| The utilization level of food nutrition by animals is an important factor that affects the economic benefits of the breeding industry.The silkworm,Bombyx mori(B.mori),is an important economic insect derived from the long-term domestication of the wild silkworm,Bombyx mandarina(B.man).Studying the molecular mechanism of silkworm nutrition utilization will lay the foundation for breeding silkworm varieties with high nutrition utilization efficiency.At the same time,the identification of important genes for silkworm nutrition and the analysis of artificial selection mechanism can also provide ideas and candidate targets for the prevention and control of lepidopteran pests.Lipase is an important digestive enzyme for insects,which is a kind of acyl hydrolase that can hydrolyze long-chain fatty acylglycerides into polar lipids.In this study,we identified a lipase gene(BmLip1)with high expression in B.mori and low expression in B.man,and its domestication characteristics and functions were studied.The following findings were obtained:1.Identification,cloning and expression characterization of BmLip1To identify the digestive enzyme genes from the midgut of silkworm,we first searched the reported amino acid sequences of the digestive enzymes in the midgut of insects,and then aligned the sequences of the silkworm to screen out the digestive enzyme genes of silkworm.Secondly,the selected digestive enzyme genes were compared with the midgut transcriptome data of the silkworm and the wild silkworm to obtain the digestive enzyme genes with high expression in the midgut of silkworm and low expression in the midgut of wild silkworm.As a result,the most typical BGIBMGA010400(BmLip1)gene was screened and further studies were carried out.BmLip1 is located on chromosome 12 and consists of 6 exons.According to the protein sequence prediction,BmLipl is a secreted protein that can express 295 amino acids and has a signal peptide sequence.PCR amplification results showed that the ORF and genome size of the gene were about 888 bp and 3500 bp,respectively.Domain prediction showed that it has a lipase activity domain.Multiple sequence alignment showed that the protein has three catalytically active sites,but serine was mutated into asparagine.Phylogenetic tree analysis shows that it is a type of neutral lipase.Spatial and temporal expression profile analysis showed that the gene was expressed in high levels in the foremidgut and the fifth instar feeding stage of silkworm.Immunofluorescence localization analysis showed that the protein was expressed in the anterior,middle and posterior parts of the midgut,and expressed more in the foregut.It was found that the protein was a kind of secreted protein.2.Analysis of the causes of differential expression of BmLipl and its domestication characteristicsFirstly,we verified the differential expression gene in the midgut transcription level of different species of silkworm and wild silkworm.The results showed that the gene in the midgut of different varieties of silkworm was generally higher than that of wild silkworm.Secondly,the protein level was also verified,and showed that the protein was significantly higher than that of wild silkworm.To explore the reasons for the differential expression of this gene,we analyzed the genome structure.It was found that there are two to three copies of the gene in different silkworm strains,while the wild silkworm has only one copy.Further genomic PCR experiments showed that the silkworm generally has multiple copies and the wild silkworm has a single copy.This is consistent with the ORF sequencing results of 932 and zhejiang wild silkworm.932 has 3 different ORF sequences,while zhejiang wild silkworm has only one.These findings indicate that BmLip1 has increased its copy number during the selection process.In addition,we also analyzed the sequence diversity of the BmLip1 gene,which showed that the genetic diversity of the BmLip1 was significantly reduced compared with the wild silkworm.Tajima’s D and πa/πs value indicate that the gene is under the pressure of positive selection in local species,it is under the pressure of purification selection in wild silkworm.3.Prokaryotic expression,purification and activity detection of BmLiplIn order to verify the function of the protein,we prokaryotically expressed the protein BmLip1 and the mutant protein BmLip1(Asn→Ser),and the recombinant protein was successfully obtained.The enzymatic activities of the two recombinant proteins were 24.43 U/L and 32.82 U/L at room temperature.The optimum temperatures of the two recombinant proteins were 20℃ and 40℃,respectively.The optimal pH were 10.We performed molecular simulation docking experiments and incubation experiments with Morachalcone A and BmLip1 and BmLip1(Asn→Ser)respectively.The results show that Morachalcone A can better inhibit the activity of BmLipl(Asn→Ser).Therefore,we conclude that silkworms express a large amount of BmLipl with low activity,which is to counteract the effect of lipase inhibitors in mulberry leaves and make silkworms better digest and absorb nutrients in food.4.Research on the overexpression of BmLipl in silkwormIn order to explore the effect of overexpression of this gene in the midgut of the silkworm on its growth and development,we obtained the corresponding transgenic line P3P:Lipl based on piggyBac transposon-mediated transgenic technology.The transgenic silkworms and wild-type silkworms were reared under the same conditions,and then phenotypic statistical analysis was performed.The results showed that there was no significant difference in feed intake of the transgenic silkworms,while the weight of male and female larvae of the overexpressed silkworm increased by 0.065g and 0.073g respectively compared with the wild-type silkworm.Compared with the wild-type,the female and male total cocoon weight of the overexpressed silkworm increased by 0.023g and 0.014g,respectively.The weight of male and female pupae of overexpressed silkworm increased by 0.021g and 0.011g,respectively,compared with that of wild-type.The rate of female and male cocoon layers of overexpressed silkworm was higher than that of wild-type,respectively increases by 0.44%and 1.02%.In summary,this study identified a lipase gene BmLip1 that was highly expressed in the midgut of the silkworm but low in the wild silkworm.Two to three copies of this gene are common in silkworms,while only one copy exists in wild silkworms.Domestication analysis shows that it is a domestication-related gene.Two recombinant proteins BmLip1 and BmLip1(Asn→Ser)were obtained,and it was confirmed that the former is not as good as the latter in binding to Morachalcone A.In addition,transgenic silkworm lines with overexpression of lipase were obtained based on piggyBac transposon mediated transgenic technology.The above results reveal the influence of artificial selection of silkworm lipase genes on the growth and development of silkworm.This study also provides theoretical reference and technical support for breeding excellent silkworm varieties based on genetic engineering. |
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