The Wildlife Toxicology Laboratory, Texas Tech University, Lubbock, TX, 79409-3290, USA.
Mol Biol Rep. 2021 Feb;48(2):1093-1101. doi: 10.1007/s11033-021-06186-3. Epub 2021 Feb 12.
Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon-intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.
许多最近的研究都集中在两种寄生虫——眼虫 Oxyspirura petrowi 和盲肠线虫 Aulonocephalus pennula——在北方鹌鹑（Colinus virginianus）中的流行和影响上。然而，很少有研究试图研究这些寄生虫对鹌鹑免疫系统的影响。这可能是由于缺乏适当的参考基因进行相对基因表达研究。3-磷酸甘油醛脱氢酶（GAPDH）是一种糖酵解酶，通常用作参考基因，在这项初步研究中，我们评估了鹌鹑 GAPDH 与其他鸟类物种的 GAPDH 的相似性，以评估其作为鹌鹑参考基因的潜力。在鹌鹑全基因组序列中鉴定出 GAPDH，并设计了多套 PCR 引物来生成重叠的 PCR 产物。然后对这些产物进行测序，然后进行比对，生成全长开放阅读框（ORF）的序列。利用这个序列，进行了系统发育分析和外显子-内含子模式的比较分析，结果表明 GAPDH 编码序列在鹌鹑和其他鸡形目动物中高度相似。此外，还利用这个 ORF 序列预测了编码蛋白及其三维结构，与系统发育分析一样，揭示了鹌鹑 GAPDH 与其他鸡形目动物的 GAPDH 相似。最后，设计、标准化了 GAPDH qPCR 引物，并在未感染和感染 O. petrowi 的鹌鹑中进行了测试，初步测试表明两组之间 GAPDH 的表达没有统计学差异。这些分析是首次在鹌鹑中研究 GAPDH。这些在系统发育、序列分析和蛋白质结构方面的努力表明，鸡形目动物的 GAPDH 有 > 97%的保守性。此外，这些计算机测试和初步 qPCR 的结果表明，GAPDH 是在鹌鹑中进行基因表达分析的有前途的候选基因。
