Screening And Artificial Regulation Of Silk Gland Related Differentially Expressed Genes Between Bombyx Mori And Bombyx Mandarina

The Silkworm,Bombyx mori(B.mori),is one of the holometabolous insects with high economic value,and is also a model insect of Lepidopterans,which has four different developmental stages,including egg,larva,pupa,and adult in its short lifespan.Silk gland of silkworm can synthesize and secret a kind of natural polymer protein fiber,the silk.Given its excellent performance,the silk can not only be used as textile clothing materials,but also be applied in fields such as medicine,beauty products and aerospace materials when processed by the modern technology.The wild silkworm,Bombyx mandarina(B.man),is the ancestor of B.mori,whcih has been domesticated for more than 5,000 years.A lot of evdiences such as the unearthed cultural relics,historical documents and modern molecular biology evidence have proved that B.mori is originated from China and then spread to other parts of the world.Due to the long-term artificial selection,B.mori has many differences compared to its ancestor,including body-color,larva size,growth cycle,flight ability of the moth and so on.The silk was mainly used to produce silk clothes in the past,so the purpose of artificial selection was to improve silk production.Therefore,as the unique organ for silk synthesis and secretion,the silk gland has been subjected to more and stronger artificial selection pressure during the domestication.The silk gland is mainly composed of three compartments,the anterior silk gland(ASG),middle silk gland(MSG)and posterior silk gland(PSG).MSG is responsible for the synthesis of sericins,and PSG synthesizes and secretes the fibroin proteins,including the fibroin heavy chain protein(Fib H),fibroin light chain protein(Fib L)and P25 proteins.Silk fibers are assembled with fibroin proteins in the inner layer and sericin proteins in the outside layer.Previous studies have been focused on the structure and function of silk gland of B.mori.However,the difference in the silk gland and cocoon between B.mori and its ancestor B.man and the domestication mechanism remain relatively unknown.In this study,we first examined the differences between B.mori and B.man cocoons,then investigated the transcriptome of both B.mori and B.man silkgland at proper timepiont to screen the differentially expressed genes.Subsequently,we verified the function of these screened genes by the method of artificial regulation.The main results of this study are as follows: 1.Investigated the difference in cocoon phenotype between B.mori and B.manTo explore the differences between these two kinds of cocoons except the silk yield,we performed experiments like SEM observation,mechanical properties test,and solubility analysis.And we found that there were more calcium oxalate crystals on the surface of B.man silk than the B.mori silk,and the cocoon layers of B.mori were relatively loose,while the cocoon layers of B.man were denser and closely adhered together.Interestingly,we observed no significant difference in the diameter between the two types silk fibers.We further performed the mechanical properties test of the two kinds of silk fibers and found that the mechanical properties of B.man silk were better than B.mori silk.The results showed no significant differences in the malleability of both silks,but the maximum stress of B.man silk is significantly greater than B.mori silk.In addition,the dissolution capacity analysis of both types of cocoons showed that the B.mori cocoon can dissolve completely in the Li SCN solution very quickly,while the B.man cocoon was difficult to dissolve with a large part remain undissolved.Through the comparison,we found significant differences in density,porosity,the mechanical properties and solubility of the silk expect the differences in color and size between B.mori cocoon and B.man cocoon.2.Transcriptome analysis of silk gland between B.mori and B.manWe dissected the intact MSG and PSG of B.mori and B.man to extracted total RNAs,then performed transcriptome sequenced by using an Illumina Hi Seq2000 platform.A total number of 20,268 annotated Unigenes were obtained by transcriptome sequencing.The differentially expressed genes(DEGs)analysis showed that there were 9,752 Unigenes differentially expressed in MSG,3,074 Unigenes were up-regulated in B.man MSG,while 6,678 Unigenes were up-regulated in B.mori MSG.In PSG,8,207 Unigenes were differentially expressed,2,052 of