| Bombyx mori,B.mori,as a representative of lepidopteran insects,has maintained close contact with humans for thousands of years.Despite the vicissitudes of dynasties,she has not changed her original aspiration as before,and has written the chapter of life for generations.B.mori is famous for its silk.B.mori silk is bright,beautiful,smooth,comfortable,and breathable.It is known as the "fiber queen" and "the second skin of the human body".The main components of silk are silk fibroin and sericin,and silk fibroin is composed of FibH,Fib L and P25.FibH has a molecular weight of 350 KDa and has5264 amino acids.Its coding gene is 16788 bp in length,including two exons and one intron.Exon 1 is only 57 bp and exon 2 is 15750 bp in length.Silk has degradability,biocompatibility and excellent mechanical properties and is widely used in textile,medical,electronics,beauty,and other fields.The mechanical properties of silk are affected by many factors such as spinning rate,metal ions,shearing force,and p H.The structure determines the properties.The mechanical properties of silk are mainly determined by the sequence of the silk fibroin heavy chain,but the relationship between the two is still unknown.There are many varieties of B.mori,with clear genetic background,complete genome information,and mature genetic operation platform.The comparison of different B.mori or FibH gene-edited B.mori is an important method to study the mechanical properties of silk and FibH sequence.However,there are too few known B.mori varieties with the complete FibH gene sequence,and only the B.mori p50 T is known.More than 20 years have passed since the FibH gene of p50 T was published in2000.The main reason for the slow progress in the research of FibH gene sequence is that the FibH gene is long,low complex and has rich GC,and cannot be amplified by conventional PCR.For obtaining FibH gene sequence,traditional BAC library construction and sequencing cycle is long,and single molecule real-time(SMRT)sequencing is expensive and the sequencing quality is not guaranteed.In view of the difficulties faced by the FibH gene,this study took the B.mori as the research object,combined with the next-generation gene editing technology CRISPR/Cas9 and Pac Bio sequencing to clone and sequence the FibH genes of B.mori(domesticated silkworm)and B.mandarina(wild silkworm).The repetitive core sequence of the FibH gene of the domesticated silkworms was edited,and the FibH gene-edited mutants were obtained.The main results and conclusions of this research are as follows:1.The cloning and sequencing method of FibH gene was establishedSince 2000,molecular biology technology has made great progress,but FibH gene sequence related research progress is still slow.The main reason is that FibH gene sequence is too long to be amplified and sequenced by conventional PCR and Sanger sequencing.To break through the technical bottleneck,this research has explored and tried from three aspects: PCR amplification,enrichment sequencing,and cloning sequencing.Firstly,we selected four high fidelity DNA polymerases including Primestar max(TAKARA),Hieff canace high fidelity(YEASEN),Q5 high fidelity(NEB)and KOD-plus-NEO(Toyobo)to amplify FibH gene by PCR with B.mori genomic DNA as template.FibH gene could not be amplified by adding DMSO,changing annealing temperature,changing genomic template or primer.Then,we established a CEO enrichment sequencing method based on Cpf1 and ONT,which mainly included four steps: dephosphorylation of B.mori genomic DNA,Cpf1/cr RNA cleavage,ligating adapters,ONT sequencing.FibH gene was enriched,and its enrichment ratio was 2.29 times via CEO.The FibH gene sequence of Dazao was assembled by bioinformatics software.The similarity between it and KWMTBOMO15365 was 99.98%,and the difference was the deletion of three bases in the intron.T-A cloning sequencing revealed that the three missing bases were not SNPS,but sequencing errors.In view of the limitations of the first two methods,we established a CACER cloning and sequencing method based on Cas9 and Pac Bio.The principle is to ligate the genomic DNA cleaved by Cas9/g RNA in vitro to the cloning vector p15 A through Gibson assembly to form a recombinant clone plasmid.Then,the recombinant plasmid was detected by PCR,restriction endonuclease digestion and pulse field gel electrophoresis.Finally,the complete FibH gene sequence of Dazao was obtained by Pac Bio sequencing and bioinformatics software,which was identical with KWMTBOMO15365 sequence.2.The complete FibH gene sequences of different silkworms were systematically analyzedUsing the "CACER" method,we obtained the complete FibH gene sequence of 7wild silkworms and 17 domesticated silkworms.The FibH gene of all wild silkworms is longer than that of the domesticated silkworms,and the FibH gene of local varieties is generally longer than that of breeding strains.Among them,the FibH gene of HCBm is as long as 20 kb,and that of Handan is the shortest,which is only 15.2 kb.According to the similarity of the sequence,all FibH genes can be divided into three categories: wild silkworm,local varieties,and breeding strains including mutant grr and nds.The FibH gene of wild silkworms has the largest difference,followed by local varieties and the smallest breeding strains.The protein sequence of FibH gene was deduced by sequence analysis software.The FibH of all silkworms is composed of conserved NTD,CTD and changeable