LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:

TSUGANE, Kazuo

Although transposons occupying a large portion of thegenome in various plants were once thought to be junkDNA, they play an important role in genome reorganization and evolution. Active DNA transposons are important tools for gene functional analysis. The endogenous
non-autonomous transposon, nDart1, in rice is said togenerate various transposon-insertion mutants because
nDart1 elements tend to insert into genic regions undernatural growth conditions. The transpositions of nDart1
were promoted by an active autonomous element, aDart127, on chromosome 6. By using the endogenous nDart1/
aDart1-27 system in rice, a large-scale nDart-inserted
mutant population was easily generated under normal
field conditions, and the resulting tagged lines were freeof somaclonal variation. The nDart1/aDart1-27 system
was introduced into a rice variety, Koshihikari (Oryza
sativa subsp. japonica), and Basmati (Oryza sativa subsp.
indica). Various mutations caused by the insertion ofnDart have been screened for characteristic phenotypes.

bore slightly larger grains (Figure 1) as a dominant inheritance. Transposon-display identified the insertion site ofnDart1 in the Lgg mutant.

II. Analysis of Lgg mutants

The identified LGG gene shows similarity to RNA
binding proteins. To investigate the subcellular localization of LGG protein, green fluorescent protein (GFP)fused constructs driven by 35S promoter, 35S:LGGNPGFP was transformed into rice calli. GFP fluorescencespots were observed in the nuclei in calli. These results
suggest that LGG is localized to the nucleus. To verify
that LGG was responsible for the long grain phenotype, knockout (GE) and overexpression (OE) lines were
produced by transforming a CRISPR/Cas9 constructtargeting the RNA recognition motif region of LGG andan LGG construct with its WT promoter into into cv.
Nipponbare (NP), respectively (Figure 2(A)). Several
independent GE lines showed significantly increased
grain length, and three independent OE lines had shorter
grains than NP. Any significant differences of panicle
length, number of panicles per plant and number of spikelets per panicle were not observed among NP, GE andOE except for culm length. Observations of longitudinal
sections from lemmas of GE and OE lines revealed thatthe cell sizes of GE and OE lines were comparable to NP(Figure2 B-C), suggesting that LGG might regulate thelongitudinal cell number of spikelet hull and thus grain
length.

I. Large grain (Lgg) mutation in rice

Seed size and number were controlled by various genes
in the plants. It was reported that expression changes inhigh contribution genes for seed size, number and panicle
shape resulted in a decrease of the total yield. A strategy
for boosting rice yield based on molecular biology is tostack the finely tuned gene expressions. The Lgg mutant
which was isolated from Koshihikari-nDart tagging line

Figure 1. Phenotype of Large gain (Lgg). Harvested panicle and seeds.

Figure 2. The spikelets morphology and longitudinal sections and cell
length of the lemma of NP, GE, and OE, respectively. (A) The spikelets.
Bar = 5 mm. (B) The Section of lemma in transgenic plants. The red andblack arrows indicate adaxial epidermis and chlorophyll-contained parenchyma cell. (C) Cell length of lemma in NP, GE and OE. n = 3. mean ± SD.

65