作物学报Issue(9)：1361-1371,11.DOI:10.3724/SP.J.1006.2015.01361
桑树多聚半乳糖醛酸酶抑制蛋白基因MaPGIP1的克隆及功能分析
Molecular Cloning and Functional Analysis of Polygalacturonase-Inhibiting Protein GeneMaPGIP1 from Mulberry (Morus atropurpureaRoxb.)
摘要
Abstract
Polygalacturonase-inhibiting protein (PGIP) is a defense protein found in plant cell wall. It is involved in plant defense against infection of pathogens by modulating/inhibiting the activity of endo-polygalacturonase. In this test, a pair of specific primers were designed based on PGIP genes of mulberry (Morus notabilis) in Morus Genome Database. The cDNA of Jialing 40 PGIPgene was amplified from fruit by RT-PCR. The sequences of mulberry PGIP, physic-chemical parameters of PGIP protein and phylogenetic relationship were analyzed by bioinformatics softwares. Using pET-28a(+) as a fused expression vector, a re-combinant plasmid pET28a-PGIPcontaining the mature peptide of PGIPwas constructed. Then its expression was induced in Escherichia coliBL21 (DE3) with IPTG. The samples induced at different times were collected and SDS-PAGE was used to ana-lyze the protein expression inE. coliBL21 (DE3). After purification of the protein by Ni-Hind affinity column and Western blot, the PGIPgene expressed inE. coliBL21 (DE3). Finally its enzymatic activity was tested by bacteriostatic experiment. The full-length cDNA ofPGIPfrom Jialing 40 fruitwas obtained. Sequence analysis showed that the fragment contains an open read-ing frame of 1017 bp encoding 338 amino acid residues with a molecular mass of 37.9 kD, namedMaPGIP1. This deduced pro-tein has a pI of 6.65, a hydrophobic region of 26 amino acid residues in the N-terminal which was considered to be a signal pep-tide, with four potential N-glycosylation sites, and it center LRR structural domain is composed of nine tandem LRR motifs. Phy-logenetic tree showed that Jialing 40 had the closest evolutionary relationship withM. notabilis. The prokaryotic expression re-sults showed that efficient expression of PGIP protein could be realized after induction with 0.5 mmol L–1 IPTG inE. coli BL21 (DE3) for five hours at 28 °C. The SDS-PAGE displayed that the recombinant proteins only appeared as inclusion bodies. The inclusion bodies protein was purified by Ni-NTP affinity column and confirmed by Western blot. Soluble product could be re-folded though stepwise dialysis strategies. The recombinant protein concentration was 0.58μgμL–1tested by Bradford method. MaPGIP1 partially inhibited CsPG with an optimum pH between 4.5 and 5.0, and an optimum temperature of 30°C. The prelimi-nary infection experiment result showed that MaPGIP1 protein after renaturation had a certain inhibiting effect on Hypertrophy Sorosis Sclerotenisis infected byCiboria shiraiana.关键词
桑树/多聚半乳糖醛酸酶抑制蛋白/序列分析/原核表达/蛋白活性Key words
Mulberry/PGIP/Sequence analysis/Prokaryotic expression/Protein activity引用本文复制引用
王晓红,朱攀攀,梁燕梅,韩淑梅,赵爱春,王传宏,鲁成,余茂德..桑树多聚半乳糖醛酸酶抑制蛋白基因MaPGIP1的克隆及功能分析[J].作物学报,2015,(9):1361-1371,11.基金项目
本研究由国家农业部公益性行业(农业)科研专项(201403064),国家自然科学基金项目(31360190)和国家现代农业产业技术体系建设专项(CARS-22)资助。 （农业）
