Rapid cloning, identification, and application of one novel crystal protein gene cry30Fa1 from Bacillus thuringiensis.
In this study, a fast and efficient strategy has been developed for identifying and isolating novel cry genes from Bacillus thuringiensis by combining the PCR-restriction fragment length polymorphism and the single-oligonucleotide nested-PCR method. Using this method, one novel holotype cry gene, cry30Fa1, encoding a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa1, was cloned from the B. thuringiensis strain BtMC28. Furthermore, the cry30Fa1 gene was successfully expressed in Escherichia coli BL21 (DE3). The Cry30Fa1 proteins, isolated from the cultures of recombinant E. coli, had remarkable insecticidal effects against Plutella xylostella and Aedes aegypti with LC50 at 6.477 and 15.359 mug mL(-1), respectively. Our results strongly suggest that this strategy is highly efficient and advantageous in terms of rapid cloning of holotype cry genes that have minimal identity to known genes. The cloning of the cry30Fa1 gene would be useful in the resources of the insecticidal crystal genes and may serve as an alternative choice of an insecticide for potential problems associated with insect resistance.
Tan
Furong
F
Biotechnology Research Institute, Shanghai Academy of Agriculture Sciences, Shanghai, China.
Zheng
Aiping
A
Zhu
Jun
J
Wang
Lingxia
L
Li
Shuangcheng
S
Deng
Qiming
Q
Wang
Shiquan
S
Li
Ping
P
Tang
Xueming
X
D000330:Aedes / Q000187:drug effects*; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004731:Endotoxins / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006460:Hemolysin Proteins / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics*; D007300:Insect Control; D007306:Insecticides / Q000008:administration & dosage*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis