Genetic Resources Evaluation And Chicken CCT6A Overexpression In

Part I: Analysis on genetic diversity of Wenshang Barred chickenWenshang Barred chicken is located in Wenshang, Shandong province and the nearby areas. This breed of chicken gains the name “Luhua” due to its plumage decorated with the same width, black and white streak. Wenshang Barred chicken is one of the fine local poultry varieties of Shandong province and widely accepted by local farmers because of its good performance in crude feed tolerance, meat quality, disease resistance, and egg production. In October of 2002, Wenshang Barred chicken was enrolled in the list of Fine Varieties of Poultry under Special Protection by Shandong Province. Of course, the cultivation of this Wenshang Barred breed is a long story. Microsatellite DNA, also called short tandem repeats(STR) or simple sequence repeats(SSR), is the second generation of molecular genetic markers after RFLPs. Due to some features including but not limited to large quantity, wide distribution, uniformity in genome, abundant polymorphism, dominant inheritance, and test simplification, microsatellite DNA is widely used in genetic diversity detection of domestic animal population, analysis of genetic distance, research of origin and evolution relationship, genome scanning, etc.In this study, we used 27 pairs of microsatellite marker, recommended by the United Nations Food and Agriculture Organization(FAO) and the International Society of Animal Genetics(ISAG), to detect genetic diversity among 100 Wenshang Barred chickens(60 females, 40 males) from Jining, and analyzed the genetic diversity of this population at DNA level, finally put forward feasible conservation measures.The results are as follows:1. 86 alleles are detected in 27 microsatellite loci. Each microsatellite loci contains 2-6 alleles respectively. The average number of alleles, the mean expected heterozygosity, and the polymorphism information content is 3.185, 0.4655, and 0.4023 respectively.2. The analysis to genetic distance among three populations located in different places shows that the genetic distance is 0.05-0.10.In summary, the genetic diversity of Wenshang Barred chicken between populations is low, and there are inbreeding and loss of heterozygosity to some degree. Thus, it is advised to take the method combined conservation with development to save the local breed resources. On one hand, using the profit gained from hybridization to promote conservation, on the other hand, providing security for the development and utilization through breeding and the expansion of conservation population.Part II: Research on chicken CCT6 A gene over-expressionCCT is a kind of molecular chaperone existing in the cytoplasm of eukaryotes, and plays important roles in the assembly and folding of actin and tubulin. The chicken CCT6 A gene is located on chromosome 19, consisting of 14 exons, and its CDS region is 1593 bp long, encoding 530 amino acids. Previous studies on Jining Bairi chickens in our laboratory showed that, from the age of 70 days to 300 days, the expression of CCT6 A increases significantly, but from the age of 300 days to 500 day, the expression decreases significantly, which unraveled the possibility that CCT6 A plays an important role in sexual maturation.In this study, our objective is to explore the effect of CCT6 A gene on theca cells by building chicken theca cell in vitro models and over-expressing chicken CCT6 A gene. The results of this study will be theoretical bases for further exploration- searching the effect of CCT6 A gene on chicken follicular development.The results are as follows:1. Results of RT-q PCR and Western Blotting show that, compared to the control group(pc DNA3.1), after transfected with recombinant plasmid(pc DNA3.1-CCT6A), the expression of CCT6 A m RNA and protein both increase significantly(P<0.05).2. Results of RT-q PCR show that, compared to the control group(pc DNA3.1), after transfected with recombinant plasmid(pc DNA3.1-CCT6A), the expression of BCL-2 gene and Caspase-3 gene increase and decrease respectively, but the differences are not significant(P>0.05).In summary, the first result above shows that over-expression vectors are successfully transfected into chicken theca cells. After over-expression of CCT6 A, the increased expression of BCL-2(apoptosis-inhibiting gene) in chicken theca cells and the decreased expression of Caspase-3 gene show that theca cells are in a state of apoptosis inhibition, and their abilities of proliferation are enhanced. In conclusion, during the process of chicken follicular development, CCT6 A may influence the proliferation and apoptosis of chicken theca cells through affecting the expression of BCL-2 and Caspase-3, and then influence the development of chicken follicle. But the specific regulatory mechanism remains to be studied. Key words:Wenshang Barred chicken, microsatellite marker, genetic variability, CCT6 A gene,...