Cloning and characterization of lipoxygenase gene from germinating seedlings of green gram (Vigna radiata L.)
Cloning and characterization of lipoxygenase gene from germinating seedlings of green gram (Vigna radiata L.)
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DOI：
10.1007/s40502-015-0186-z
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
Kasi Viswanath Kotapati;Bhagath Kumar Palaka;R. Anangi;Thyagaraju Kedam;D. Ampasala
中科院分区：
文献类型：
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作者：
Kasi Viswanath Kotapati;Bhagath Kumar Palaka;R. Anangi;Thyagaraju Kedam;D. Ampasala
植物LOXs在植物生长、衰老、防御和胁迫应答等生理过程中发挥着重要作用。本研究利用5′-和3′- race PCR方法从绿克萌发苗中克隆了全长脂氧合酶cDNA (VrLOX)，并命名为VrLOX。ORF（2601）编码867个氨基酸残基，分子量约为97.43 kDa，等电点为5.93。该vrlox由一个推测的PLAT结构域和一个铁结合催化结构域组成，该序列与蛋白质数据库中已知的植物lox具有显著的同源性。生物信息学分析表明，该基因很可能是LOX推测的13-LOX型蛋白，与glycine maxLOX-10具有最高的同源性（91%）。酶测结果表明，萌发后第3天pH值为6.5时，LOX活性较高，第5天MDA含量较高。半定量RT-PCR分析显示，vrloxmrna在萌发后第3天表达量最高，可以通过茉莉酸甲酯（MeJA）诱导。利用计算机方法和分子动力学模拟对vrlox的三维结构进行了分析。
Plant LOXs play a significant role in plant physiological processes such as growth, senescence, defence and stress-related responses. In the present study a full length lipoxygenase cDNA (VrLOX) has been cloned from green gram germinating seedlings using 5′- and 3′-RACE PCR method and nomenclaturedVrLOX. The ORF (2601) encodes 867 amino acid residues with an approximate molecular weight of 97.43 kDa and an isoelectric point of 5.93. TheVrLOXcomprises of a putative PLAT domain and an iron-binding catalytic domain, and the sequence displayed a significant identity to known plant LOXs deposited in Protein Data Bank. Bioinformatics analysis demonstrated that this gene is most probably a LOX putative protein of type 13-LOX, and shared highest identity (91 %) with theGlycine maxLOX-10. Enzymatic assay showed LOX activity was more on day 3 at pH 6.5 and higher MDA levels were observed on day 5 after germination. Semi-quantitative RT-PCR analysis indicated highestVrLOXmRNA expression on day 3 after germination, and could be induced by methyl jasmonate (MeJA). The 3-dimensional structure of theVrLOXwas elucidated using in silico approach and molecular dynamics simulations.