摘要
植物螯合肽合成酶(phytochelatin synthase, PCS)催化谷胱甘肽(glutathione, GSH)合成植物螯合肽(phytochelatins, PCs),在植物重金属解毒方面具有重要作用。苎麻(Boehmeria nivea) PCS1 (命名为BnPCS1)主要在叶片中表达,镉胁迫下表达上调,但其参与苎麻耐受重金属的分子机制尚不明确。本实验对BnPCS1进行了生物信息学分析,构建了苎麻叶片均一化cDNA文库和诱饵载体pGBKT7-BnPCS1,利用酵母双杂交系统进行BnPCS1互作蛋白的筛选。结果显示苎麻BnPCS1是等电点为6.76的不稳定蛋白质和亲水蛋白质,亚细胞定位预测结果表明BnPCS1位于细胞质。苎麻叶片均一化cDNA文库库容是7.12×10^(6)cfu,文库滴度是6.98×10^(6)cfu·mL^(-1),均一化程度均大于10^(6)。将pGBKT7-BnPCS1诱饵载体转化至Y2HGold酵母菌株检测发现,其无毒性和自激活现象,通过构建的诱饵载体pGBKT7-BnPCS1与苎麻均一化cDNA文库杂交,鉴定出两个BnPCS1的互作蛋白,分别为伴侣蛋白ClpB3和前体mRNA加工因子39。这为进一步研究BnPCS1的功能及分子机制提供了参考依据。
Phytochelatin synthase(PCS) catalyzes the synthesis of glutathione(GSH) into phytochelatins(PCs), and plays an important role in detoxification of heavy metals in plants. Ramie PCS1(named as BnPCS1) is mainly expressed in leaves, and its expression is up-regulated under cadmium stress, but its molecular mechanism involved in ramie tolerance to heavy metals is still unclear. In this experiment, the bioinformatics analysis of BnPCS1 was carried out, and the homogenized cDNA library of ramie leaves and the bait vector pGBKT7-BnPCS1 were constructed. The yeast two-hybrid system was used to screen the BnPCS1 interaction proteins. The results showed that BnPCS1 of ramie was an unstable protein and a hydrophilic protein with an isoelectric point of 6.76. The prediction results of subcellular localization indicated that BnPCS1 was located in the cytoplasm. The capacity of the normalized cDNA library of ramie leaves was 7.12×10^(6)cfu, the titer of the library was 6.98×10^(6)cfu·mL^(-1), and the degree of homogenization was greater than 10^(6). The bait vector pGBKT7-BnPCS1 was transformed into the Y2HGold yeast strain and it was found to be non-toxic and nonself-activated. By hybridizing the constructed bait vector pGBKT7-BnPCS1 with the ramie homogenized cDNA library, two interacting proteins of BnPCS1 were identified, namely chaperone ClpB3 and pre-mRNA processing factor 39, respectively. This provides a reference for further researches on the function and molecular mechanism of BnPCS1.
作者
全芮萍
刘婕仪
周倩文
杨瑞芳
付虹雨
佘玮
崔国贤
QUAN Ruiping;LIU Jieyi;ZHOU Qianwen;YANG Ruifang;FU Hongyu;SHE Wei;CUI Guoxian(College of Agronomy,Hunan Agricultural University,Changsha 410128,China)
出处
《植物生理学报》
CAS
CSCD
北大核心
2021年第12期2301-2309,共9页
Plant Physiology Journal
基金
国家自然科学基金(31871673)
财政部、农业农村部国家现代农业产业技术体系-国家麻类产业技术体系(CARS-16-E11)。
