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鱼腥藻 PCC7120外膜蛋白的鉴定及功能研究
Alternative TitleIdentification and functional studies of outer membrane proteins of Anabaena sp. PCC 7120
Thesis Advisor徐旭东
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword鱼腥藻 Pcc7120 外膜蛋白 铁离子转运 钙离子转运
Abstract鱼腥藻和其他蓝藻的外膜是吸收营养物质的第一道屏障,在鱼腥藻中还可能直接影响异形胞包被的形成,但迄今为止,有关蓝藻外膜蛋白的功能尚缺乏研究。本研究分离纯化了鱼腥藻 PCC7120的外膜,通过双向电泳(2-DE) 和质谱鉴定了外膜蛋白,利用luxAB 报告基因研究了编码基因的表达。 主要研究内容和研究结果分3部分: (一)利用双相法结合蔗糖密度梯度超离心纯化了鱼腥藻PCC7120的外膜;利用2-DE分离外膜蛋白,以MOLDI-TOF或ESI-Q-TOF鉴定了8种蛋白。 (二)利用2-DE分析了在有铁和无铁两种培养条件下鱼腥藻的外膜蛋白,发现在缺铁条件下外膜蛋白中有5种表达明显上调,与铁离子转运相关的8种外膜蛋白基因在转录水平也发生调控; (三)对外膜蛋白基因all3983 和alr2269的突变株进行研究,发现在alr2269突变株中外膜蛋白All3983水平增加,外膜蛋白Alr2269和All3983可能与钙离子吸收有密切关系。 本研究得到以下主要结论: (1) 利用双相法结合蔗糖密度梯度超离心可以有效纯化鱼腥藻 PCC7120的外膜; (2) 在缺铁诱导的条件下,鱼腥藻 PCC7120有5种外膜蛋白显著增加,并且与铁离子转运相关的8种外膜蛋白基因也在转录水平发生调控; (3) 外膜蛋白Alr2269 和All3983 可能与胞外钙离子的吸收和转运有关。
Other AbstractOuter membrane of Anabaena and other cyanobacteria is the first barrier for nutrition uptake, and in Anabaena even affects the development of heterocyst envelope. But until now, few functional studies have been done about outer membrane proteins of cyanobacteria. In this study, outer membrane of Anabaena sp. PCC7120 was isolated, and some outer membrane proteins were identified using 2-DE and mass spectrometry. Reporter gene of bacterial luciferase (luxAB) was used to analyze transcription of genes encoding the outer membrane proteins. The experimentation and results are described in 3 parts: (1) Purification of the outer membrane and identification of outer membrane proteins from Anabaena sp. PCC7120. The outer membrane from Anabaena 7120 was isolated using aqueous polymer two-phase partitioning in combination with sucrose density gradient ultra-centrifugation with very few cross-contaminations by plasma and thylakoid membranes. Eight proteins were identified using 2-DE and MOLDI-TOF MS or ESI-Q-TOF MS. (2) The study of outer membrane proteins involved in iron uptake. 2-DE was used to analyze outer membrane proteins from cultures grown under iron-sufficient and iron-deficient conditions, and the results showed that five of eight outer membrane proteins involved in iron uptake were up-regulated by iron deprivation, and transcriptional complementarity relationship existed among the eight genes. (3) The study of outer membrane proteins involved in calcium uptake. The results indicated that in NO3--deficient media, mutant all3983::C.CE2 grew at a slightly lower rate than the wild type. The expression of all3983 was up-regulated in another mutant alr2269::C.CE2. Removal of individual nutrient element from BG-11 showed that the regulation of all3983 was mainly dependent on the Ca2+ concentration. All these showed that outer membrane proteins of Alr2269 and All3983 probably played an important role in Ca2+ uptake.   The main conclusions of this study are as follows: (1) Outer membrane from the Anabaena PCC 7120 could be isolated efficiently by aqueous polymer two-phase partitioning in combination with sucrose density gradient ultra-centrifugation. (2) Five of eight outer membrane proteins involved in iron uptake were up-regulated by iron deprivation, and in transcriptional level the regulation of these genes appeared to be complementary with each other. (3) Alr2269 and All3983 were probably involved in Ca2+ uptake.
Document Type学位论文
Recommended Citation
GB/T 7714
董妍玲. 鱼腥藻 PCC7120外膜蛋白的鉴定及功能研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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