| People began to use silk as early as 5,000 years ago and it has been the main raw material for weaving high-end silk fabrics for a long time.But with the development of synthetic fibers,the uses of the silk are partly replaced.On the one hand,the synthetic fibers have excellent performances and low price;on the other hand,the fineness of the silk is concentrated at the ordinary fineness level,and the single fineness can't fully adapt and meet the diversified demand of the market.Since the silk is a natural protein fiber,there are still many excellent properties that the synthetic fiber cannot substitution,such as the bio-compatibility.Therefore,to develop the silk with different fineness should be able to break the bottleneck of the narrow application of it to some extent.At present,researchers have developed a certain amount of silk with different fineness by the traditional breeding methods,but it takes a long time and it's hard to meet the variations of market.Although the technology of molecular breeding can greatly shorten the breeding time and increase the breeding rate of excellent strains,however,the molecular mechanism of silk formation remains to be studied,and the molecular modified method is difficult to implement.Spinner which has an effect on the structure and performance of silk plays a crucial role in the silk formation process.Based on this,in this study,we observed the overall morphology and structure of the spinneret and its related structure,and analyzed its influence on the silk formation process.The different expressed genes which have the influence on the silk fineness and properties were screened out by transcriptome too.The main results are shown as the follows:1.Anatomical observation of the overall structure and morphology of the spinneretThe silkworms of Xiafang,Qiubai,Suxiu and Chunfeng were selected as the research object.The overall morphology of silkworm spinneret was observed by scanning electron microscope and stereomicroscope.After removing of the cuticles around the spinneret carefully,the complete three-dimensional morphology and distribution of muscles around the silk press were observed.Removing the muscles and the cuticles surrounding the spinneret,three parts of the spinneret were clearly exhibited,namely,the common tube,the silk press and the spinning tube.2.The differences and changes of the cross-sections of the spinneretObserving the cross-sections of the spinneret,it found that the lumen shapes were varied in the spinneret,especially in the silk press.In the common tube,the shape of the lumen remains elliptical substantially.The lumen shape in the silk press changes from triangle to semi-crescent,and the shapes of the semi-crescent are not uniform.The shape of the lumen is gradually changed into oval in the spinning tube,and the closer to the spigot,the larger of the lumen size.The cross section dimensions of the lumen were calculated.It found that the change rule of the lumen shapes in Xiafang and Qiubai(the ordinary denier strain)were similar among different samples,but the lumen shapes were varied in Suxiu and Chunfeng(the fine denier strain)among different samples,especially in the silk press.It means that the lumen shape of ordinary denier strains is relatively stable.In addition,to compare with the ordinary denier strains,it found that the lumen shapes of the fine denier strains had a large change,which means that the shape deformation of the fine denier strain is easier.3.Observation of the formation process of silk in the spinneretThe morphological changes of silk fibroin were observed by serial sections.It is found that the cross section morphology of silk protein fiber close to circle before they enter into the common tube,the two single filaments merged together in the common tube and the shape changed into ellipse.When the silk flow pass through the silk press,the morphology of the single filaments gradually became triangular under the contraction of the muscles around the silk press.At last,the silk protein enter into the spinning tube,the extrusion effect of macromolecule material occurred,and it spun out after adjusted in this area.It can be seen that the spinneret,especially the silk press has a significant effect on the morphology and size of the silk.In order to ensure the cross section shape of the silk,the muscles around the silk press should have a certain contraction frequency.According the spinning speed and the length of the silk press,the contraction frequency of the surrounding muscles is estimated to be 11.42 Hz to 50Hz.According to the uneven thickness and the in-homogeneity cross-section shape of the silk,it is also can speculate that the muscle contraction around the silk press is irregular too.The irregular contraction of the muscles can affect the secondary structure of the silk,resulting in differences in its mechanical properties.4.A preliminary analysis on the relationship between silk fineness and lumen areasBy comparing the cross-section areas of silk protein fibers before they entered into the common tube of the four strains,it found that the silk size were smaller in the ordinary denier strains which indicates that the silk size has been changed.It is known that the silk morphology and size are greatly changed in the silk press which indicates that there are differences in the activities of the spinneret between the ordinary denier strain and the fine denier strain which has an important effect on the silk fineness.It has not been found the corresponding relationship between the silk fineness and the lumen area,there are still some doubts need to be studied.5.Transcriptomic analysis of the silk press in denier varieties strainsA transcriptome study was carried out on the silk press and its surrounding muscles of the four strains in day W1.According to the difference expressed analysis,there were 889 common differentially expressed genes(DEGs)between Xiafang and Chunfeng,and 373 between Xiafang and Suxiu,thus,there were 192 DEGs between Xiafang and two fine denier strains.There were 525 common DEGs between Qiubai and Chunfeng,and 506 between Qiubai and Suxiu,thus,there were 170 DEGs between Qiubai and two fine denier strains.Thus,there are 36 common DEGs in the four strains,with 18 up-regulated and the other 18 down-regulated.Gene function analysis was performed the 36 selected DEGs.According to the functional annotation of the genes,we screened three genes that may have a large influence on the silk fineness.(1)BGIBMGA008861(troponin C).This gene is involved in the regulating of muscles'contraction.Therefore,when it is up-regulated in the fine denier strain,the contraction activity of the muscles around the silk press is relatively active.This will lead to a large change in the lumen size in the fine denier strains,and the compression effect on silk is enhanced too.(2)BGIBMGA005899(Chitooligosaccharidolytic beta-N-acetylglucosaminidase),which is involved in the degradation of chitin.There is a stiff chitin plate on the back of the silk press,and it is connected to the dorsal muscles.It participates in the regulation of lumen size under the control of the dorsal muscles.Therefore,when the expression up-regulated in the fine denier strain which will lead a reducing of the chitin in the fine denier strain which maybe make the deformation of the lumen easier.This result is consistent with the big changes of the lumen shapes in the fine denier strains.The chitin plate provides a shear force in silk fiber forming.When it is up-regulated in fine denier strains,the shear force is provided smaller which lead to the mechanical properties of the fine denier strains of silk is slightly weaker than the ordinary denier.(3)BGIBMGA000983(TWiK family of potassium channels protein 7),it has potassium ion trans-membrane transport function.Existing studies have shown that the increase of K~+can promote the formation of?-sheet in the regenerated fibroin protein.So when this gene up-regulated in the ordinary denier strains,it may promote more?-sheets formed in ordinary denier silk,which lead a improvement of the mechanical properties of the ordinary denier silk,this result is consistent with the experimental data.In conclusion,this study exhibits a more clear morphology and its structure.Secondly,the influence of spinneret on the morphology and some properties of silk were analyzed too.What's more,three genes that may have an influence on the silk fineness were screened out by transcriptome,which provides a valuable reference for the subsequent study of the regulatory mechanism of the silk fineness. |
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