Identification And Molecular Mechanism Of The Naked Pupa (Nd) Mutation In Silkworm

Animal silk fibers are natural polymer with attractive bioactive properties and have been widely used in biomedical applications.The silks produced by silkworm and spider are by far the most striking and well-studied among numerous silk fibers in the animal kingdom.Silk gene structure,silk protein synthesis,silk protein assembly,silk protein secretion,silk protein transport,and silk gland development are important topics in silk fiber study,but still contain many unknowns.Here,the silk-related mutant,named Naked pupa?Nd?,was studied to enrich the understanding of the synthesis and secretion of silk protein.Nd mutation is single gene controlled dominant inheritance and a classical genetic marker of chromosome 25^th?0.0 cM?.Previous studies showed that Nd mutants'posterior silk gland?PSG?was degenerated,most of them formed naked pupa,and little produced sericin cocoon.In this study,we uncover the unknown molecular nature of Nd mutation by phenotype observation,genetic identification,pathway analysis and functional verification.The main results are as follows:1.Fibroin secretion deficiency affects spinning behavior of the Nd mutantNd mutants spun no silk thread from the beginning of larval stage,and until wandering stage,they could spin small amount of sericin to form thin and easily broken cocoons.The PSG of Nd mutants was extremely degenerate but the middle silk gland?MSG?was normal,implying that the fibroin synthesis ability of PSG was deficient but the sericin synthesis ability of MSG was normal.The cocoon of Nd mutants was completely dissolved in 8 M urea solution,suggesting the cocoon consisted mostly of sericin.These results showed that,in Nd mutants,the fibroin secretion ability was deficient from beginning to end,and the posterior silk gland was poorly developed.In addition,different mounting conditions?normal plastic collapsible mountages and suitable cardboard frame mountages?analysis showed that the mounting condition is very important to the stability of Nd phenotype,and under suitable mounting condition,absolute 100%of Nd mutants formed sericin cocoon.2.Repetitive region R08–10 of FibH mutation encodes a CTD-deleted Fib H in the Nd mutantWe performed linkage mapping using single nucleotide polymorphism?SNP?markers,and narrowed 573 kb genomic region on nscaf2823 of chromosome 25^th,which included thirteen predicted genes.Among these genes,only BGIBMGA005111?fibroin heavy chain gene,FibH?was differentially expressed.Sequencing analysis showed that,in Nd mutants and WT/Dazao,the NTD and CTD encoding regions of FibH were identical.However,over 15kb of FibH are highly repetitive GC rich sequence,making it hard to amplify and sequencing.Therefore,we detected mutations in protein level.LC-MS analysis showed that only peptides of Fib H NTD,but none of FibH CTD were identified in the PSG of Nd mutants,suggesting that this CTD-deleted FibH was premature translationally terminated or frameshift mutant.To further identify the mutational pattern,based on the FibH amino acid sequence,we prepared four antibodies against FibH NTD,A01,A02–10 and CTD.Immunofluorescence analysis showed that most of the fluorescence signals for Nd mutant FibH existed in PSG cells,and not in PSG lumen,suggesting that the CTD-deleted FibH was secretion-deficient.In PSG cells of Nd mutants and WT/Dazao,the NTD and A01 fluorescence intensities were in similar level;however,the A02–10 fluorescence intensity was significantly decreased in Nd mutants;and moreover,none of the CTD fluorescence signals were detected in Nd mutants.These results indicated that the amino acid sequence between FibH A02 and A10 was changed and the antigen peptide number in FibH A02–10 was decreased in Nd mutants.Western blot analysis showed that two immune bands around the 390 kDa FibH?between 240 to390 kDa?were detected in the PSG of Nd mutants.We analysed the three-frame translation sequence of FibH and found that A07 encoding region,and its downstream sequence mutation could form a mutant FibH with 240 to 390 kDa molecular weight.We therefore concluded that FibH encoding region between A07 and A10?corresponding R08–10?mutation resulted in CTD-deleted and truncated FibH.3.Secretion-deficient mutant FibH causes cellular stress response and PSG degenerationTransmission electron microscope observation of PSG cell showed that an increased number of spotted autophagosomes,distended endoplasmic reticulums?ER?and poorly developed Golgi complexes?Go?were in Nd mutant PSG cells'cytoplasm.The fibroin intracellular transport and secretion system was broken in Nd mutant PSG cells'cytoplasm.Western blot analysis showed that,the fibroins couldn't be transported from the PSG to the MSG lumen in Nd mutants,suggesting that CTD was important to FibH secretion.To precisely determine how the secretion-deficient FibH causes the PSG subcellular structural change,we performed RNA-seq to identify the