PDF(Pubmed)
Abstract：
In plants, serpins are a superfamily of serine and cysteine protease inhibitors involved in stress and defense mechanisms, with potential for controlling agricultural pests, making them important biotechnological tools. The objective of this study was to characterize a serpin from Theobroma cacao, called TcSERPIN, to identify its endogenous targets and determine its function and biotechnological potential. TcSERPIN has 390 amino acid residues and shows conservation of the main active site, RCL. Cis-elements related to light, stress, hormones, anaerobic induction, cell cycle regulation and defense have been identified in the gene\'s regulatory region. TcSERPIN transcripts are accumulated in different tissues of Theobroma cacao. Furthermore, in plants infected with Moniliophtora perniciosa and Phytophthora palmivora, the expression of TcSERPIN was positively regulated. The protein spectrum, rTcSERPIN, reveals a typical β-sheet pattern and is thermostable at pH 8, but loses its structure with temperature increases above 66°C at pH 7. At the molar ratios of 0.65 and 0.49, rTcSERPIN inhibited 55 and 28% of the activity of papain from Carica papaya and trypsin from Sus scrofa, respectively. The protease trap containing immobilized rTcSERPIN captured endogenous defense proteins from cocoa extracts that are related to metabolic pathways, stress and defense. The evaluation of the biotechnological potential against geohelminth larvae showed that rTcSERPIN and rTcCYS4 (Theobroma cacao cystatin 4) reduced the movement of larvae after 24 hours. The results of this work show that TcSERPIN has ideal biochemical characteristics for biotechnological applications, as well as potential for studies of resistance to phytopathogens of agricultural crops.
摘要：
在植物中,serpin是参与应激和防御机制的丝氨酸和半胱氨酸蛋白酶抑制剂超家族，具有控制农业害虫的潜力，使它们成为重要的生物技术工具。这项研究的目的是表征来自可可可可的serpin，叫做TcSERPIN，确定其内源性靶标并确定其功能和生物技术潜力。TcSERPIN具有390个氨基酸残基，显示出主要活性位点的保守性，RCL。与光相关的顺式元素，压力，荷尔蒙，厌氧诱导,细胞周期调控和防御已在基因的调控区得到鉴定。TcSERPIN转录物积累在可可的不同组织中。此外，在感染了Perniciophtora和棕榈疫霉的植物中，TcSERPIN的表达呈正调控。蛋白质谱，rTcSERPIN,揭示了典型的β-折叠图案并且在pH8下是热稳定的,但是在pH7下随着温度升高到66°C以上而失去其结构。在0.65和0.49的摩尔比下，rTcSERPIN抑制了番木瓜木瓜的木瓜蛋白酶和Susscrofa的胰蛋白酶的55％和28％的活性，分别。含有固定化rTcSERPIN的蛋白酶陷阱从可可提取物中捕获与代谢途径相关的内源性防御蛋白，压力和防御。对地球蠕虫幼虫的生物技术潜力的评估表明，rTcSERPIN和rTcCYS4（Theobromacacao胱抑素4）在24小时后降低了幼虫的运动。这项工作的结果表明，TcSERPIN具有生物技术应用的理想生化特性，以及对农作物植物病原体抗性研究的潜力。
