近日，苏州大学李兵课题组于Journal of Agricultural and Food Chemistry发表题为Cloning and Functional Analysis of CncC and Keap1 Genes in Silkworm的最新研究文章。
哺乳动物细胞核转录因子Nrf2 (nuclear factor erythroid 2 related factor 2)在细胞解除外源应激毒性和缓解氧化应激中有重要的作用。近年研究表明，Nrf2/keap1(kelch-like ECH-associate protein 1)信号通路能有效治疗肝中毒、癌症、炎症等疾病。Nrf2特异性识别抗氧化调控元件(ARE)调控很多解毒基因和抗氧化基因的转录。
Nrf2分为6个关键功能域，其中Neh1、Neh3、Neh4和Neh5的作用都是让进入细胞核的Nrf2识别ARE，调控基因的转录。Neh2和Neh6是调控Nrf2的泛素化降解的关键区域，Neh2能与keap1的双甘氨酸重复区(DGR)识别结合。
Keap1含有3个结构域，分别为干预区(IVR)、BTB区和DGR。BTB区通过BTB-Cullin3连接酶与Cullin3结合到一起，IVR区半胱氨酸的氧化修饰在keap1的亲电反应中起着重要作用。
在昆虫中，Nrf2被更名为CncC(cap ‘n’ collar isoform-C)，果蝇(Drosophila melanogaster)的CncC/Keap1信号通路主要功能是选择性与ARE结合。CncC具有调控氧化还原的稳态、防止衰老、调控细胞增殖、解毒、增强外界抗逆性等作用。CncC/keap1-ARE信号通路能够调控CYP450家族基因、GST(glutathione S-transferase)、HO-(Heme oxygenas-1)1和Superoxide Dismutase(SOD)的表达，增强细胞自身对外界应激的抗性。
对DDT抗性的果蝇品系中，CncC的含量水平高于对照组，cyp6G1, cyp6A2和cyp12D1的表达水平均提高。干扰果蝇CncC的表达，使解毒相关的基因CYP6a8和CYP6a2均出现显著下调。CncC-Maf复合体能增强赤拟谷盗(Tribolium castaneum)P450家族cyp6BQ6、cyp6BQ7和cyp6BQ9的转录水平。当keap1表达水平降低，或者受到急性外源性压力时，CncC的转录水平上调。CncC受到线粒体产生的ROS、二乙酯、H2O2和重金属等刺激时，其会调控下游基因的转录。CncC诱导昆虫解毒酶基因和抗氧化基因的表达，调控对农药的抗性。
家蚕是重要的经济昆虫，中国生产的生丝占全球的78%以上，也是重要的鳞翅目模式昆虫。近年由于农药的广泛使用，桑叶残留的农药会引起家蚕中毒，导致吐丝量减少，或者不吐丝，对蚕丝业生产造成严重的打击。辛硫磷是目前被广泛使用的有机磷农药之一，微量中毒会造成线粒体损伤，产生过量的ROS，导致氧化应激。目前还没有鳞翅目昆虫的CncC/keap1-ARE信号通路在调控解毒酶CYP450家族基因和抗氧化酶HO-1表达的研究报道。研究家蚕CncC/keap1的信号通路的功能，同样有利于阐明鳞翅目昆虫代谢解毒和抗氧化的调控机制。
本研究分别克隆了家蚕CncC(BmCncC)和keap1(Bmkeap1)基因，分析其氨基酸序列，得到了各个功能区基序的定位。通过进化树分析和多物种序列对比发现，CncC的Neh1基序在所有被比较物种中高度保守，首次发现鳞翅目昆虫Neh2的DMG基序代替DLG基序。
蛋白质三维构象分析表明， BmCncC的Neh1形成发夹结构，结合到DNA上，Bmkeap1的DGR区域含有丰富的β-折叠参与对Neh2的识别。转录表达分析表明，BmCncC和Bmkeap1均在一龄幼虫体内高表达，两个基因在生殖腺、脂肪体和头等组织中表达较高。微量辛硫磷添食家蚕幼虫后，脂肪体内Akt and BmCncC的转录水平和蛋白表达水平显著上调，Bmkeap1的表达量下调。同时受CncC调控的下游解毒酶GST, cyp4M5, cyp6AE2 and cyp9G3的转录水平分别上调4.026倍、5.246倍、3.821倍和9.787倍，GST和CYP450的酶活性增加1.52倍和1.23倍。结果表明，BmCncC/Bmkeap1信号通路在辛硫磷诱导后被激活，调节下游解毒酶基因的表达，参与家蚕对辛硫磷的代谢解毒。
英文摘要：CncC/keap1–ARE is an important signaling pathway for detoxification and antioxidation in Diptera and Coleoptera insects. However, such a signaling pathway has not been studied in Bombyx mori. In this study, BmCncC and Bmkeap1 genes were cloned, their amino acid sequences were analyzed, and each functional domain was mapped. Through phylogenetic analysis and sequence comparison among multiple species, we found that the Neh1 motif of CncC was highly conserved and the DLG motif was replaced by the DMG motif in Neh2. Conformational analysis showed that Neh1 of BmCncC forms a hairpin structure to bind DNA. The DGR region of Bmkeap1 contained abundant β sheets, which was involved in the recognition of Neh2. The transcription and expression analyses showed that both BmCncC and Bmkeap1 were highly expressed in the first instar larvae, and these two genes were expressed at a high level in the reproductive gland, fat body, and head. The transcriptional and expression levels of Akt and BmCncC in the fat body were significantly upregulated, and the expression of Bmkeap1 was downregulated after the phoxim treatment in silkworm. The transcriptional levels of CncC-regulated detoxification enzymes GST, cyp4M5, cyp6AE2, and cyp9G3 were increased by 4.026-, 5.246-, 3.821-, and 9.787-fold, respectively, while the activities of GST and CYP450 were increased by 1.521- and 1.231-fold, respectively, after phoxim treatment. These results indicated that the BmCncC/Bmkeap1 signaling pathway was activated by phoxim, leading to the expression of downstream detoxifying enzymes and detoxification of phoxim in silkworm.
