PCR-aided Synthesis and
Stable Expression in E.coli of the cryIA(c)Bt Gene
PENG Ri-He, XIONG Ai-Sheng, LI Xian, FAN Hui-Qin, HUANG
Xiao-Min, YAO Quan-Hong*
( Shanghai Key Laboratory of Agricultural Genetics and Breeding； Agro biotechnology Research
Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China )
Abstract
Insecticidal crystal proteins, known as δ-endotoxins, from a gram-positive bacterium
Bacillus thuringiensis have been used as bio-pesticides for over 3
decades. By using a successive PCR method, the 1.8 kb cryIA(c)Bt gene
coding for the fragment of protoxin was synthesized. Different from B.thuringiensis
subsp.kurstaki Hd 73 cry gene, the synthesized gene has
the codon usage pattern of an average Pseudomonas spp gene. 614
nucleotides were changed in the synthesized cryIA(c)Bt gene and the G＋C content was increased from
37.2% to 64%. The synthesized cryIA(c)Bt gene was cloned into pUT56
vector under the tac promoter and T1-T2 terminator. SDS-PAGE
analysis revealed that 66 kD protein of the modified cryIA(c)Bt gene
was expressed in E.coli and accounted for about 30% of total protein
in the bacterial cells. Bioassays using crude expression products from host
strains indicated that they had high toxicity to third instar larvae of the
cabbage butterfly (Pieris brassicae). The LD50 was
calculated to be0.024 μg/cm2.
Key words chemical synthesis； cryIA(c)Bt gene； gene stable expression； insecticide activity
*Corresponding author： Tel, 86-21-62208660-3207； Fax, 86-21-62209988； e-mail, [email protected]