论文部分内容阅读
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。
In order to further understand the structure and expression characteristics of plant endogenous xylanase genes, an endogenous xylanase gene was cloned and expressed from rice. The sequence of conserved regions was determined by sequence alignment analysis. The conserved region sequences amplified by PCR were selected from the possible target genes of many “fishing” in the predicted sequence of rice xylanase and primers were designed by using this gene as template The gene fragment osx of 1 443 bp in length was amplified by RT-PCR. The sequencing result was 99% identical to the predicted sequence of rice xylanase gene NM_001061680. The sequence encoded 480 amino acids and no signal peptide sequence was predicted. The expression vector pET28-a (+) - osx was constructed and introduced into E. coli BL21 for inducing by IPTG. No xylanase activity was detected in this expression product. The precursors of endogenous xylanase gene expressed by barley and maize are the inspirations that the inactive proteins need to be sheared. By further analyzing the on-line protein homology modeling, primers are designed to remove about 120 amino acids from the N-terminus of the protein, and C-terminal 40 amino acids, retaining the “Glyco-hydro-10” domain. The expressed protein was about 40 kDa and the activity of xylanase was 11.53 IU / mL.