Molecular Evolution Analysis Of TIME-EA4 And Molecular Cloning, Evolution And Feedback Loop Study Of Circadian Rhythm Biological Clock Genes In Bombyx Mori

Bombyx mori is the model insect of Lepioloptera with diapausing during the egg stage, the activation of diapause eggs is closely related to diapause-duration clock esterase A4 (Time Interval Measuring Enzyme Esterase A4, TIME-EA4). TIME-EA4 continues stable Cu/Zn superoxide dismutase (SOD) activity and one-time transitory esterase ATPase activity (time interval measuring activity), which appeared only in the last stage of diapauses.Circadian rhythm is the most common form of biothythm phenomenon, which regulated by genetic clock genes. The diapause during the egg stage of Bombyx mori is a kind of phenomenon to response the circadian rhythm zeitgeber (Temperature and light etc), which offer us a good reference to study the circadian clock genes of silkworm. In order to further study the molecular mechanism of TIME-EA4, the paper has studied the mechanism of TIME-EA4 dual enzyme activity?from the perspective of molecular evolution of protein;? at the same time, in order to in-depth study the relationship between the time measuring activity of TIME-EA4 and the signaling pathways of circadian biological clock genes, the paper cloned, analysised and preliminarily integrated the circadian rhythm biological clock genes in Bombyx mori.First, in the paper, the homologous genes of Time-ea4 (Time-likes) in 17 species were cloned, the protein sequences of Cu/Zn SOD, carboxylesterase and acetylcholinesterase in 17–20 species provided by NCBI were synthesized. The methods used were sequence alignment, MOTIFS analysis, evolutionary trace analysis, scansite and cluster analysis to determine the molecular evolution of TIME-EA4. Second, the cDNA sequences of Bmcry1 and Bmcry2 were cloned by RT-PCR and RACE, the ORF sequences of Bmvri, Bmcyc, Bmtim2, Bmpdp and ORF sequence fragments of Bmclk were cloned by in silico. On this basis, the structure, chromosomal location and molecular evolution of the above genes and their expression products were studied. Finally, the silkworm circadian clock feedback loop was integrated preliminarily according to the structure characteristics of the clock genes and their expression products, and combining the existing data resources.1 TIME-EA4 is derived from Cu/Zn SODTIME-EA4 (or TIME-likes) and Cu/Zn SOD have a high degree of homology (33%¹00%), they all have the Cu/Zn SOD characteristic sequences (SOD_CU_ZN₁ and SOD_CU_ZN₂ motifs), they belong to the Cu/Zn SOD super family. TIME-EA4 has stable Cu/Zn SOD activity and Cu/Zn ion binding sites. 91.2% of the 34 relatively important residues (coverage < 25%) that experience stronger evolutionary pressure in TIME-EA4 are identical with Cu/Zn SOD. The TIME-EA4 homologous proteins (TIME-likes) were Cu/Zn SOD in some species of non-insect. TIME-EA4 (or TIME-like) and Cu/Zn SOD in the same insect species might be paralogs, whereas Cu/Zn SOD among species might be orthologs. With regard to evolutionary history, the appearance of Cu/Zn SOD was triggered by the proliferation of photosynthetic organisms that began to produce oxygen about 2 billion years ago. That means there were Cu/Zn SODs before species splits in biological evolution. The results of this study showed that TIME-EA4 appeared later than Cu/Zn SOD in evolutionary history because TIME-like that is different from their own Cu/Zn SOD has been found in only some insects. Therefore, we speculate that TIME-EA4 is derived from Cu/Zn SOD.2 TIME-EA4 is a novel esteraseTIME-EA4 was purified as an esterase and can be colored by the esterase idio-dye. The N-terminus of TIME-EA4 is the specific esterase ATPase domain that has a time interval measuring function. That means TIME-EA4 has evolved novel functions even though it originated from Cu/Zn SOD. We knew that there were some differences, such as composition, amino acid residue polarity and bonded atoms, in Cu/Zn ion binding sites between TIME-EA4 and Cu/Zn SOD. Further, TIME-EA4 and TIME-like proteins in the Lepidoptera have some characteristic sequence sites of esterase except the sites of their own Cu/Zn SOD. Further, the