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

doi:10.3724/SP.J.1006.2015.01361

摘要/Abstract

摘要： 多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种特异性结合和抑制真菌内切多聚半乳糖醛酸酶(endo-PG)活性的细胞壁结合蛋白。采用RT-PCR从嘉陵40 (Morus atropurpurea Roxb.)果实中扩增PGIP基因cDNA, 利用生物信息学的方法分析其编码蛋白的结构和功能。结果表明, 嘉陵40PGIP开放阅读框全长1017 bp, 编码338个氨基酸残基, 被命名为MaPGIP1。MaPGIP1蛋白分子量37.9 kD, 等电点为为6.65, 信号肽为N端26个氨基酸残基, 具有4个潜在的N-糖基化位点。MaPGIP1蛋白的核心区域由9个串联的LRRs基序组成。原核表达产物经SDS-PAGE分析, MaPGIP1蛋白以包涵体形式出现, Western blot证实了重组蛋白的特异性, 经过Ni-NTA柱纯化和分步透析复性后获得可溶性蛋白, 该蛋白能部分抑制果桑肥大性菌核病菌(Ciboria shiraiana) PG (CsPG)活性, 其最适pH值为4.5~5.0, 最适温度30℃。抑菌试验结果表明, MaPGIP1蛋白在果桑肥大性菌核病菌菌丝侵染油菜叶片过程中具有一定的抑制效果。

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

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 expression of PGIP protein could be realized after induction with 0.5 mmol L^-1 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^-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 preliminary infection experiment result showed that MaPGIP1 protein after renaturation had a certain inhibiting effect on Hypertrophy Sorosis Sclerotenisis infected by Ciboria shiraiana.

Key words: Mulberry, PGIP, Sequence analysis, Prokaryotic expression, Protein activity

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, 41(09): 1361-1371.

WANG Xiao-Hong, ZHU Pan-Pan, LIANG Yan-Mei, HAN Shu-Mei, ZHAO Ai-Chun, WANG Chuan-Hong, LU Cheng, YU Mao-De. Molecular Cloning and Functional Analysis of Polygalacturonase-Inhibiting Protein Gene MaPGIP1 from Mulberry (Morus atropurpurea Roxb.)[J]. Acta Agron Sin, 2015, 41(09): 1361-1371.

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