Function Of Sericin3 In The Process Of Silkprotein Synthesis And Cocoon Shell Adhesion Ofbombyx.mori

Bombyx mori is a typical lepidopteran insect,and the protein product of cocoon silk has important economic value.A cocoon silk is mainly composed of two silk fibroin fibers in the center and sericin wrapped around the periphery.Fibroin protein and sericin protein is mainly synthesized by the silk gland,which is the mainly silk-producing organ of the silkworm.The silk glands are divided into anterior silk gland(ASG),middle silk gland(MSG)and posterior silk gland(PSG)according to their morphological characteristics.The posterior silk gland mainly secrete fibroin,including fibroin heavy chain protein(Fib-H),fibroin light chain protein(Fib-L)and P25 protein;the middle silk gland mainly secrete sericins,including Sericin1(Ser1),Sericin2(Ser2)and Sericin3(Ser3).The silk fibroin secreted by the posterior silk gland is wrapped by the sericin secreted by the middle silk gland,transported through the anterior silk gland,and finally processed in the spinning part to form cocoon silk.According to the previous research,the sericin protein,especially the Ser3 protein plays an important role in the process of cocoon silk formation and cocoon layer adhesion.Previously,some scholars carried out the proteomic analysis of cocoons of silkworm 872 strain with tightly adhered cocoons and cocoons of Floosy(Fl)strain with fluffy cocoons,founding that the content of Ser3 protein in the Fl strain cocoon is higher than that in the 872 strain.The relationship between the amount of Ser3 protein content and the adhesion of the cocoon layer is not clear now.Based on it,the Ser3 antibody prepared in our laboratory was used to localize the expression of Ser3 protein in different silk gland sites at different developmental periods by immunofluorescence experiment.To study the function of Ser3 protein in the silk protein synthesis and cocoon layer adhesion,we used the CRISPR/Cas9 system to knock out the Ser3 gene in the silkworm.The following findings were obtained:1.The expression of Ser3 protein in silk gland of the silkworm gradually increases in the middle and late 5th instarIt has been shown that the Ser3 gene is mainly expressed in the anterior part of the middle silk gland(A-MSG)in the 5th instar of the silkworm,but the research on the synthesis and secretion of Ser3 protein at the protein level is still lack.Therefore,in order to localize the expression of Ser3 protein more precisely,the antibody of Ser3 protein was used to localize the Ser3 protein in different regions and time points of silk glands of the silkworm.Both the ASG and A-MSG were divided into three parts,ASGⅠ,ASGⅡ,ASGⅢ and A-MSGⅠ,A-MSGⅡ,A-MSGⅢ.In terms of developmental period,the ASG and A-MSG parts at the 1st,3rd and 5th days of 5th instar,and the ASG,A-MSG,the middle part of the middle silk gland(M-MSG)and posterior part of the middle silk gland(P-MSG)at the 7th day of 5th instar were investigated.The above materials were collected and fixed,and paraffin sections,HE staining and immunofluorescence experiments were performed.The results showed that the Ser3 protein was expressed in the cell layer of A-MSG III from the third day of instar 5.With the development of time,Ser3 protein is gradually secreted into the glandular cavity.Ser3 protein is secreted from the P-MSG,M-MSG and A-MSG II/III cell layers into the glandular cavity and transported to the ASG at the 7th of 5th instar.The fluorescence signal of Ser3 protein in the glandular cavity is located at the periphery of the Ser1 protein layer,which eventually mixes at the ASG.2.Knockout of the Ser3 gene in the silkworm reduces silk production and causes cocoon fluffingAccording to the previous research,Ser3 protein is specifically expressed in silk gland.To better study the function of Ser3 protein,we constructed Ser3 knockout strain by CRISPR/Cas9 knockout system.Firstly,three sg RNAs were designed on the sg RNA design website and synthesized in vitro.Secondly,the synthesized sg RNAs were mixed with Cas9 protein and injected into early embryos.The injected silkworm eggs hatched,normally raised to the moths and then self-fertilized to obtain G1 individuals.A small amount of G1 eggs were taken for genome extraction and carried out the knockout validation,and part of moth circles were found to have knockout individuals.The results of the genomic testing showed that the genome of knockout individuals had 4 bp and 2 bp insertions,respectively.The results of the m RNA were consistent.Prediction of protein translation on the m RNA of knockout individuals revealed the presence of stop codons within the first 200 nucleotides.Western blot assays on the silk gland proteins of the knockout individuals failed to detect the signal of Ser3 protein.The HE staining and immunofluorescence assays of silk glands