杨树根特异性表达β-果糖苷酶抑制子的功能性验证

doi:10.3969/j.issn.1000-2006.201911062

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6): 169-174.doi: 10.3969/j.issn.1000-2006.201911062

杨树根特异性表达β-果糖苷酶抑制子的功能性验证

苏涛¹(), 周怀烨¹(), 周碧瑶¹, 石婉婷¹, 张琪²

1.南京林业大学,南方现代林业协同创新中心, 南京林业大学生物与环境学院, 江苏南京 210037
2.南京林业大学林学院, 江苏南京 210037

收稿日期:2019-11-29 修回日期:2020-02-28 出版日期:2020-11-30 发布日期:2020-12-07
基金资助:
国家自然科学基金项目(31870589);南京林业大学高层次引进人才科研启动基金项目(GXL2017011);南京林业大学大学生实践创新训练计划项目(2018NFUSPITP044)

The enzyme purification and functional evaluation of a root-expressed invertase inhibitor in poplar

SU Tao¹(), ZHOU Huaiye¹(), ZHOU Biyao¹, SHI Wanting¹, ZHANG Qi²

1. Co-Innovation Center for the Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China
2. College of Forest, Nanjing Forestry University, Nanjing 210037, China

Received:2019-11-29 Revised:2020-02-28 Online:2020-11-30 Published:2020-12-07

摘要/Abstract

摘要：

【目的】植物细胞壁转化酶(CWI)和液泡转化酶(VI)是分配碳水化合物、维持库器官强度和环境胁迫应答的重要因子。大量证据表明CWI和VI由β-果糖苷酶蛋白抑制子的翻译后调控机制介导。然而,关于C/VIFs家族的分子和遗传学基础以及生化特性研究目前还不清楚。【方法】应用生物信息学分析基因结构特征和系统进化关系,并利用实时荧光定量PCR研究转录本在组织中的丰富度。同时,结合烟草叶片瞬时和拟南芥遗传转化法的荧光表达,以及重组蛋白系统分别对PtC/VIF1亚细胞定位特征和体外抑制活性进行深入鉴定。【结果】共39个PtC/VIF候选编码基因家族被筛选出来。PtC/VIF1在根中具有高转录水平;共聚焦显微镜观察证实PtC/VIF1是分泌到细胞外空间。重组蛋白活性检测表明其对CWI蛋白具有特异的亲和抑制活性,说明PtC/VIF1是定位于质外体的β-果糖苷酶抑制子。【结论】对C/VIF编码基因家族生物信息学分析和PtC/VIF1在杨树中的功能性鉴定属于首次报道,可为后期进一步深入探索该基因的生理学作用和参与抗病防御路径以及环境胁迫响应的潜在功能提供理论基础和研究依据。

关键词: 杨树, β-果糖苷酶抑制子;, 翻译后调控, 细胞壁和液泡转化酶, 蔗糖, 质外体, 生物胁迫

Abstract:

【Objective】Cell wall invertase (CWI) and vacuolar invertase (VI) act as the essential regulators in photosynthate distribution, maintenance of sink strength and stress responses. A large body of evidence prompted that the tight control of CWI and VI substantially subjects to the post-translational mechanisms mediated by small proteinaceous inhibitors (C/VIFs, inhibitor of β-fructosidases). However, the survey of the molecular information and gene transcript abundance, as well as biochemical characteristics of C/VIFs is largely unknown in a woody model plant, black cottonwood (Populus trichocarpa). 【Method】In this work, the bioinformatics and qRT-PCR were performed to analyze the phylogenetic tree and gene tissue-specific expression profiling. The transient and stable transformation systems and enzyme assays were conducted to evaluate the subcellular target and inhibitory function of PtC/VIF1. 【Result】A total of 39 genes encoding C/VIF were identified. PtC/VIF1 showing root-specific expression was characterized and its extracellular localization was verified. Evaluation of the recombinant enzyme suggested that PtC/VIF1 showed patterns of the typical apoplastic invertase inhibitor. 【Conclusion】It is the first report on the functional isolation of the C/VIF in poplar. These results pointed out that PtC/VIF1 may play potential roles in defense- and stress-related responses.

Key words: poplar, β-fructosidases invertase inhibitor;, post-translational regulation mechanism, cell wall invertase and vacuolar invertase, sucrose, apoplast, biotic stress

中图分类号:

苏涛,周怀烨,周碧瑶,等. 杨树根特异性表达β-果糖苷酶抑制子的功能性验证[J]. 南京林业大学学报（自然科学版）, 2020, 44(6): 169-174.

SU Tao, ZHOU Huaiye, ZHOU Biyao, SHI Wanting, ZHANG Qi. The enzyme purification and functional evaluation of a root-expressed invertase inhibitor in poplar[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(6): 169-174.DOI: 10.3969/j.issn.1000-2006.201911062.

图/表 3

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参考文献 23

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杨树根特异性表达β-果糖苷酶抑制子的功能性验证

The enzyme purification and functional evaluation of a root-expressed invertase inhibitor in poplar

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