Abstract: There is the potential for Anabaena variabilis ATCC29413 to be developed into a model strain for studies of heterocyst-forming cyanobacteria that can grow heterotrophically. However, partially due to the restriction-modification systems in cells, the efficiency of gene transfer into this strain is extremely low. To this end, we cloned the two methylase genes, ava_3181 (M. AvaI) and ava_4359 (M. AvrII), of A. variabilis ATCC 29413 and constructed the helper plasmid pHB6088 to protect cargo plasmids from cleavage by restriction enzymes. Taking ava_1237 and ava_4412 as the examples, this study showed that conjugation with this helper plasmid can allow genes to be interrupted in one step through homologous recombination in A. variabilis ATCC 29413.