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从月季中分离得到的一个富含晚期胚胎蛋白基因 RcLEA,赋予大肠杆菌和拟南芥的耐受性,并在各种胁迫下稳定酶活性。

RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses.

机构信息

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai, 200433, China.

出版信息

Plant Mol Biol. 2014 Jul;85(4-5):333-47. doi: 10.1007/s11103-014-0192-y. Epub 2014 Apr 24.

Abstract

The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers.

摘要

晚期胚胎丰富(LEA)蛋白家族是一个大型蛋白家族,与许多生物体(如植物、细菌和动物)对非生物胁迫的抗性密切相关。在这项研究中,我们从玫瑰中分离出一个细胞质定位的 LEA 基因 RcLEA。通过 RT-PCR 发现 RcLEA 受到高温诱导。与对照相比,在高温、低温、NaCl 和氧化应激条件下,RcLEA 在大肠杆菌中的过表达提高了其生长性能。RcLEA 也在拟南芥中过表达。转基因拟南芥在高温和低温处理后表现出更好的生长,根据 3,3-二氨基联苯胺染色显示过氧化物含量较少。然而,RcLEA 并没有提高拟南芥对 NaCl 或渗透胁迫的耐受性。体外分析表明,RcLEA 能够防止冻融诱导的各种底物(如乳酸脱氢酶和柠檬酸合酶)失活或热诱导聚集。它还可以保护大肠杆菌的蛋白质组在蛋白质受热冲击或处于酸性条件下不发生变性。此外,双分子荧光互补测定表明,RcLEA 蛋白通过形成同源二聚体以复杂的方式发挥作用。

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