Highly phosphorylated functionalized rice starch produced by transgenic rice expressing the potato GWD1 gene

Abstract

Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.

Figure 1

(A) Expression of StGWD1 in the developing endosperm of stable offspring lines of transgenic rice (RGD1-6) at the transcript level determined by qRT-PCR (up: StGWD1, down: actin), and (B) StGWD1 protein determined by western blotting (up: StGWD1, down: the total protein). The gels were cropped to only show the relevant bands. M: marker, 1: control, 2-7: RGD1-6.

Figure 2

(A) Content of starch bound Glc-6-P and Glc-3-P (nmol mg⁻¹ starch) in starch from RGD1-6 lines, and (B) Apparent amylose content (%) in starch from the same lines as in A.

Figure 3

Starch granules visualized by Scanning Electron Microscopy at 3000×. (A) Control (Zhonghua 11). (B) RGD3. (C) RGD5. White arrows indicate pores in the starch granule. Selected starch granules in central part are magnified at 5000× and shown at the lower -right (A) and upper-right corners (B and C).