differentially expressed gene?DEG?between Nd mutant PSG and WT/Dazao PSG on the V3^rdD,and 2,178 DEGs?1,254 up-regulated and 924 down-regulated?were identified.The KEGG analysis showed that three pathways related to neurodegenerative diseases?Huntington's disease,Alzheimer's disease and Parkinson's disease?,and four pathways related to cellular quality-control system,which was quality control strategy to avert dangers from damaged protein,were significantly enriched.These results suggested that Nd mutant PSG cell initiated stress response to secretion-deficient FibH through cellular quality-control system.We investigated the silk gland development patterns during the whole larval stage,and found there was no significant difference in the cell number of the MSG and PSG between Nd mutants and WT/Dazao,However,the PSG length and PSG nuclear ramification of Nd mutants increasing was significantly lower than that of WT/Dazao starting from the IV1^stD.At the end of the larval stage,the amount of DNA in Nd mutant PSG was only about one-third of what it in WT/Dazao PSG.qRT-PCR analysis showed that,as the larva growth and FibH expression increasing,the ATG8/LC3,E3 and Hsp19.9 expression levels in Nd mutant PSG were significantly higher than in WT/Dazao PSG,suggesting that the progressive PSG degeneration was the result of continuous cellular stress response triggered by mutant FibH.4.CRISPR/dCas9 and genetic hybrid validations of Nd mutation mechanismUsing the CRISPR/dCas9 system,we validated secretion-deficient causing cellular stress response in BmE cells,which didn't express FibH and without FibH secretion condition.We designed three sgRNA and cotransfected BmE cells with activator fused dCas9?dCas9-VPR?,and found that,under the conditions that transfection ratio of sgRNA and dCas9-VPR was 9:1 and the transfection time was up to 60 hours,the endogenous FibH expression reached the maximum,which induced the ATG8/LC3,E3 and Hsp19.9expression significant increasing.In addition,the Lyso-Tracker staining and the monodansylcadaverine?MDC?staining,a fluorescent marker to label autophagy,showed that the FibH activated cells were stained positively by both two stains.These results suggested that the cellular stress respone was successfully activated by secretion-deficient FibH.The PSGs of homozygous Nd?repetitive region mutant FibH?mutants and Nd-s?mutant FibL?mutants were short and degenerated,but the PSG of homozygous fibH-k?NTD mutant FibH?mutants were normal and forded,compared with WT/Dazao PSG.All the mutants had significantly decreased FibH expression levels in common.However,the expression level of the ATG8/LC3,E3 and Hsp19.9,which were involved in the cellular quality-control system,were significantly increased only in degenerated PSG?Nd mutants and Nd-s mutants?and non-significant in that of the fibH-k mutant PSG.We therefore concluded that PSG degeneration was relative to the activated cellular stress response.In heterozygous mutants,Nd/+mutants showed a short and degenerated PSG,the fibH-k/+mutants showed a perfectly normal PSG as that of WT/Dazao,and Nd-s/+mutants showed a semi-degenerated?long?and semi-normal?less folded?PSG in between that of Nd/+mutants and fibH-k/+mutants.The degenerated PSG?Nd/+mutants?also showed a significantly decreased FibH expression level and significantly increased ATG8/LC3,E3 and Hsp19.9 expression level compared with the normal PSG?fibH-k/+mutants?.Intriguingly,in semi-degenerated and semi-normal PSG?Nd-s/+mutants?only E3 and Hsp19.9 were significantly decreased compared with the normal PSG?fibH-k/+mutants?.We therefore concluded that the serious PSG degeneration was caused by serious cellular stress response.5.Nd mutant dominant produces fibroin-free sericin cocoonThe cocoons of Nd mutants,Nd-s mutants,fibH-k mutants were thin,easy to break,and were completely dissolved in 8 M urea solution.In heterozygous mutants,only cocoon of Nd/+mutants was completely dissolved,suggesting Nd mutant dominant produced sericin cocoon.The cocoons of Nd-s/+mutants and fibH-k/+mutants contain fibroin.Western blot analysis showed that,only with Nd mutation,the cocoon of mutant parental generation and its hybridize generation did not contain any of the three fibroins?FibH,Fib L and P25?.Thus,Nd mutants and any type of its progeny could produce entirely fibroin-free sericin cocoon.By using this sericin cocoon,we next compared the integrity of sericin extracted with three conventional methods).The SDS-PAGE analysis showed the intact and undegraded sericin was obtained with the LiSCN extraction method,which could be used to prepare elastic hydrogel with the addition of ethanol rather than chemicals or irradiation crosslinking agent,suggesting that intact sericin extraction requires LiSCN method and sericin cocoon.Insummary,thesefindingssuggestedthatanysilkwormstrain?commercial/bioengineered?crossed with Nd mutants could serve as a special sericin maker who promoted various sericin biomaterials were available.