| As one member of lipin family, Lpin2 is the key gene which regulate adipocyte development and triacylglycerol (TAG) storage in adipose tissue. At present, research about Lpin2 was mainly concentrated on human, mice and pig, we can only searched Lpin2 gene mRNA from the database, study on chicken Lpin2 is hardly found.We carried out transcripts choning, gene structure and spatiotemporal expression characteristics analysis of chicken Lpin2 gene, and the association between Lpin2 polymorphisms and fat traits of gushi resource group. We adopted the method of EST database searching combining with RT-PCR, and finally got a 3,269-bp cDNA sequence of chicken Lpin2, which contains a 2,664-bp open reading frame ?anked by an 176-bp 5'UTR and a 429-bp 3'UTR. The CDS was predicted encoding 887 amino acids protein, and the similarities with human, mouse, Sus scrofa, xenopus Lpin2 CDS and Lpin1 gene were 74.35%, 74.59%, 72.86% ,69.73% and 54%, respectively.Based on the CDS information we have gained, we corrected chicken Lpin2 genomic structure ,and the result exhibited that chicken Lpin2 gene consisted of 20 exons spanning about 49 kb and 19 introns, the translation initiation codon is in exon 2 and the stop codon is in exon 20. All of exon 1 and most of exon 20 are untranslated, representing the 5'and 3'untranslated regions, respectively.In order to detect the chicken total Lpin2 gene expression difference in various tissues, we choose mature black-feather chicken(a hybrid variety of native chicken) as the object in this study, and detected the relative amount of chicken total Lpin2 gene mRNA in 15 tissues. The result showed that Lpin2 was expressed in all tissues examined. Lpin2 mRNA was very high in liver and ovary, and was expressed at high levels in brain, heart, spleen, lung, kidney, and was present at low levels in gizzard, small intestine, abdominal fat, sebum, skin, pectoralis, the Lpin2 mRNA level was very low in pancreas and crureus. Then we verified Lpin2 expression levels by TaqMan real-time PCR, and the result showed that the highest chicken Lpin2 gene expression level were in ovry and liver, respectively, which were higher than other tissues significantly(p<0.05), and the lowest expression level was found in pectoralis and crureus, which was basically consistent with that of semi-quantitative RT-PCR. But when tissues from the two varieties were plotted together, the differences between every tissue were unsignificant (p>0.05), although the difference between the weight of black-feather and yellow-spotted chickens was significant.As for different growth stages, the results of real-time PCR showed that 0 weeks-old exhibited highest Lpin2 mRNA levels in all tissues, the differences among five stages in liver and skin were unsignificant(p>0.05), while in muscle, the Lpin2 mRNA of 0 weeks-old was markedly higher(p<0.05) than 4 and 16 weeks-old. As for different tissues of the same stage, Lpin2 mRNA level in liver of 12 (p<0.05)and 16weeks-old(p<0.01) higher than that in skin and muscle.We found 15 SNPs in silky fowl, broiler, black-feather and yellow-spotted chicken CDS by direct sequencing during chicken Lpin2 CDS verification. c.444G>A in Exon4 and c.1730A >T in Exon13 lead to the change in restriction enzyme sites and amino acids, respectively. Moreover, we also found a c.-599G>A variation in promoter region. Therefore, we designed primers according to the three SNPs, and tested their distribution in different varities and gushi resource group. We also carried out traits association analysis. The result indicated that the three SNPs of chicken Lpin2 gene were significantly associated with liver weight, sebum weight, sebum rate, intermuscular fat width and triglyceride, low-density lipoprotein, which were precursor substances of lipid synthesis(p<0.05).This experiment lay the foundation for studying the role and interpreting the molecule mechanism of adipose deposition of the Chicken Lpin2 gene. |
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