Tang Mu-Jin, Yuan Mei-Jin, Chen Jian-Wu, Shi Yong-Xia, Zeng Shao-Ling, Yu Jian-Xiu, Pang Yi
State Key Laboratory for Biocontrol, Zhongshan University, Guangzhou 510275, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):566-71.
The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.
Cry1Ab与其他相关杀虫晶体蛋白（ICPs）的最大差异在于其缺少包含四个半胱氨酸的28个氨基酸的羧基末端；因此它不太稳定。我们报道辅助蛋白P20在Cry1Ab的表达和结晶过程中发挥作用。基于穿梭载体pHT3101构建了三种Cry1Ab表达质粒pT1B、pP1B和pDP1B。载体pT1B不含p20基因，pP1B携带p20，pDP1B含有带有cry1A(c)启动子的p20。通过将质粒电穿孔导入苏云金芽孢杆菌无晶体突变体CryB获得转化体。蛋白质免疫印迹表明，crylAb在所有转化体中均表达为130 kD的蛋白，并且部分蛋白被部分降解为60 kD的肽段。蛋白质定量分析表明，130 kD蛋白的量在转化体中有所不同，PT1B、pP1B和pDP1B的比例分别为1:1.4:1.5。对于60 kD的蛋白，比例为1:1.1:1.6。显微镜检查显示，三个转化体中典型的棱柱状晶体大小顺序为T1B < P1B < DP1B。生物测定表明，T1B、P1B和DP1B对棉铃虫幼虫均有毒性，LC50相似。本研究表明P20在Cry1Ab的表达和结晶过程中发挥作用。
