Molecule Clone, Expression And The Association Analysis In Reproduction Traits Of The Chicken Crbps

In this study, taking Erlang Mountainous Chickens as materials, the chicken CRBP1, CRBP2, CRBP3 and CRBP4 gene entire coding sequence were cloned, and the correlations of the bioinformatics analysis were done. Taking Roman egg shell powder (D) as control, we used SYBR Green I quantitative PCR to analysis CRBPs gene family members expression patterns on SD02, SD03 two lines of Erlang Mountainous Chickens at 12,20, 32 and 45 weeks and seven tissues (heart, liver, kidney, fallopian tubes, ovaries, pituitary and hypothalamus). We also detected the SNPs of CRBPs exon and adjacent intron sequence using PCR-SSCP and sequence methods in SD02 and SD03 two strains of Erlang mountain chicken totally 349 individuals, and meanwhile, association analysis was conducted combined reproductive traits with SNP loci, or haplotypes of traits analyzed, the results are as follows:We successfully obtained the coding sequence of CRBP1, CRBP2, CRBP3 and CRBP4 gene. Based on these four cDNA sequences, four amino acid sequences were obtained, their lengths were 135aa,135aa,159aa and 127aa. We found CRBP1, CRBP2, CRBP3 and CRBP4 maybe a hydrophilic but Non-transmembrane protein. Meanwhile, except for CRBP3, they were of common characteristic for Lipocalin superfamily. The chicken CRBPs amino acid sequences had high identity with that of other animal by bioinformative analysis. Between CRBP1 and CRBP4, the sequences had a higher identity (57.30%), yet between CRBP2 and CRBP1, the identity of sequences was lower (43.50%).The quantitative real-time PCR expression pattern of CRBP1, CRBP2, CRBP3 and CRBP4 gene indicated that in all tissues at four growth points there was advantageous tissue for gene expression at each growth point. Expression of CRBP1 was detected that the ovarian, hypothalamus and liver had the tissue differences between detected breeds. The heart was advantageous tissue for CRBP1 gene, and hypophysis and hypothalamus had the highest expression level in 20week, the liver was 12week, while the others were all 32weeks. For CRBP2, hypothalamus and kidney had the tissues differences in different breeds, and the heart was advantageous tissues. Except that liver had the highest expression level at 12week, others were 32week. The expression level of CRBP3 in liver had difference in different breed. The kidney, liver and ovary were advantageous tissues for CRBP3. In ovary and oviduct, the expression level at 32week was the highest, the liver and kidney was at 45week, but the expression level of other tissues was 20week. And for CRBP4, we found the kidney and oviduct had the breed differences, the advantageous tissues were heart, kidney and oviduct. Except that in hypothalamus, the expression level at 20week was the highest, the liver was 45week, other tissues were all at 32week, the expression was highest. In addition, we also detected that the CRBP2 gene expression is the lowest in CRBPs member on all tissues at 4 growth points.For SNPs analysis, we found that a SNP was detected at 14604bp (G/T) (GenBank NO:NC＿006096.2) in CRBP1 gene. It was located in the 3rd intron. It was a novel SNP. Association analysis showed that the SNP had significant genetic effect on egg-shape index, the body weight at first egg, the weight at first egg, age at first egg and the total number of eggs with 300 age (P<0.01). Age at first egg early or late will lead to lower total number of eggs. Egg-shape index too large or too small will affect on the hatching rate. The relative size of age at first egg and egg-shape index is as for the specific types of chicken breeds and the economy. Therefore, the SNP locus may be used as a candidate molecular marker for the total number of eggs with 300 age of Erlang Mountainous chicken.A SNP was detected at 9028bp (C/T) (GenBank NO:NC₀06096.2) in CRBP2 gene. The SNPs detected in the current study was novel and a nonsynonymous SNP, which caused an amino acid (AA) replacement (p.117Tyr>His). The association analysis results showed that SNPs locus had highly significant genetic effect on age at first egg, the total number of eggs with 300 age, average egg-laying interval and shell thickness egg-shape index (P<0.01). Age at first egg and total number of eggs was positively correlated. The average egg-laying interval was shorter, the higher total egg production. Therefore, the SNP locus may be used as a candidate molecular marker for the total number of eggs with 300 age of Erlang Mountainous chicken, and the CC was an advantage genotype.Two SNPs were detected in CRBP3 gene. They were located in the 2nd intron and 3rd intron. SNP1 (C/G) was at position 934bp and SNP2 (A/G) was at position 1324bp (GenBank NO:NC＿006088.2). Four haplotypes were constructed from the two SNPs which were HI (C-G),H2 (C-A),H3 (G-G) and H4(G-A). The association analysis results showed that Haplotype H2H2 (C-A-C-A) had a positive effect on the total number of eggs with 300 age, Haplotype H1H1 (C-G-C-G) had a negative effect on the total number of eggs with 300 age.Three SNPs were obtained in CRBP4 gene. They were located in the 2nd exon and 3rd exon. SNP1 (C/T) was located at position 826bp, SNP2 (C/T) was at position 1240bp and SNP3 (A/G) was at position 1241 bp (GenBank NO:NC₀06108.2). The SNP2 was synonymous SNPs. SNP1 and SNP3 were nonsynonymous, which caused amino acids (AA) replacement, respectively p.49Ser>Leu and 97He>Val. Four halplotypes were constructed from 3 SNPs and four haplotypes including H1 (T-T-G,H2 (T-C-A),H3 (C-T-G) and H4 (C-C-A) which were accounted for 100% of all the observations. The association analysis results showed that haplotype had significant genetic effect on age at first egg and the total number of eggs with 300 age. Haplotype H4H4 (C-C-A-C-C-A) had a positive effect on the total number of eggs with 300 age. Haplotype H1H1 (T-T-G-T-T-G) had a negative effect on the total number of eggs with 300 age. So it was concluded that the CRBP4 may be used as a candidate molecular marker for Erlang Mountainous chicken of the total number of eggs with 300 age.