Molecular Cloning and Functional Analysis of Polygalacturonase-Inhibiting Protein Gene MaPGIP1 from Mulberry( Morus atropurpurea Roxb.)

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 PGIP gene 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 recombinant plasmid pET28a-PGIP containing the mature peptide of PGIP was constructed. Then its expression was induced in Escherichia coli BL21 (DE3) with IPTG. The samples induced at different times were collected and SDS-PAGE was used to analyze the protein expression in E. coli BL21 (DE3). After purification of the protein by Ni-Hind affinity column and Western blot, the PGIP gene expressed in E. coli BL21 (DE3). Finally its enzymatic activity was tested by bacteriostatic experiment. The full-length cDNA of PGIP from Jialing 40 fruit was obtained. Sequence analysis showed that the fragment contains an open reading frame of 1017 bp encoding 338 amino acid residues with a molecular mass of 37.9 kD, named MaPGIP1. This deduced protein 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 peptide, with four potential N-glycosylation sites, and it center LRR structural domain is composed of nine tandem LRR motifs. Phylogenetic tree showed that Jialing 40 had the closest evolutionary relationship with M. notabilis. The prokaryotic expression results showed that efficient 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。 * 通讯作者(Corresponding author): 余茂德, E-mail: yumd@163.com, Tel: 023-68250191 第一作者联系方式: E-mail: swuwxhong@163.com Received(收稿日期): 2015-01-25; Accepted(接受日期): 2015-05-04; Published online(网络出版日期): 2015-06-03. URL: http://www.cnki.net/kcms/detail/11.1809.S.20150603.0901.004.html 2 expression of PGIP protein could be realized after induction with 0.5 mmol L IPTG in E. 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 refolded though stepwise dialysis strategies. The recombinant protein concentration was 0.58 μg μL tested 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 preliminary infection experiment result showed that MaPGIP1 protein after renaturation had a certain inhibiting effect on Hypertrophy Sorosis Sclerotenisis infected by Ciboria shiraiana.