Isolation and characterisation of cDNAs encoding the large and small subunits of ADP-glucose pyrophosphorylase from cassava (Manihot esculenta Crantz)

Screening of a tuber specific cassava cDNA library resulted in the isolatio n of full length cDNA clones with homology to the genes encoding the small and large subunits of ADP glucose pyrophosphoryalse. Sequence analysis reve aled that AGPase B the clone with homology to the small subunit shared 54% homology at amino acid level with the AGPase S clone that is more closely r elated to the large subunit. Segregation analysis of a cross between the ca ssava cultivars TMS 30572 and CM 2177-2 revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the ca ssava genetic map. AGPase B is a low copy gene of which one member is local ised on the female derived linkage group P. The two genes are expressed in all cassava tissues but AGPase B exhibits a higher steady state mRNA level than AGPase S and is highly expressed in leaf and tuber tissue. The AGPase enzyme activity was much higher in young cassava leaves as compared to olde r leaves and tubers. Cassava AGPase was activated by 3-PGA and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme wa s relatively unaffected by 3PGA but was highly inhibited by Pi. Transformat ion of potato (Solanum tuberosum) plants with an antisense AGPase B constru ct resulted in 10 out of 134 antisense AGPase B plants having on average 3. 5 times more tubers than the control non transgenic plants. Analysis of the se transgenic plants revealed they had greatly reduced levels of AGPase B m RNA, 1.5 to 3 times less starch, and five times higher levels of soluble su gars, sucrose, glucose and fructose, to those found in control plants.