Molecular cloning and characterization of pigeon (Columba liva) ubiquitin and ubiquitin-conjugating enzyme genes from pituitary gland library.

In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54x10(6) pfu/mL and 1.80x10(6) pfu/mL and the titers of amplified libraries were 1.89x10(8) pfu/mL and 2.32x10(9 )pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a(+) expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.

[Study on the calculation methods of confidence intervals of genetic parameter estimates by method R and number of repeating estimations].

The objective of the study was to probe the calculation methods of confidence intervals of genetic parameter estimates by Method R and influences of number of repeating estimations (NORE). Four models were used to generate the datasets by simulation. The datasets originated from 200 sires and 2 000 dams and simulation progressed by BLUP( Best Linear Unbiased Prediction) selection for five overlapping generations. Variance components were estimated by using multivariate multiplicative iteration (MMI), combined with preconditioned conjugate gradient (PCG) to solve the mixed model equations. Parameter estimates and their standard errors and confidence intervals were computed by classical method, classical method after Box-Cox transformation and bootstrapping. The results showed that when NORE was larger, all three methods were feasible, but when NORE was fewer, bootstrapping was recommended. Fewer NORE was feasible under simple models, but more NORE was needed for complex models. Direct heritabilities were overestimated with the increase of number of random effects, but underestimation was found for many parameters with the increase of iteration numbers of PCG and MMI.