Validation of housekeeping genes as internal controls for gene expression studies on biofilm formation in Bacillus velezensis

Bacillus velezensis is an important bacterium widely applied in agriculture and industry, and biofilms play critical roles in its environmental tolerance. The appropriate choice of reference genes is essential for key gene expression studies. Multiple internal control genes were selected and validated from the 21 housekeeping genes of B. velezensis by expression stability evaluation during biofilm formation and were used to study the expression of key genes involved in the process. The results showed that pyk, gyrA, recA, and gyrB were stably expressed, and the expression of pyk was the most stable during biofilm formation. A pair of two genes, pyk and gyrA, provided high-quality data when used as internal controls, and the combination of three genes, pyk, gyrA, and recA, was even better. The expression levels of pyk, gyrA, and recA approximated those of five key genes, abrB, epsD, kinC, sinR, and tasA, in biofilm formation, meeting the requirements of ideal internal control genes. The expression patterns of 5 key genes were studied with 16S, pyk, the pair of 2 genes, pyk and gyrA, and the combination of 3 genes, pyk, gyrA, and recA, as internal controls during the biofilm formation process. The results proved that pyk was a suitable internal control, as were the pair of 2 genes, pyk and gyrA, and the combination of 3 genes, pyk, gyrA, and recA. This study provided genes and gene combinations which were validated as suitable internal controls for gene expression studies, especially those on the mechanism of biofilm formation in B. velezensis or even other Bacillus spp.