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之前，我们向大家简单介绍了关于线虫与各种非生物和生物胁迫的内容（传送门1：https://mp.weixin.qq.com/s/CdGGWZXWESkO7z-9R8U0tA）以及线虫中荧光蛋白的使用（传送门2：https://mp.weixin.qq.com/s/Yk_VkNCWHXJXbu-7U2FKzA）。但是无声的秀丽线虫如何告诉我们它们的切身感受呢？在本文中我们结合之前的内容，向大家展示了如何利用特异性启动子表达荧光蛋白或与蛋白融合表达的方式来真实地看见线虫的应激反馈。
01
内质网胁迫
内质网胁迫会诱导热休克蛋白HSP-4在肠道细胞中上调表达[1]。因此，HSP-4::GFP可指示线虫所处的内质网胁迫状态。Caenorhabditis Genetics Center （CGC）的代表线虫品系有：SJ4005 zcIs4 [hsp-4::GFP] V。
图1 使用衣霉素诱导的内质网应激[1]
02
线粒体胁迫
在线虫中通过选择性地干扰线粒体多复合物组装或用RNAi的方式敲降线粒体分子伴侣和蛋白酶会激活hsp-6和hsp-60的表达，而上述这些均会导致线粒体中蛋白折叠和加工缺陷。hsp-6和hsp-60对扰乱线粒体蛋白处理的诱导是特异性的，不受热激、内质网应激、损伤线粒体中间代谢或ATP合成等操作的影响[2]。CGC的代表线虫品系有：GL347（或SJ4100） zcIs13 [hsp-6p::GFP + lin-15(+)] V；SJ4058 zcIs9 [hsp-60::GFP + lin-15(+)]。
图2 通过抗霉素A诱导线粒体UPR并激活hsp-6特异性启动子的GFP表达[3]
03
氧化胁迫
在线虫中，Phase 2去氧化基因是受保守的Nrf 同源蛋白SKN-1转录因子调控的[4]。而作为其靶基因的gcs-1（gamma GlutamylCysteine Synthetase）和gst-4（Glutathione S-Transferase）转录能反映线虫处于氧化环境中SKN-1在去氧化过程中发挥的积极作用[5]。相对于gst-4受SKN-1调控[5]（也有报道认为gst-4还受到EGF信号通路的转录因子EOR-1调控[6]），gcs-1还存在不依赖SKN-1的调控通路，体现复杂的细胞过程和信号通路。CGC的代表线虫品系有：CL2166 dvIs19 [(pAF15)gst-4p::GFP::NLS] III；LD1171 ldIs3 [gcs-1p::GFP + rol-6(su1006)]
04
低氧胁迫
之前，我们介绍过在研究线虫低氧胁迫时发现低氧诱导因子（HIF-1），主要掌控调节细胞内对低氧环境的反馈，线虫在低氧环境中会广泛诱导上调表达HIF-1[7]。因此，HIF-1::GFP可作为线虫处于低氧胁迫状态的指示标志。CGC的代表线虫品系有：UL1447 unc-119(ed3) III; leEx1447 [hif-1::GFP + unc-119(+)]。
05
热胁迫
线虫处于热激或其他环境压力时，会诱导表达分子伴侣蛋白HSP-16.2。将含有hsp-16.2启动子表达GFP的线虫在35 ℃热激3小时，并随后在20℃恢复1小时即可观察到线虫多组织的绿色荧光[8]。CGC的代表线虫品系有：CL2070 dvIs70 [hsp-16.2p::GFP + rol-6(su1006)]。
06
冷胁迫
在低温条件下，细胞为维持其细胞膜的流动性需要增加细胞膜不饱和脂肪酸的总体比例。TMEM-135能够正向调控细胞的脂肪存储及寿命。将GFP与TMEM-135融合表达的线虫在4℃下培养1小时即可诱导其荧光表达。代表线虫品系：MAB124[9]。
图3 在低温下TMEM-135::GFP信号增强[9]
07
重金属（镉）胁迫
NUMR-1和NUMR-2 (nuclear localized metal responsive) 在发育阶段中的肠道、头部、尾部神经和vulva肌肉组织形成表达；当暴露于金属胁迫时，线虫会被诱导在咽部和肠道表达NUMR-1及NUMR-2。但其他环境胁迫并不会影响它们的转录，因此表明它们是对金属特异性的反馈基因[10]。CGC的代表线虫品系有：JF88 mtEx63 [numr-1p::numr-1::GFP + rol-6(su1006)]。
图4 线虫在无金属和在镉胁迫下的荧光变化[10]
08
高渗胁迫
细胞对受到高渗透胁迫时的水分流失会做出必要的应激反馈，调整有关基因上调表达，例如调节有机渗透物积累以及作为化学伴侣功能和恢复渗透压平衡的溶质。将含有gpdh-1启动子表达GFP的线虫仅在高渗条件下诱导表达绿色荧光蛋白，其他胁迫条件并没有响应[11]。CGC的代表线虫品系有：VP198 kbIs5 [gpdh-1p::GFP + rol-6(su1006)]。
图5 gpdh-1p::GFP线虫在高渗条件下诱导表达荧光[11]
09
生物胁迫
为了应对病原体侵染，线虫会产生多种的抗菌蛋白。BCF-1（bacterial colonization factor）是一种假定多糖-蛋白质复合物成分，在病原生物（P. luminescens Hb和P. aeruginosa PA14）暴露24小时下诱导L4线虫（f57f4.4p::gfp）增加绿色荧光蛋白表达[12]。CGC的代表线虫品系有：IG1335 frEx479 [F57F4.4p::GFP + col-12p::DsRed]。
图6 f57f4.4p::gfp线虫在病原菌暴露下的荧光表达[12]
另外，lys-7（lysozyme）表达daf-16依赖的溶菌酶样抗菌因子[13]，广泛表达于头部神经、肠道以及直肠腺。在PA14侵染条件下，线虫（lys-7::GFP）荧光强度反而降低，并以此来反映受PA14病原体侵染程度。CGC的代表线虫品系有：SAL105 pha-1(e2123) III; denEx2[lys-7::GFP + pha-1(+)]。
图7 LYS-7::GFP线虫在被PA14侵染后荧光减弱[14]
10
其他胁迫反馈报告基因---DAF-16/FOXO
DAF-16/FOXO是线虫细胞内重要转录因子，调控细胞内的多个代谢过程，包括应激反应。在处于环境压力条件下，通过观察DAF-16::GFP从细胞质转入细胞核激活靶基因表达。CGC的代表线虫品系有：TJ356 zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]。
图8 DAF-16::GFP荧光转移变化体现在各种胁迫刺激[15]
参考文献
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撰稿：卞文印
审核：陈岚彬
编辑：刘乐丰
