| The adaptability of insects to the environment plays an important role in their survival and reproduction.Insects are poikilothermic animals,and are more sensitive to the changes of environmental temperature than thermostatic animals.The changes of ambient temperature and humidity will have a significant impact on their behavior,metabolism,growth and development.Therefore,it is of great significance to analyze the mechanism of insect resistance to high temperature and humidity for effectively controlling harmful insects and strengthening the utilization of economic insects.The silkworm,Bombyx mori,is an excellent research object,because it is not only an insect with important economic value,but also an internationally recognized research model insect of Lepidoptera,the main group of agricultural and forestry pests.As one of the traditional experimental animals of genetics,silkworm has rich genetic resources,especially the silkworm strains with different resistance to different environmental conditions,which provides valuable materials for the study of its resistance mechanism.In this study,two silkworm strains with significant difference in high temperature and high humidity tolerance were selected by comparing different resistant strains.The key genes of high temperature and high humidity resistance were identified byassociation analysis of transcriptome and methylome of the two stains after high temperature and high humidity shock,and the function and mechanism of candidate genes were studied.The results we obtained are as follows:1.Identification and genetic analysis of high temperature and high humidity resistant materials in silkwormBy comparing and analyzing the 10-year rearing results and silk quality of 400silkworm germplasms preserved by the Institute of sericulture,Sichuan Academy of Agricultural Sciences,we found that the health and yield quality indexes of different silkworm strains,such as the fifth instar course,the whole instar course,the fourth instar cocooning rate,the pupa life rate,the dead cage rate,the whole cocoon quantity,the cocoon layer quantity,the cocoon layer ratio,the cocoon yield of ten thousand silkworms and the silk yield of ten thousand silkworms.There were significant differences between the strains with strong stress resistance and those with weak stress resistance.In the case of the same genetic background,the directional selection and the change of feeding methods will also lead to the change of strains'stress resistance,and the economic indicators will show significant differences.We found that the differences of health indexes among strains and systems were not only related to the external environment,mulberry leaf quality and feeding technology level,but also closely related to the disease resistance,environmental adaptability and immune response mechanism of varieties.Through genetic analysis and high temperature and high humidity condition shock experiment,we found that the bivoltine strain 7532 and the bivoltine strain Knobbed showed resistance and sensitivity to high temperature and high humidity respectively,and the difference was very significant,which was a good material to identify the genes related to high temperature and high humidity resistance and analyze its mechanism.Furthermore,through the investigation and Analysis on the tolerance of 7532 and Knobbed to high temperature and high humidity,both the experimental group and the control group showed strong resistance to high temperature and high humidity.In the control group,the 7-day mortality of 250 silkworms in 5 areas was 4,and the mortality rate was 1.60%;in the experimental group,the 7-day mortality of 250 silkworms in 5 areas was 3,and the mortality rate was 1.20%.The difference between Knobbed experimental group and control group was obvious.The death rate of250 silkworms in control group was 100 in 7 days,with a mortality rate of 40.00%;250silkworm deaths in 5 areas of experimental group were 250,and a large number of deaths occurred on the second day,all of them died on the 5th day,with a mortality rate of 100.00%.In conclusion,7532 showed strong resistance to high temperature and high humidity,however,Knobbed was very sensitive to high temperature and high humidity.We used them as experimental materials to identify the genes of high temperature and high humidity resistance in silkworm.2.Transcriptome analysis of high temperature and humidity resistance genes in silkwormIn order to systematically analyze the differences of resistance to high temperature and high humidity between silkworm strain 7532 and Knobbed,explore the immune response mechanism of silkworm to high temperature and high humidity environment,and screen the key genes of resistance to high