摘要
鱼腥藻PCC 7120是一种丝状蓝细菌,在培养基中缺失化合态氮源的条件下可分化形成异形胞执行固氮的功能。鱼腥藻PCC 7120的全基因组序列可从CyanoBase数据库中获得。T型裂合酶CpcT2(编码基因alr0647)和CpcT1(编码基因all5339)具有较高同源性,经实验室体外重组研究可知CpcT1可催化藻蓝色素PCB与半胱氨酸155位进行连接;而CpcT2无活性,即CpcT2无催化功能;但RT-PCR及western blot实验表明该基因在无氮情况下可以转录和翻译,从而通过进一步研究确定其为固氮基因。本研究的主要目的是通过对基因alr0647的定向敲除研究该基因的功能。
本研究经分子生物学方法通过基因敲除对alr0647进行缺失失活,从鱼腥藻PCC 7120总DNA中调取alr0647基因片段,将基因片段缺失并替换为壮观霉素(Spectinomycin,简称Sp)抗性基因片段,按照三亲本杂交的方法将构建好的质粒转入鱼腥藻PCC 7120,利用抗性筛选得到同源重组成功的阳性转化子。由于蓝藻基因组含有多个拷贝,经分子生物学方法检测,证明得到的菌株为部分同源双交换的突变株。
缺氮条件下从生长曲线等相关生理指标对突变株与野生株进行研究。结果表明,突变株的生长速度较野生株慢,但缺氮24小时后野生株和突变株均产生异形胞。由此可推测alr0647可能与生物固氮有关,但它的缺失并不会从根本上影响鱼腥藻PCC 7120的固氮功能。
通过对alr0647的启动子分析,该基因前500bp可能没有完全包含alr0647的启动子序列,需对基因前的序列加长来进一步确定启动子的位置。对基因alr0647的定位分析,已知启动子PpetE与alr0647和gfp(克隆于穿梭载体pHB912)融合质粒的构建部分已经完成。PpetE是鱼腥藻PCC 7120中的一段序列,其已被证实可以启动蓝藻中的目的基因,通过将重组质粒转入鱼腥藻PCC 7120后,缺氮条件下观察绿色荧光的位置,可对alr0647进行定位,为alr0647的固氮功能提供理论依据。因此对于alr0647的定位及启动子分析有待进一步研究。
Anabaena PCC 7120 is a filamentous cyanobacterium capable of both photosynthesis and dinitrogen fixation under aerobic conditions. Genome sequences of Anabaena PCC 7120 are available at the CyanoBase databank. alr0647 (CpcT2) and all5339 (CpcT1) are homologous to the lyase CpcT. CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine-155. No lyase activity was found for the lyase homologues CpcT2, but the gene coding the inactive homologues CpcT2 which was proved by RT-PCR and western blot can been transcribed and translated in N-starved Nostoc.
Through molecular biology technique, the gene alr0647 was amplified by PCR. alr0647 was deleted and replaced by an spectinomycin resistance cassette to generate plasmid, which were used to transform the Anabaena PCC 7120 wild-type strain by triparental mating.By resistance screening, the mutant-type strain with spectinomycin resistance was obtained. Because the genome of anabaena PCC 7120 is multicopy.
The mutants were examined for phenotypes after nitrogen depletion .The result showed that the mutant-type growed slower than the wild-type through the result of growth curve.But both mutant-type and wild-type can differentiate heterocysts after 24 hours. So we suppose that alr0647 should relate to the biological nitrogen fixation but not effect on the fuction of it radically.
Through analysis of the promotor of alr0647, the upstream 500bp of alr0647 didn’t contain any promoter.We should lengthen the sequence before alr0647 to confirm the localization of the promoter. PpetE from Anabaena PCC 7120 and the gfp from pHB912 were cloned respectively by doing polymerase chain reaction (PCR), and the PpetE was positioned upstreamof gfp. The PpetE-alr0647-gfp construct was integrated into the genome of Anabaena PCC 7120 via homologous recombinations. Observing whether the green fluorescence in the heterocyst or in the nutritive cell to locate the gene alr0647, which lay a foundation for the function of alr0647 is nitrogen fixation.
引文
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