水稻内源木聚糖酶osx基因的克隆及表达分析

Cloning and Expression Analysis of Endoxylanase Gene osx from Oryza sativa

为进一步了解植物内源木聚糖酶基因结构及其表达特性,从水稻中克隆和表达了一种内源木聚糖酶基因。通过序列比对分析确定保守区序列,以PCR扩增得到的保守区序列从众多水稻木聚糖酶预测序列中"钓取"可能的目标基因,并以此基因为模板设计引物,通过RT-PCR扩增得到一条长度为1 443 bp的基因片段osx,测序结果与水稻木聚糖酶基因预测序列NM_001061680一致性高达99%,该序列编码480个氨基酸,经预测不存在信号肽序列。构建表达载体pET28-a(+)-osx,导入大肠杆菌BL21进行IPTG诱导表达,该表达产物未检测到木聚糖酶活性。受大麦和玉米内源木聚糖酶基因表达的前体为无活性蛋白需要剪切加工的启示,进一步通过在线蛋白同源建模分析,设计引物去掉蛋白N端约120个氨基酸,C端约40个氨基酸,保留"Glyco-hydro-10"结构域,使其成功实现了原核活性表达,表达蛋白约40 kDa,木聚糖酶活性为11.53 IU/mL。

X＇ylanase is a key enzyme in the degradation of xylan, but study on the endogenous xylanase of plant is limited. To further understand the gene structure and expression characteristics of the endogenous xylanase of plant, an endogenous xylanase gene from rice was first cloned and expressioned. Frist, conservative region was found out according to the blast analysis between the predictive xylanase sequences from rice and the known xylanase sequences from other plants. Then, using the conservative sequence to fish the target gene from the rice gene database, and designing the primers based on the target gene （NM_001061680） to clone the endogenous xylanase gene from rice, a 1 443 bp sequence （osx） was obtained with a identity up to 99% to target gene, and was encoded 480 amino acids without a signal peptide. A prokaryotic expression vector pET28-a（ ＋ ）-osx was constructed, and was introduced into E. coli BL21 for induced expressing by IPTG. Expression product was analyzed by SDS-PAGE electrophoresis, a specific band about 56 kDa was obtained; but no xylanase activity was detected. So, enzyme activity reformation of rice xylanase was performed, based on analyzation of the 3D structure of expression protein by online homology modeling. Primers were designed to remove the N- and C-end of the inactive protein about 120aa, 40aa, respectively, and performed another prokaryotic expression strategy for the truncated xylanase gene, which only contain the ＂ Glyco-hydro-10＂ structural domain. Both SDS-PAGE electrophoresis and western-blotting confirmed that the truncated protein could he successfully expressed in E. coli, which molecule weight was about 40 kDa, and the xylanase enzyme activity was detected, up to 11.53 IU/ml.