Generation of constructs and transformation
To generate the OE construct of Os10g39300 , the full-length cDNA
was amplified from the resistant line RBPH16, and cloned into
pCAMBIA1301A with the CaMV35S promoter.
To knockout gene Os10g39300 , two target sites (31–49 bp and
910–927 bp) were selected in the exon region to generate the construct
using the CRISPR-Cas9 technique (http://skl.scau.edu.cn/). The gRNA
target oligonucleotides were synthesized by the Beijing Genomics
Institute. Secondary structures of both sgRNAs were developed using
CRISPR-P (version 2.0; http://crispr.hzau.edu.cn/CRISPR2/). The
annealing products of target sense and target antisense oligonucleotides
of sgRNA1 and sgRNA2 were ligated with pYLgRNA-OsU6a and pYLgRNA-OsU6b,
respectively, using specific primers. Both PCR products were combined
and purified using the TaKaRa MiniBEST Purification Kit Ver.4.0. The
expression cassette was pooled, and the restriction ligation reaction
was set up using the pYLCRISPR/Cas9 plasmid. The constructs were
individually transformed into Agrobacterium strain EHA105.