Cloning And Expression Of NPY And HCRTR1 Gene In E.coli And Their Genetic Polymorphism In Cattle

The hypothalamus being the central feeding and energy equation organ mediates regulation of dietary intake via synthesis of various orexigenic and anorectic neuropeptides. Neuropeptide Y was the key factor of nervous central in appetite regulation and distributed mainly in arcurate nucleus. HCRTR1 was the peripheral peptide and involved in appetite regulation. By now, fewer papers about bovine NPY and HCRTR1 gene were reported.PCR-SSCP and sequencing assay technologies were applied to detect SNPs of NPY and HCRTR1 gene in four Chiense cattle breeds (Nanyang cattle, 100; Jiaxian cattle, 130; Qinchuan cattle, 68; and Jinnan cattle, 40). The associations between these polymorphisms and some cattle economic traits were analyzed using general linear model (GLM) with software SPSS 12.0. Cloning and expression of NPY and HCRTR1gene in E.coli were also studied in Qinchuan cattle. This work would lay a foundation for cattle breeding. Results were obtained as follows:1 Polymorphisms of HCRTR1 gene in four breeds of Chinese cattle338 cattle from four Chinese four cattle breeds were used for SNPs discovery in the complete coding region of HCRTR1 gene using PCR-SSCP and sequencing methods. Eleven SNPs were detected in the complete coding region of HCRTR1 gene. The variations at 322, 481, 631 and 736 bp caused amino acid replacements and variations at the other 7 SNPs were synonymous. Not all of the loci were at Hardy-Weinberg equilibration(P>0.05).2 Effects of HCRTR1 gene polymorphisms on traits in cattleNo significant association was found between genotypes of exon 2 and the growth traits of Qinchuan and Nanyang cattle(P>0.05). For Jiaxian cattle, the value of body length and cannon circumference in B, C and D genotype was larger than that in A genotype (P<0.05). The value of heart girth and rump length in D genotype was larger than that in A genotype (P<0.05). The value of withers height and body weight in B and D genotype was larger than that in A genotype at the age of 1-8 years old (P<0.05). From above, cattle in D genotype grew faster than those in A genotype. SNPs at 384, 420 and 423bp can be fit for genetic markers in Jiaxian cattle breeding.3 Polymorphisms of NPY gene in four breeds of Chinese cattle 338 cattle from four Chinese cattle breeds were used for SNPs discovery in the region of exon 1, 2, 3 and their bandary sequence of NPY gene using PCR-SSCP and sequencing methods. Five SNPs were discovered. The variations at the 29th bp of exon 2 caused amino acid replacement: from threonine to asparagines. Most of loci were not at Hardy-Weinberg equilibration(P <0.05)in these four populations. PIC of the locus at 71th bp in the 3'end of NPY gene in Qinchuan cattle and the locus at 1725th bp in intron 2 of NPY gene in Nanyang cattle were low polymorphic and the other loci were moderate polymorphic.4 Effects of polymorphisms of NPY gene on traits in cattleIn locus of exon3 region, the value of body length and heart girth of Nanyang cattle in A genotype was longer than that in B genotype at the age of six, twelve and eighteen months age (P <0.05). At the age of 24 months the value of this two traits in A genotype was still larger than that in B genotype though the significant level can't be attained to. The SNP at 71th bp of 3'end of NPY gene can be used as a genetic marker for breeding.5 Cloning and expression of NPY and HCRTR1 gene in Qinchuan cattleThe coding sequence of NPY and HCRTR1 gene of Qinchuan cattle was amplified by overlap-PCR and confirmed with sequencing assay. The recombinant expressive plasmid pET32a+-NPY and pET32a+-HCRTR1 were transduced into E.coli strain BL21(DE3) and were induced to express by IPTG. In result, the recombinant plasmid pET32a+-NPY were over-expressed in E.coli as solubility and inclusion body with molecular weight 30 kD or so. But the recombinant plasmid pET32a+-HCRTR1 were not expressed in E.coli. We immunized rabbit using the purified fusion protein. The result of animal experiment showed that the fusion protein could stimulate animals to produce special and sensitive antibody. Using the obtained antiserum as a testing reagent combining with immunity histochemistry technology, we identified NPY as a coding protein which expressed specially in cellular cytoplast and cellular membrane of neuron in hypothalamic arcuate nucleus of cattle.