Genomic DNA was extracted from L. bicolor using a FastPure Plant DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd.) to obtain the full-length LbHLH promoter. The reference sequence of the LbHLH promoter was obtained from the L. bicolor genome (unpublished) and the about 2-kb target sequence upstream of the start codon was retained to be considered the promoter sequence for reference. The promoter sequence was cloned using primers LbHLH-P-S and LbHLH-P-A (Table S1). Elements in the promoter were predicted using PlantCARE (, and maps were drawn using CSDS 2.0 (

To replace the 35S promoter with the LbHLH promoter in pCAMBIA3301, HindIII and NcoI were used to excise the CaMV 35S promoter from pCAMBIA3301 and to obtain a linear vector. The promoter was cloned into the vector using primers 3301-LbHLH-P-S and 3301-LbHLH-P-A (Table S1) to add HindIII and NcoI digestion sites in advance. The linear vector pCAMBIA3301 and the inserted LbHLH promoter were ligated together using an In-Fusion HD Cloning Kit (Takara) to construct the recombinant vector.

Agrobacterium tumefaciens GV3101 cells were transformed with the recombinant plasmid and used to infect Arabidopsis thaliana Col-0 to generate Col::pLbHLH-GUS. The transgenic seedlings were continuously screened with herbicides (0.1%, v/v), and homozygous plants of the T3 generation were subjected to histochemical staining. Ten-day-old seedlings were immersed in GUS staining solution and incubated at 37 °C overnight with shaking. The stained plant materials were decolorized by incubating in 70% ethanol 2–3 times and observed under a dissecting microscope (Nikon, Japan)[30].

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