| In order to amplify the genome of ovine orexin,the total RNA of orexin was extracted from hypothalamus tissue of domestic ovine.A 246bp cDNA fragment was amplified with primers designed based on the conservative sequences for the Mammal (human, rat, mouse, pig, dog and so on) genes via RT-PCR and sequencesed.Gene specific primers derived from this partial sequence were used for the amplification of the 3′ends of the cDNA by RACE method,and this lead to a 244bp fragment of 3′ends.These two fragments were spliced to be a 468bp fragment which contains complete 3′noncoding region. The orexin sequence obtained was compared with the other published orexin sequences such as AF136225, AF153615, DQ838493, EF680297 and EF680315,and sequence analysis revealed that the clones derived from the ovine hypothalamus were more than 80% identical to others. Analysis and forecast the orexin A and B peptide which the ovine orexin genintercalary fragment coded separately by bioinformatics,the results showed that ovine orexin A is an small peptide protein coded by 33 amino acid,its molecular formula is C152H249N47O45S4, molecular weight is 3583.1u,theoretical isoelectric point pI is 7.90,and is made up by two spirals,its included angle is 59.1, the terminatio of NH2 has two disulfide bonds;orexin B is an small peptide protein coded by 28 amino acid,its molecular formula is C125H213N43O35S1,molecular weight is 2910.3u,theoretical isoelectric point pI is 12.30, and is made up by two spirals,its included angle is 90.3. We successfully identified in ovine orexin genintercalary fragment sequence in hypothalamus genomic DNA. The resource work has provided a wide range of information useful for expression genomics and annotation of genomic DNA sequence. |
