Functional Identification Of Intramuscular Fat Deposition-related Genes Of Banna Mini-pig Inbred Lines

Intramuscular fat (IMF) content is a major determinant of meat quality, which is positively correlated with tenderness, juiciness and flavor. In recent three decades, measures of breed improvement and growth promotion were used to pursue increased lean meat and decreased backfat thickness, however, IMF content and pig meat quality decreased significantly at the same time. How to improve IMF content not affecting lean meat production and pig growth performance, has been an urgent scientific question of animal scientists.In this study, the effect of overexpression of heart fatty acid binding protein (H-FABP, FABP3) derived from the fat Banna mini-pig inbred line (BMIL) on IMF content was investigated, at the cellular and whole organism levels, aimed to elucidate the possible molecular mechanisms for IMF deposition. This work may provide scientific basis for the development of pigs with higher IMF content and improved pork quality. The main results are as follows:1. FABP3was separately inserted into pcDNA3, pIRES2-EGFP and piggyBac to generate eukaryotic expression vectors of pcDNA3-FABP3, pIRES2-EGFP-FABP3and PB[EGFP, FABP3]. Western blot analysis showed that FABP3protein can be successfully expressed in transformed MDCK, BHK-21, HepG2and3T3-L1cells.3T3-L1preadipocytes stably transfected with pcDNA3-FABP3were selected by G418, RT-PCR and western blot analysis indicated that a cell line stably expressing FABP3(3T3-L1FABP3) was established.2.3T3-L1FABP3and control cells were induced to differentiate by standard differentiation cocktail (IBMX+DEX+FBS), showing the expression of FABP3facilitated cell differentiation and triacylglycerol (TAG) deposition. Real-time PCR analysis indicated that mRNAs for PPARy2, C/EBPa,422/aP2, ACC1, ACS, GPDH, LPL, FAS were all increased and HSL was decreased in3T3-L1FABP3cells. In particular, relative mRNA expression levels of PPAR/2,422/aP2and GPDH achieved significant levels statistically (P<0.05).3. Founder transgenic mice were produced by pronuclear injection with PB[EGFP, FABP3] and allowed to mate with wild type (WT) mice, giving birth to the F1generation of transgenic (TG) mice which was identified by PCR-based genotyping. RT-PCR and western blot analysis showed that FABP3can be expressed in skeletal muscle, adipose tissue, liver, heart, lung and kidney of TG mice. The enhanced green fluorescent originating from the bodies of the TG mice was detected using in vivo imaging system.4. Body weight of transgenic offspring was measured weekly for a further10weeks, results showed that there was no difference between TG and WT mice (P>0.05). TAG content and frozen section of soleus muscle showed that FABP3overexpression resulted in increased accumulation of lipid droplets in skeletal muscle (P<0.01). Marker genes for TAG metabolism in soleus muscle were examined by real-time PCR in4-month-old TG and WT mice kept on a regular chow diet. Results indicated that mRNA levels of almost all enzymes (PPARy2,422/aP2, CD36, LPL, ACC1, FAS and DGAT1), particularly FAS and ACC1, were significantly elevated in TG mice.5. TAG metabolism-related genes in liver tissue of TG mice was examined by real-time PCR showing up-regulated expression of422/aP2and FAS, however, more TAG accumulation was not observed in liver sections. This may be due to the fact that fatty acids synthetic in liver was transferred to other tissues for reservation or utilization.This work may lay the foundation for functional identification of IMF deposition related genes and provide gene resources for breeding transgenic pigs with higher IMF content and better pork quality.