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ISSN 1672-9145                                                 Acta Biochim Biophys Sin 2004, 36(9): 644–648                                                      CN 31-1940/Q

Preparation of Polyclonal Antibodies of Rubisco Large and Small Subunits and Their Application in the Functional Analysis of the Genes

 

 

Peng-Da MA1, Tian-Cheng LU1, Xiao-Fu ZHOU1,2, Xiao-Juan ZHU1, and Xing-Zhi WANG1*

 

1The Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China;

2The Institute of Gene Technology, Jilin Normal University, Siping 136000, China

 

Abstract        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 mg 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.

 

Key words        ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); rbcL; rbcS; polyclonal antibody; virus-induced gene silencing (VIGS)

 

 

 

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Received: May 27, 2004        Accepted: August 3, 2004

This work was supported by a grant from the National Natural Science Foundation of China (No. 30370767)

Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; rbcL, large subunits of Rubisco; rbcS, small subunits of Rubisco; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVX, potato virus X; ELISA, enzyme-linked immunosorbent assay; VIGS, virus-induced gene silencing

*Corresponding author: Tel, 86-431-5269769; E-mail, [email protected]