The Mechanism Of VEGF And FGF5 On Regulating Cashmere Goat Hair Growth

Textiles made from cashmere have the characteristics of light,gauzy and lissome,and have excellent keeping warm effect.Besides,cashmere is low produced,so it has high economic value.Inner Mongolia cashmere goat Albas type is mainly distributed in the northwest of Inner Mongolia.It has high reproduction rate,large size,thin cashmere fiber,and can meet the requirements of high-end textiles for raw materials.Because the cashmere has the characteristics of fine and short,the binding force and friction between fibers are less,which is easy to cause pilling.Increasing the length of the cashmere fibers can effectively solve this problem.VEGF can promote the growth of cashmere by inducing angiogenesis,while FGF5 can affect the length of cashmere fiber by regulating the growth cycle of hair follicles,and the two have positive and negative regulation effects on hair growth,respectively.In this study,we used the VEGF gene integration at FGF5 site cashmere goat as an animal model to study the effect of gene editing on cashmere goat phenotype.In addition,a variety of high-throughput sequencing technologies combined with molecular biology methods were used to explore the mechanism of VEGF and FGF5 on regulating hair growth,exploring downstream functional genes and key acting molecules,so that provide a theoretical basis for the cultivation of new cashmere goat varieties with high yield and quality.Chapter 1:Identification and detection of the integration of VEGF gene at FGF5site gene-edited cashmere goat1.Identification of gene integration in the gene-edited cashmere goatAt first,the gene integration of the gene edited cashmere goat was identified by PCR and Southern Blot at the DNA level.The specific bands with the correct sequence and length were found,which proved that VEGF was integrated at FGF5 site.Real-time PCR was used to detect the expression of VEGF and FGF5 in cashmere goat skin tissue,which were 1.63 times and 0.41 times of the control group,respectively.The results were consistent with Western blot,indicating that VEGF had high expression and reduced the expression of FGF5.In addition,the safety of the gene edited cashmere goat was tested from the aspects of off-target analysis and health status.The gene edited cashmere goat did not occur off-target and was in good health.2.Cashmere production performance test of the gene-edited cashmere goatThe cashmere production performance of gene edited cashmere goats was tested from macroscopic and microscopic aspects.Macroscopically,the cashmere goat showed the cluster growth phenotype of multi position cashmere.Compared with the control,the cashmere yield increased by 31.5%,the length of cashmere fiber increased by 35%,and did not affect the fineness and strength of cashmere fiber.Microscopically,it was found that the number of secondary hair follicles in the skin tissue of the cashmere goat increased by 19%compared with the control,and the average diameter of secondary hair follicles increased by 48.3%by paraffin section and he staining.This cashmere goat had a good cashmere production performance,and could be used as an animal model to study the mechanism of VEGF and FGF5 regulating cashmere growth of cashmere goats,and could be used as an excellent breeding stock for high-yield and high-quality cashmere goats.Chapter 2:Multi-omics analysis of the integration of VEGF gene at FGF5 site gene-edited cashmere goat1.Transcriptomics studiesThe gene-edited cashmere goat was used as the experimental group,and a non-gene-edited somatic cell nuclear transfer cashmere goat and a wild-type cashmere goat produced by natural mating were used as the control group.The differentially expressed genes（DEGs）in skin tissues were studied by transcriptome sequencing.According to the criteria of|log₂FC|≥0.848 and p-adjust<0.05,1246 DEGs were screened,including 843 up-regulated genes and 403 down-regulated genes.Go and KEGG were used to study the function of the differentially expressed genes,which were mainly enriched in growth factor binding,the PI3K-Akt signaling pathway,and ECM receptor interaction.Moreover,PI3K-Akt signaling pathway and VEGF signaling pathway had a confluence,and acted on arachidonic acid metabolism together.2.TMT proteomics studiesThrough TMT labeling,the proteins in cashmere goat skin tissue were studied,and 178 differentially expressed proteins were screened,including 108 up-regulated proteins and 70 down-regulated proteins.In GO enrichment,differential proteins were mainly enriched in arachidonic acid metabolism,endoplasmic reticulum,and iron binding;In KEGG enrichment,the differential proteins were mainly enriched in other polysaccharide degradation,steroid biosynthesis,and arachidonic acid metabolism.In addition,the enrichment of differential proteins in arachidonic acid metabolism showed the characteristics of high expression protein more and scattered,low expression protein less and concentrated.3.LC-MS untargeted metabolomics studiesThrough non targeted metabolomics analysis of the types and contents of metabolites in cashmere goat skin tissue,a total of 122 named differential metabolites were screened,including 73 cations and 49 anions.In KEGG enrichment,the differential metabolites were mainly enriched in linoleic acid metabolism,arachidonic acid metabolism and thyroxine synthesis.Also,the position of metabolites in arachidonic acid metabolism was consistent with that of proteome.Previous studies have shown that the increase of VEGF expression and the decrease of FGF5 can both increase p-Akt expression,proving that the PI3K-Akt signaling pathway is activated.Combined with the high-throughput sequencing results in this study,it was suggested that the high expression of VEGF and the knockout of FGF5 jointly activated the PI3K-Akt signaling pathway,thus affecting the metabolic process of arachidonic acid,and ultimately affecting the growth of cashmere goats.Chapter 3:Regulation of VEGF and FGF5 on the cashmere growth of cashmere goatsUsing GFFs as experimental materials,we analyzed the effects of PI3K-Akt signal pathway activator 740Y-P and inhibitor NU7441 on the proliferation,apoptosis,and cell cycle of GFFs.The expression of Akt,p-Akt and PLA2G4D,PTGS2,CYP2B,and EPHX2 in arachidonic acid metabolic pathway were detected by real-time PCR and Western blot to explore the effect of VEGF and FGF5 on arachidonic acid metabolic pathway.The results showed that the expression trend of these genes in GFFs treated with PI3K-Akt signaling pathway activator 740Y-P was consistent with the integration of VEGF gene at FGF5 site gene-edited cashmere goat skin tissue.These results indicate that VEGF and FGF5 affected arachidonic acid metabolism by affecting the activation state of PI3K-Akt signaling pathway,and hindered the transformation of arachidonic acid to EETs and DHETs,and thereby promoted the cashmere growth of cashmere goats.This study had preliminarily clarified the mechanism of VEGF and FGF5regulating the hair growth of cashmere,provided breeding materials for high-yield and high-quality cashmere goats,and provided theoretical basis for the exploration of downstream functional genes and core action molecules.