temperature and high humidity,we used RNA-seq technology to sequence the transcriptome of experimental samples of 7532and Knobbed under normal feeding conditions and high temperature and high humidity shock conditions.We selected and collected the m RNA of silkworm larvae from 7532and Knobbed experimental groups and control groups at different time points(1,6,24and 48h),and constructed 16 transcriptome databases.The percentage of each sample with Phred value greater than 30 base to the total base(Q30)was at least 87.04%.After data filtering,the clean reads of 16 samples ranged from 6.00 Gb to 7.82 Gb,and the coverage of reference genome ranged from 61.34%to 74.32%,indicating that the obtained data can be used for subsequent analysis.Through data processing and analysis,4944 differential expression genes were identified,including 4390 annotation genes and554 novel genes.In order to understand the expression pattern of differentially expressed genes,the cluster analysis of differentially expressed genes based on log10(FPKM+1)value of all samples showed that the highly resistant strain 7532 and the sensitive strain Knobbed were very regularly aggregated,and the differentially expressed genes showed regular distribution in different experimental materials and different treatment methods.The difference of gene expression was the most obvious when 7532 was treated for 48h,and the difference of gene expression was the most obvious when Knobbed was treated for 6h,which indicated that 48h might be the key time point of stress response to high temperature and high humidity.In order to analyze the genes related to hygrothermal tolerance better,we classified the differentially expressed genes in 7532 and Knobbed samples treated for 48h by GO and analyzed the enrichment of KEGG pathway.A total of 747 differentially expressed genes were identified.The GO classification analysis showed that they were enriched in three functional groups:biological process,cellular component and molecular function,including metabolic process,proteolysis,aminoglycan metabolic process Extracellular region,serine-type peptidase activity,serine hydrolase activity,serine-type endopeptidase activity,peptidase activity-acting on L-amino acid peptides,peptidase activity,catalytic activity,endopeptidase activityand hydrolase activity.KEGG pathway enrichment analysis also confirmed the go classification results,and the differentially expressed genes were significantly enriched in the metabolic pathway.By constructing a variety of patterns of Venn diagram for gene screening and comparative analysis,we found that BGIBMGA005701 and BGIBMGA010163 genes were differentially expressed at four time points,and six genes were differentially expressed at 6h,24h and48h after treatment,which were BGIBMGA000387,BGIBMGA004579,BGIBMGA004675,BGIBMGA004910,BGIBMGA011699 and BGIBMGA013893,respectively.In the control group,BGIBMGA003739,BGIBMGA005876,BGIBMGA011821 and novel01749 were differentially expressed at four time points,and these genes will be the focus of future research.In addition,we selected 11differentially expressed genes for q RT-PCR analysis,the results showed that the gene expression trend was consistent with RNA-seq data,which proved the reliability of transcriptome comparative analysis.3.Identification of temperature and humidity resistance genes in silkworm based on whole genome methylationIn order to explore the role of DNA methylation in the response of silkworm to high temperature and high humidity stress,we used whole genome bisulfite sequencing to compare and analyze the DNA methylation levels of two silkworm strains with significantly different heat-humidity resistance.Genomic DNA libraries were constructed from 8 samples of 7532 and Knobbed strains at 24h and 48h(control group and treatment group respectively),and 15.85-21.31Gb original bases were obtained from 8 libraries.After quality control and data filtering,15.11-20.21Gb of clean bases were obtained in all samples,of which 41.07%-49.13%of the clean data only matched the reference genome of silkworm,and the repetition rate was 13.14%-17.22%.In addition,the sulfite conversion rates were higher than 99.7%,indicating the integrity and accuracy of WGBS.In order to further analyze genome-wide DNA methylation patterns among groups,we calculated cytosine methylation levels in CG,CHG and CHH context.About 0.14%of the genomic C sites were methylated in the resistant strains,while about 0.12%in the sensitive strains.At the same time,in CG context,the average level of cytosine methylation in resistant and sensitive groups was 0.66%and0.54%,respectively,while in CHG and CHH background,the level of cytosine methylation in all groups was almost negligible.Through the