had the same results,indicating that we successfully constructed the pure knockout strains of Ser3 protein.We conducted phenotypic observations and economic traits statistics on the cocoons of the knockout lines,and the results showed that the cocoons of the knockout lines showed a fluffy state,and the cocoon weight and cocoon layer rate were significantly lower compared with the wild type.3.Knockout of the Ser3 gene in the silkworm results in altered cocoon silk protein compositionSince the cocoon weight of Ser3 knockout individuals is changed,we speculated that Ser3 knockout will affect the synthesis of the silk protein.Therefore,proteomic analysis was performed on the cocoon silk proteins of wild type and knockout strains.It was found that the top ten proteins among the two strains were mainly silk proteins.Only one of the top ten proteins had significant difference,and that was the Ser3 protein with a 216.59-fold difference.The result further confirmed that the knockout strains contained essentially no Ser3 protein,but instead a protein containing the HTH-psq domain.Moreover,although Ser1 protein was found to be in the first place in both strains,the amount of Ser1 protein in the cocoon of the Ser3 knockout strain was about 1.5 times higher than that of the wild type,indicating that individuals of the Ser3 knockout strain were able to synthesize more Ser1 protein.Moreover,the content of Fib-H in silk fibroin in the knockout cocoon silk was only 3/4 of that of the wild type,and the protein content of Fib-L and P25 was slightly higher than that of the wild type.The proteins with significant difference were analyzed,founding that the protein with the most significant difference between the two strains was still the Ser3 protein.The specific expression proteins in the two strains were analyzed,founding that Ser2 protein was specifically and highly expressed in the cocoon of the Ser3 gene knockout line compared to the wild type cocoon.It could be seen that the knockout of Ser3 gene would affect the synthesis of silk protein,further affect the composition of cocoon silk protein,and finally affect the production of the cocoon silk.4.The role of Ser3 protein in cocoon layer adhesion in silkwormThe typical phenotype of cocoon of Ser3 knockout strain is cocoon layer fluffy,indicating that the lack of Ser3 protein affects the cocoon layer adhesion.However,the content of Ser3 protein was more in cocoons of the Fl strain with a fluffy cocoon layer,than in cocoons of the 872 strain with a dense cocoon layer.In order to investigate how the Ser3 protein affects cocoon layer adhesion,we chose to investigate the cocoon of 872,Fl,WT and Ser3 knockout strains.In this experiment,the original thickness and the compressed thickness of the cocoons of the above strains were measured and the difference was calculated to indicate the fluffiness.The results showed that the fluffiness of the cocoon of the above strains was ranked as Ser3 knockout strain > Fl > WT > 872.Ser3 protein is mainly distributed in the outermost layer of the cocoon silk.In order to determine its content on the surface of cocoon silk,cocoons of the above strains were layered in cocoon layers,and immunohistochemical experiments were carried out using sericin antibody.The experimental results showed that,except for the cocoon of the Ser3 gene knockout line,the signals of Ser1 and Ser3 proteins were observed on the surface of each cocoon layer of the other three lines,but no signal of Ser2 protein was detected.while the Ser3 knockout strain cocoons were only observed with Ser1 protein signals.The result indicated that there was not only Ser3 protein but also Ser1 protein on the surface of the cocoon silk.To further confirm that the signal of Ser1 protein in cocoon slices was from the outer layer of cocoon,the cocoon slices were subjected to frozen section and found that the antibody of sericin was bound only to the outermost layer of cocoon silk.The antibody signal was analyzed in gray scale,and the ratio of Ser1 protein signal to Ser3 protein signal was calculated after uniform deduction of Ig G control.The results showed that the order of the ratio size of the signal values was872<WT<Fl<Ser3 knockout strain,which was opposite to the fluffiness of the cocoon layer.The above results suggested that the cocoon layer adhesion is not solely determined by the content of Ser3 protein in the outer layer of cocoon silk,but is related to the ratio of Ser1 protein and Ser3 protein in the outer layer of cocoon silk.The smaller the ratio,the better the cocoon layer adhesion effect..This result is consistent with the result that Ser1 protein and Ser3 protein were mixed in ASG on the day of silkworm clustering.The outermost sericin of cocoon silk is a mixed protein of Ser1 and Ser3,and its relative content is related to the compactness of cocoon layer.