Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes.
Spinach Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 microg antigens were used and 0.3 ml anti-sera with titer of 1:5000 were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthamiana was performed. The expression level of rbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.
Ma
Peng-Da
PD
The Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China.
Lu
Tian-Cheng
TC
Zhou
Xiao-Fu
XF
Zhu
Xiao-Juan
XJ
Wang
Xing-Zhi
XZ
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000302:isolation & purification; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D051379:Mice; D008807:Mice, Inbred BALB C; D018515:Plant Leaves / Q000235:genetics; D021122:Protein Subunits / Q000276:immunology*; D012231:Rhizobium / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D018724:Spinacia oleracea / Q000737:chemistry; D014026:Nicotiana / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D021122:Protein Subunits; D012273:Ribulose-Bisphosphate Carboxylase