Sharma Shruti, Gupta Ravi, Deswal Renu
Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, India.
Department of Plant Bioscience, Pusan National University, Miryang, South Korea.
Plant Physiol Biochem. 2017 May;114:128-137. doi: 10.1016/j.plaphy.2017.03.001. Epub 2017 Mar 6.
In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins.
在薯蓣属植物中，薯蓣贮藏蛋白（31 kDa）是主要的贮藏蛋白，占块茎总蛋白的85%。采用蛋白质组学与生化相结合的方法，以了解薯蓣贮藏蛋白在两个不同生长阶段（发芽块茎和成熟块茎）的生理作用。高效液相色谱分析表明，发芽块茎中甘露醇含量降低了3倍，蔗糖和麦芽糖含量分别增加了12.88倍和l.24倍。发芽块茎中蔗糖磷酸合酶和甘露醇脱氢酶活性分别增加了1.8倍和3倍，而成熟块茎中活性较高的转化酶可能降低了蔗糖积累。成熟块茎和发芽块茎的SDS-PAGE和二维图谱证实，发芽时薯蓣贮藏蛋白减少（超过50%）。采用离子交换和凝胶过滤色谱法纯化薯蓣贮藏蛋白，纯化倍数为43.32，得率为38.16%。在31 kDa的35个条带中，纯化组分的二维凝胶中仅存在12个条带（鉴定为薯蓣贮藏蛋白异构体）。为了寻找除薯蓣贮藏蛋白之外的其他块茎蛋白，对DEAE柱的未结合组分进行了双向凝胶电泳分析。在薯蓣属蛋白质组中，可能首次鉴定出DREB 1A、咖啡酸3 - O - 甲基转移酶和Rab - 1小GTP结合蛋白。相互作用组分析表明，这些蛋白参与氧化应激、类胡萝卜素合成和囊泡运输。这可能是首次尝试鉴定块茎蛋白质组（尽管有限）并了解这些蛋白质的生理意义。
