Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.
Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.
Berdichevets
Iryna N
IN
Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, 119991 Moscow, Russia.
Shimshilashvili
Hristina R
HR
Gerasymenko
Iryna M
IM
Sindarovska
Yana R
YR
Sheludko
Yuriy V
YV
Goldenkova-Pavlova
Irina V
IV
D000064:Acclimatization / Q000235:genetics; D048013:Acetivibrio thermocellus / Q000201:enzymology; D003080:Cold Temperature; D044943:Fatty Acid Desaturases / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D046940:Synechococcus / Q000235:genetics; D046939:Synechocystis / Q000235:genetics; D019076:Transgenes
D011993:Recombinant Fusion Proteins; D044943:Fatty Acid Desaturases; D006026:Glycoside Hydrolases; C028020:licheninase