【作者】 高春芳；

【导师】 吴庆余；

【作者基本信息】 清华大学 ， 生物学， 2012， 博士

【摘要】 Chlorella protothecoides既可以自养生长也可以异养生长，异养细胞油脂含量达到50%，同时细胞密度可提高20倍以上。C. protothecoides的高产油特性使其适宜于制备生物柴油。为建立C. protothecoides分子研究平台并探索进一步提高微藻产油率的方法，本研究在优化微藻培养生产生物柴油的基础上，进行C.protothecoides基因组测序并对自养和异养藻的全蛋白做差异表达分析。全基因组测序采用Solexa Paird-End、Solexa Mate-Pair和454三种方法，过滤后序列的总覆盖度为120x。最终拼装得到406个Scaffold，N50为24，全长22.9Mb。显示三个主要的特点：第一，基因组小，基因组比近缘藻Chlorella variabilisNC64A小一倍，基因数目7,039，在已测序绿藻中最少。第二，发现一类在小球藻属及近缘藻中独立进化而来的编码己糖转运蛋白的基因，C. protothecoides中有三个，可能参与异养细胞对糖信号的感应及葡萄糖的吸收。第三，C. protothecoides是唯一缺少编码硝酸盐转运蛋白基因的真核绿藻，但编码氨基酸转运蛋白的基因很丰富，显示出对不同氮源的偏好性。对自养和异养C. protothecoides的全细胞蛋白做质谱鉴定，共鉴定到1,931个蛋白，占总预测蛋白的27.4%，其中异养细胞上调蛋白206个，下调288个。对差异表达蛋白的功能分析显示出代谢通路的变化，糖酵解、TCA循环和脂肪合成过程均增强，光合作用和碳固定显著降低。首先，说明异养细胞的能量来源从光合磷酸化转变为氧化磷酸化；其次，糖酵解的酶成为异养细胞含量最高的蛋白，较高的糖酵解能力为脂肪酸的合成提供前体物质，而脂肪合成的酶上调为导致油脂积累的直接原因。另外，发现异养状态下脱落酸（ABA）信号转导蛋白表达上调，说明ABA在异养细胞中起到很重要的调控作用。本研究建立了C. protothecoides的基因组和蛋白质组数据库，为分子研究奠定了基础。同时，揭示出C. protothecoides基因组的特点并阐述异养细胞代谢途径的变化及脂肪积累的机理，分析得到一些可能的调控蛋白及信号，为进一步研究微藻产油的机制提供了新的思路。更多还原

【Abstract】 Chlorella protothecoides can grow in both autotrophic and heterotrophic conditions.In heterotrophic cells, the oil content reached to50%, the cell density was increasedmore than20-fold. The high oil yield of C. protothecoides makes it an importantresource for biodeisel production. In order to eatablish the platform for molecularresearch and explore methods to improve oil production of microalgae, we sequencedthe genome of C. protothecoides and investigated the proteomic changes betweenautotrophic and heterotrophic cells.The genome of C. protothecoides was sequenced by Solexa Paird-End, Solexa MatePair and Roche454method, the coverage of the clean data was120x. After assembly406scaffolds (>100bp) were obtained, and the N50was24. The size of the genome is22.9Mb, and7.039genes are predicted. By comparative analysis of the genome, threemain characteristics were found. Firstly, the C. protothecoides has a small genome insize, and about half of what its close relative Chlorella variabilis NC64A has.Furthermore, it encodes the fewest number of genes among all sequenced green algae,and the decrease of total gene is partly because the decrease of genes with the similarfunction. Secondly, a family of hexose/H+cotransporter that restricts to Chlorella wasfound, and there are three such genes in C. protothecoides, which may participated inthe transform of glucose across the plasma membrane. Thirdly, C. protothecoides is theonly eukaryotic green algae that lack nitrate transporter, but represented a biaseddistribution of amino acid transporters, indicating that C. protothecoides prefer to useamino acid as nitrogen source.The proteins of autotrophic and heterotrophic C. protothecoides were identified byLC/MS/MS.1,931proteins were identified which accounted for27.4%of the totalpredicted proteins. Compared with autotrophic cells,206proteins were up regulatedand288proteins were down regulated. The functional analysis of the differentlyexpressed proteins revealed the changes of the metabolic pathways. The process ofglycolysis, TCA cycle and the synthesis of oil are all enhanced, meanwhile, thephotosynthesis and carbon fixation were inhibited. The results illustrated the transitionof energy biosythesis from photophosphorylation to oxidative phosphorylation in heterotrophic cells. The enzymes in glycolysis are the most abundant proteins inheterotrphic cells. High glycolytic capacity ensured the cell to meet the demand ofprecursors for fatty acid synthesis, and the increase of the fatty acid synthesis enzymesdirectly lead to oil accumulation. The proteins of PP2C and SnRK2in abscisic acid(ABA) signal transduction were also upregulated in heterotrophic cells. ABA may playan important role in heterotrophic growth and oil accumulation.This study established the molecular research platform of C. protothecoides throughthe completement of genomic sequencing and proteomic research. Meantime, the studyrevealed the characteristics of C. protothecoides genome, and the changes of metabolicpathways in autotrophic and heterotrophic cells were illustrated. The results indicatedthe mechanism of oil accumulation in heterotrophic cells. Moreover, the possiblesignal transduction and regulatory proteins were found. All these provide new ideas forfurther research in mechanism of microalgal oil production.更多还原