Studies Of The Sequence Cloned And Genetic Variation Of AA-NAT And TPH Gene From Liaoning Cashmere Goats

It is confimed that melatonin promotes the growth of goat cashmere.Aromatic-N-alkylamine acetyltransferase (AA-NAT) is a major rate-limiting enzyme in theprocess of melatonin biosynthesis pathway, while TPH is involed. In this study, thesequence of AA-NAT was cloned from Liaoning cashmere goats, and its protein functi waspredicted。The genetic variation of AA-NAT and TPH were analysed by PCR-SSCP andsequencing, and the correlation between the genetic variation and cashmere traits weredetected, which would provide marker assisted selection(MAS) sites in Liaoning cashmeregoats breeding. The results as follows:1, Partial sequences(1389bp) of AA-NAT gene were cloned, which included first exon(163bp), the first intron (290bp), the second exon (155bp),the second intron (338bp), thethirdly exon (306bp) and partial3’flanking region (-137bp) andencoded207amino acids,The first intron and secod intron were amplified for the first time.2, AA-NAT gene polymorphisms were analyzed. Results showed that in AA-NAT geneexisted17polymorphisms sites, in exon1:129bp G�'C (codon GAG�'GAC, amino acidsGlu�'Asp),132bp C�'T and141bp C�'T; in exon2:590bp C�'T; in exon3:977bpA�'G(codon CAC�'CGC, amino acids His�'Arg),1060bpC�'T(codon CAC�'TAC, amino acidsHis�'Tyr),1091bp G�'A,1161bp A�'G and1163bp G�'A (codon TGT�'TAT, aminoacids Cys�'Tyr); in intron1:356bp G�'C,372bp C�'T,416bp G�'A,418bp C�'G and425bp C�'T; in intron2:625bp C�'A,631bp T�'C and638bp G�'del. Primier TPH-exon2and TPH-exon5of TPH gene were not existed polymorphisms sites, but primierTPH-exon10were existed a T�'C mutation, and has three genetic SS、SO�'�OO.3, Protein function prediction analysis showed that the relative molecular weight ofAA-NAT protein as23.03kDa and theoretical isoelectric point as8.56. ProtScale of theExPASyde program predicted AA-NAT protein hydrophobicity, results showed AA-NATprotein contains four distinct hydrophobic region, which are55～61,71～98,155～169and171～187. And the minimum as-2.633and the maximum as2.689. TMPRED predictionanalysis software on line predicted the AA-NAT gene encods protein transmembranestructure, the results shows that the AA-NAT protein transmembrane direction from inside to outside and from outside to inside with two sets of values,2region located in89～99and173～191might be the transmembrane regions.4, Population genetic analysis showed that the AA and CC genotype frequencey of theprimers AA-exon1and AA-intron1loci were significantly higher than AB and CDrespectvetely, which were the dominant genotypes and the A and C allele as dominant allele,the genotype frequencies of the other AA-exon2, AA-exon3and TPH-exon10loci werenosignificantly change; Hardy-Weinberg equilibrium test showed that only AA-exon3siteswas of imbalance in five sites, the rest points were in Hardy-Weinberg equilibrium;polymorphism information content analysis showed, AA-exon1and AA-intron1locus werein low polymorphism (P <0.25); AA-exon2and TPH-exon10were in moderatelypolymorphic (0.25<P<0.5.); and AA-exon3sites were in highly polymorphic (P>0.5).5, Correlation between AA-NAT and TPH gene polymorphism and cashmereperformance and MLT levelswere analysed by the general linear model. The results showed:the AA genotype of AA-exon1locus had highly significant positive effect on cashmereyield, cashmere fiber length and MLT content (P <0.01), and the average cashmere yield ofAA type goat was higher than AB type49.59g; CD genotype of AA-intron1sites had asignificant positive effecton the net cashmere rate (P <0.05); The FF genotype of AA-exon2locus had a very highly significant positive effect on the cashmere yield, cashmere fiberlength and MLT content (P <0.01), and cashmere yield of the FF type goat was higher thanEE and EF types, increased97.52g and81.95g respectively. The GG genotype of AA-exon3locus had a very highly significant positive effect on cashmere yield (P <0.01), and thecashmere yield of GG genotype was higher than GH, HH, and HT genetype, increased57.48g,69.84g and71.3g respectively, while MLT content of the GG type was alsosignificantly higher than HT genotype (P <0.01); Each genotype of TPH-exon10locus hadno significant effect on cashmere diameter, the net cashmere rate and MLT content, only SSgenotype had a significant effect (P <0.05) on cashmere yield and cashmere length.