these genes were up-regulated in B.man and 6,155 of them were up-regulated in B.mori.Basically,we found that B.mori had more Unigenes with higher expression level,no matter in MSG or in PSG.To further explore the functions of these differentially expressed genes,we performed GO enrichment analysis of differentially expressed genes.The results showed that most DEGs in MSG and PSG were both significantly enriched in GO classification to the catalytic activity,suggesting that B.mori and B.man may have differences in silk synthesis and secretion process.We also found that many differentially expressed genes were annotated as enzymes,indicating that there might be some differences in secretion and processing of silk proteins,which may affect the structure of silk proteins.In addition,the KEGG pathway enrichment analysis showed that the differentially expressed genes in the MSG and PSG are involved in protein synthesis and transport,amino acid metabolism,energy metabolism,etc.,which suggested that there might be differences in protein synthesis and utilization in silk glands between B.mori and B.man,and this probably is the reason for why they show big difference in silk production capacity.Besides,we found that Unigene CL1003.Contig2?All(BGIBMGA003330)was differentially expressed in both MSG and PSG,and the expression of BGIBMGA003330 was higher in B.man than that in B.mori.Through co-expression network analysis,we found that the gene especially show co-expression correlation with the silk fibroin heavy chain gene(Fib-H)in PSG.BGIBMGA003330 encodes a putative inosine-uridine preferring nucleoside hydrolase,which can decompose uridine into ribose and uracil.Nucleoside hydrolase participates in generation and utilization of pyrimidine nucleotides,which palys an important role in the growth and development of organisms.Therefore,we speculated that this gene could affect the development and growth of silk glands and the secretion and synthesis of silk proteins.3.Establishment of CRISPRa and CRISPRi system in Bm E cellsIn order to explore the function of the screened genes,we first constructed a CRISPRa and CRISPRi-mediated gene activating or repressing system in the Bm E cell line.For the CRISPRa activation system,we constructed two activation vectors,d Cas9-VP64 and d Cas9-VPR.Our experiments demonstrated that both activators can efficiently activate the expression of the target genes,while the d Cas9-VPR is much more efficient than d Cas9-VP64.The d Cas9-VPR system can efficiently activate the target gene without causing off-target effects,and it can simultaneously activate the expression of three target genes,demonstrating that it can be used in the simultaneous activation expression of multiple genes.Meanwhile,we also found that d Cas9-VPR have different activation capacity for each of the three target genes.The activation effect is stronger for lowly-expressed genes.For genes that are already expressed,its activation capacity is relatively weaker,showing a negative correlation.In the CRISPRi system,we selected a total of five different transcription repression factors for vector construction: KRAB,SRDX,ERD,hairy,and SID.Firstly,we confirmed that these five different suppression systems can specifically and efficiently down-regulate the expression of target genes and with no significant difference among them.Subsequently,we also demonstrated that the CRISPRi system can be applied to the simultaneous down-regulation of multiple genes.We also found two factors that affect the suppression efficiency.One of them is that the distance between the sg RNA and the transcriptional start site(TSS)could affect CRISPRi efficiency.The closer it is to the TSS,the better CRISPRi efficiency is.The other is that the transduction ratio of d Cas9 and sg RNA vectors could also affect the efficiency.In our experiment,the molar ratio of 1:4 is remarkable for high repression.Our results also confirmed that the CRISPRa and CRISPRi systems are feasible and efficient for study of gene functions in B.mori and even lepidopteran insects,and also provide evidence for the promising application of the system in silkworm individuals.4.Functional verification of differentially expressed genes using CRSIRPa or CRISPRi transgenic silkworm linesFor the first time,we applied the the CRISPRa and CRISPRi systems on silkworm individuals based on piggy Bac transposon to make transgenic lines.In PSG,the CRISPR activator or repressor(p Bac-Fib H-d Cas9-VPR and p Bac-Fib H-d Cas9-ERD)were