可剪切多拷贝抗菌肽融合表达载体的构建

抗菌肽是生物体防御系统产生的一类对外源病菌具有高效杀灭活性的小分子多肽, 在植物抗病基因工程中具有重要的应用价值。Thanatin是刺肩蝽(Podisus maculiventris)成虫经诱导产生的一种抗菌肽, 由21个氨基酸残基组成, 该抗菌肽对革兰阳性、革兰阴性菌以及真菌都有很强的抗菌活性。为研究该抗菌肽转入油菜对菌核病抗性提高的效果, 采用同尾酶反复酶切连接的方法构建了分别含1~5拷贝的Thanatin串联融合表达载体, 并导入农杆菌用于油菜的遗传转化。研究采用引物重叠法扩增并克隆了抗菌肽基因, 并采用了一种在植物体内可被特异性切割的短肽作为连接肽, 使多拷贝融合表达的抗菌肽在植物体内可自动剪切为有功能活性的单个抗菌肽单元, 以增加抗菌肽表达丰度和抗菌肽的稳定性。研究还采用了大豆几丁质酶的信号肽作为引导肽引导多拷贝融合表达的抗菌肽分泌到细胞间隙, 以增强抗菌肽作用效果。

Construction of vectors for expression of cleavable tandem repeat Thanatin fusion protein in plants

Thanatin,a 21-residue antimicrobial peptide,is an inducible insect peptide with a broad range of activity against bacteria and fungi. It can improve the expression level of the antimicrobial peptide in plants to express the peptide as tan-dem repeat fusion protein containing multiple Thanatin copies. However,the fusion protein has no antimicrobial activity. To make the fusion protein automatically break into single Thanatin unit with antimicrobial activity,the fused Thanatin was spaced by a linker peptide,which was cleavable in vivo. The soybean(Glysine max L.) chitinase signal peptide was fused to the N end of the fusion protein to induce the antimicrobial peptide accumulated in intercellular space. To construct the vec-tors for expression of the fusion protein,the overlapped primers were used to clone the antimicrobial peptide gene and the co-adhesive end restriction and ligation strategy was used to add the repeat unit one by one. Vectors containing 1 to 5 repeat units of Thanatin were constructed respectively. These vectors were being used to transform plants to improv plant disease resistance.