The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis
Abstract
:1. Introduction
2. Results
2.1. Genotyping for cry2 Gene
2.2. Sequencing of the cry2ac7 Gene
2.3. Hyperexpression of cry2ac7 Gene in Escherichia coli (BL21 Codon Plus)
2.4. Partial Purification of Cry2Ac7 Inclusion Bodies
2.5. Toxicity Assay of Cry2Ac7 Toxin against Helicoverpa armigera
3. Discussion
3.1. Profile of Cry2A Subtypes and Toxicity
3.2. Sequence Homology of Cry2Ac7 Protein with Other Related Proteins
4. Materials and Methods
4.1. Sampling and Isolation of Bacillus thuringiensis Strains
4.2. Ribotyping
4.3. DNA Isolation and Genotyping
4.4. Cloning and Sequencing of the cry2ac7 Gene
- pET21a-N 5′ GCTCGGTACCTCGCTGAATGCATCTAGCATATGAATAC 3′
- M13 R 5′-d(GAGCGGATAACAATTTCACACAGG)-3′)
4.5. Sub Cloning in Expression Vector pET22b
4.6. Expression of cry2ac7 Gene in Escherichia coli (BL21C+)
4.7. Purification of Cry2Ac7
4.8. Toxicity Assay of Cloned Toxin against H. armigera and M. domestica
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Conflicts of Interest
References
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y2Ac5 | MNNVLNSGRNTTCHAHNVVAHDPFSFEHKSLNTIEKEWKEWRRTDHSLYVAPIVGTVGSF |
Cry2Ac7 | MNTVLNNGRNTTCHAHNVVAHDPFSFEHKSLNTIEKEWKEWKRTDHSLYVAPIVGTVGSF |
Cry2Ac1 | MNTVLNNGRNTTCHAHNVVAHDPFSFEHKSLNTIEKEWKEWKRTDHSLYVAPIVGTVGSF |
**.***.**********************************:****************** | |
Cry2Ac5 | LLKKVGSLVGKRILSELQNLIFPSGSIDLMQEILRATEQFINQRLNADTLGRVNAELAGL |
Cry2Ac7 | LLKKVGSLVGKRILSELQNLIFPGGSIDLMQEILRATEQFINQRLNADTLGRVNAELAGL |
Cry2Ac1 | LLKKVGSLVGKRILSELQNLIFPSGSIDLMQEILRATEQFINQRLNADTLGRVNAELAGL |
***********************.************************************ | |
Cry2Ac5 | QANVAEFNRQVDNFLNLNQNPVPLAIIDSVNTLQQLFLSRLPQFQIQGYQLLLLPLFAQA |
Cry2Ac7 | QANVAEFNRQVDNFLNPNQNPVPLAIIDSVNTLQQLFLSRLPQFQIQGYQLLLLPLFAQA |
Cry2Ac1 | QANVAEFNRQVDNFLNPNQNPVPLAIIDSVNTLQQLFLSRLPQFQIQGYQLLLLPLFAQA |
**************** ******************************************* | |
Cry2Ac5 | ANLHLSFIRDVILNADEWGISAATVRTYRDHLRNFTRDYSNYCINTYQTAFRGLNTRLHD |
Cry2Ac7 | ANLHLSFIRDVILNADEWGISAATVRTYRDHLRNFTRDYSNYCINTYQTAFRGLNTRLHD |
Cry2Ac1 | ANFNLSFIRGVILNADEWGISAATVRTYRDHLRKFHRDYSNYCINPYQTAFRGLNHRLPD |
**::*****.***********************:* *********.********* ** * | |
Cry2Ac5 | MLEFRTYMFLNVFEYVSIWSLFKYQSLLVSSGANLYASGSGPTQSFTAQNWPFLYSLFQV |
Cry2Ac7 | MLEFRTYMFLNVFEYVSIWSLFKYQSLLVSSGANLYASGSGPTQSFTAQNWPFLYSLFQV |
Cry2Ac1 | MLEFRTYMFLNVFEYVSIWSLFKYQSLLVSSGANLYASGSGPTQSFTAQNWPFLYSLFQV |
************************************************************ | |
Cry2Ac5 | NSNYVLNGLSGARTTITFPNIGGLPGSTTTQTLHFARINYRGGVSSSRIGQANLNQNFNI |
