【作者】 王建林；

【导师】 陈思礼； 赵开弘；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 2004年Shen G等在Synechococcus PCC7002中辩别出一组裂合酶编码基因cpcS、cpcT、cpcU和cpcV,认为这些基因所编码的蛋白质能够催化藻蓝色素和藻蓝蛋白β亚基的结合。本实验通过同源性分析软件在鱼腥藻基因组中获取了cpcS另一个同源编码基因all5292和cpcT的同源编码基因alr0647。通过all5292和alr0647两个基因的克隆和两者所编码蛋白产物的表达,初步获取了目的基因片段并对其表达产物分子量的大小进行了鉴定。为了研究二者所编码蛋白表达产物是否具有与其同源蛋白——裂合酶相同或相似的催化色素和脱辅基蛋白共价结合的活性,本实验构建了基因表达产物不携带His-tag标记的重组表达质粒pETDuet-all5292和pCDFDuet-alr0647,然后在大肠杆菌细胞内对PCB和藻蓝蛋白(藻红蓝蛋白)β亚基进行了异源体内重组,并对重组产物进行了光谱测定。实验结果显示,其编码的蛋白质产物All5292和Alr0647并没有催化PCB结合到藻蓝蛋白或藻红蓝蛋白β亚基上的功能。随后,通过SDS-PAGE和Western blot技术对异源体内重组系统各表达组分进行了检测和验证。结果发现,cpcB、pecB、ho1、pcyA、all5292和alr0647各基因均在相应的异源体内重组系统得到表达。只不过cpcB、pecB、ho1、pcyA和all5292基因的表达产物存在与异源体内细胞上清中,而alr0647基因的表达产物却存在与异源体内细胞沉淀中。由此可以确定,各体内重组系统细胞中不存在因元件缺失而导致没有藻胆蛋白生成,这两个基因编码的裂合酶同系物并不具备与CpcS和CpcT相同或者相似的催化功能。另一方面,本文对all5292和alr0647这两个基因在鱼腥藻中的表达情况进行了初步研究。通过对处于对数生长期生长的鱼腥藻进行正常生理条件和缺氮条件下的对比培养,然后在48 h后收集细胞并提取RNA。RT-PCR实验显示,这两个基因的表达与培养基中有无氮源有一定关联。二者均在经过缺氮处理后的蓝藻进行了转录表达,而在正常生理条件下培养的蓝藻中却没有发现这两个基因的转录产物存在。更多还原

【Abstract】 Recently, a group of four genes (cpcS, cpcT, cpcU, and cpcV) has been identified in Synechococcus sp. PCC7002 that code for lyases that attach PCB to cysteine-84 (consensus numbering) of theβ-subunits of CPC and possibly allophycocyanin, homologous genes are also found in other cyanobacteria[1]. Genes all5292 and alr0647 in the cyanobacterium, Anabaena (Nostoc) PCC7120, are homologous to the biliprotein lyase cpcS and cpcT. The functions of the encoded proteins were tested in vitro, and in a heterologous E. coli system with plasmids conferring biosynthesis of the phycocyanobilin (PCB) chromophore and of the acceptor proteins,β-phycoerythrocyanin (PecB) orβ-phycocyanin(CpcB). The reconstituted products were determined with absorption spectra and fluorescence spectra. The resuLts showed that no lyase activity was found for the lyase homologues, All5292 and Alr0647.Subsequently, synthesis of the desired proteins was checked by SDS-PAGE and Western blot. The resuLts showed that all genes, including all5292 and alr0647, were successfuL expressed in every heterologous E. coli system. And in this process, we also found that All5292 is soluble but the other is insoluble in a heterologous E. coli system.In this work, the expression of genes all5292 and alr0647 from Anabaena sp. PCC 7120 was studied by reverse transcription PCR. RT-PCR analysis showed that all5292 and alr0647 are transcribed when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition. But these observations require more detailed investigations because it is conceivable that both homologues are present during transients, or under other physiological conditions.更多还原