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根据本实验室蛋白质组学研究鉴定的EST序列,克隆了一个可能在棉花纤维起始和伸长发育阶段起调控作用的棉花14-3-3蛋白质的全长cDNA,定名为Gh14-3-3L2。Gh14-3-3L2蛋白毒性大,难以通过酵母双杂交途径鉴定其互作蛋白质组。因此,本研究首先经原核表达并纯化出添加六联组氨酸标签的Gh14-3-3L2蛋白,以此蛋白质为诱饵,以开花前3 d至开花后6 d正常野生型、徐州142无纤维突变体和Li-1无长绒纤维突变体胚珠或纤维混合蛋白质为猎物样品,采用Pull-down技术分离、富集Gh14-3-3L2相互作用蛋白质,再经2-DE和MALDI-MS/MS串联质谱分析,鉴定了7个可能与Gh14-3-3L2相互作用的蛋白,其功能有作为分子伴侣、参与微囊的运输、代谢、信号转导等。为进一步解析Gh14-3-3L2的功能以及棉花纤维发育的分子调控网络奠定了基础。
According to the EST sequence identified by our laboratory proteomics, we cloned a full-length cDNA of 14-3-3 cotton protein, which may play a regulatory role in the initiation and development of cotton fiber, and named Gh14-3-3L2. The Gh14-3-3L2 protein is so toxic that it is difficult to identify its interacting proteome by the yeast two-hybrid approach. Therefore, in the present study, Gh14-3-3L2 protein with hexa-histidine tag was expressed and purified by prokaryotic expression. The protein was used as bait. The wild-type Gh14-3-3L2 protein The mutants and Li-1 non-cashmere mutant ovule or fiber mixed protein were used as prey samples, and the proteins of Gh14-3-3L2 interaction were enriched by Pull-down technique. The proteins of 2-DE and MALDI-MS / MS In tandem mass spectrometry, seven proteins that may interact with Gh14-3-3L2 were identified, and their function as molecular chaperones involved in the transport, metabolism, signal transduction of the microcapsules and the like. Which laid the foundation for further analysis of the function of Gh14-3-3L2 and the molecular regulation network of cotton fiber development.