Cry2Ac7 | NSNYVLNGLSGARTTITFPNIGGLPGSTTTQTLHFARINYRGGVSSSRIGQANLNQNFNI |
Cry2Ac1 | NSNYVLNGLSGARTTITFPNIGGLP-VYHNSTLHFARINYRGGVSSSRIGQANLNQNFNI |
************************* ..***************************** | |
Cry2Ac5 | STLFNPLQTPFIRSWLDSGTDREGVATSTNWQSGAFETTLLRFSIFSARGNSNFFPDYFI |
Cry2Ac7 | STLFNPLQTPFIRSWLDSGTDREGVATSTNWQSGAFETTLLRFSIFSARGNSNFFPDYFI |
Cry2Ac1 | STLFNPLQTPFIRSWLDSGTDREGVATSTNWQSGAFETTLLRFSIFSARGNSNFFPDYFI |
************************************************************ | |
Cry2Ac5 | RNISGVVGTISNADLARPLHFNEIRDIGTTAVASLVTVHNRKNNIYDTHENGTMIHLAPN |
Cry2Ac7 | RNISGVVGTISNADLARPLHFNEIRDIGTTAVASLVTVHNRKNNIYDTHENGTMIHLAPN |
Cry2Ac1 | RNISGVVGTISNADLARPLHFNEIRDIGTTAVASLVTVHNRKNNIYDTHENGTMIHLAPN |
************************************************************ | |
Cry2Ac5 | DYTGFTVSPIHATQVNNQIRTFISEKYGNQGDSLRFELSNTTARHTLRGNGNSYNLYLRV |
Cry2Ac7 | DYTGFTVSPIHATQVNNQIRTFISEKYGNQGDSLRFELSNTTARYTLRGNGNSYNLYLRV |
Cry2Ac1 | DYTGFTVSPIHATQVNNQIRTFISEKYGNQGDSLRFELSNPTARYTLRGNGNSYNLYLRV |
****************************************.***:*************** | |
Cry2Ac5 | SSIGSSTIRVTINGRVYTANVNTTTNNDGVLDNGARFSDINIGNVVASANTNVPLDIQVT |
Cry2Ac7 | SSIGSSTIRVTINGRVYTANVNTTTNNDGVLDNGARFSDINIGNVVASANTNVPLDIQVT |
Cry2Ac1 | SSIGSSTIRVTINGRVYTANVNTTTNNDGVLDNGARFSDINIGNVVASANTNVPLDIQVT |
************************************************************ | |
Cry2Ac5 | FNGNPQFELMNIMFVPTNIPPLY |
Cry2Ac7 | FNGNPQFELMNIMFVPTNLPPLY |
Cry2Ac1 | FNGNPQFELMNIMFVPTNLPPLY |
******************:**** |
Amino Acid | Cry2Ac1 | Cry2Ac5 | Cry2Ac7 |
---|---|---|---|
3 | Asn | Thr | |
7 | Ser | Asn | |
42 | Arg | Lys | |
84 | Ser | Ser | Gly |
137 | Leu | Pro | |
183 | Phe | Leu | |
184 | Asn | His | |
190 | Gly | Asp | |
214 | Lys | Asn | |
216 | His | Thr | |
226 | Pro | Thr | |
236 | His | Thr | |
239 | Pro | His | |
326 | Insertion | Gly | |
327 | Val | Ser | |
328 | Tyr | Thr | |
329 | His | Thr | |
330 | Asn | Thr | |
331 | Ser | Gln | |
521 | Pro | Thr | |
525 | His | Tyr | |
619 | Ile | Leu |
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Saleem, F.; Shakoori, A.R. The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis. Toxins 2017, 9, 358. https://doi.org/10.3390/toxins9110358
Saleem F, Shakoori AR. The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis. Toxins. 2017; 9(11):358. https://doi.org/10.3390/toxins9110358
Chicago/Turabian StyleSaleem, Faiza, and Abdul Rauf Shakoori. 2017. "The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis" Toxins 9, no. 11: 358. https://doi.org/10.3390/toxins9110358
APA StyleSaleem, F., & Shakoori, A. R. (2017). The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis. Toxins, 9(11), 358. https://doi.org/10.3390/toxins9110358