Screening Method for Novel Rice Starch Mutant Lines Prepared by Introducing Gene Encoding Starch Synthase IIa and Granule-bound Starch Synthase I from Indica Cultivar into a Branching Enzyme IIb-Deficient Mutant Line

Abstract

Rice is a staple food for over half the world s population. 1) The standard rice consumed in Japan is steamed rice from japonica cultivars. Numerous good-tasting japonica cultivars with excellent agricultural traits have been developed at agricultural stations. The starch properties in japonica rice cultivars are quite different from those of indica rice cultivars. These differences are mainly explained by the single nucleotide polymorphisms (SNPs) in the starch synthase IIa and granule-bound starch synthase I genes (SSIIa and GBSSI, respectively) in japonica cultivars, whereas indica cultivars are thought to have wild-type alleles of these genes. 2,3) Glutinous rice cultivars are gbss1 null mutant lines containing amylose-free starch. Thus, spontaneous mutant lines with distinct starch traits in the endosperm have long been used for human consumption. Since 2000, numerous rice starch mutant lines have been generated to elucidate the functions of starch biosynthetic enzymes. Among these, the starch properties in the endosperm of SSI, SSIIa, SSIIIa, GBSSI, BEI, BEIIb, and ISAI-deficient mutant lines are quite different from those of the wild type. 4,5) Double mutant lines representing different combinations of these single mutant lines are also different from the wild type and their parental mutant lines. 6 10) These mutant lines are in the japonica background (Nipponbare, Taichung 65, or Kinmaze), suggesting that the introduction of SSIIa and GBSSI from indica cultivars into these mutant lines would increase the diversity of starch properties. When screening for such mutants, obtaining the homozygous genotypes of the target genes is indispensable. Here, we describe an effective, accurate method for screening novel starch rice lines, for example, a novel mutant line containing SSIIa and GBSSI genes introduced from indica cultivars into a starch branching enzyme IIb (BEIIb)deficient mutant line (SSIIa I /GBSSI I /be2b). The novel mutant line, SSIIa I /GBSSI I /be2b, was generated by crossing a BEIIb-deficient mutant (EM10, SSIIa J / GBSSI J /be2b) 11) with an indica rice cultivar, Kasalath (SSIIa I / GBSSI I /BEIIb). GBSSI I (corresponding to Wx a) represents wild-type GBSSI derived from indica cultivar, Kasalath, in this study, and is highly expressed in developing endosperm. 12) On the other hand, GBSSI J (corresponding to Wx b) represents leaky gbss1 mutant with low expression levels, which was derived from japonica cultivar, Kinmaze, in this study. 3) SSIIa J from japonica cultivars is almost inactive, whereas SSIIa I from indica cultivars is active. 13) After screening, the rice plants were grown during the summer of 2014 in an experimental paddy field at Akita Prefectural University under natural environmental conditions. Total protein was extracted from 1/4 sections of mature rice endosperm and immunoblotting was performed to screen F2 seeds using rice BEIIb anti-serum 11) as described in Crofts et al. 14) Extraction of soluble, loosely-bound and tightly-bound starch granules proteins from mature F3 seeds an...

Active-type starch synthase (SS) IIa from indica rice partially complements the sugary-1 phenotype in japonica rice endosperm

“…The SS activity staining (Fig. 1d) showed that mobility of SSI between ss2a L gbss1 L ISA1 (T65) and SS2a GBSS1 ISA1 (IR36) were different, which is in agreement with previous studies (Chen and Bao 2016;Itoh et al 2016;Crofts et al 2017). SS2a gbss1 L isa1 (#1305A) and ss2a L GBSS1 isa1 (#1305B) lines showed similar mobility of SSI to ss2a L gbss1 L ISA1 (T65), while SS2a GBSS1 isa1 (#1305C) showed similar mobility of SSI to SS2a GBSS1 ISA1 (IR36).…”

“…Activity bands of PUL were assigned according to Fujita et al 2009. The mobility of PUL were different between ss2a L gbss1 L ISA1 (T65) and SS2a GBSS1 ISA1 (IR36), and ss2a L GBSS1 isa1 (#1305B) lines showed similar mobility of PUL to either ss2a L gbss1 L ISA1 (T65) or SS2a GBSS1 ISA1 (IR36), which is in agreement with previous studies (Chen and Bao 2016;Itoh et al 2016;Crofts et al 2017). Pho1 elongates glucans using glucose-1-phospate as a substrate, but its activity can be detected in reverse direction on native-PAGE, which hydrolyzes α-1,4 linked glucans (Satoh et al 2008).…”