Kumari Khushboo, Rai Monika Prakash, Bansal Navita, Prashat G Rama, Kumari Sweta, Srivathsa Rohini, Dahuja Anil, Sachdev Archana, Praveen Shelly, Vinutha T
1Division of Biochemistry, ICAR-Indian Agricultural Research Institue, New Delhi, 110012 India.
2Amity University, Noida, Uttar Pradesh 201313 India.
3 Biotech. 2020 Mar;10(3):110. doi: 10.1007/s13205-020-2086-9. Epub 2020 Feb 11.
Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc-the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of and were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, --TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress.
γ-生育酚甲基转移酶（γ-TMT）将γ-生育酚转化为α-生育酚，这是生育酚生物合成中的限速步骤。测序结果显示，[具体基因1]和[具体基因2]的编码区彼此高度相似（氨基酸水平上为93%）。基于N端氨基酸的差异，γ-TMT蛋白可分为三种亚型：γ-TMT1、γ-TMT2和γ-TMT3。计算机结构分析显示，γ-TMT1和γ-TMT3蛋白中存在叶绿体转运肽（cTP）。然而，仅在γ-TMT3蛋白中发现了转运肽的其他特性，如N端前三个位置存在疏水氨基酸以及酸性氨基酸水平较低。利用共聚焦显微镜在[具体植物名称]中研究了35S启动子下绿色荧光蛋白（GFP）融合的γ-TMT1和γ-TMT3的亚细胞定位。结果表明，γ-TMT1存在于细胞质中，而γ-TMT3则定位于细胞质和叶绿体中。此外，通过超高效液相色谱（UPLC）定量α-生育酚，验证了γ-TMT3在叶绿体中的存在。因此，本研究中γ-TMT1和γ-TMT3蛋白的细胞质定位以及γ-TMT3的叶绿体定位，将有助于揭示γ-TMT编码的α-生育酚在保护叶绿体和细胞膜免受植物氧化应激方面的重要性。
