【作者】 刘谊；

【导师】 乔代蓉；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 在很多真核生物线粒体和细菌中，复合体Ⅰ是呼吸链上的第一个酶。它将电子从NADH传递到辅酶O，同时伴随着质子跨膜的转移。变形菌门、革兰氏阳性菌、栖热菌／奇异球菌门、嗜热菌复合体Ⅰ的主要结构是已经知道的。细菌复合体Ⅰ一般主要由14个不同的亚基组成，代表了复合体Ⅰ的一个最小结构域。这14个亚基一共约有530 kDa。但是对于它们的功能知之甚少，可能与醌还原和质子转移有关。另外真核细胞线粒体复合体Ⅰ与细菌复合体Ⅰ的14个亚基同源，只是增加了29个亚基。其分子量约为1 MDa。与细菌复合体Ⅰ七个疏水亚基同源的线粒体复合体Ⅰ亚基都是由线粒体编码的。线粒体复合体Ⅰ其它亚基的功能就不清楚了。它们都是由核基因编码，在牛心和粗糙脉孢菌中，同源性也不高。它们也许是在14个亚基组成的最小结构域外围构建一个骨架，来避免活性氧带来的危害。另外两个亚基可能与生物合成有关。一个是酰基转运蛋白，另一个有40kDa，与NADPH紧密结合，属于还原酶／异构酶的异类家族。盐生杜氏藻(Dunaliella salina)是一种极其耐盐的单细胞绿藻，能在0.1-5.5mol／L NaCl条件下生存。其极强的耐盐能力决定了盐生杜氏藻的呼吸链需要提供更大的能量来满足自身的能量需求。线粒体复合体Ⅰ正是呼吸链的第一个关键酶，所以盐生杜氏藻的抗逆性就与复合体Ⅰ的活性有密切的关系。本文在已经构建了盐生杜氏藻cDNA文库的基础上，完成了以下研究内容：1、克隆盐生杜氏藻线粒体复合体Ⅰ19kD亚基。通过3’RACE和5’RACE得到全长基因。在3’RACE过程中，克隆到两条不同的完整3端序列。拼接得到两条完整19kD亚基cDNA序列，分别为682bp和727bp，编码两种不同的蛋白。同时在基因的非翻译区设计特异引物克隆全长。意外的是，RT-PCR得到另外两条不同的19kD亚基cDNA序列。大片段680bp，小片段1128bp。分别将这四条不同的cDNA序列由小到大依次命名为variant A，B，C和D。2、确定盐生杜氏藻线粒体复合体Ⅰ19kD亚基存在选择性剪切方式。利用在非翻译区设计的特异引物扩增到19kD亚基的基因组序列，确定盐生杜氏藻线粒体复合体Ⅰ19kD亚基是通过选择性剪切方式来调控。对基因组序列的分析可知19kD亚基的不同mRNA都是由同一基因组序列剪切而来。3、生物信息学分析。对盐生杜氏藻线粒体复合体Ⅰ19kD亚基基因及其推演出的蛋白质序列进行了相关的生物信息学分析，预测它们的功能，发现了一些特性。例如盐生杜氏藻的线粒体复合体Ⅰ19kD亚基与衣藻相应亚基同源性最高。4、盐生杜氏藻线粒体复合体Ⅰ19kD亚基在不同胁迫条件下mRNA转录水平差异的研究。通过荧光定量PCR的方法，表明所有的19kDmRNA都被氧、盐、鱼藤酮胁迫调控。5、测定盐生杜氏藻线粒体复合体Ⅰ在不同胁迫条件下的酶活。发现：线粒体复合体Ⅰ19kD亚基很可能与线粒体复合体Ⅰ的生物作用过程；盐生杜氏藻线粒体复合体Ⅰ对盐胁迫的损害具有较强的抵抗能力，盐胁迫通过氧胁迫来损伤盐生杜氏藻线粒体复合体Ⅰ的活性；盐生杜氏藻线粒体复合体Ⅰ为鱼藤酮敏感型，受鱼藤酮的特异抑制。通过对盐生杜氏藻线粒体复合体Ⅰ19kD亚基的克隆，第一次在海藻中发现该亚基是通过选择性剪切方式调控。同时通过利用荧光定量PCR的方法，进行初步研究。在mRNA水平发现了复合体Ⅰ19kD亚基与复合体Ⅰ的关系。为更进一步研究复合体Ⅰ的结构和功能奠定基础。更多还原

【Abstract】 The complexⅠis the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes.It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The primary structures of the complexⅠsubunits from several proteobacteria, Gram positive bacteria, members of the Thermus/Deinococcus group, and from the genus Aquifex are available now. The bacterial complex I, in general,is made up of 14 different subunits, representing a minimal structural form of a proton-pumping NADH:ubiquinone oxidoreductase. The 14 subunits sum up to a molecular mass of approx. 530 kDa. Little is known about their function, but they are most likely involved in quinone reduction and proton translocation.In addition to the homologues of the 14 bacterial complexⅠsubunits, the mitochondrial complexⅠof eukaryotes contains up to 29 additional proteins. They add up to a molecular mass of approx.1 MDa. The homologues of the seven hydrophobic bacterial subunits are mitochondrially encoded in all eukaryotes. The function of the additional subunits in the mitochondrial complexⅠis not clear.They are all nuclear-encoded and show a weak but distinct sequence similarity between bovine and Neurospora crassa. They might build a scaffold around the 14 minimal subunits preventing the high energy electrons from escaping the complex and forming reactive oxygen species. Two of the additional subunits appear to have a biosynthetic function.One is an acyl carrier protein, the other is a 40 kDa subunit with a tightly bound