Study On The Cloning, Polymorphism And Organization Expression Of AA-NAT, HIOMT And TAC3Gene In Small Tail Han Sheep

The year-round estrous in sheep contain tremendous economic value, it helps to improve the annual little size and improve the economic benefit. Small Tail Han sheep can estrus and be mating lambing all the year round, with high fecundity, so it is unique genetic resources in China. The aim of the present research was to find new genes associated with year-round estrous of sheep and its mechanism of action, and provide some useful information for the molecular breeding to cultivate high fecunndity and multi fetal sheep. The results are as follows.1.Five pairs of primers (P1to P5) of AA-NAT gene were designed to clone Small Tail Han sheep AA-NAT, a2138bp DNA sequence was obtained. By sequence alignment with Tan sheep, four mutations were found in exon2(A705G, T733C) and exon4(C1690T, C1796T). These polymorphisms were detected in year-round estrous sheep breeds (Small Tail Han sheep, White Dorper sheep) and seasonal estrous sheep breeds (Tan, Suffolk and Texel sheep) by PCR-RFLP. For the705locus, only one genotype (AG) was detected in Texel sheep, two genotypes (AG and GG) were in the remaining four breeds. Small Tail Han sheep with genotype GG had0.36kids more than those with genotype AG (P<0.05). For the733locus, only two genotypes (CC and CT) were detected in Small Tail Han sheep, Tan and Texel sheep, three genotypes (CC, CT and TT) were in the remaining two breeds. No significant difference (P>0.05) was found in litter size between CC and CT genotypes in Small Tail Han ewes. For the1690locus, three genotypes (CC, CT and TT) were detected in Small Tail Han sheep, White Dorper and Tan sheep, two genotypes (CC and CT) were detected in Suffolk sheep, only one genotype (CT) was in Texel sheep. No significant difference (P>0.05) was found in litter size among CC, CT and TT genotypes in Small Tail Han ewes. For the1796locus, three genotypes (CC, CT and TT) were detected in Tan, Suffolk and Texel sheep, two genotypes (CC and CT) were detected in Small Tail Han sheep and Suffolk sheep, there were significant differences (P<0.05) in the mutation site in different genotypes distribution in year-round estrous sheep breeds and seasonal estrous sheep breeds. Small Tail Han sheep with genotype CC had0.48kids more than those with genotype CT (P<0.05). The present study preliminarily indicated that there may be some association between705and1796loci of AA-NAT gene and year-round estrous in sheep, and alleles G and C of AA-NAT gene were potential DNA markers for improving litter size in sheep.2.Eight pairs of primers (H1to H8) of HIOMT gene were designed to clone part of exon sequences in Small Tail Han sheep and two mutations were detected in exon5(G606A) and exon6(A737C). No significant difference (P>0.05) was found in litter size in Small Tail Han ewes for606and737loci.3.Five pairs of primers (C1to C5) of TAC3gene were designed to clone Small Tail Han sheep TAC3DNA sequence without exon1, a3296bp DNA sequence was obtained. By sequence alignment two mutations were found in intron1(C327T) and exon2(G1803A). No significant difference (P>0.05) was found in litter size in Small Tail Han ewes for327and1803loci.4.This research was study on spring estrus, spring diestrus and fall estrus in female year-round estrous sheep breeds Small Tail Han sheep (two and a half years old) and spring anestrus, fall estrus and fall diestrus in female seasonal estrous sheep breed Tan sheep (two and a half years old). We used real-time PCR technology to examine the mRNA expression of AA-NAT and TAC3genes in hypothalamus, pituitary gland, ovary, uterus body, adrenal gland and pineal, aimed to know the association between the genes and year-round estrous of sheep. Quantitative results showed that AA-NAT and TAC3genes expressed in the six tissues of different developmental stages, the ovary and uterus body expression level were very significantly lower in Small Tail Han sheep spring and fall estrus than that in Tan sheep fall estrus (P<0.01), but the pineal expression level was very significantly higher in Small Tail Han sheep spring and fall estrus than that in Tan sheep fall estrus (P<0.01). The results showed that the expression level of AA-NAT in ovary, uterus body and pineal may have an effect on the year-round estrous of sheep, and the ovary and uterus body expression level of TAC3may have an effect on the year-round estrous of sheep.