Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato

Abstract

A tuber-specific cDNA library of cassava (Manihot esculenta Crantz) was constructed and a full-length cDNA for granule-bound starch synthase (GBSS, also known as waxy protein), the enzyme responsible for the synthesis of amylose in reserve starch, was cloned. Sequencing of the cloned cDNA showed that it has 74% identity with potato GBSS and 60-72% identity with GBSS from other plant species. The cDNA encodes a 608 amino acid protein of which 78 amino acids form a chloroplast/amyloplast transit peptide of 8.37 kDa. The mature protein has a predicted molecular mass of 58.61 kDa (530 amino acids). Comparison of the GBSS proteins of various plant species and glycogen synthase of bacteria showed extensive identity among the mature form of plant GBSS proteins, in which the monocots and dicots form two separate branches in the evolutionary tree. From analysis of the genomic DNA of allotetraploid cassava, it is shown that GBSS is a low-copy-number gene. GBSS transcript is synthesized in a number of different organs, but most abundantly in tubers. Potato plants were transformed with the cassava GBSS cDNA in antisense orientation fused between the potato GBSS promoter and the nopaline synthase terminator. The expression of the endogenous GBSS gene in these transgenic potato plants was partially or completely inhibited. Complete inhibition of GBSS activity by the cassava antisense gene resulted in absence of GBSS protein and amylose giving rise to almost complete amylose-free potato starch. This shows that also heterologous genes can be used to achieve antisense effects in other plant species.

Discovery of an Amylose-free Starch Mutant in Cassava (Manihot esculenta Crantz)

“…Compared with other root and tuber tropical crops, cassava starch and its biosynthesis have been better studied (3)(4)(5). The starch granules are generally round (oval), with a flat surface on one side (truncated) and range from 5 to about 40 µm in size.…”

Characterisation and role of <i>Isoamylase1 (Meisa1</i>) gene in cassava

“…This suggests that Meisa1 gene occurs in at least two copies in cassava genome. Starch granule bound synthase1 (GbssI) has been reported to occur in two copies in cassava genome (Salehuzzaman et al, 1993). The occurrence of two or more copies of Meisa1 and GbssI genes could be due to the ploidy level of cassava genome (Awoleye et al, 1994).…”

Identification of the gene encoding granule-bound starch synthase I in sweet potato (Ipomoea batatas (L.) Lam.).

“…It is unclear at present whether this result is due to the hexaploid and heterozygous nature of sweet potato. The complicated band pattern also has been seen in the analysis of the GBSSI gene of cassava, a tetraploid plant (Salehuzzaman et al, 1993). It would be interesting to clarify the homology between/among these copies of GBSSI and whether they have the same strengths and patterns of expression.…”