李霞 潘春柳 苏桂军 詹洁 王爱勤 肖冬 何龙飞

摘  要：h型硫氧还蛋白（TRX h）是最多的一类硫氧还蛋白，在种子萌发、氧化还原、信号传递以及响应生物和非生物胁迫等多种途径中起重要作用。以花生铝敏感品种‘中花2号和耐铝品种‘99-1507为材料，对h型硫氧还蛋白亚族成员AhTRX h进行克隆和表达分析。结果表明：该基因包含长为429 bp的完整开放阅读框，编码142个氨基酸;进化树分析表明，花生TRX h与鹰嘴豆TRX h、拟南芥TH9亲缘关系较近，且含有保守活性位点WCGPC;亚细胞定位显示，AhTRX h定位于叶绿体;构建原核表达重组质粒pGEX-6p-1-AhTRX h，转化大肠杆菌Rosetta菌种，在上清和沉淀均有表达，纯化获得可溶的具活性的重组蛋白，体外检测重组蛋白没有明显的发生亚硝基化的趋势。酵母双杂显示，AhTRX h蛋白没有自激活性，筛选花生文库获得互作蛋白——钙网蛋白和金属硫蛋白。该结果表明AhTRX h可能通过与钙网蛋白和金属硫蛋白相互作用，参与花生对铝胁迫响应。

关键词：花生;硫氧还蛋白;铝胁迫;基因克隆

中图分类号：S565.2      文献标识码：A

Abstract： Thioredoxin plays an important role in seed germination， redox homeostasis， signal transduction and res-ponses to biotic and abiotic stresses. In this study， AhTRX h， a member of h-thioredoxin subfamily， was cloned from peanut varieties ZH2 （Al-sensitive） and 99-1507 （Al-tolerant）. Sequence analysis showed that AhTRX h contained an open reading frame （ORF） of 429 bp encoding 142 amino acids with a conservative active site-WCGPC. Phylogenetic analysis showed that AhTRX h was closely related to TRX h in Cicer arietinum and Th9 in Arabidopsis. AhTRX h was located in chloroplast. The recombinant plasmid pGEX-6p-1-AhTRX h was transformed into Escherichia coli （Rosetta）， recombinant proteins induced by IPTG were founded in supernatant and precipitate. Soluble recombinant proteins with activity were purified， and had no obvious trend of S-nitrosylation in vitro. The results of yeast two hybrid showed that potentially interactional proteins were calreticulin and metallothionein. The results indicate that AhTRX h play an important role in response to Al toxicity by likely interacting with calreticulin and metallothionein.

Keywords： Arachis hypogaea; thioredoxin; aluminum stress; gene cloning

DOI： 10.3969/j.issn.1000-2561.2021.05.008

活性氧（ROS）為细胞正常代谢过程的一种代谢产物，以极低的含量存在，但在逆境胁迫条件下ROS含量迸发，影响植物代谢，对植物产生胁迫等，如在干旱、高温、强光等逆境胁迫条件下，植物会产生大量ROS[1]。ROS含量受到众多调控因子的调节，氧化还原系统是主要的调控因子之一。硫氧还蛋白（thioredoxin，TRX）是一类普遍存在的二硫化物还原酶，在保守基序WC[G/P]PC中存在2个活性半胱氨酸位点，是氧化还原系统中重要的成员之一。……