correlation analysis of methylation levels among samples,it was found that the correlation between samples of the same strain was very high(R2>0.95),while the correlation between different strains was relatively low,ranging from 0.80 to 0.83.On the basis of correlation analysis,we further carried out cluster analysis.The results showed that the samples of the same strain were clustered together,but the samples of resistant strains were clustered together after treatment,while the samples of sensitive strains were not clustered together,indicating that high temperature and high humidity had a greater impact on DNA methylation of resistant strains.We analyzed DNA methylation levels in different genomic functional regions in RT?48hvs ST?48h and RNT?48hvs SNT?48h on CG sites.Our results showed that the DNA methylation levels were different in different functional regions.The methylation level was the highest in exon regions,followed by intron regions,and relatively low in promoter and repeat regions.Furthermore,methylation peaked in the gene body region near the transcription start site(TSS).We compared the differentially methylated regions(DMRs)and DMR-relatedgenes(DMGs)of the two strains after treatment for 48 h(RT?48h and ST?48h).The results revealed that a total of 2934 DMRs were identified under the CG context,which corresponded to 1230 DMGs.GO and KEGG analysis showed that these DMGs were most enriched in binding,cellular metabolism and RNA transport pathways.In order to further study the relationship between the differentially expressed genes corresponding to the methylation region and the differentially expressed genes obtained from the transcriptome data,we analyzed the association between the two gene data.The results showed that four genes BGIBMGA007132,BGIBMGA008110,BGIBMGA01255 and BGIBMGA014048 were down regulated due to hypermethylation,and three genes BGIBMGA008664,BGIBMGA010840 and BGIBMGA012129 were up regulated due to hypomethylation.In the untreated group,three genes BGIBMGA007132,BGIBMGA001151 and BGIBMGA014048 were down regulated due to hypermethylation,and three genes BGIBMGA001644,BGIBMGA008664 and BGIBMGA009130 were up regulated due to hypomethylation.Three genes,BGIBMGA007132,BGIBMGA014048 and BGIBMGA008664,were identified in the two methods.In addition,we also verified the transcriptome and methylation sites by q RT-PCR and pyrosequencing,respectively,and the results were consistent with the omic data,indicating the reliability of our data.These results indicate that DNA methylation plays an important role in the response of Bombyx mori to environmental stress,and provide important clues for identifying key resistance genes of silkworm under high temperature and high humidity stress.4.The mechanism of S-adenosylmethionine synthetase gene(BmSAMS)regulating high temperature and high humidity in silkwormIn order to analyze the expression characteristics of candidate genes selected by transcriptomics and methylomics after high temperature treatment in resistant and sensitive strains,the expression characteristics of candidate genes in different strains were analyzed by q RT-PCR.The results showed that the candidate gene BmSAMS was highly expressed in the high temperature and humidity resistant strains,but was relatively low in the low temperature and humidity resistant strains.In order to analyze the potential functional characteristics of BmSAMS gene at protein level,the structure,expression and localization of BmSAMS gene were analyzed by bioinformatics.It was confirmed that BmSAMS gene was highly conserved in insects and had a typical S-Ado Met domain.BmSAMS gene was highly expressed in the epidermis,midgut,malpighian tubule and fat body of posterior silk gland of silkworm,and highly expressed in cytoplasm,nuclear,mitochondria and Golgi apparatus.Overexpression of BmSAMS gene significantly increased the proliferation activity of cells after treatment at27?and 35?,which could improve the proliferation activity of cells to resist the disadvantage of cells under extreme temperature conditions and make them survive stably.The possible interaction proteins of BmSAMS were screened by immunocoprecipitation.The results showed that BmSAMS potentially interacted with ribosomal protein S3(RPS3),heterogeneous nuclear ribonucleoprotein K(hn RNPK),r RNA processing protein Ebp2,bmp-2 protein isoform X1(BMP-2),ribosomal protein S3(rps3a)and actin.We further explored the relationship between BmSAMS gene and their regulation.The results showed that overexpression of BmSAMS gene could significantly promote the expression of Bm PPE,Bm Actin and Bm RPS3 genes,and significantly inhibit Bm HNRK,Bm RPS3aand Bm BMP2 genes. |
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