Padaria Jasdeep Chatrath, Vishwakarma Harinder, Biswas Koushik, Jasrotia Rahul Singh, Singh Gyanendra Pratap
Biotechnology and Climate Change Laboratory, National Research Centre on Plant Biotechnology, Pusa Campu, New Delhi 110012, India.
BMC Res Notes. 2014 Oct 10;7:713. doi: 10.1186/1756-0500-7-713.
Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India.
qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting.
TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.
热胁迫导致活性氧（ROS）加速产生，这会对植物的细胞成分造成大量氧化损伤。许多与热胁迫相关的基因，如热休克蛋白（HSPs）、过氧化氢酶、过氧化物酶，在胁迫时会过度表达。从耐热小麦品种Raj3765花期的热胁迫（42°C）响应消减cDNA文库中获得了一个有效的胁迫响应基因——过氧化物酶体抗坏血酸过氧化物酶（TapAPX），对其进行了克隆、表征，并通过蛋白质组学和计算机模拟研究验证了其在热胁迫下的作用。在本研究中，我们报告了从印度耐热小麦品种中分离出的过氧化物酶体TapAPX基因在分子和计算机模拟水平上的表征。
TapAPX基因的qPCR研究显示，在42°C热胁迫处理下，其表达水平高达203倍。通过RACE获得的876 bp全长cDNA推导得到一个由292个氨基酸残基组成的蛋白质，通过同源建模得到了pAPX完整的三维结构。TapAPX cDNA被克隆到表达载体pET28（a+）中，在大肠杆菌BL21中过表达的重组蛋白经肽质量指纹图谱分析显示与APX蛋白具有最高的同源性。
小麦品种Raj3765的TapAPX基因在赋予植物耐热性方面具有独特作用，因此可用于作物改良计划，以培育耐高温作物。
