摘要
分别构建含有GBNBS基因全长序列以及去除富含亮氨酸重复区(Leucine-rich repeat,LRR)的部分序列的原核表达载体P3161和NBCD,SDS-PAGE凝胶电泳检测发现在IPTG诱导下,重组载体P3161和NBCD在BL21菌株中可以分别表达90 kDa和60 kDa的目的蛋白,Western blot证明它们为目的基因表达产物。分离纯化这两种蛋白并分析酶活功能,发现GBNBS全长蛋白具有ATPase活性,而去除LRR的缺失蛋白的ATPase活性则大幅降低。GBNBS基因的原核表达以及表达产物的活性研究为进一步了解该基因抗性功能与抗性机制奠定基础。
GBNBS is a NBS-LRR gene derived from Gossypium barbadense and the full length of its open reading flame comprises 2584 bp and encodes a deduced protein of 861 amino acid residues with molecular weight of 93 kDa. In order to characterize the function of GBNBS, the fused prokaryotic expression vector P3161 containing the full GBNBS gene and vector NBCD containing partial GBNBS gene without the Leucine-Rich Repeat were constructed respectively. SDS-PAGE analysis showed 90 kDa and 60 kDa fusion protein was expressed by P3161 and NBCD in E.coli BL21 after IPTG induction, respectively. Western blot further verified the fusion proteins were object proteins coded by vector P3161 and NBCD, respectively. The object proteins were further isolated and purified for ATPase activity analysis, which illustrated the full protein has the ATP hydrolysis activity while the partial protein deleting the LRR region dramatically lost the ATP hydrolysis activity. This study laid a basis for understanding the resistant function and mechanism of GBNBS gene.
出处
《棉花学报》
CSCD
北大核心
2012年第6期524-528,共5页
Cotton Science
基金
转基因生物新品种培育重大专项"转基因抗黄萎病棉花新种质研制"(2009ZX08005-003B)
关键词
棉花
NBS—LRR
原核表达
ATPASE
island cotton（Gossypium barbadense）
NBS-LRR
prokaryotic expression
ATPase
