To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. Thes...To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.展开更多
Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium f...Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium for genetic transformation was successfully achieved for well known date palm （Phoenix dactylifera L.） cv. Medjool and Khalas using callus as explant. Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction （PCR） was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene （forward and reverse）, a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.展开更多
文摘To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.
文摘Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium for genetic transformation was successfully achieved for well known date palm （Phoenix dactylifera L.） cv. Medjool and Khalas using callus as explant. Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction （PCR） was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene （forward and reverse）, a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.
文摘苏云金芽胞杆菌(Bacillus thuringiensis,Bt)产生的Cry3Aa毒素在许多鞘翅目害虫的防治中发挥了重要作用。随着昆虫抗性的产生,如何通过大肠杆菌高通量表达筛选cry3Aa基因改造突变蛋白成为了研究的重点。然而,cry3Aa基因在大肠杆菌BL21(DE3)中诱导表达后其蛋白酶活化效果及杀虫活性均受到一定程度影响,对于影响的程度仍知之甚少。为探索cry3Aa基因所表达的融合蛋白GST-Cry3Aa中GST标签对Cry蛋白结构和功能的影响,本研究通过利用丝氨酸类蛋白酶(胰蛋白酶,糜蛋白酶)对纯化后的Cry3Aa融合蛋白进行水解条件研究分析,结果表明:在温度为11~37℃、GST-Cry3Aa与胰蛋白酶的质量比为4:1~30:1时,均可被水解出约55 k D的活性片段,而在同样条件下糜蛋白酶则无法对GST-Cry3Aa进行有效的水解活化。此外,结合I-TASSER和Swiss model对Cry3Aa与GST-Cry3Aa进行三维结构分析比对,发现GST-Cry3Aa在其结构域Ⅰ内有部分改变,推测其可能为两种丝氨酸类蛋白酶对该融合蛋白水解效果产生差异的原因。为后期构建Cry3Aa毒素高通量分子改造平台提供了理论基础。
