| The Krüppel-like Factor (KLF) family of zinc-finger transcription factors, which has the feature of threehighly conserved classical Cys2/His2zinc fingers, plays a critical role in cell differentiation, phenotypicmodulation and physiologic function. KLF15, as a critical member, has been proposed to regulateadipogenesis and gluconeogenesis.In this study, to investigate the evolutionary origin of KLF15, a phylogenetic tree was constructedwith the amino acid sequences of chicken and18other species. The gene expression of chicken KLF15gene was detected in the different tissues of Gushi chicken with different age. An F2resource populationof Gushi chicken crossed with Anka broilers was used to investigate the genetic effects of the chickenKLF15gene. DNA pool sequencing method was performed to scan the SNPs of the chicken KLF15gene;PCR-RFLP method was used to analyze the SNP distribution in the Gushi x Anka F2resource population.According to the screening results, a total of20SNPs were found within the chicken KLF15gene, and2were located in exons and18in introns. A novel2-bp indel located in the intron2was selected to do thefurther analysis. The association between the2-bp indel and the chicken growth traits, carcass traits,muscle fiber traits and blood biochemical indexes was analyzed using the F2generation resourcepopulation. Subsequently, the Xichuan chicken was used to verify this kind of association, and then theDual-Luciferase Reporter Assay System and qRT-PCR were utilized to perform the further functionalanalysis of this mutation. The main results are as follows,1. The phylogenetic tree showedtwo big groups. The Danio rerio and the other species composed thefirst group. The12kinds of bird species were put in the same cluster. For the bird group, the Gallusgallus, Columba liviaand Melopsittacus undulatesbelong to one group, which indicated that chickenKLF15is evolutionarily closer to that in these species than the others.2. The chicken KLF15gene expression pattern in10tissues at2stages (1-day and36-weeks) ofGushi chicken was characterized by qRT-PCR. The results showed that the chicken KLF15gene widelyexpressed in different tissues. For the1-day-chicken, this gene highly expressed in liver and has lowestgene expression in breast muscle. For the36-weeks-chicken, the glandular stomach showed the highestgene expression and the breast muscle showed lowest as well.3. For the novel2-bp indel, genotypic distribution and allelic frequencies in the F0, F1and F2generations were calculated and showed that the insAG/insAG allelic frequency was higher than that ofthe delAG/delAG in chickens of all three generations. The association analysis showed that this SNP wassignificantly associated with chicken growth and carcass traits. For the growth traits, significantassociations with body weights at0,2,4,6,10and12weeks of age were observed (P<0.05). There werealso significant associations between the mutation and some body size indexes including shank length at8and12weeks of age, shank girth at4and8weeks, breast bone length at4and12weeks and bodyslanting length at8and12weeks (P<0.05).For the chicken carcass traits, the mutation was significantly associated with carcass weight, semi-evisceration weight, evisceration weight, head weight, feet weight,wings weight, leg weight, breast muscle weight and spleen weight (P<0.05).As for the effect of thegenotype on growth and carcass traits, we found birds carrying the insAG/insAG genotype consistentlyshowed the highest value except for body weight and shank length at0weeks of age. However, as for themuscle fiber traits and blood biochemical indexes, there was no obvious pattern for the associationanalysis.4. Gene expression for each genotype showed that birds carrying insAG/insAG had the highestexpression level than the other genotypes. The results of the Dual-Luciferase Reporter Assay Systemindicated that, in the DF1cell line, the luciferase activity of the pGL3-Pro-insAG were significantlylower than that of the pGL3-Pro. And, there was no significant difference between the pGL3-Pro-delAGand pGL3-Pro-insAG. For the Hela cell line, the luciferase activity of pGL3-Pro-insAG was significantlygreater than that of the others.These results suggested that the2-bp indel in the chicken KLF15gene may serve as a useful targetfor marker assisted selection of chicken growth and carcass traits. The ins-allele and del-allele may havethe different potential function, but the molecular mechanism warrants further investigation. |
