Cloning of polygalacturonase (PG) cDNA and inhibition effects of its antisense RNA on the expression of PG gene in transgenic tomato plants.
We have amplified a 1.5-kb PG cDNA including its complete open reading frame, using two primers specific to PG gene and PCR technique. Its fidelity and correctness were confirmed by restriction mapping and partial DNA sequencing. The PG gene was then inserted into a binary vector, pBin437, in an inverted orientation between the 35S promoter with duplicated enhancers and the Nos3' transcriptional termination sequence, and the resulting plasmid pBPG was introduced into LBA 4404. The A. tumefaciens harboring pBPG was used to transform tomato plants. Sixty kanamycin-resistant transformants were selected. PCR detection showed two-third of the transformed plants tested (30 plants) contain the exogenous PG gene. The PG activity assay and SDS-PAGE analysis of proteins from ripening fruit cell wall showed that the PG activity in transgenic tomato plants were inhibited in different extent. One of them, plant 3, gave only 7% PG activity compared with the control (100%). These results suggest that the expression of the anti-PG gene has effectively inhibited the expression of endogenous PG gene. The effect of low-level PG activity on tomato fruit ripening and storage is being investigated.
Ju
R
R
Institute of Botany, Chinese Academy of Sciences, Beijing.
Tian
Y
Y
Shen
Q
Q
Liu
C
C
Mang
K
K
D001483:Base Sequence; D007446:Chromosome Inversion; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011096:Polygalacturonase / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000235:genetics*; D014170:Transformation, Genetic
D018076:DNA, Complementary; D016372:RNA, Antisense; D011096:Polygalacturonase