Functional Study Of Singed Gene In Response To High Temperature Stress In Silkworm

Insects are the largest group of animals on the planet with a wide variety of species and diverse forms,and their tracks are found in almost every corner of the world,causing significant impacts on agricultural production and human health.Insects are variable temperature animals,and temperature is an important environmental factor that affects the growth and development of insects.Changes in environmental temperature have a serious impact on their living conditions,habitat distribution,growth and development,and metabolic behavior.After a long period of evolution,organisms have formed a complete set of antioxidant systems to protect themselves from oxidative stress.This system is mainly composed of a class of antioxidant enzymes,including superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（Gpx）.These enzymes maintain the redox balance in the organism through synergistic action.Currently,it has been identified that heat shock proteins（Hsps）and antioxidants are involved in insect heat tolerance,but the underlying mechanism of insect heat tolerance is unclear.As an important economic insect,Bombyx mori is also an important genetic model for insects following Drosophila.Currently,China’s sericulture industry is shifting from the west to the east,with the weather in the west becoming hotter and wetter.The drastic changes in ambient temperature have a serious impact on sericulture production.During the rearing process,silkworm is affected by various environmental conditions,especially temperature,which will directly affect the yield and quality of silk.Therefore,it is urgent to identify genes related to high temperature tolerance and cultivate high temperature resistant silkworm varieties.In the early stage,our research team conducted a high-temperature shock test on the high-temperature resistant strain（7532）and the sensitive strain（Knobbed）of the silkworm,and screened the Singed（Sn）gene through transcriptome,which was named BmSn.The gene was differentially expressed in the two strains at all high-temperature treatment time points,suggesting that the BmSn gene is highly sensitive to high temperature and may play an important role in the high-temperature resistance process of the silkworm.Therefore,this study took the BmSn gene as the research object.This study first clarified the molecular changes and damage of silkworm under high temperature or hydrogen peroxide stress;Secondly,the basic characteristics and functions of the BmSn gene were identified,and a BmSn overexpression transgenic strain was constructed to explore its impact on the growth and development of the silkworm;Finally,two interacting proteins of BmSn were identified and their mechanism of action was preliminarily analyzed.The identification and functional study of high-temperature resistant genes in silkworm can provide a genetic target for cultivating high-temperature resistant special varieties,as well as provide a leading theoretical basis for analyzing the adaptation mechanism of Lepidoptera insects to the environment.The main results and conclusions of this study are as follows:1.Silkworm responds to oxidative stress induced by high temperature and H₂O₂In order to explore the physiological and biochemical changes of the silkworm body under high temperature stress,indicators related to oxidative stress in fat bodies were detected after high temperature treatment.The results showed that with the extension of high temperature treatment time,the level of ROS continued to increase,and the activities of SOD and CAT enzymes showed a trend of first increasing and then decreasing.Fluorescence quantitative PCR results showed that antioxidant enzyme related genes were upregulated to varying degrees in a short period of time.In order to verify the above results,using H₂O₂ solution to directly cause oxidative stress damage to the silkworm,the expression changes of oxidative stress indicators and related genes in the silkworm adipose body were detected,and the results were consistent with those obtained by high-temperature treatment.Further exploring the impact of high temperature on silkworm cells,the results showed that with the extension of high temperature treatment time,the cell status gradually deteriorated,and clustered,a large number of deaths,and blurred edges.Then,we tested whether high temperature or H₂O₂ could cause oxidative stress on BmN-SWU1 cells,and detected the proliferative activity and ROS level changes of silkworm cells after high temperature treatment.The results showed that the cell viability was significantly inhibited,the degree of inhibition was positively correlated with treatment time,and the ROS level was significantly increased.The