Fig. 3

Construction of Botrytis cinerea transformants with different lengths of cry5Ba3Φ. a Six different lengths of cry5Ba3Φ gene amplified from the plasmid pTFCM-cry5Ba3Φ. 1: cry5Ba3Φ aa 74–698; 2: cry5Ba3Φ aa 115–698; 3: cry5Ba3Φ aa 202–698; 4: cry5Ba3Φ aa 1–572; 5: cry5Ba3Φ aa 1–560; 6: cry5Ba3Φ aa 74–572. b, c Certification of plasmids pTFCM carrying different lengths of cry5Ba3Φ gene. The arrows indicate expected DNA bands amplified from successfully recombined plasmids. d Extraction of recombinant plasmids carrying different lengths of cry5Ba3Φ gene for AGL-1 transformation. 1: plasmid pTFCM-cry5Ba3Φ aa 74–698; 2: pTFCM-cry5Ba3Φ aa 115–698; 3: pTFCM-cry5Ba3Φ aa 202–698; 4: pTFCM-cry5Ba3Φ aa 1–572; 5: pTFCM-cry5Ba3Φ aa 1–560; 6: pTFCM-cry5Ba3Φ aa 74–572. e PCR analysis of AGL-1 transformation confirmed by amplifying the hph gene. 1: AGL-1 pTFCM-cry5Ba3Φ aa 74–698; 2: AGL-1 pTFCM-cry5Ba3Φ aa 115–698; 3: AGL-1 pTFCM-cry5Ba3Φ aa 202–698; 4: AGL-1 pTFCM-cry5Ba3Φ aa 1–572; 5: AGL-1 pTFCM-cry5Ba3Φ aa 1–560; 6: AGL-1 pTFCM-cry5Ba3Φ aa 74–572. 4ʹ, 5ʹ, and 6ʹ: failed AGL-1 transformations with corresponding pTFCM plasmids. f Identification of Botrytis cinerea transformants by amplifying cry5Ba3Φ gene of corresponding lengths. g SDS-PAGE analysis of soluble proteins produced by Botrytis cinerea transformants. Asterisks indicate the protein band of truncated Cry5Ba3Φ. MW: protein molecular weight; M: pre-stained protein marker; C: Botrytis cinerea (pTFCM). In (a–f), M: DNA marker; in f and g, 1: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 74–698); 2: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 115–698); 3: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 202–698); 4: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 1–572); 5: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 1–560); 6: Botrytis cinerea (pTFCM-cry5Ba3Φ aa 74–572)