水稻Os4CL5的Val337缺失突变激活其芥子酸催化活性  

Deletion of a Single Amino Acid Residue Val337 from Rice(Oryza sativa) Os4CL5 Results in the Generation of New Substrate Specificities Toward Sinapate

作　　者：孙海燕[1,2] 罗兵[1,2] 赵敏欢 李朋朋[1] 杨志刚[1,2] 

机构地区：[1]常熟理工学院生物与食品工程学院,常熟215500 [2]国家杂交水稻工程技术研究中心常熟分中心,常熟215500

出　　处：《农业生物技术学报》2016年第9期1312-1318,共7页Journal of Agricultural Biotechnology

基　　金：江苏省青年基金项目(No.BK20140417);苏州市科技计划项目(SYN201514)

摘　　要：4-香豆酸辅酶A连接酶(4-coumarate:coenzyme A ligase,4CL)催化各种羟基肉桂酸生成相应的硫酯,从而调控木质素和黄酮类物质的生成。4CL的芥子酸催化活性一直是备受关注的一个问题,目前只有拟南芥(Arabidopsis thaliana)的At4CL4和大豆(Glycine max)的Gm4CL1具有芥子酸转化能力。本研究用水稻(Oryza sativa subsp.japonica)苗期的叶片为材料,提取总RNA并以此为模板,用反转录PCR扩增水稻的Os4CL5基因,并将其连接到原核表达载体p ET22-b(+)上,构建了原核表达载体p ET-4CL5(+Val)。通过定点突变的方法删除了水稻Os4CL5的Val337,构建了原核表达载体p ET-4CL5(-Val)。重组质粒转化大肠杆菌(Escherichia coli)BL21(ED3)并经异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)诱导表达,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测表明,融合蛋白的分子量为57.0 k D,与预测值一致。His-Spin Trap蛋白纯化系统纯化回收重组蛋白,然后对重组蛋白的酶学性质进行表征。结果表明,野生型Os4CL5对不同底物表现出了不同的催化效率,香豆酸是最适底物,其次是阿魏酸,再次是咖啡酸,对芥子酸没有活性。与野生型Os4CL5相比,突变型Os4CL5对香豆酸、咖啡酸和阿魏酸的催化效率有所提高,并且其底物特异性发生了明显改变,获得了对芥子酸的催化活性。本研究为利用基因工程手段调控木质素的生物合成提供了理论依据。4-coumarate： coenzyme A ligase （4CL） is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. The catalytic activity of 4CL towards sinapate got a lot of attention at present. Only Arabidopsis thaliana At4CL4 and soybean （Glycine max） Gm4CL1 have catalytic ability toward sinapate so far. There is a conserved valine which is located in the substrate binding pocket by sequence comparison of rice （Oryza sativa subsp, japonica） Os4CL5 with At4CL4 and Gm4CL1. The existence of valine between Pro336 and Leu338 may eliminate the activity of Os4CL5 towards sinapate. In this paper, the expression vectors of wild-type Os4CL5 and mutant Os4CL5 with the deletion of Va1337 were constructed. Then the recombinant plasmids were transformed into Escherichia coli BL21（DE3） respectively. The recombinant cells were grown and induced by isopropyl-[3-d-thiogalactoside （IPTG）. Sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） revealed that the molecular mass of the induced protein was about 57.0 kD consistent with the predicted value. After purification by affinity chromatography, the enzymatic properties of wild-type Os4CL5 and mutant Os4CL5 were primarily characterized. The results showed that wild-type Os4CL5 had different enzymatic activity towards the different substrate. The rank in order of turnover rate for different substrate was coumarate〉ferulate〉 caffeate. It should be noted that sinapate was not accepted as a substrate under experimental conditions. The deletion of Va1337 from rice Os4CL5 results in increased activity towards different substrate and the generation of new substrate specificities towards sinapate. This study provides a theoretical basis for the regulation of lignin biosynthesis by genetic engineering.

关 键 词：水稻 4-香豆酸:辅酶A连接酶 原核表达 酶活性 

分 类 号：Q71[生物学—分子生物学]