A Study Of Bmtop3-alpha’s Effects On The Phenotype Of Lethal Mutant L-4i In Fourth Instar Larvae Of Bombyx Mori

One of the oldest enzymes in nature,which is produced with the production of DNA,is the DNA topoisomerase(topo).Topo is a class of enzymes that control and modify the topological structure of double-helix DNA replication and transcription.The most basic cytological functions of this enzyme are involved in DNA replication,transcription,chromatin and chromosome transition and genome stability.In organisms,changes in the activity of this enzyme will lead to a series of abnormal life processes and thus can not complete their life history.In the analysis of the related mutation characters on the natural mutant l-4i from bivoltine silkworm P33,it has been shown that the l-4i intensive appetite loss,which leads to a growth retardation,even results in failure of finishing the whole life cycle;genetic statistics analysis showed that the recessive homozygous lethal single gene mutation on autosomal chromosome and the mutant gene was Bombyx mori topoisomerase.When the reason for the mutation of topoisomerase to l-4i has been preliminarily identified,there are still many problems to be solved about the function of topoisomerase(Bmtop3-alpha)and the mechanism of l-4i formation.1.Sequence analysis and protein expression of the mutant gene(Bmtop3 α).In this paper,the complete sequence of the gene was cloned,and the ORF length was3051 bp.Compared with the mutant sequence,the mutation site was 48 bp deletion in exon7,and there was no frameshift mutation.The Bmtop3α gene was inserted into the prokaryotic expression vector,and the expression of target protein was detected.2.Property analysis,purification of Bmtop3-alpha protein and determination of its enzyme activity in vitro.According to the prediction of physiological and biochemical properties,Bmtop3-alpha protein is a hydrophilic protein with certain solubility and without transmembrane domain,signal peptide,and a nuclear localization signal of a small peptide at the C-terminal.All of these are consistent with the function of the enzyme which performs DNA uncoiling relaxation and cell replication in the nucleus.After structural analysis,the core functional regions were intercepted for expression,and then the active enzymes were purified from the inclusion bodies.This solves the problem that Bmtop3-alpha is difficult to purify the target protein from either prokaryotic or eukaryotic expression systems.The purified enzyme relaxed the p BR322 plasmid in vitro,and the enzyme activity was greatly decreased after the deletion of 16 amino acid residues.3.Histomorphological analysis of mutants.Compared with the wild-type,the mutant is smaller in size,slower in development and unable to complete generation replacement,it is considered that there are a large number of apoptosis events happened in the mutant.The relationship between average optical density,cumulative optical density(IOD),area,diameter and apoptosis rate was analyzed by tissue frozen section and tunel fluorescence.The results showed that in the detection of apoptosis signal in the experimental range,the wild-type silkworm had no obvious change trend,while the mutant was stronger and stronger.The correlation analysis showed that there was the greatest Pearson correlation between IOD and apoptosis rate.4.Comparative transcriptome analysis of mutants was carried out by RNA-Seq.The enzyme activity of the mutant decreased due to the deletion of 16 amino acid residues,and a large number of apoptotic events were detected,which alone can not fully explain its phenotypic mechanism.The metabolic pathways of GO and KEGG were enriched by RNA-Seq method through comparative transcriptome analysis.Among the 2013 downregulated genes in the mutants,many biological processes such as splice sn RNP assembly,protein folding and protein catabolism were significantly enriched.Among the 2405 upregulated genes,several biological processes were significantly enriched,including purine base metabolism,nucleoside metabolism and ab initio IMP biosynthesis.In addition,a large number of RNA alternative splicing events were found between l-4i and P33.The analysis shows that the imbalance of various biological processes,pathways and the abnormal proteins produced by alternative splicing may lead to different phenotypes of the mutants.5.Homology analysis of two important pathways and amino acid evolution of Bmtop3-alpha protein.Through the cross-union analysis of Bmtop3α gene,it was found that the expression of related factors in the two pathways of Fanconi Anemia and Homologous Recombination changed significantly in the mapped KEGG pathway.Three representative genes were selected.Then the physiological traits similar to that of l-4i appeared in the silkworm of the experimental group after RNAi analysis of P33.Statistical analysis showed that the body weight of the experimental group also reached a very significant difference(p < 0.0001).q RTPCR confirmed that the expression of related genes was down-regulated.Through the study of three different dimensions including gene structure change,transcriptional expression and protein level,together with the results of histomorphological analysis and RNAi technique,this paper preliminarily elucidates the genomic instability caused by abnormal processes of DNA replication,recombination,transcription and repair owing to the decrease of Bmtop3-alpha enzyme activity.The accumulation of a series of chain reactions resulted in the mechanism that the l-4i mutants were stunted,ate less and failed to complete the whole life cycle.