Cloning And Expression Analysis Of A Novel LSD-1 Gene, Acclsd-1, From Apis Cerana Cerana

PAT family proteins, the major structural proteins located in the coat of lipid droplet (LD), play an important role in lipid metabolism and are involved in lipid droplets formation, decomposition and transport. Recent studies on PAT family proteins mainly foucs on the mammal, yet little is known about these in insects. In mammal, five PAT family proteins including perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT have been identified by a number of previous studies. However, only two PAT proteins, lipid storage droplet protein-1 and -2 (LSD-1 and LSD-2), are expressed by insect genomes. LSD-1, a PAT family protein located around LD in insects, is linked to control of lipolysis. In this study, we select the Apis cerana cerana as the experiment material, and a series of research have been managed on the isolation, sequence and expression profile analysis, and functional identification of AccLSD-1, which can greatly help to study the function and mechanism of LSD-1. The main results are as follows:1. Through RT-PCR and RACE-PCR methods, a novel LSD-1 gene, named as AccLSD-1 (GenBank accession no: GU722328), was isolated from Apis cerana cerana. The result of sequence analysis indicated that the full-length cDNA was 1,313 bp in size and contained an open reading frame (ORF) of 1,161 bp. There was a 40 bp 5′untranslated region (UTR) upstream of the start codon and a 112 bp 3′UTR following the stop codon. The deduced AccLSD-1 protein was 386 aa along with a calculated molecular weight (MW) of 42.6 kDa. Multiple sequence alignment showed that AccLSD-1 had high homology with other insect LSD-1s, and AccLSD-1 also contained lipid binding spot and PKA-phosphorylation sites. Phylogenetic analysis revealed that AccLSD-1 had closer relation with insect LSD-1 than other mammal PAT family proteins. The genomic DNA of AccLSD-1 was 5,224 bp in length, including 8 exons and 7 introns. Interestingly, the first intron of AccLSD-1 was located in the 5′UTR, which suggested that the first intron might play an important role in transcription of AccLSD-1.The promoter sequence of the AccLSD-1 gene was obtained by LA PCR and reverse PCR. Using the analysis soft TFBIND, we find that in addition to the typical TATA-box and CAAT-box, the binding sites of PPARγand CAAT/enhancer binding proteinα(C/EBPα), which are the major and determining adipogenic transcription factors, were found in this region. In addition, several important transcription factors such as heat shock factors (HSF) and T-cell factor required for regulating various environmental stresses were predicted.2. The expression profile of AccLSD-1 at the different developmental stages was detected by real-time quantitative PCR. The results indicated that AccLSD-1 constitutively expressed throughout feeding larval, pupal and adult stages. In the larval feeding stages (L3-L5), the AccLSD-1 transcripts showed a very steep growth as increasing ages of larvae. In adult stage, the expression of AccLSD-1 exhibited much stronger in youth workers (25 day-old) than childhood (2 day-old) and old age ones (50 day-old). Howerver, AccLSD-1 was transcribed in a parabola fashion rather than persistently reduced trend in nonfeeding pupal stage and exhibt a highest level in brown-eyed pupae (Pb) stage. Moreover, there was an abrupt decrease in the quantity of transcripts in both larval-pupal transition and pupal-adult transition.4. The effects of various concentrations of conjugated linoleic acid (CLA) or rosiglitazone (Rosi) diets on the expression pattern of AccLSD-1 were investigated through rearing honeybee in laboratory. The results revealed that the transcripts of AccLSD-1 could be up-regulated by Rosi and down-regulated by CLA. The congenerous application of CLA and Rosi showed obvious alteration of the AccLSD-1 expression that was significantly higher than that of CLA alone group.5. A 600 aa 5′fragment of AccLSD-1 was selected to construct an E.coli expression vector pET- AccLSD-1-600, and then it was induced by IPTG to express in E.coli strain BL21 (DE3). The strong induced fusion protein bands were collected into PBS solution and immuned smart mouse to obtain antiserum. The value of antibody reaches 1:2000.This study identified the structural characterization and functional implication of a novel AccLSD-1 gene, showing that AccLSD-1 plays a considerable role in A. cerana cerana development, especially during pupal metamorphosis, and can be regulated by CLA or Rosi possibly via activating peroxisome proliferator–activated receptor-γ(PPARγ).