Through direct sequencing of PCR products with pooled DNA, we totally identified 12 single nucleotide polymorphisms (SNPs) within the KLF6 gene. The set of SNPs encompasses 7 located in 5\' flanking region, 2 located in exon 2 and 3 located in 3\' untranslated region (UTR). Of these, the g.44601035G > A is a missense mutation that resulting in the replacement of arginine (CGG) with glutamine (CAG), consequently leading to alterations in the secondary structure of the KLF6 protein, as predicted by SOPMA. The remaining 7 regulatory SNPs significantly impacted the transcriptional activity of KLF6 following mutation (P < 0.005), manifesting as changes in transcription factor binding sites. Additionally, 4 SNPs located in both the UTR and exons were predicted to influence the secondary structure of KLF6 mRNA using the RNAfold web server. Furthermore, we performed the genotype-phenotype association analysis using SAS 9.2 which found all the 12 SNPs were significantly correlated to milk yield, fat yield, fat percentage, protein yield and protein percentage within both the first and second lactations (P < 0.0001 ~ 0.0441). Also, with Haploview 4.2 software, we found the 12 SNPs linked closely and formed a haplotype block, which was strongly associated with five milk traits (P < 0.0001 ~ 0.0203).
In summary, our study represented the KLF6 gene has significant impacts on milk yield and composition traits in dairy cattle. Among the identified SNPs, 7 were implicated in modulating milk traits by impacting transcriptional activity, 4 by altering mRNA secondary structure, and 1 by affecting the protein secondary structure of KLF6. These findings provided valuable molecular insights for genomic selection program of dairy cattle.
结果:通过对合并DNA的PCR产物进行直接测序,我们在KLF6基因中鉴定出12个单核苷酸多态性(SNPs).SNP集包含7个位于5'侧翼区,2位于外显子2和3位于3'非翻译区(UTR)。其中,g.44601035G>A是一种错义突变,导致用谷氨酰胺(CAG)替换精氨酸(CGG),因此导致KLF6蛋白二级结构的改变,正如SOPMA预测的那样。其余7个调节性SNP显著影响KLF6突变后的转录活性(P<0.005),表现为转录因子结合位点的变化。此外,使用RNAfold网络服务器预测位于UTR和外显子中的4个SNP会影响KLF6mRNA的二级结构。此外,我们使用SAS9.2进行基因型-表型关联分析,发现所有12个SNP与产奶量显著相关,脂肪产量,脂肪百分比,第一次和第二次泌乳中的蛋白质产量和蛋白质百分比(P<0.0001〜0.0441)。此外,使用Haploview4.2软件,我们发现12个SNPs紧密相连,形成了一个单倍型区块,与5个乳性状密切相关(P<0.0001~0.0203)。
结论:总之,我们的研究表明,KLF6基因对奶牛的产奶量和组成性状具有显着影响。在确定的SNP中,7参与通过影响转录活性来调节牛奶性状,4通过改变mRNA二级结构,1通过影响KLF6的蛋白质二级结构。这些发现为奶牛基因组选择程序提供了有价值的分子见解。