Screening Of Temperature-responsive Marker Genes In Silk Protein Synthesis And Metabolism Of Guiseri

Silkworms,the greatest contribution is the synthesis of silk proteins and then silk to provide humans with gorgeous and comfortable silk fabrics.Silk glands are important glands for synthesizing and secreting silk proteins in silkworm.Temperature plays an important role in regulating the process of synthesizing silk proteins in silk glands,and has an important effect on the yield and quality of silk.Revealing the temperature of silk protein gene expression regulation function can effectively regulate the silk production and silk quality of mature silkworm.In this study,the practical silkworm variety ’Guican 7’ is taken as the research object.The practical silkworm variety ’Guican 7’ is fed at different temperatures of 20 ℃,25 ℃ and 30 ℃ respectively,at the key period of silk gland development and silk protein synthesis(5 instar).By means of phenotypic traits investigation,enzyme-related immunoassay,transcriptional data analysis and q RT-PCR analysis,the effects of temperature on silk gland development and silk protein synthesis were discuss,and screen marker genes.The main results are as follows:1.Effect of temperature on fifth instar silk glands and cocoonsElectronic precision balance was used to weigh the silk glands from the second day to the seventh day of the fifth instar at different temperatures(20 ℃、25 ℃、30 ℃).The daily increase of silk gland weight was the largest at 25 ℃,and the development state of silk glands was better.The quantitative characters of cocoons were investigated after cocooning at 25 ℃.The female cocoon layer rate was 21.58%,the male cocoon layer rate was 26.02%,and the average male and female cocoon layer rate was 23.80%,which were superior to other temperature groups.2.Effect of temperature on the type and content of silk proteinsThe types and contents of silk proteins at different temperatures were determined by enzyma-linked immunoassay.The contents of heavy chain Fib-H protein,light chain Fib-L protein,P25 protein,ser-1 protein and ser-2 protein of sericin were the highest at 25 ℃,and the results of comparison among different temperature groups were significantly different.3.Screening of pathways and marker genes related to silk protein synthesisTranscriptome analysis of posterior silk glands at different temperatures at final ripening stage showed that 67 differentially expressed genes were significantly up-regulated and 196 were significantly down-regulated in group H.There were 101 significantly up-regulated differentially expressed genes and 124 significantly down-regulated differentially expressed genes in group L.GO and KEGG enrichment analysis showed that the differentially expressed genes in group H were mainly concentrated in amino acid metabolism,juvenile hormone esterase activity and other signal pathways related to silk protein synthesis,while the differentially expressed genes in group L were mainly concentrated in phenylpropyl biosynthesis,glutathione metabolism and other signal pathways related to substance metabolism,and the co-enriched pathways were concentrated in substance transport and energy metabolism.It is preliminarily speculated that high temperature may affect the development of silk glands and the synthesis of silk proteins at the same time,while low temperature may indirectly affect the synthesis of silk proteins mainly through energy metabolism.Six important genes,enzymes and proteins that may be involved in silk protein synthesis were preliminarily screened.4.Temperature response of filament protein coding genes and marker genesFluorescence quantitative PCR was used to detect the relative expressions of the encoding silk protein-related genes fib-H,fib-L,P25,Ser-1 and Ser-2 in the posterior silk gland of the day before spinning at 20 ℃,25 ℃ and 30 ℃.The results showed that,The expression levels of fibroin fib-H,fib-L,P25 gene and Sericin Ser-2 gene at 20 ℃ and 30 ℃ were lower than those at25 ℃.The expression profile of BGIBMGA004726 marker gene at 25 ℃ showed that the expression level of BGIBMGA004726 was the highest on the sixth day of the fifth age,and the expression level in the posterior silk glands on the seventh day of the fifth age was higher than that in the head tissue and midgut tissue.The correlation between relative gene expression and individual silk protein production under temperature response was preliminarily verified: The expression patterns of the encoding silk protein-related genes and BGIBMGA004726 genes at25 ℃ were positively correlated with the investigation data of the silk gland performance and cocoon traits at 25 ℃,which was consistent with the highest values of the species and content of filamentin detected at 25 ℃.In this study,we analyzed the transcriptome of silk glands and the gene expression levels of synthetic silk proteins under different temperature conditions.The relevant results laid the theoretical and technical foundation for breeding silkworm with high silk yield.