The Role Of LKB1 Gene In Regulating The Differentiation Of Goat Intramuscular Adipocytes And Its Mechanism Exploration

Intramuscular fat is one of the important factors affecting the meat quality of livestock,which can affect the tenderness and juiciness of meat.This process is regulated by many complex factors,but the key regulatory factors involved in this process are still not completely clear.Although liver kinase B1 has been reported to be involved in the development of skeletal muscle,white adipose tissue and brown adipose tissue in other animals,the role of LKB1 in goat IMF deposition needs to be further elucidated because of the special location of intramuscular adipocytes deposited between or within muscle bundles.Here,the goat LKB1 gene sequence was cloned by PCR technology,and then the real-time fluorescence Real-time Quantitative PCR technique was used to detect the expression pattern of LKB1 in various tissues and intramuscular adipocytes of Jianzhou big ear goat.Furthermore,RNA interference and adenovirus overexpression were used to determine the regulatory effect of LKB1 on the differentiation of goat intramuscular adipocytes.Finally,RNA-seq was used to screen the transcriptional spectrum changes and signal pathways of goat intramuscular adipocytes regulated by LKB1,in order to clarify the potential molecular mechanism of LKB1 regulating the differentiation of goat intramuscular adipocytes.The results are as follows:(1)Cloning and expression Analysis of Goat LKB1 GeneIn this study,the LKB1 sequence 1317 bp including goat ORF was cloned,which encodes 438 amino acids.LKB1 protein has a typical(serine/ threonine kinase catalytic)S-TKC domain,which is a hydrophilic protein with nucleocytoplasmic shuttle and phosphorylation/ dephosphorylation.LKB1 gene is widely expressed in the tested tissues(heart,liver,spleen,kidney,large intestine,rumen,subcutaneous fat,visceral fat,longissimus dorsi,biceps femoris,triceps brachii),and has a high expression level in kidney,visceral fat and subcutaneous fat.LKB1 gene was expressed during the differentiation of goat intramuscular adipocytes,and the expression decreased at the early stage,reached the lowest point at 24 hours after induction,and increased gradually at the later stage.(2)Effect of LKB1 on the differentiation of goat intramuscular adipocytesCompared with the control group,the interference LKB1 group significantly promoted the lipid accumulation of intramuscular adipocytes,and dramatically increased the expression levels of differentiation marker genes PPARγ,CEBPα,CEBPβand fat synthesis related genes FASN,DGAT2 and lipolysis gene LPL.Adenovirusmediated LKB1 gene overexpression significantly inhibited lipid accumulation in intramuscular adipocytes,down-regulated the expression of adipocyte differentiation marker genes PPARγ and SREBP1,and significantly decreased the expression levels of adipose synthesis-related genes and lipolysis genes FASN,DGAT2 and lipolysis gene LPL.(3)Exploration of the potential mechanism of LKB1 regulating intramuscular adipocyte differentiation in goatsRNA-seq technique was used to reveal the profiles that interferes with the regulation of genome-wide transcripts by LKB1,and 1043 differentially expressed genes(DEGs)were found.Compared with the control group,425 differential genes were up-regulated and 618 down-regulated genes by LKB1 interference in intramuscular adipocytes.KEGG analysis showed that differentially expressed genes(DEGs)were mainly enriched in adhesion molecule signal pathway(Focal adhesion pathway)and its classical downstream PI3K-Akt signal pathway.FAK is a key member of adhesion molecule signaling.Further analysis showed that knockdown of LKB1 increased the expression level of FAK in cells,and vice versa.The specific FAK inhibitor(PF573228)was used to block the adhesion molecule signal to restore the phenotype of adipogenic differentiation promoted by LKB1 interference.In summary,LKB1 has a role in inhibiting intramuscular adipocyte differentiation in goats,and transcriptome sequencing showed that the differential genes were mainly enriched in the FAK signaling pathway and its classical downstream PI3K-Akt signaling pathway.Additionally,the promotion phenotype induced by LKB1 loss of function was rescued by FAK specific inhibitor treatment in intramuscular adipocytes.The results of this study expanded new evidence for the genetic regulatory network of goat IMF deposition in goats,and provided a theoretical support for improving human health and meat quality from the perspective of intramuscular fat deposition.