dropped to a much lower level). mth-like1, mth-like2-1, mth-like2-2 and mth-like2-3 were responded to oxidative stress indicating that they were likely to involved in the resistance of oxidation; mth-like2-1, mth-like2-2 and mth-like2-3 were responded to heat stress indicating that they were likely to involved in the resistance of high temperature; All the six mth-likes were responded to the stress of starvation indicating that they were likely to involved in the resistance of starvation.