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3d至开花后6d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.%From an EST sequence identified by previous proteomic analysis in our lab we cloned a full-length cDNA, named Ghl4-3-3L2, which encodes a cotton (Gossypium hirsutum) 14-3-3 protein that probably plays a regulatory role in cotton fiber initiation and elongation. To unravel the target gene networks of this protein, the interactome of Ghl4-3-3L2 was studied using the yeast two-hybrid system and a Pull-down assay. However, this protein was severely toxic to yeast. Thus, this paper focuses on the Pull-down results. Six x His tagged Ghl 4-3-3L2 was prokaryotically expressed in E. coli and purified for use as bait protein. Ghl4-3-3L2 associated prey proteins were isolated and enriched from mixed protein samples derived from ovules 3, 1 or 0 days before anthesis and fibers 3 or 6 days after anthesis of wild-type upland cotton cv. Xuzhoul42, the lintless mutant Xuzhoul42, and the Ligon lintless 1 mutant using Pull-down technology. The resulting proteins were further isolated by 2-DE and identified by MALDI-MS/MS analysis. Seven GM4-3-3L2 associated proteins were identified. The putative functions of these proteins included molecular chaperoning, vacuolar trafficking, metabolism, and signal transduction. Some of these proteins, including annexin, vacuolar H+-ATPase and Ran GTPase 3, have been previously linked with fiber development. Our study provides novel data for further elucidation of the molecular functions of Ghl4-3-3L2 and the regulatory mechanisms governing cotton fiber development.
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