Effects Of PDGFD Gene On Tail Fat Deposition In Tan Sheep

China has a vast territory,a complex and diverse ecological environment and abundant animal genetic resources.Tan sheep produced in Ningxia Hui Autonomous Region is not only a famous fur sheep breed in China,but also has excellent meat production performance.Tan sheep is famous both at home and abroad for its delicate meat and light odor.Tan sheep belong to fat tail type sheep,the thick tail not only seriously reduces the carcass quality,but also brings some difficulties for its breeding.It is of great significance for the development of Tan sheep industry to study and improve the traits of fat tail of Tan sheep and improve the slaughter rate under the premise of keeping the good traits of Tan sheep.Studies have shown that PDGFD is a potential key gene affecting the tail type of sheep,and is closely related to the process of tail fat generation in sheep.However,there are few studies on PDGFD gene in sheep tail cells,and its mechanism of action remains unclear.This study verified the key role of PDGFD gene in tail fat deposition of Tan sheep from genome-wide association analysis,tail fat cell level and individual level,in order to provide theoretical basis for tan sheep breeding.At the same time,a batch of Tan sheep with PDGFD point mutation deletion were created and used as breeding material of Tan sheep.The main results are as follows:1.Analysis of candidate genes related to fat deposition in Tail of Tan SheepThe whole genome sequencing data of fat-tailed sheep breeds such as Tan sheep and large-tailed Han sheep and thin-tailed sheep breeds such as Diqing sheep and Hanzhong sheep were compared to the latest version of sheep reference genome OAR_Rambouillet_V1.0,and THE SNVs were calculated by GATK.On this basis,through genome-wide association analysis of tail size,It was found that BMP2,PDGFD and PLA2G16 genes were significantly correlated with tail size traits of sheep,and PDGFD gene was one of the key candidate genes for this trait.2.Effects of PDGFD gene on tail adipocyte proliferation in Tan SheepAdipose tissue was collected from the tail of Tan sheep,and the precursor adip-ose cells were successfully isolated and cultured in vitro.The preadipocytes were induced and differentiated by the adipogenic induction differentiation medium of rat adipocytes mesenchymal stem cells.The adipocytes at the 2nd,4th,6th and 8th day of differentiation were collected and the expression level of the PDGFD gene was detected to plot the PDGFD gene expression profile during the differentiation of preadipocytes.The expression of PDGFD decreased with induction time.Overexpression vectors pcDNA3.1(+)-c-EGFP-PDGFD-X1,pc DNA3.1(+)-c-EGFP-PDGFD-X2,pc DNA3.1(+)-C-EGFP-PDGFD-X3 were constructed and transfected into tan sheep adipose cells.Finally,PDGFD gene expression was upregulated to 177.29 times,PCNA expression was upregulated to 89.26 times,Bcl2: BAX was upregulated to 3.05 times,and BAX was significantly down-regulated.Compared with the key genes of cell proliferation and apoptosis,the overexpression of PDGFD gene did not significantly upregulate the key gene of adipose differentiation,PPARγ.The growth curve of PDGFD overexpression precursor adipocytes was plotted.The results showed that PDGFD overexpression could accelerate the proliferation rate of adipocytes.These results indicate that PDGFD gene can promote the proliferation of fat precursors,and is a key gene in regulating tail fat deposition and improving tail type of Tan sheep.3.Effect of mutation inactivation of the first exon target of PDGFD gene in Tan SheepA sg RNA was designed on the first exon of the PDGFD gene in sheep,and point mutation(A > G)of the start codon of PDGFD gene was carried out by ABE base editor system at the cellular level,which successfully inactivated PDGFD gene with editing efficiency of 68.7%.On this basis,combined with embryo microinjection technology,we generated PDGFD gene edited Tan sheep and obtained 9 newborn lambs.Blood samples of newborn lambs were collected and genomes were extracted.The 10 predicted editing miss sites were amplified and detected,and no miss was detected.The skin,muscle and tail adipose tissues of newborn lambs were collected for targeted deep sequencing,and target site editing was detected in the above parts of the genome.Adipose tissue from the tail of gene-edited Tan sheep and wild-type Tan sheep were collected and made H&E staining sections.No significant difference was observed in adipose tissue morphology between PDGFD gene-edited Tan sheep and wild-type Tan sheep,and the morphology of adipose cells was normal.The tail size and body weight growth data of PDGFD gene-edited Tan sheep were measured and recorded for 6 months and compared.It was found that the tail size data of PDGFD gene-edited Tan sheep was significantly smaller than that of wild type Tan sheep,but there was no significant difference in body weight growth trend between PDGFD gene-edited Tan sheep and wild types.These results indicated that PDGFD gene editing could reduce tail fat deposition and improve carcass quality while ensuring normal growth and development of Tan sheep.In conclusion,at the genomic level,this study identified PDGFD gene as the key gene of sheep tail fat deposition by genome-wide association analysis.At the cellular level,the effect of PDGFD gene on the proliferation of sheep tail adipocytes was systematically verified.At the individual level,PDGFD gene edited tan sheep was created by ABE base editor,and its tail was significantly smaller than wild tan sheep.The biosafety of PDGFD gene editing Tan sheep was verified by off-target detection and other methods.The above research results are of great significance for breeding improvement of new tan sheep and provide excellent germplasm resources and technical support for breeding work.