NADPH belonging to a heterogeneous family of reductases/isomerases.Dunaliella salina is a unicellular green alga which is unique in its ability to adapt to hypersaline environment, from 0.1 mol/L to 5.5mol/L. The character of hypersaline make that respiratory chain offer more energy for themselves. The complexⅠis the first and key of the respiratory chain complexes,so it is involved in resisting the stresses. Based on the construction of cDNA library in Dunaliella salina, in this study we got these results:Ⅰ. Cloning full length 19-kD subunit of ComplexⅠgene in Dunaliella salina. We also got the full length cDNA of this EST by 3’ RACE and 5’ RACE. During 3’RACE experiments of 19-kD subunit gene, two different full 3’ ends were amplified and sequenced. Thus, Dunaliella salina has at least 2 full length of the 19-kD subunit, 682 bp and 727 bp, respectively.And encoding two distinct protein of the 19-kD subunit. Therefore we designed the reverse primer from the 3’-UTR of the 19-kD subunit cDNA. The RT-PCR screening for the 19-kD subunit used Dunaliella salina cells RNA and the specific primers. Surprisingly, the RT-PCR result gave rise to two bands after agarose gel electrophoresis. In addition to the smaller fragment (680 bp) a larger fragment (1128 bp) was present. The four genes are named variants A,B,C and D of 19-kD subunit, respectively.Ⅱ. Make sure that 19-kD subunit of ComplexⅠgene in Dunaliella salina regulate by alternative splicing.The genomic DNA was amplified by PCR using a specific primer set corresponding to the 5’ and 3’ untranslated regions. The genomic DNA sequence of 19-kD subunit indicate that all the variant transcripts of 19-kD subunit are spliced by this genomic DNA sequence.Ⅲ. Bioinformatics analysis. The deduced proteins are analyed, and some features are found. For example, Analysis showed that these clones shared high identities with ComplexⅠsubunit from Chlamydomonas reinhardtii.Ⅳ. Short-term changes in 19-kD subunit of ComplexⅠgene mRNA levels corresponding to different stresses. This research was studyed by Real-Time Quantitative PCR. The results indicate that all the transcripts were down regulated under oxygen deficiency, salt treatment, and rotenone treatment.Ⅴ. Enzyme activity assay in different stresses. Found that 19-kD subunit may participate in the function of ComplexⅠ. ComplexⅠfrom Dunaliella salina can strongly resist salinity treatment, and NaCl stress damaged ComplexⅠvia oxidative stress. ComplexⅠfrom Dunaliella salina is rotenone-insensitive and specifically restrain by rotenone.We have cloned the gene of 19-kD subunit of ComplexⅠfrom Dunaliella salina. It is the first time to find that the complexⅠsubunit gene expresses four distinct mature transcripts by alternative splicing in algae. And used Real-Time Quantitative PCR analyse this phenomenon and found that the relation between 19-kD subunit and ComplexⅠfrom Dunaliella salina, it will be important to understand the structure and function of ComplexⅠ.更多还原