| 果桑肥大性菌核病菌多聚半乳糖醛酸酶基因(CsPG1)的克隆及功能分析 |
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| 引用本文： | 李孟娇,吕蕊花,余建,蔡雨翔,王传宏,赵爱春,鲁成,余茂德.果桑肥大性菌核病菌多聚半乳糖醛酸酶基因(CsPG1)的克隆及功能分析[J].作物学报,2016,42(2):199-200. |
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| 作者姓名： | 李孟娇 吕蕊花 余建 蔡雨翔 王传宏 赵爱春 鲁成 余茂德 |
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| 作者单位： | 西南大学生物技术学院，重庆 400716 |
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| 基金项目： | 本研究由国家农业部公益性行业(农业)科研专项经费项目(201403064), 国家自然科学基金项目(31360190), 国家现代农业产业技术体系建设专项(CARS-22)资助。 |
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| 摘 要： | 多聚半乳糖醛酸酶(polygalacturonase,PG)是一种细胞壁结构蛋白,可以催化果胶分子多聚α-(1,4)-聚半乳糖醛酸的裂解,使细胞壁结构解体,导致果实软化。本文采用q RT-PCR方法,分析不同生长阶段菌丝侵染的油菜叶片中多聚半乳糖醛酸酶基因的表达模式。结果表明:(1)采用RT-PCR从果桑肥大性菌核病菌(Ciboria shiraiana)菌核中扩增多聚半乳糖醛酸酶基因c DNA,其全长为1143 bp,命名为CsPG1(Gen Bank登录号为KR296662),编码380个氨基酸残基。根据侵染油菜叶片的表达量,确认CsPG1为基因家族的主效基因;(2)将CsPG1基因连接p ET28a(+)载体并转化大肠杆菌E.coli BL21(DE3),获得包涵体形式,但对底物(聚半乳糖醛酸)没有活性的蛋白;(3)最适CsPG1包涵体溶解的尿素浓度6 mol L~(–1),采用缓慢稀释低温复性的方法对重组蛋白CsPG1进行了重折叠复性试验,经高亲和力的Ni-NTA树脂纯化后为得到单一条带,获得可溶性蛋白,复性后的多聚半乳糖醛酸酶比活力为5.02 U mg–1;(4)生物试验表明,CsPG1蛋白能够加速嘉陵40(Morus atropurpurea Roxb.)桑椹的成熟和软化,推测该蛋白与肥大性菌核病菌对桑椹的侵染有关。这一结果揭示了不同果桑品种对菌核病敏感性的差异,为果桑生产中菌核病的防控提供了分子证据。
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| 关 键 词： | 果桑 肥大性菌核病菌 CsPG1 蛋白表达活性 |
| 收稿时间： | 2015-06-11 |
Cloning and Functional Analysis of Polygalacturonase Genes from Ciboria shiraiana |
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| LI Meng-Jiao,Lü Rui-Hua,YU Jian,CAI Yu-Xiang,WANG Chuan-Hong,ZHAO Ai-Chun,LU Cheng,YU Mao-De.Cloning and Functional Analysis of Polygalacturonase Genes from Ciboria shiraiana[J].Acta Agronomica Sinica,2016,42(2):199-200. |
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| Authors: | LI Meng-Jiao Lü Rui-Hua YU Jian CAI Yu-Xiang WANG Chuan-Hong ZHAO Ai-Chun LU Cheng YU Mao-De |
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| Institution: | College of Biotechnology, Southwest University, Chongqing 400715, China |
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| Abstract: | Polygalactuionase (PG) is a kind of cell wall structural proteins that can catalyes the decompocition of alpha- (1,4)-polymer of galacturonia acid , which makes the cell wall structure disintegration and fruit softening. In the paper, we analyzed the expression patterns of PG genes in the leaf infection by the hypha of C. shiraiana at different growth stages by using qRT-PCR methods. The results showed that the cDNA of CsPG1 gene was amplified from the sclerotia of C. shiraiana by RT-PCR, namely CsPG1 (GenBank accession number: KR296662) with 1143 bp of full length, encoding 380 amino acid residues. On the basis of the highest level of gene expression in the process of infecting rape leaf, the main gene of the CsPG gene family was confirmed. CsPG1 was cloned into pET-28a(+) vector and expressed in E. coli BL21 (DE3). The recombinant CsPG1 protein was expressed in the form of inclusion bodies without activity towards polygalaturonic acid. The optimal urea concentration for dissolving CsPG1 inclusion bodies was 6 mol L–1, CsPG1was renaturated by dilution gradiently at low temperature. We sorted out single band after purified by High-Affinity Ni-NTA Resin, and obtained soluble protein. The specific activity of renatured CsPG1 was 5.02 U mg–1. The result of biological test showed that the recombinant protein accelerated the fruit maturity and softening of Jialing 40 (Morus atropurpurea Roxb.). So we speculated that the CsPG1 protein is related to the infection of C. shiraiana to mulberry fruits. The result reveals the sensitivity difference among mulberry varieties to sclerotial disease, providing the molecular evidence for preventing and controlling the sclerotial disease in mulberry cultivation. |
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| Keywords: | Mulberry Ciboria shiraiana CsPG1 Protein expression activity |
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