| Cashmere is an important raw material for textiles,and cashmere requirements on production and quality are also increasing as the expanding of cashmere market all over the world.Cashmere goat,as a fine domestic animal breed in China,is famous for excellent cashmere quality.However,in the purpose of improving cashmere yield,blind hybridization performed many years in cashmere goat breeding has resulted in the decrease of average cashmere yield and cashmere quality,and the low proportion of traditional excellent varieties and improved varieties.Recently molecular breeding with modern biotechnology has become an important direction in cashmere goat breeding.Thymosin ?4 has a variety of biological functions,and its role on promoting hair growth has been confirmed in many studies,but the mechanism has not been clear.In this study,T?4 gene site-specific integrated cashmere goats obtained by gene editing technology were used,to identify and evaluate the health status,and to observe the effect of T?4 gene on cashmere growth.The T?4 gene integrated goats were enlarged quantity by MOET technique and the offsprings were detected.Further transcriptome and proteomics analysis were combined to explore the mechanism of T?4 gene promoting the growth of cashmere,so as to provide theoretical basis for breeding high-yield and high-quality cashmere goats with gene editing technology in the future.1.Detection of T?4 gene site-specific integrated cashmere goat(1).Identification and health evaluation of T?4 gene site-specific integrated cashmere goatIn this study,the T?4 gene site-specific integrated cashmere goat obtained by CRISPR/Cas9 combined with nuclear transfer technology were used as research object,and Southern blot,real-time PCR and immunocytochemistry were used to detect it.The results showed that the goat achieved site-specific integration of T?4 gene at the CCR5 locus.In addition,the safety and health status of gene-editing goat were detected.The detection indexes mainly included off-target analysis,mRNA levels of genes adjacent to the CCR5,growth monitoring and blood routine detection.The results showed that there was no off-target detected in potential off-target sites.The expression of CCR5 adjacent genes was not affected,and there was no significant difference in the growth status and blood index of the goat compared with control group.This indicated that site-specific integration of T?4 gene did not affect the expression of endogenous genes around the integration site,and the growth and health status of the gene editing goats were good.(2).Cashmere production performance of T?4 gene site-specific integrated cashmere goatThe performance of cashmere production was tested,and the test indexes mainly included the strength extension,fineness,the quality ratio of cashmere to wool,and cashmere yield.The results showed that the strength extension and fineness was no significant difference between gene editing goat and controls,the quality ratio of cashmere to wool of gene editing goat was significantly higher than that of controls.The cashmere yield of gene editing goat was increased by 74.5% compared with controls.At the same time,HE staining and immunohistochemistry were also performed on the skin tissue of the goats.The results showed that the S/P of gene editing goat was higher than that of controls.The results of immunohistochemistry showed that T?4 gene was mainly expressed in secondary follicles,and the expression of T?4 gene in secondary follicles of gene editing goat was higher than that in controls.The results indicated that T?4 gene mainly acted on SHF,increased the number of SHF to raise cashmere yield.(3).Propagation and offspring detection of T?4 gene site-specific integrated cashmere goatTwenty embryos of 2-8 cell stage were obtained by superovulation of T?4 gene site-specific integrated goat.These embryos were transferred into four recipient goats,and seven offspring were obtained,three of which were died after birth.The results of PCR identification showed that two of the dead kids and three of the survived kids achieved site-specific integrated of T?4 gene.Paraffin sections,HE staining,qPCR and immunohistochemistry were performed on the skin tissues of the four surviving kids.It was found that the S/P and the expression of T?4 genes in secondary hair follicles of T?4 gene integrated goat were significantly higher than that of controls.The mRNA expression level of T?4 gene in each organ of the three died lambs was detected.The results showed that the mRNA expression of T?4 gene in each organ of the three kids was no significant difference.The site-specific integration of T?4 gene could only highly expresse in skin tissue without affecting the expression of T?4 gene in other organs.The results indicated that T?4 gene integrated goat could stably transmit the fine traits to the next generation meanwhile ensure its security.2.The mechanism of T?4 gene on promoting hair growth(1).Transcriptome analysis of T?4 gene site-specific integrated cashmere goatHigh-throughput transcriptome sequencing was performed on the skin tissues of T?4 gene integrated goat and controls.A total of 1076 differentially expressed genes were screened,of which 463 up-regulated genes and 613 down-regulated genes.The up-regulated differential genes are mainly enriched in the biological process of negative regulation of peptidase activity,the establishment of skin barrier and the regulation of skin water loss,and enriched in the molecular functions of growth factor receptor.The down-regulated differential genes are mainly enriched in the biological process of multicellular regulation and cell adhesion,and enriched in the molecular functions of calcium ions combination.In the KEGG enrichment analysis,the differentially expressed genes were significantly enriched in the signaling pathways involving in vascular smooth muscle contraction,cGMP-PKG,cell adhesion,renin secretion,and leukocyte transendothelial cell migration.Further analysis of the signaling pathways enriched by differentially expressed genes and screens of key differentially expressed genes,revealed that these differentially expressed genes were mainly involved in intercellular connectivity,regulating intercellular movement,and affecting angiogenesis,vasodilation,vascular permeability,and maintenance of vascular stability.These differentially expressed genes were verified by qPCR,and the results were consistent with the transcriptome sequencing.Those results suggested that T?4 might affect the cell movement by affecting the connection between cells,and also affect the formation and vasodilation of blood vessels around the hair follicles,and finally promote the growth of cashmere.(2).Proteomics and correlation analysis of T?4 gene site-specific integrated cashmere goatITRAQ technology was used to analyze the different protein in the skin tissues of T?4 integrated goat and controls.A total of 875 differentially expressed proteins were screened,of which 666 were up-regulated and 209 were down-regulated.The up-regulated differential proteins are mainly enriched in the biological process of ion transmembrane transport involved,and enriched in the molecular functions of organic anion transmembrane transporter.The down regulated differential proteins are mainly enriched in the process of aldehyde biosynthesis and histone mRNA metabolism,and enriched in the molecular function of lithium ion binding and alkali metal ion binding.In the KEGG enrichment analysis,differentially expressed proteins were significantly enriched in the SNARE interactions in vesicular transport and the inflammatory mediator regulation of TRP channel.The key differential proteins were screened including MAPK,CAMK and PKC.Proteomic analysis was correlated with transcriptometric analysis to identify common up-regulated or down-regulated differential genes/proteins.Combined with the signaling pathway,the results suggested that the overexpression of T?4 reduced the expression of TIMPs and increased the expression of MMPs,promoted the degradation of the basement membrane and extracellular matrix,released the non-covalent bond of VEGF with proteoglycan,and activated the p38 MAPK signaling pathway by the combination of VEGF and VEGFR-2.In addition,T?4 also activated the PKC signaling pathway.It is suggested that T?4 affected actin assembly,cell migration,cell proliferation and differentiation through MAPK and PKC signaling pathway,and finally promoted hair growth. |
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