results of the physiological and biochemical test kit showed that high temperature stress could lead to an increase in the levels of H₂O₂ and MDA（malondialdehyde）in the silkworm cells,as well as an increase in the activities of SOD and CAT antioxidant enzymes.The fluorescence quantitative PCR results showed that all genes related to oxidative stress were up-regulated in the silkworm cells after high temperature stress.In order to better elucidate the molecular mechanism of oxidative stress,BmN-SWU1 cells were treated with different concentrations of H₂O₂ and their proliferative activity was measured.The results showed that the cells treated with1000μM or more concentrations of H₂O₂ exhibited an irreversible decline in activity,while ROS levels significantly increased;With the increase of H₂O₂ concentration,SOD,CAT enzyme activity,and MDA content significantly increased,and the expression of genes related to oxidative stress gradually increased with the increase of H₂O₂ concentration.The above results indicate that high temperature and H₂O₂ stress can cause oxidative stress reactions in silkworm and silkworm cells,and significantly upregulate the expression of BmSn gene.2.Functional analysis of BmSn gene in BmN-SWU1 cellsFirstly,the BmSn gene was cloned,identified,and bioinformatics analyzed.The CDS sequence of the BmSn gene has a total length of 1536 bp,encoding 511 amino acids,a predicted molecular weight of 57 k Da,and an isoelectric point of 6.25.It has a typical Fascin domain.Multiple species amino acid sequence homologous protein sequence alignment found that the overall conservation of BmSn gene is high;The phylogenetic tree shows that BmSn is clustered with Lepidoptera insects and has the closest affinity with tobacco moth.By analyzing the spatiotemporal expression pattern of BmSn gene,it was found that the highest expression level of BmSn gene was found in fat bodies;The highest expression level was found in fat bodies at the third day of fifth instar,and it was highly expressed during sleep,possibly related to growth and molting;Immunofluorescence results showed that the BmSn protein was localized in the nucleus.Further constructed BmSn gene overexpression and interference vectors,and analyzed their functions in cells.The results showed that overexpression of BmSn gene promoted cell proliferation,while interference with BmSn gene inhibited cell proliferation;After overexpression of BmSn,compared with the control group,the ROS level of cells significantly decreased,DNA damage significantly weakened,and the survival rate of cells subjected to oxidative stress significantly increased after high-temperature treatment.This indicates that BmSn gene can reduce oxidative stress damage to silkworm cells.3.Analysis on the mechanism of BmSn gene regulating silkworm’s resistance to high temperatureIn order to explore the regulatory mechanism of BmSn gene on the high-temperature resistance of silkworm,a transgenic strain with BmSn gene overexpression was created through transgenic injection technology,and was named BmSn-OE strain.Through the investigation and analysis of the high temperature resistance of BmSn-OE strain and the control large product strain,it was found that BmSn-OE strain showed stronger resistance to high temperature.The adipose tissue of the BmSn-OE strain and the large silkworm strain treated with high temperature was observed.It was found that as time went on,there were differences in the morphology of the adipose tissue and the dissociation of adipose cells between the BmSn-OE strain and the control group.The cell dissociation of the BmSn-OE strain was slower than that of the control group under high temperature;The results of q RT-PCR showed that the oxidative stress related genes in adipose tissue were significantly lower than those in the control group.By analyzing the body size,body weight,total cocoon weight,pupa weight,cocoon layer weight,and cocoon layer rate of transgenic lines,it was found that after overexpression of BmSn gene in silkworm,the appearance of cocoons and pupae increased compared to the control line,and the total cocoon weight,pupa weight,cocoon layer weight,and cocoon layer rate were higher than the control line.To further analyze the mechanism and regulatory network of BmSn gene resistance to high temperature,four candidate interacting proteins were screened through protein mass spectrometry,and two interacting proteins,BmPSMB7and BmCYP450,were identified.They play an important role in regulating cell cycle,DNA damage repair,drug detoxification,fine cell metabolism,and body balance;The BmSn gene can upregulate the expression of BmPSMB7 and BmCYP450,and the BmPSMB7 and BmCYP450 genes can also enhance the proliferative activity of cells in high-temperature environments.