Cloning And Analysis Of Buffalo Alpha-whey Protein Gene Sequence

Biodiversity is the basis for human survival and development, which is also a veryimportant factor affecting the stability of the ecosystem. The diversities in domestic animals, thepart most directly related with human being, are important components of biodiversity, and areprecious resource accumulated during the long period of evolution. The effective protection ofbiodiversity involves in the survival and development of human beings in the future.China is one of the countries that have the richest animal breed resources in the world, butbecause of a variety of reasons, their diversities are reducing virtually, and a variety of foreignprotein have been expressed in the mammary glands of transgenic animals directed by the 5'regulatory sequence of bovineα-lactalbumin gene. In this study the authors hoped to clone thewhole sequence of theα-lactalbumin gene in Chinese water buffalo. This research wouldprovide some conditions for the studies related with animal classification, origin, and evolutionbased on the nucleotide homologies, also support the mammary gland bioreactor studies usingthe regulatory region of the bovineα-lactalbumin gene as promoter.In this study four pairs of primers for PCR were designed according to referencespublished, the oligonucleotides were as follow:Primer 1: the upstream primer, 5'-TATTTAGTGGTATTGGTGGTTGG-3'the down-stream primer, 5'-TATTCTGTGCTGTCATTGTTTTG-3', this pair primer is designed to amplifya fragment of 1225bp; Primer 2: the upstream primer, 5'-TCGTCTTTCTTTCAGGG GTC-3', thedownstream primer, 5'-AACTTCATCAACCAACTTAG-3', this pair primer is designed toamplify a fragment of 451bp; Primer 3: the upstream primer, 5'-GACCAGAACCCTCACTCAAGC-3', the downstream primer, 5'-CAGAACCAGCAAAGACAGCAG-3', thispair primer is designed to amplify a fragment of 1188bp; Primer 4: the upstream primer, 5'-CCATAAAGCACTCTGTCTG-3'the downstream primer, 5'-CCTTGTGGGCTACCGTCTA T-3',this pair primer is designed to amplify a fragment of 634bp. After 30 cycles of PCR procedureswith annealing temperature at 61℃, 60℃, 68℃, and 61℃respectively, our fragments wereamplified with genomic DNA isolated from muscle tissue of water buffalo and were inserted intoa sequencing plasmid pMD18-T vector and sequenced.The sequencing results indicated that among the 3038bp of the nucleotides in thea-lactalbumin gene of the homology between Bubalus bubalis and diary cattle was 97.57%, thatbetween Bubalus bubalis and yak was 97.17%.