新疆黄精凝集素基因的克隆、序列分析和表达  

Cloning,Sequencing Analysis and Expression of a Putative Mannose-binding Lectin Gene from Polygonatum roseum in Xinjiang

作　　者：孙素荣[1] 张智[1] 李素丽[1] 胡俊[1] 张富春[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《生物工程学报》2008年第3期387-394,共8页Chinese Journal of Biotechnology

基　　金：新疆高校科研计划重点项目(No.XJEDU2004I10);新疆高校创新研究群体基金(No.XJEDU2004G02)~~

摘　　要：用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明,克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin,PRA)基因完整的ORF片段大小为550 bp,编码1条长159个氨基酸肽链,没有内含子,其中N端的28个氨基酸是信号肽。对新疆黄精凝集素cDNA序列同源性的分析比较发现,黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL同源模建分析表明,PRA由12个β-折叠片组成的β-桶结构,具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA和pMAL-p2x-PRA,分别转化E.coli BL21进行原核表达,新疆黄精凝集素能够以可溶性融合蛋白形式表达,分子量约为14kD。构建真核表达载体pcDNA3-PRA,免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带,证明该抗血清具有针对PRA抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备,为进一步研究凝集素蛋白的性质和功能,并为植物抗病虫基因工程研究提供有用的实验材料。The genomic DNA were extracted from the leaves of Polygonatum roseum （Liliaceae） in Xinjiang and the primers were designed according to conservative sequences of Polygonatum lectins gene. The complete ORF of Polygonatum roseum agglufinin （PRA） gene was amplified as a fragment of 550 bp, which was identical with predicted size. Like most of the plant lectin genes, there was no intron in the PRA gene. The ORF of the gene encoded 159 amino acid residues, in which included a signal sequence of 28 amino acid residues at its N-terminus. The cDNA sequence had 92% identifies compared with the published sequence. The amino acid sequence and SWISS-MODEL analysis indicated that the three-dimensional structure of PRA strongly resembled with that of monocot mannose-binding lecfins, which comprised with three anfiparallel four-stranded β-sheets arranged as a 12-stranded β-barrel. The recombinant pGEX4T-1-PRA and pMAL-p2x-PRA prokaryotic expression vectors were constructed to produce GST-PRA and MBP-PRA fusion proteins in E. coli, respectively. SDS-PAGE of the fusion protein demonstrated that the PRA lectin protein migrated at a size of 14 kD. The immunization was performed by intra-muscular injection of pcDNA3-PRA, and the antiserum was detected by ELISA. Western blotting analysis showed the antiserum specifically bound the lectin protein. The establishment of such an expression system might provide materials for further investigation of the properties and functions of PRA proteins. It also laid the basis for plant genetic engineering on its defensive functions to pests and diseases.

关 键 词：新疆黄精 凝集素 序列比较 原核表达 

分 类 号：Q943.2[生物学—植物学]