2.4 Vector Construction and Transformation of Rice
Rice genomic DNA was prepared from young fully expanded leaves using
cetyltrimethylammonium bromide as described previously (Morita, Hatanak,
Misoo, & Fukayama, 2014). To construct the promoter::GUSchimeric gene, 5’-flanking region including 5’-UTR and partial coding
region of glucose 6-phosphate/phosphate translocator 2(OsGPT2 ; -2428 to +654, numbered from the translation initiation
site), OsAGPL1 (-3758 to +200), AGPase small subunit 1(OsAGPS1 ; -1943 to +851), OsBE1 (-2049 to +263) andPho1 (OsPho1 ; -2037 to +1431) were amplified by PCR using
gene specific primers (Table S1). The amplified DNAs were fused upstream
of GUS into a binary vector pBI-Hm.
For overexpression of FLAG-CRCT, the 3xFLAG fragment was amplified by
PCR using the primers shown in Table S1 and the p3XFLAG-CMV plasmid
(Sigma-Aldrich, https://www.sigmaaldrich.com) as a template. The
obtained 3xFLAG fragment was fused to the rice Actin promoter and
cloned into a binary vector pBI-Hm. First-strand cDNA was synthesized
from total RNA with an oligo (dT)18 primer using
PrimeScript II first-strand cDNA synthesis kit (Takara) as above. The
coding region of CRCT was amplified by RT-PCR using gene specific
primers (Table S1) and fused downstream of 3xFLAG into pBI-Hm.
The plasmid construction for the CRCT knockout by CRISPR/Cas9 was
carried out according to Mikami, Toki, & Endo, (2015). A target
sequence created from two primers (Table S1) was cloned into the vector
pZK_OsU6-gRNA. The connected DNA fragment of OsU6 promoter,
target sequence, sgRNA scaffold and poly(T) was obtained from the
digestion of the vector by I-SceI and inserted into I-SceIsite of binary vector pZH_MMCas9.
These constructs were introduced into rice viaAgrobacterium -mediated gene transfer. Antibiotic-resistant
transgenic rice plants were regenerated and grown in soil as stated
above.