Molecular Cloning And Expression Of Genes Related With Laying Quail Reproduction And Their Associations With Egg Performances

Egg production has always been an important trait in the poultry industry and had a great effect on farmer earnings. Laying quail is one of the most important poultry in poultry production and has become a very influential industry in recent years. Laying quail have some characters including strong adaptability, fast growth, early sexual maturation, high egg production, low material consumption, short production-phase etc, Thus, it is known as the "second poultry industry" after chickens. Many Laying quail breeds(stiains) has been successively breeding through the traditional breeding method since 1980 in China. And some molecular researches have been done. However, compred with chicken and livestock, molecular biology research of laying quail in China had just begun, especially there are few marker of molecular biology, which greatly limits the application of molecular breeding technology in laying quail breeding process. In the long term, there were useful method for thorough understanding of quail breeding characters, mechanism of formation, develop and applicate molecular markers through using the method of candidate genes and draw some lessons from some successful experience in pigs, chicken and human ect.In this study, H and L population of Shen DanⅠquail of and a wild quail group which was catched from Henan were used as experimental materials. Four genes including ESR1, GnRH1, PRLR, VIPR-1 were selected as candidate genes of laying quail egg production traits and conducted gene cloning, expression, single nucleotide polymorphism(SNP) and correlation analysis research. The main research results are as follows: 1.The PCR- RFLP and sequence technique were used to screen the SNPs in partial CDS of quail ESR1 gene. Five new SNPs loci were detected in ESR1 exon 1, exon 4 and exon 8. Three SNPs including C312 T loci, C50 T loci and C91 T loci respectively were digested by PvuⅡ, Hpy18 I and Acc I. The PCR-RFLP results showed that the genotypic frequencies and allelic frequencies of the three sites were significant differences in different quail populations. Association analysis demons trated that there was significant correlation with age at first egg and the mutation genotype of the C312 T site which was a silent mutation on exon 1(P<0.05). Associate the mutation genotype of C50 T site on exon 4 with Age at first egg and 20 weeks egg number was also significant difference(P<0.05). Associate the mutation genotype of C91 T site on exon 8 with age at first egg, 20 weeks egg number and egg weight was significant correlation and significant difference respectively(P<0.05). Correlation of different type haplotype individual H1H1(CTCT) or H1H3(CTTT) and first laying quail age was significant difference(P<0.05).2.Partial cDNA sequence of quail GnRH1 which included 359 bp was cloned by comparative genomic method. The bioinformatics analysis result illustrated that the protein of quail GnRH1 gene have a cross structure domain and a signal peptide. SqRT-PCR analysis results showed that the expression of quail GnRH1 gene expression was differences in all examinated organization. Some issue including heart, lungs and leg muscles, chest muscle have expression and others have no expression. There was higher expression in spleen, the hypothalamus and the expression of liver was the lowest. Six SNPs loci were detected by PCR-RFLP and sequence in exon 2-3. A mutation C108 T was located in the exon 2, but others were located in the introns. Translation comparison found that the mutation is synonymous mutations. Howere, there were no Restriction Enzyme cutting site.3.A full-length cDNA of quail PRLR gene which included 3106 bp was cloned by comparative genomic and RACE method. The analyzed results showed one PRLR transcripts which have been identified. Bioinformatics analysis showed that the CDS of PRLR transcripts had a cross structure domain and five contains N terminal glycosylation sites. SqRT-PCR analysis results showed that the quail PRLR gene widely expressed in all testing organization. The expression quantity of bowel issue was the highest, then the hypothalamus, the expression amount of heart, chest, leg muscle is minimum. Character correlation analysis results show that three genotypes of mutation C104 T age at first day and 20 weeks egg number of quail were significant differences respectively(P < 0.05).4.PCR-RFLP and sequencing were used to screen the SNPs in the CDS of quail VIPR-1 and 36 mutations were detected in the research. Restriction analysis showed that in allele frequency of two loci in the three quail group differences was different. Alleles T on G373 T loci for H group was superior gene; but for A313 G loci, alleles G was a dominant genes for three groups. The association between genotypes and quail egg production, which in the H series and L series is signicant different between two mutations(A313G and G373T) and egg weight; Correlation of different type haplotype individual(H1H1 or H1H4) and quail egg weight were significant difference(P < 0.05).