毛竹PePGIP基因的克隆及表达分析

Cloning and Expression Analysis of Pe PGIP from Phyllostachys edulis

多聚半乳糖醛酸酶抑制蛋白是一种重要的植物防御蛋白,在植物对真菌防御中发挥着重要作用。利用同源序列比对方法从毛竹(Phyllostachys edulis)全长c DNA文库中获得1个PGIP同源基因序列(FP100022),命名为Pe PGIP。该基因序列全长1370 bp,开放阅读框1008 bp,编码335个氨基酸,分子量为36.0 k Da。蛋白序列分析表明,该序列包含8个典型的亮氨酸重复序列,属于植物胞外蛋白e LRR超家族。Pe PGIP与其他禾本科植物的PGIP均具有较高的同源性,聚类分析与来自二穗短柄草和水稻的PGIP聚在较近的分支。半定量RT-PCR分析结果表明,Pe PGIP在毛竹的根、茎、叶中均表达,且根中表达量最高。构建p EASY-Pe PGIP原核表达载体,转化大肠杆菌BL21(DE3),经IPTG诱导获得了重组蛋白,分子量约为42 k Da。本研究为深入了解Pe PGIP的基因功能奠定了基础。

Polygalacturonase inhibiting protein（ PGIP） is one of important plant defense proteins,which plays an important role in the defense of fungi. Based on the sequence alignment method,a homologous gene of PGIP was obtained from the full length c DNA database of Phyllostachys edulis and named as Pe PGIP（ FP100022）. The full length of c DNA of Pe PGIP is 1370 bp including an open reading frame（ ORF） of 1008 bp,which encodes a predicted protein of 335 amino acids with a calculated molecular weight of 36. 0 k Da. Sequence analysis showed that there were 8 conserved leucine-rich repeats,indicating that it belongs to the e LRR superfamily. Pe PGIP had higher homology as PGIPs from other gramineous plants did,which was clustered in the clade with those of Brachypodium distachyum and Oryza sativa. RT-PCR analysis showed that Pe PGIP was expressed in roots,culms and leaves of P.edulis,with the highest level in roots. The prokaryotic expression vector plasmid of p EASY-Pe PGIP was constructed and then transformed into Escherichia coli strain BL21（ DE3）. The analysis of SDS-PAGE showed that the recombinant protein was around 42 k Da after being induced by IPTG. This study lays a foundation for an in-depth understanding of Pe PGIP function.