[Construction of expression vector with antisense Rubisco activase gene and its genetic transformation in rice].
In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.
Jin
Song-Heng
SH
State key lab of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, China. jsh2002@163.com
Wong
Xiao-Yan
XY
Wang
Ni-Yan
NY
Li
Xue-Qin
XQ
Mao
Wei-Hua
WH
Jiang
De-An
DA
D016960:Agrobacterium tumefaciens / Q000235:genetics; D016375:Antisense Elements (Genetics) / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D012275:Oryza / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D016375:Antisense Elements (Genetics); D010940:Plant Proteins; C054765:rca protein, plant; D012273:Ribulose-Bisphosphate Carboxylase