ABSTRACT
Objective To identify the common Sarcophagidae species of necrophagous flies in Luoyang by DNA barcoding and 28S ribosomal RNA(28S rRNA) gene and evaluate its effectiveness for forensic practice. Methods Eighteen Sarcosaprophagous flies were collected and classified by entomologists with traditional morphological characteristics. The DNA of flies was extracted with Chelex-100 method. The fragments of mitochondrial cytochromec oxidase subunit I (COI) and 28S rRNA gene were amplified and sequenced. Twenty corresponding species (China and South Korea) were loaded from Barcode of Life Data System (BOLD) and added to the alignment. All the sequences were analyzed by MEGA 7.0 software package for nucleotide composition, genetic distance computation and phylogenetic tree construction. Results Eighteen Sarcosaprophagous flies were classified into 5 species of 3 genera. The result of amplification with 18 samples showed that length of the obtained COI and 28S rRNA gene sequences were 646bp and 721bp, respectively. And the result of alignment on BLAST online showed that index of similarity of the same species was above 99%. The thirty-eight COI sequences of Sarcosaprophagous flies were clustered into five groups by a neighbor-joining (NJ) tree on value of Bootstrap 1000. The intraspecific difference in COI was 0 to 0.022 while the interspecific difference ranged from 0.057 to 0.090 excluding Sarcophaga Africa and Sarcophaga haemorrhoidalis, which was 0~0.086. The NJ tree of 28S rRNA showed Sarcophaga peregrine and Sarcophaga portschinskyi sequences were obviously clustered into two groups and the others a group. Conclusion For the five sarcophagous flies in this study, the DNA barcoding based on COI gene were able to effectively identify the Sarcophaga peregrine, Sarcophaga dux and Sarcophaga portschinskyi, while 28S rRNA gene can only differentiate Sarcophaga peregrine from others. DNA barcoding based on COI gene and 28S rRNA gene can be used as supplemental molecular markers for identifying these species.
ABSTRACT
The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.