Abstract：
Recent studies have demonstrated the crucial role of Cytochrome P450 enzymes (CYPs) in the production of secondary metabolites, phytohormones and antioxidants in plants. However, their functional characterization specifically under alkaline stress remains elusive. CYP82C4 was the key gene screened from a family of wild soybean CYPs in our previous studies. The aim of this present study was to clone the Glycine soja GsCYP82C4 gene and characterize its functions in Arabidopsis and Glycine max. The results showed that the GsCYP82C4 gene displayed a high expression in different plant tissues at mature stages compared to young stages. Further, higher temporal expression of the GsCYP82C4 gene was noted at 6, 12 and 24 h time points after alkali treatment in leaves compared to roots. In addition, overexpression of GsCYP82C4 improved alkaline stress tolerance in Arabidopsis via increased root lengths and fresh biomass and strengthened the antioxidant defense system via a reduction in superoxide radicals in transgenic lines compared to wild type (WT) and atcyp82c4 mutants. Further, the expression levels of stress-related marker genes were up-regulated in GsCYP82C4 OX lines under alkali stress. The functional analysis of GsCYP82C4 overexpression in soybean displayed better hairy root growth, increased fresh weight, higher antioxidant enzyme activities and reduced lipid peroxidation rates in OX lines compared to the soybean WT (K599) line. In total, our study displayed positive roles of GsCYP82C4 overexpression in both Arabidopsis and Glycine max to alleviate alkaline stress via altering expression abundance of stress responsive genes, stronger roots, higher antioxidant enzyme activities as well as reduced rates of lipid peroxidation and superoxide radicals.
摘要：
最近的研究已经证明了细胞色素P450酶（CYPs）在次级代谢产物的产生中的关键作用，植物中的植物激素和抗氧化剂。然而,它们的功能表征，特别是在碱性胁迫下仍然难以捉摸。CYP82C4是我们先前研究中从一个野生大豆CYP家族中筛选的关键基因。本研究的目的是克隆大豆GsCYP82C4基因，并表征其在拟南芥和大豆中的功能。结果表明，与幼年期相比，GsCYP82C4基因在成熟期的不同植物组织中显示出高表达。Further,与根相比，在碱处理后的6、12和24小时时间点，叶片中GsCYP82C4基因的瞬时表达更高。此外,与野生型（WT）和atcyp82c4突变体相比，GsCYP82C4的过表达通过增加根长和新鲜生物量来提高拟南芥的碱性胁迫耐受性，并通过减少转基因品系中的超氧化物自由基来增强抗氧化防御系统。Further,在碱胁迫下，GsCYP82C4OX品系的胁迫相关标记基因表达水平上调。GsCYP82C4过表达大豆的功能分析显示出较好的毛状根生长,增加新鲜的重量，与大豆WT（K599）品系相比，OX品系具有更高的抗氧化酶活性和降低的脂质过氧化率。总的来说,我们的研究显示GsCYP82C4过表达在拟南芥和甘氨酸max中通过改变应激反应基因的表达丰度来缓解碱性胁迫的积极作用，更强的根，更高的抗氧化酶活性以及降低脂质过氧化和超氧自由基的速率。
