Cloning, SNPs Detection Of MKK6 And AKT2 Gene And Their Association With Economic Traits

The combination of traditional breeding method and molecular breeding has become a major way of breeding for morden livestock.Looking for major genes and molecular markers that are relevant to important economic characters is the foundation of molecular breeding.MKK6 and AKT2 gene were selected as candidate genes affecting the quality and characters of pig muscle according to muscle development network regulation,knowledge of PHysiology and biochemistry and research results obtained in species such as human and rat.MKK6 and AKT2 gene were separated and indentified by genome comparision and electronic cloning.Studies of tissue expression profile were conducted on target genes by half-quatitive RT-PCR method.Direct sequencing was adopted for the analysis of polymorPHism of the separated fragments and the character correlation analysis was carried out in "Yorkshire×Meishan" F₂ resource population.The results of the study were as follows.1.MKK6 gene(1) cDNA sequence of 1516bp of the MKK6 gene was obtained including CDS.ORF is 1005bp and encodes 334 amino acids,which is 91%and 90%homologous with human and rat respectively.The third and the eighth intervening sequence were obtained.(2) PolymorPHism test was conduct on the obtained sequence and the PCR-FspBI-RFLP typing technology at 667bp of the eighth intron of the MKK6 gene was established.(3) The analysis results of genetical markers and important economic characters showed that the difference between the fat meat percentage,lean meat percentage,ratio of fat meat and lean meat and the area of eye muscle of different genotype combinations is significant.The fat meat percentage of AA genotype is significantly higher than AB genotype(p＜0.05).The lean meat percentage of AA genotype is very significantly lower than BB genotype(p＜0.01).The ratio of fat meat and lean meat of AA genotype is significantly lower than BB genotype(p＜0.05).The area of eye muscle of AA genotype is significantly lower than BB genotype(p＜0.05).(4) The expression situation of MKK6 gene in 11 tissues of 4 months old mei san pig namely liver,uterus, ovaries,lung,stomach,heart,small intestine,kidney,fat,spleen and muscle was analysed by half-quatitive RT-PCR method.The expression level of MKK6 was the heightest in muscle.Expression was also detected in heart and kidney while no expression was detected in the rest of the tissues.2.AKT2 gene (1) cDNA sequence of 1589bp of the AKT2 gene was obtained including CDS. ORF is 1344bp and encodes 447 amino acids,which is 92%and 93%homologous with human and rat respectively.The fifth and the seventh intervening sequence were obtained.(2) PolymorPHism test was conduct on the obtained sequence and the PCR-FspBI-RFLP typing technology at 348bp of the tenth intron of the AKT2 gene was established.(3) The analysis results of genetical markers and important economic characters showed that the difference between the fat thickness of the thick of shoulder,PH of the muscle(BF),holding water rate(LD) and water(LD) of different genotype combinations is significant.The fat thickness of the thick of shoulder of AA genotype is significantly lower than BB genotype(p＜0.05).The ratio PH of the muscle(BF) of AA genotype is significantly higher than BB genotype(p＜0.05).The holding water rate(LD) of AA genotype is significantly higher than AB genotype(p＜0.05). The water(LD) of AA genotype is significantly higher than BB genotype(p＜0.05).(4) The expression situation of AKT2 gene in 11 tissues of 4 months old mei san pig namely liver, uterus,ovaries,lung,stomach,heart,small intestine,kidney,fat,spleen and muscle was analysed by half-quatitive RT-PCR method.The expression level of AKT2 was the heightest in muscle,uterus and heart.Expression was also detected in liver and kidney while no expression was detected in the rest of the tissues.