Identification And Cloning Of Mastitis Resistant Genes In Dairy Cows Using Suppression Subtractive Hybridization

Bovine mastitis is a complex disease,which remains the most costly disease to the worldwide dairy industry, despite the widespread implementation of mastitis control strateges.Genetic differences of mastitis resistance between breeds and within breed in dairy cows are significant, so genetic selection to improve mastitis resistance may be the best long-term stratege bacause it offers the possibility for permanent resolution of this complex disease problem.Since the molecular mechanisms, gene systems and expression regulation of mastitis resistance in dairy cows remain unclear, it has seriously impeded our ability to substantially improve overall resistance to mastitis through therapies, vaccination programs and genetic selection schemes. The aim of the current research was to isolate and clone the mastitis resistant genes to look into molecular events associated with mastitis resistance and establish fundamental materials and techniques for further study and genetic selection. The main results obtained are listed bellow.To screen and clone mastitis resistant genes in dairy cows, differentially expressed cDNA library of peripheral blood leukocyte were constructed using suppression subtractive hybridization through following protocol: poly (A)+ RNA were purified using Oligotex mRNA Kits (Qiagen) from peripheral blood leukocytes of 20 Holstein cows with mastitis and 20 health Holstein cows in lactation as control; single- and double-stranded cDNA were synthesized from the poly(A)+ RNA using PCR-SelectTM cDNA Subtraction Kit (Clontech) and further digested using Rsa I into cDNA fragments sized from 400 to 600 bp (dscDNA), dscDNAs from the mastitis cows (as tester) were divided into two portions which were ligated separately with a different cDNA adaptor; the ligated cDNA were hybridized twice with the dscDNA of the healthy cows at 68℃for 8 h each; the products after double hybridizations were diluted by 200 times and then used for suppression PCR twice; the secondary PCR products was inserted into pGM-T Easy vector and transformed into E. coli TOP10 competent cells; 610 positive clones were obtained; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments of 250-750 bp in the 16 randomly selected positive clones, which would provide useful baseline for the screening and cloning of specific mastitis resistant genes and understanding the molecular mechanism of mastitis resistance in dairy cow.Differentially screening was performed using the reverse northern dot-blotting...