| With the rapid development of industrialization and urbanization,the environment pollution has been continuously deteriorated.Multiple pollution factors have hazarded the whole ecosystem via food chains.As the largest quantity of animal population,insects are the first to be gravely affected in pollution.The environmental severe deterioration has induced insect species extinction and rapid variation,which also causes irreversible damage for human production and life.Therefore,exploring the pollutants' toxicological effect and mechanism has important theoretical value and practical significance.As an ideal model for Lepidoptera,the silkworm,Bombyx mori,has been completed about its genome sequencing,and it owns clear genetic background and good breeding system.Moreover,in recent years,researchers have constructed a series of genetic operating platforms,such as transgenic overexpression,RNA interference,and genome editing in silkworm.Alternatively,after thousands of years of artificial domestication,silkworm has degenerated on drug resistance and disease resistance and been sensitive to environmental adverse factors with directional selection.Therefore,it is a suitable specie for toxicity mechanism research of environmental pollutants.In practice,as a significant economic insect,silkworm was easily susceptible of pollutants during large-scale breeding,which has a severely direct effect on the economic benefit and the whole sericulture industry.The huge yield loss on sericulture has been reported many times,and once got the attention of the Ministry of Commerce.Hence,analyzing the pollutants toxicity mechanism,creating the resistant variety,and mitigating the harm of environmental contamination are vital for the flourish of whole sericulture.Soil heavy metal pollution and atmospheric pollution are the two major pollutants.We selected two typical environmental pollutants,fluoride from atmospheric pollution and cadmium from heavy metal pollution.The former is the first halogen element and has a serious impact on animal and plant growth,human health,and ecosystem function,the latter is the trace heavy metal elements that has the greatest and widest impact on animals,plants,and human health.We studied the toxicological mechanism of fluoride/cadmium and created resistant strains in silkworm.The main results and conclusions of this study are as follows:1.Creating fluoride/cadmium high tolerance silkworm strains by transgenic heterologous overexpressionThe researches about toxicity mechanisms of environmental pollutants in organisms have a time-honored history.The researchers have been conducted studies about resisting atmospheric contamination and heavy metal ion pollution in silkworm since 1980s.The early approach was hybrid breeding and high concentration pollutants exposure treatment.In recent years,enzyme activity determination,transcriptome sequencing,and proteomics analysis have been applied to explore the toxicity mechanisms of pollutants in silkworm.However,the main results were focused on the change in physiological and biochemical index and the difference in genes and proteins,which was limit help to explore the target genes and develop resistant varieties.Therefore,we firstly attempted to find out the pollutant targets,analyze the response mechanism and develop resistant varieties in silkworm by gene modification.In this section,we firstly identified a Bombyx mori Metallothionein(BmMT)gene via the silkworm larval midgut transcriptomics analysis with cadmium exposure.BmMT possesses the typical protein characteristic of Metallothionein,including ten Cys,no aromatic amino acid,and it was significantly down-regulated by cadmium exposure.The prokaryotic expression of BmMT in E.coli could improve the cadmium and copper tolerance.The overexpression of BmMT in BmE cells observably enhanced the cadmium-tolerance.Therefore,BmMT might be an ideal metal-tolerant protein.Nevertheless,the BmMT-overexpression strain showed serious growth and development defect because of its other unknown functions.Based on this,we chose to overexpress hDMT protein,which was a combined protein from human and Drosophila melanogaster metallothioneins and has been reported about its anti-heavy metal function.The transgenic strain could raise the cadmium tolerance in larva.To improve the fluoride tolerance,we have attempted to search FEX gene in silkworm by sequence alignment and transcriptome analysis but no result.Therefore,we selected FEX-At and FEX-Aq from Arabidopsis thaliana and Amphimedon queenslandica.Their overexpression could increase the fluoride tolerance in BmE cells and provide targets for resistance strains construction.Transgenic overexpression pattern was able to improve the pollutant tolerance to some