intermediate repetitive core sequence composed of multiple repetitive domains and amorphous regions.The number of repetitive domains in FibH of wild silkworms is more than that of domesticated silkworms,and there are shorter repetitive domains.The number of repetitive domains of FibH of HCBm is the largest,as many as 19.All the repetitive domains of FibH are made up of unequal number of subdomains,and the subdomains are composed of polypeptides GAGAGS,GAGAGY,GAGYGA,GAGAGA,and so on,among which the content of GAGAGS is the largest,and is positively correlated with the length of FibH.The repetitive core sequence of FibH is composed of repetitive domains and amorphous regions alternately arranged.The last amorphous region of all silkworms is identical,with 44 amino acids,the typical amino acid is RRE;the first amorphous region has 44 amino acids,but there are two amino acid substitutions,and the typical amino acid is RSD;The middle amorphous region has 43-44 amino acids and may be repeated several times.The first amorphous region of TLBm and BBBm was repeated twice in the repetitive core sequence of FibH,while the first amorphous region of other silkworms only appeared once.3.The relationship between FibH sequence length and mechanical properties of silk was establishedWild silkworms were collected from different areas and raised outdoors and covered with gauze.At 12 h after spinning,the silk of wild and domesticated silkworms was obtained by strong drawing.The determination of mechanical properties found that the breaking strength of wild silkworm was generally higher than that of domesticated silkworms,and that of local varieties was higher than that of breeding strains,which may be the result of breeding economic characters and ignoring mechanical properties in the long-term domestication process.Combining the mechanical properties of silkworm silk and the FibH gene sequence,we established a model of the relationship between mechanical properties and sequence.The breaking strength of silk was positively correlated with the length of FibH,repeating domains and the number of hexapeptide GAGAGS,and negatively correlated with GC content,while the elastic modulus,extensibility and toughness of silk had no correlation with FibH gene sequence.It was worth mentioning that although all breeding strains had the same FibH gene,their mechanical properties were not the same,which showed that the mechanical properties of silk vary depending on the silkworm species.4.The FibH gene-edited B.mori mutants were preparedTo eliminate the influence of B.mori strains and further confirm the relationship between breaking strength and FibH,this study designed Rg RNA and Ag RNA targeting the repetitive domain and amorphous region of FibH gene respectively and obtained g RNA transgenic silkworm strains by microinjection.This strain was crossed with the Cas9 strain to obtain the F1 hybrid line.Its silkworm cocoons have three phenotypes:thick cocoons,thin cocoons,and naked pupae.We selected thick-cocooned silkworm moths to self-bred for multiple generations and obtained inbred strains of F2-F7 generations.In the F5 generation,we selected 30 FibH gene-edited mutants for nanopore whole-genome sequencing and found that the FibH gene of most of the mutants showed multiple editing forms,including the deletion and insertion of long sequence.Moreover,most of the mutants were heterozygotes,with two FibH genes of different lengths.There was no obvious regularity between mechanical properties and sequence of silk fibers.We obtained two homozygous mutants from 30 mutants,lw01 and lw07,whose FibH genes were 17230 bp and 5124 bp in length,respectively.The FibH gene sequence of lw01 was long,and the average sequencing depth of whole-genome sequencing was only 10 x,so we couldn’t get the fine sequence of its FibH gene;the FibH gene sequence of lw07 was short,and the manually extracted sequencing reads was equivalent to the length of FibH gene assembled by bioinformatics software,and there was no frameshift mutation.The phenotypic observation showed that the cocoon of lw01 was similar to the wild type,while the cocoon of lw07 was thin and not stratified,and the inner surface of silkworm cocoons was rich in sericin.The cocoon shell rate of male and female mutant silkworm cocoons of lw01 is about twice that of lw07.The measurement of mechanical properties found that the breaking strength and toughness of lw01 were 1.75 times and 2 times that of lw07,respectively,and there was no significant difference in their extensibility and stiffness.The editing of the repetitive core sequence of the FibH gene further confirmed the relationship between the breaking strength of silk and the length of FibH,that is,the breaking strength is positively correlated with the length of FibH.In summary,this study established a CACER cloning and sequencing method based on Cas9 and Pac Bio,which solved the difficulty of FibH gene cloning for many years;The complete FibH gene sequences of different silkworms were analyzed via CACER;The repetitive core sequence of the gene was edited,and a new material of FibH gene editing mutant was prepared,which confirmed the positive correlation between the breaking strength of silk fiber and the length of the FibH sequence.This study provides a reference for the design and modification of FibH gene,as well as other silk protein genes. |
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