Li Xu, Liu Yawen, Liu Hongtao
National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, Shanghai, P. R. China.
Methods Mol Biol. 2021;2297:161-166. doi: 10.1007/978-1-0716-1370-2_17.
Cryptochromes are photolyase-like blue-light receptors found in all major evolutionary lineages (Ahmad and Cashmore, Nature 366:162-166, 1993; Lin, Plant Physiol 110:1047, 1996; Cashmore, Cell 114:537-543, 2003; Partch and Sancar, Methods Enzymol 393:726-745, 2005). Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate primarily blue-light inhibition of hypocotyl elongation and photoperiodic control of floral initiation (Ahmad and Cashmore, Nature 366:162-166, 1993; Somers et al., Science, 282:1488-1490, 1998; Guo et al., Science 279 (5355):1360-1363, 1998; Yu et al., Arabidopsis Book 8:e0135, 2010). It has been proposed that phototransduction of cryptochromes involves the blue-light-dependent protein interactions, such as AtCRY2-CIB1 (CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1), AtCRY1-PIF4 (PHYTOCHROME INTERACTING FACTOR 4) modules, sequentially mediate gene expression and plant growth (Liu et al., Science 322 (5907):1535-1539, 2008; Ma et al., Proc Natl Acad Sci U S A 113 (1):224-229, 2016; Wang et al., Science 354:343-347, 2016). Cryptochromes also showed blue light response in vitro when expressed in Sf9 insect cells using the baculovirus expression system, thus the wavelength-specific CRY2-CIB1 interaction can also be observed in Semi-in-vivo pull-down assay (Li et al., Proc Natl Acad Sci U S A 108 (51):20844-20849, 2011; Liu et al., EMBO Reports, 2018). Here, we describe the detailed process of blue light-dependent CRY2-CIB1 interaction in Semi-in-vivo conditions.
隐花色素是一种类似于光解酶的蓝光受体，存在于所有主要的进化谱系中（艾哈迈德和卡什莫尔，《自然》366:162 - 166，1993；林，《植物生理学》110:1047，1996；卡什莫尔，《细胞》114:537 - 543，2003；帕奇和桑卡尔，《酶学方法》393:726 - 745，2005）。拟南芥隐花色素1（CRY1）和隐花色素2（CRY2）主要介导蓝光对下胚轴伸长的抑制以及花期起始的光周期控制（艾哈迈德和卡什莫尔，《自然》366:162 - 166，1993；萨默斯等人，《科学》，282:1488 - 1490，1998；郭等人，《科学》279 (5355):1360 - 1363，1998；于等人，《拟南芥全书》8:e0135，2010）。有人提出，隐花色素的光信号转导涉及蓝光依赖性蛋白质相互作用，如AtCRY2 - CIB1（隐花色素相互作用碱性螺旋 - 环 - 螺旋1）、AtCRY1 - PIF4（光敏色素相互作用因子4）模块，依次介导基因表达和植物生长（刘等人，《科学》322 (5907):1535 - 1539，2008；马等人，《美国国家科学院院刊》113 (1):224 - 229，2016；王等人，《科学》354:343 - 347，2016）。当使用杆状病毒表达系统在Sf9昆虫细胞中表达时，隐花色素在体外也表现出蓝光反应，因此在半体内下拉试验中也可以观察到波长特异性的CRY2 - CIB1相互作用（李等人，《美国国家科学院院刊》108 (51):20844 - 20849，2011；刘等人，《欧洲分子生物学组织报告》，2018）。在此，我们描述了在半体内条件下蓝光依赖性CRY2 - CIB1相互作用的详细过程。
