Fig. 1

Construction of the recombinant plasmid for gene knockout. First, using plasmid pSilent-1 as the template, hygromycin resistance gene was amplified by PCR. An approximately 1-kb upstream DNA fragment before the tri3, tri4, and tir11 gene start codon and another 1-kb upstream DNA fragment downstream of the tri3, tri4, and tir11 gene stop codon were amplified to obtain homology L and R, respectively. Second, the above three linearized fragments were equimolarly mixed and cycled in a fusion PCR to generate a gene knockout fragment. Third, the corresponding gene knockout fragments were cloned into the BstXI/XmaI sites of pCAMBIA0380 to generate the corresponding vectors designated as pKT3, pKT4, and pKT11