Validation of a Reference Gene (BdFIM) for Quantifying Transgene Copy Numbers in Brachypodium distachyon by Real-Time PCR

Abstract

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.

Genetic characteristics of polycistronic system‑mediated randomly‑inserted multi‑transgenes in miniature pigs and mice

“…The copy numbers of randomly integrated transgenes in multi-transgenic mice and miniature pigs were determined. In the present study, the quantitative dissociation curves were unimodal, confirming the specificity of the primers ( 15 , 35 , 36 ). In the development of the gradient copy standard curve, it was assumed that PCR efficiency is generally equal between a plasmid template and genomic DNA ( 7 , 8 , 31 ).…”

“…It is important to characterize multi-transgenic animal integration. Fortunately, exogenous gene copy numbers are one of the important factors affecting the level of expression and genetic stability ( 15 , 16 ). The transgene copy number indicates the number of genomic transgene copies ( 17 ).…”

Salicylic acid‐dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon

“…PCR for R. solani DNAs (28S rDNA for MAFF305230, a tubulin gene for MAFF305256) was performed using a KAPA SYBR Fast qPCR Kit (Kapa Biosystems, Woburn, MA, USA) with a GVP‐9600 instrument (Shimadzu, Kyoto, Japan) or SYBR Premix Ex Taq II (Takara Bio) with an Applied Biosystems 7500 System (Thermo Fisher Scientific, Waltham, MA, USA). The B. distachyon BdFIM gene was used for normalization (Zhu et al ., ). Primers are listed in Supporting Information Table S1.…”

Colonization dynamic and distribution of the endophytic fungus Microdochium bolleyi in plants measured by qPCR

“…The quantitative PCR mix consisted of 1× SYBR Green (Top-Bio, Vestec, Czech Republic), 0.2 μM forward and reverse primers (the best final primers MbqITSF/R were selected from a preliminary screen of the designed primers; ( S1 Table )), 10 ng DNA (2 μL), and water to final volume 15 μL. The reference gene for wheat was TaPAL [ 43 ], and for Bd , it was BdFIM [ 44 ]. The control samples consisted of DNA of wheat or Bd plants colonized by the known level of root colonization by Mb obtained from the previous experiment; it will be further described in this article as a standard sample (sample of roots 1 cm under crown collected 90 days after sowing; wheat 20.6% and Bd with 23.4% of colonization measured by light microscopy method according to Trouvelot et al (1986) [ 40 ]).…”