drived by the Fib H promoter.While in MSG,the CRISPRa and CRISPRi systems were drived by the Ser1 promoter,including p Bac-Ser1-d Cas9-VPR and p Bac-Ser1-d Cas9-ERD.In addition,we designed three pairs of sg RNAs at different positions for the screened differentially expressed genes according to the sg RNA designing principle and injected them into the silkworm embryos.We first selected positives transgenic line for the BGIBMGA003330 gene targeting sg RNA from the mixed sg RNA library individuals and crossed them with p Bac-Fib Hd Cas9-VPR(CRISPRa transgenic line)or p Bac-Fib H-d Cas9-ERD(CRISPRi transgenic line),or with the previously established transgenic line of p Bac-Na Nos-Cas9(CRISPR knockout line).Thereafter,we screened their offsprings for double positive individuals.We also checked the expression level of BGIBMGA003330 gene by q RT-PCR on day 3 of fifth-instar larvae of the transgenic hybrid offspring larvae.The results showed that BGIBMGA003330 expression was significantly increased in the hybrid transgenic line of CRISPRa and BGIBMGA003330 sg RNA when comparing to the control,with the cocoon weight and cocoon layer rate of female transgenic progenies increased significantly.Meanwhile,the hybrid offspring of the knockout line of the BGIBMGA003330 gene promoter by p Bac-Na Nos-Cas9 were also examined,and PCR sequencing revealed that it was successfully knocked out at the sg RNA targeting site.Subsequently,we also tested the expression level of the gene in knockout individuals at day 3 of fifth-instar larvae.We found that BGIBMGA003330 expression level of knockout individuals was significantly lower than that in the control group,demonstrating that Nanos-Cas9 can successfully knock out the promoter of the BGIBMGA003330 gene which lead to the down-regulation of its expression.Then,we performed a character analysis on the cocoons of the knockout individuals,which showed that the cocoon weight,pupa weight,and the cocoon layer rate of the knockout individuals decreased significantly in contrast to the control group.q RTPCR was used to detect the expression of Fib H in the silk glands of knockout individuals,it showed a significant decrease compared to the control group,but there was no significant difference in Fib L and P25.This also indicated that the knockout of BGIBMGA003330 gene promoter caused a decrease in gene expression and affect the silk protein synthesis and secretion.In order to explore whether the down-regulation of the gene expression has an effect on silk performance,we also tested the mechanical properties of cocoon silk in the knockout individuals.While it showed that the downregulation of BGIBMGA003330 gene expression did not affect the mechanical properties of silk.Moreover,we also screened for hybrid transgenic positive lines for other genes by crossing their sg RNA transgenic positive line with CRISPRa or CRISPRi transgenic line.Among them,BGIBMGA009814 is a gene that encodes a serine/threonine protein kinase.When we crossed its sg RNA transgenic line with CRIPSRa transgenic line,we detect the expression level in PSG by q RT-PCR on day 3 of fifth-instar larvae.This proved that the CRISPRa system can successfully upregulate the expression of genes of interest in B.mori.We also tested the cocoon weight,pupa weight,and cocoon layer rate of the hybrid transgenic positive individuals,and we found that the up-regulation of the BGIBMGA009814 gene can not affect the characteristics of the cocoons of the offspring.However,SEM observations of cocoons showed that the cocoon layer of the positive individuals appeared to be denser,which was more similar to the cocoons of B.man.In summary,we analyzed the differences of cocoons between domestic silkworms and wild silkworms,and screened differentially expressed genes that might be related to these differences by transcriptome sequencing of silk gland in this study.And we constructed a CRISPRa activation system and a CRISPRi suppression system at silkworm individual level,providing a new method for studying gene function in silkworm.At the same time,we also confirmed that the BGIBMGA003330 gene is related to the silk production of silkworms,which strongly suggested that it suffered artificial selection during the domestication process of silkworms.Importantly,our study provided molecular basis for understanding the domestication mechanism of silkworms and the process of silk synthesis and secretion,and also proposed new ideas and methods fot improving the silk quality.