Figure 3. PCR analysis and phenotypes of mutants.

(A) PCR verification of gene deletion. Lane 1, a 42,725 bp fragment was amplified using primers ZB287/ZB288. The size of the fragment was too large and could not be PCR-amplified, thus no band was seen in lane 1. Lane 2, a 1981 bp fragment was amplified using primers ZB287/ZB288. The CDA biosynthetic gene cluster was replaced with the aac(3)IV gene. Lane3, a 927 bp fragment was amplified using primers ZB287/ZB288, and the resistance gene was removed. Lane 4, a 4073 bp fragment was amplified with primers ZB289/ZB290. Lanes 5 and 6 showed the bands before and after removing the aphII resistance gene. Lane 7, a 928 bp fragment was amplified with primers ZB285/ZB286. Lanes 8 and 9 showed thebands before and after removing the aphII resistance gene. WT, wild type strain; ZB1∼ZB8, serial mutant strains. (B) PCR verification of gene deletion. Lane 1: the fragment was amplified with primers ZB145/ZB146 and primers were both within sco3229. Lane 2: the fragment was amplified with primers ZB195/ZB196 and primerZB195 was within sco5089 and primer ZB196 was within sco5090. Lane 3: the fragment was amplified with primers ZB184/ZB186 and primerZB184 was within sco1103 and primer ZB184 was within sco1104. Lane 4: the fragment as a positive control was amplified with primers ZB147/ZB148 and primers were both within sco5888. M: 150 bp ladder. (C) PCR verification of the resistance removing. Lane 1: M145; Lane 2: ZB1; Lane 3: ZB2. The three fragments were amplified with primers ZB189/ZB190, primers were both within the acc(3)IV gene. Lane 4: M 145; Lane 5: ZB3; Lane 6: ZB4; Lane 7: ZB7; Lane 8: ZB8. The five fragments were amplified with primers ZB187/ZB188, primers were both within the aphII gene. M: 150 bp ladder. (D) Bioassay of CDA extracts from the WT strain and CDA-null mutant strain ZB2. (E) Phenotypes of wild-type and three mutant strains. The spores were cultured for 2 days (left) and 5 days (right) on R2YE agar.