从植物cDNA表达文库中筛选钙调素结合蛋白-生物化学专业论文.docxVIP

nL南开大学硕士生毕业论文 nL Abstract Through the interact i.on wi th p 1enty o[ binding protcins ，CaM can regula1e a wide variety of cellular proccsses. Therefore ， the rcsearch of CaM BPs (ca1modulin binding proteins) wil] help 10 exp1rrin how Ca2+1 CrrM mediates signal trrrnsduction pathways in organic systems. So rar very litt1e is known about the numher ， 1oca1ion，and iden1ification of CaM BP in plant 只 comparing wi th those in animals. 1n plants ，much more need to he done to understand the CaM binding domains of CaM BP and the prccise mechrrnisms by which th 已y regulate CaM upon binding. 1n this thesis ， CaM was 1abeled by biotin . The biotiny1rrted calmodulin can detect 100ng CaM BP-l0 with high specificity. In order to fi nd more CaM BPs ，especially CrrM BP-l0 (a Q训 BP found by our research group) ， a cDNA library was screened wi th biotiny 1atcd crrlmodulin as a probe. Nine positive clones were isolatcd. After re-clone and puri fication ，the fusion proteins of these clo凹 es were extracted. Colloidrrl Go1d-CaM Ge1 ?ver1ay of [usion prolein indicated that a11 positive clones synthesized peptídes that could b ind calmodu 1in. 。口e fusion protein that was encoded by Y7 clone can bind CaM independent of Ca2+. Y7 rrnd Y9 clone were excised in vivo; then two pl asmids pBluescript contain ing insert cDNA sequence came 1口to being. Now two p1asmids are heing sequenced. The research of CaM BP in plants wi 11 come up to a new leveJ by this thesis and further works. Keywords Ca.lmodulin binding protein ， expression 1ibrary， hiotinylated 前言 钙鸽子 (Ca2 .) 作为细胞信号传导信使的作用己被许多研究所证实 (Poovaiah 和 Reddy ，1993)。目前就植物而言， Ca2+仍是唯一己知并且普遍 存在的信号传寻信使，因此具有极其重要的研究价值 (Poov创ah 等， 1987，Roberts 和 Harmon ，1992，Bush，1ω5)。植物体内的许多生理活动 都与 Ca2 有关，如离子平衡、细胞质流动、器官极性发育、花粉管伸长、 + 碳水化合物代谢、有丝分裂、基因表达以及对生长素和损伤的反应 C Hetherington 和 Trewavas，1982)。在静息条件下，植物细胞内 Ca主+浓 度在lO -mo1/L-1O-6mo l/L 之间，细胞受到剌激后，会导致胞内 Ca2 浓度 + 的迅速上升，从而引发一系列生理生化反应 C Roberts 和 l