Tracking Of Exogenous Spider Silk Proteins Of Biologically Modified Silk And Study On Silk Properties

Silk is a natural material with superior performance.It has a long history of application in traditional textile fields and has broad application prospects in modern materials,medical treatment,health and other fields.In order to make up for the inherent defects of silk,endow silk with new properties,and expand the application field of silk,researchers continue to explore the modification of silk.Common modification methods include physical and chemical methods that directly target silk,as well as biological engineering methods and feeding methods for silkworm.Among them,the bioengineering method changes the characteristics of silkworm and silk from the source by introducing foreign genes or knocking out inherent genes.It is expected to achieve once and for all,low-cost and high-efficiency production of modified silk.It is a silk modification that has attracted much attention in recent years method.The introduction of foreign genes based on piggybac(PB)transposon is the most commonly used silkworm transgenic method.Our laboratory used PB transposon as a vector to successfully construct a transgenic silkworm(RES)containing the MaSp1 protein gene of the spider silk.In the long-term subculture,we found the silkworm individuals(WES)with the missing red compound eyes report traits and the white-eyed traits.In order to verify the impact of reported trait loss on transgenic silkworm and cocoon silk,this study was followed at the gene level,protein expression level and silk material level.The main research contents are as follows:(1)Genetic testing and genetic analysis of Bombyx mori individuals with missing reported traits.In the experiment,primers for MaSp1 gene and PB transposon were designed respectively,and PCR amplification reaction was carried out with RES and WES silkworm genomic DNA as templates.The amplified products were recovered for T vector cloning and sequencing analysis.The results showed that the MaSp1 gene has been lost in the WES silkworm genome,the L arm of the PB transposon was lost and the R arm part was present.The MaSp1 gene and PB transposon in the genome of the RES silkworm were intact.This indicates that the retransposition behavior of foreign genes has occurred in the WES silkworm,which implies that the PB transposon as a silkworm transgene vector is not completely stable,and there is a risk of retransposition.The results of further hybridization experiments and genetic analysis showed that the inheritance of white eye traits caused by gene loss conformed to Mendelian inheritance rules.In the past studies of silkworm transgenes,they often focused on the smooth expression of the transferred genes,and lacked tracking of the genetic stability of foreign genes.This study found and tracked the retransposition behavior of PB transposons in silkworms for the first time,and proposed an important research direction for perfecting PB-based silkworm transgenic technology.(2)The effect of gene loss on silkworm growth and cocoon qualityRES and WES silkworms were reared under the same conditions.The daily weight change,total cocoon weight,cocoon layer weight,cocoon layer rate,cocoon silk diameter,and tensile stress and strain of the five-instar silkworms of the two silkworms were investigated and analyzed.The results showed that compared with RES,the development of WES silkworm was extended by 1.5days,the weight gain was slower in the fifth instar early stage and the body weight was heavier in the later period.The cocoon layer rate and cocoon silk diameterof WES silkworm were reduced by1%and 11%respectively compared with RES.Existing studies rarely involve detailed tracking of the growth and development of transgenic silkworms,and there are no research reports on the effects of gene loss on silkworms.This study traced for the first time that the loss of exogenous genes in the transgenic silkworm significantly affected the growth and development of the silkworm body,cocoon quality and cocoon silk diameter and other production traits,which should be paid more attention to in the breeding of transgenic silkworm varieties.(3)Research on the properties of biologically modified cocoon silk and its regenerated silk protein membraneThe purpose of biological modification in this study is to express foreign proteins in silk glands of silkworms in order to obtain new silks with foreign protein properties.It is important to report whether the loss of traits is accompanied by changes in the composition and properties of modified silk.In the experiment,RES and WES silkworm cocoons were used as materials,and ordinary silkworm cocoons were used as controls.The structure and performance of degummed and non-degummed cocoon silk and silk protein membranes were tested and analyzed.The results of structural analysis showed that the?-sheet content of RES silk in the ungummed samples was 4%higher than that of WES,the?-sheet content of RES filaments in the degummed samples was significantly lower than that of WES.The mechanical performance test results show that the breaking stress of the degummed RES yarn is greater than that of WES,the breaking stress of degummed RES silk is much smaller than WES,and the strain at the breaking point of degummed and undegummed RES silk is slightly larger than WES.It is speculated that the exogenous spider silk protein located in the sericin layer is lost along with the sericin along with the degumming operation,resulting in the changes in the above-mentioned structure and properties of the cocoon silk.The thermal stability test results show that the initial decomposition temperature of RES wire is 3-5 ~oC higher than that of WES.The results of ultraviolet radiation indicate that RES silk is more resistant to ultraviolet rays than WES silk.The breaking point stress of RES silk protein membrane can reach 18.65±0.98MPa,which is better than WES and the control group,and has better thermal stability.The blending of glycerin can improve the tensile elongation of the silk protein film,and particularly increase the elongation at break of the RES silk protein film.The introduction of exogenous spider silk protein into sericin has a significant contribution to the improvement of cocoon silk and silk protein membrane structure and performance,which provides experimental basis for expanding the application of silk and its protein in materials,medical and other fields.