cluster analysis showed that TIME-EA4 and its homologous proteins in insects (TIME-like) are grouped with esterase. TIME-EA4 and Bma-TIME-like are closer to the mammalian carboxylesterase, but TIME-like proteins in other insects are grouped with some invertebrate esterase and Cu/Zn SOD. The results revealed that TIME-EA4 and TIME-like of insect species were derived from Cu/Zn SOD and are evolving to esterase at different evolutionary rates. In Bombyx, domestication for several thousand years accelerated this evolutionary process. In summary, we speculate that TIME-EA4 in the silkworm is a novel esterase that is derived from Cu/Zn SOD according to the available information and data analysis in our laboratory.3 Bombyx mori has two types of circadian core clock Cry gene:Bmcry1 and Bmcry2We cloned the cDNA sequences of Bmcry1 (2 166 bp, HM747059) and Bmcry2 (2 389 bp, HM747060) by in silico, RT-PCR and RACE, and obtained their gene sequences (GenBank accession no. HM747057 and HM747058, respectively) by sequence alignment and assembly. Bmcry1 have 12 exons and 11 introns, while Bmcry2 have 9 exons and 8 introns. Chromosome mapping showed that Bmcry1 and Bmcry2 were located in chr.17 and chr.15 respectively. We predicted the three-dimensional structures of BmCRY1 and BmCRY2 by homology modeling, their FAD entrances are large and deep, which consistent to the fact that CRY proteins don't bind with pyrimidine dimers, the surfaces of BmCRY1 and BmCRY2 are more negatively charged, only FAD entrances have accumulated positive charge. The molecular evolution of BmCRY1 and BmCRY2 were researched by multiple sequence alignment, protein motif, functional domain and cluster analysis. BmCRY1 and BmCRY2 belonged to insect CRY1 and CRY2 respectively, and were closely to the corresponding proteins in other lepidopteran insects like Antheraea pernyi. BmCRY1 and BmCRY2 have DNA photolysis enzyme domain and FAD binding domain that similar to the CRY proteins in other insects. However, these two domains have different conservative sites and different protein motifs between CRY1 and CRY2 in insect species. This experiment provided a basis for further investigating the molecular evolution mechanism and function of CRY1 and CRY2 in B. mori. 4 The genes excavation and feedback loop preliminary integration of circadian clock in Bombyx moriThe ORF sequences of Bmvri, Bmcyc, Bmtim2, Bmpdp and ORF sequence fragment of Bmclk were cloned by in silico. Genetic structure analysis and chromosomal location show: Bmvri have 2 exons and 1 intron, be located in nscaf3072 of chr.27; Bmpdp have 7 exons and 6 introns, be located in nscaf2734 of chr.1; Bmtim2 have 21 exons and 20 introns, be located in nscaf3048 of chr.9; Bmcyc have 10 exons and 9 introns, be located in nscaf2734 of chr.1. Bmpdp and Bmcyc located in the same nscaf of same chromosome, but their exact locations are different.Phylogenetic analysis showed that VRI, PDP, PER, TIM1, TIM2 and CYC belonged to corresponding protein groups of insect separately. The homologs of same insect medica are closely in evolutionary distance, which is fit for paternity.Whatever evolution distance or function domain, TIM2 in insect are similar to TIM1 in vertebrate. Thus the homologs in insects of vertebrate TIM1 are TIM2, not TIM1.Function domain and MOTIFS analysis showed that DNA-binding domains of BmVRI and BmPDP are very similar, which means they can combine same DNA sequences (Clk gene promoter). There are no DNA-binding domains in BmPER and BmTIM, but they all have protein interaction motifs, which realized signalling molecules of BmPER and BmTIM. BmCYC has transcription activated domain besides protein interaction motifs(bHLH/PAS/PAC), which is highly conservative.The homologous genes of all circadian clock genes in Drosophila are almost exist in silkworm, the silkworm circadian clock feedback loop was integrated preliminarily according to structure characteristics of the clock gene expression products, and combining the existing data resources. The loop combines the characteristics of feedback loop in fly and Danaus plexippus, their core biological clock gene transcriptional-translation feedback loops are very conservative.