| Silk is a natural protein that contains a collection of complex micro fibers,which is widely used and studied for its excellent properties.However,some in its mechanical defects in mechanical properties make the application of silk a limitation.Therefore,the modification of silk fiber is the top priority to expand the application and prosper industry.Silk fiber modification research has a long history,the modification methods can be divided into physical modification,chemical modification and genetic modification.Physical and chemical modification methods of just by modifying the silk,not essentially change silk protein properties,and the cost is high,the process cumbersome.Using the genetic operation technology for artificial silk gene transformation,new lines be obtained with more excellent performance and stable genetic transformation.Spider silk is the best mechanical properties in known animal fiber,its protein characteristics,composition and spinning process and the similarity to silk fiber,so some scholars constantly to try make spidroin expressed in the silk gland of silkworm to improve the mechanical properties of silk,but the low amount of exogenous gene expression is still a difficult problem to overcome.It will be the most cost-effective way to improve the performance of silk by editing the genes of silk itself.The study found that the repeat area sequence of spidroin is more regular and orderly than fibroin,which may be the important reason for that spider silk is stronger than silk fiber.The Fib-H gene is the most important structural gene for silk fibroin,contains long tadem repeat regions,which determines the mechanical properties of silk.The researchers found that the mechanical properties of silkworm silk were worse when the Fib-H was cut short truncated.Then,it is likely that super strong artificial silk retain its natural properties will be obtained if transformed the Fib-H to make its sequence more regular and more longer.The characteristic of repetitive sequences and high GC content of silk heavy chain makes it difficult to easily clone and edit using conventional PCR technique and sequencing technology.Therefore,we make tentative exploration and attempt on this assumption adopting the method of manual assembly gene.In this paper the main achievements are as follows:1?The design of artificial silk heavy chain(artFib H)According to the characteristics of Fib-H gene sequence,we select the longest seventh repeat region(R07)and the sixth amorphous region(A06)as a repeating unit,called "A06R07".In order to ensure the artificial silk heavy chain to keep the original sequence characteristics,we selected two IIs type restriction endonucleases: BbsI and Bsa I,each chain of gene containing one recognition sequence of Bbs I and one recognition sequenc of Bsa I,and are designed on the plasmid vector.The recognition sequences of BbsI are designed in downstream A06R07 sequence,one of them has two bases distance from "A06R07",TTGT in 3'end of A06R07 sequence came out after digestion;Bsa I identification sequences designed in both ends of A06R07 sequences,one is designed in the upstream of A06R07 sequence for five bases distance,AACA before 5'ends of A06R07 sequence formed after digestion.There are 7 bases in the middle of another recognition sequence of BbsI and BsaI,and they share the same enzyme loci,can cut out one pair of sticky end just complementary base.By using enzyme digestion and ligation method to realize the seamless connection and doubling of the repeat unit.2?Construction of transgenic silkworm strains of artFibHBy enzyme digestion,we successfully constructed the artificial Fib-H gene piggyBac transgenic vectors driven by the Fib-H promoter as follows: pBac[R01A06R07R12],pBac[R01(A06R07)3R12],pBac[R01(A06R07)5R12],pBac[R01(A06R07)5-last-R12],pBac[R01(A06R07)3-Red-R12],pBac[R01(A06R07)3-SELR13-R12].The silkworm Bombyx mori 932 was used as receptor material,and the transgenic silkworm was prepared by microinjection.The positive individuals of pBac[R01(A06R07)3R12] and pBac[R01(A06R07)5R12] were screened out by fluorescence observation.3?Correlation detection of transgenic silk of art FibH silkThrough the analysis of 3? and 5? artificial Fib-H protein by protein prediction software,their size were 203 KDa and 295 KDa,which less than natural Fib-H protein(350KDa).By SDS-PAGE silver staining and Western Blot assay,the results show that the predicted band is not obvious,thus we speculate that may be due to its protein molecular weight is too big and has characteristic of self-assembly,stranded in the sample points in the hole;By freezing slice technology for single cross-sectional diameter size of cocoon silk,the results show that there is no obvious difference in morphology and diameter between wild type and artificial silk,suggests artFibH insert will not affect the normal morphology and diameter size of cocoon filament;Through the stress-strain tension test.results show that,compared with WT,elongation of manual 3? type and 5? type increase slightly,strength and toughness and elastic modulus decrease slightly,suggests that because of the uncertainty of the genetically modified insertion site,insertion of artFibH induce the mechanical properties of silk;By Fourier infrared spectrum experiment,we further analyze the secondary structure of cocoon silk protein,and the results show that compared with WT,in the silk protein conformation of manual 3? type and 5? type,beta sheet,alpha helix and random coil content increased slightly,beta turn content is slightly lower,shows that in the spinning process artFibH can promote the formation of the beta sheet. |
PDF Full Text Request