extent.However,this pattern could not be large-scale promoted and applicated as the Genetically Modified Organism(GMO)policy restrictions.Hence,we carried out the following work centered on CRISPR genomic knockout technology.2.Screening fluoride/cadmium target genes by genome-wide CRISPR editing library in BmE cellsBased on the completion of genome sequencing and assembling,genome-wide high-throughput screening for loss of function has been evolved a pivotal technology to analyze a series of biological problems containing resistance gene,drug targets,and hormone regulation.Especially in recent years,the genome-wide CRISPR screening has realized quick development and comprehensive application.In our previous work,we have established genome-wide CRISPR screening library in silkworm cells.And it has been utilized to explore the indispensable genes/nonessential genes,temperature responded genes,and BmNPV target genes in silkworm,which suggested the high-efficiency of CRISPR screening in silkworm.In this section,we utilized the genome-wide CRISPR screening library to explore the toxicity mechanism of cadmium/fluoride in BmE cells.Firstly,we detected the cytotoxicity of cadmium and fluoride.The concentration and poisoning phenotype made a big difference,suggesting the different toxicological mechanisms.Cell activity test showed that the IC50 of NaF was 8.64 mM,while the IC50 of CdCl2 was 3.08 ?M.Then,we amplified the CRISPR editing cell library and divided it into three groups,each group including about 4 x 107 cells.One group was treated with normal culture as negative group,one was treated with 4.32 mM NaF and the other was treated with 1.59 ?M CdCl2.With seven days treatment,the negative cells amplified 2-3 times,while 10%cells survived in the two experiment groups.We gathered the survival cells and extracted the genome for deep sequencing to detect the changes in sgRNA abundance.The screening results showed that 757 resistance genes and 725 sensitive genes in cadmium group,751 resistance genes and 753 sensitive genes in fluoride group.Single gene knockout and qRT-PCR verified that these top genes in screening were assuredly involved in the exposure process of cadmium/fluoride.We also confirmed that MAPK signal pathway and DNA damage and repair pathways were relative to this process.Meanwhile,we constructed top 50 genes knockout strains.Pollutant exposure assay showed that the knockout of BGIBMGA003267 improved larval cadmium tolerance,while the knockout of BGIBMGA007400 improved larval fluoride tolerance.Therefore,the cell library screening draws the toxicological networks of fluoride/cadmium in silkworm,and provided the targets for resistance strains construction.3.Screening fluoride/cadmium resistant strain by silkworm mutant libraryThe genome-wide CRISPR screening library in cell level has more advantage in finding out target genes and analyzing toxicity mechanism with simplicity and convenience.While the mutant individual library screening is more beneficial to find out resistant strain with heavy workload and long period.Therefore,we performed silkworm mutant individual library screening after genome-wide CRISPR screening in BmE cells.During the earlier stage of establishing the BmE cell library,we also constructed a sgRNA library suitable for mutant creating.After microinjection,fluorescent screening,hybridization with Cas9 strain,twice fluorescent screening,we got 520 mutants.The survival individuals were gathered after cadmium or fluoride exposure,respectively.The sequencing result demonstrated that a sgRNA were enriched by 20.75%survivals with cadmium exposure,which targeted BGIBMGA000475.Under cadmium exposure,the mutant strain could significantly improve the tolerance for cadmium.Combining the cell screening and mutant library screening results,we verified that BGIBMGA003267 and BGIBMGA000475 could be induced by cadmium and activate endoplasmic reticulum pressure,resulting the Ca2+ signal dysfunction and cell death.Meanwhile,there was 13.11%survivals enriched a sgRNA targeting BGIBMGA013244.The gene was induced to activate TGF-? and JNK signals,leading cell death.Individual mutant library screening helped to identify resistant strain,and complete the toxicity mechanism of cadmium/fluoride.In summary,in this study we have identified a series of fluoride/cadmium target genes and completed the toxicity mechanism in silkworm by transgenic heterologous overexpression,genome-wide CRISPR screening in BmE cells,and individual mutant library screening.Meanwhile,our study has also proved that the superiority high-efficiency of using genome-wide CRISPR screening system to reveal the pollutants toxicological mechanism and create insect resistant strains. |
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