利用高通量测序技术检测在不同基因表达水平和不同环境中蛋白编码基因的点突变对适应度的影响

已经被研究了超过半个世纪的蛋白质序列进化速率，是重构进化历史和构建演化机制的一个关键因素。随着基因组数据的增加，包括我们前期工作的研究发现，基因的表达水平，比一直被广泛认为最能限制蛋白质序列进化速率的蛋白质功能重要性，起着更重要的决定性作用，并提出了基因表达水平影响蛋白质序列进化速率的多种假说。由于部分假说一直缺乏直接实验证据的支持，本研究计划通过高通量测序技术，对多个蛋白质编码基因在不同的表达水平以及不同的培养环境下，检测涉及所有可能点突变的影响酵母生长速率的遗传效应。该研究将全面测定特定蛋白编码基因的点突变适应度景观；提供蛋白编码基因进化模式的前瞻性预测；鉴定基因表达量和基因功能重要性对适应度景观的影响；检验决定蛋白质进化速率的各种分子机制模型的正确性和适用范围。该研究结果将实现蛋白质序列进化模式的研究创新，并为研究病原生物的流行性与耐药性的演化过程提供新的技术与理论基础。

The rate of protein sequence evolution has been studied for half a century and is critical to the reconstruction of evolutionary history and mechanisms. Following the increased availability of genomic data, the functional importance of a protein, widely thought to approximate the level of functional constraint, has only a minor role on the protein evolutionary rate, whereas protein expression level is found to be a major determinant. Subsequent studies by us and others identified multiple reasons behind the impact of expression level on the rate of protein sequence evolution. Because some of the above studies have been scrutinized by direct experimental tests, we plan to synthesize all single mutant sequences of specific proteins, and use High Throughput Sequencing to measure the genetic effects of these mutations on yeast growth rate under various expression levels of the mutated gene and different environments. This study will (1) give rise to a comprehensive single mutant fitness landscape of protein coding genes; (2) facilitate prospective prediction of protein sequence evolution through single point mutations; (3) reveal the dependency of fitness landscape on the expression level and functional importance of the mutated gene; (4) detect the validity and applicability of the mechanical hypotheses for the determinant of protein sequence evolutionary rate. Results of this research will improve our understandings of the molecular mechanisms governing protein sequence evolution, and establish novel strategies and in studies of protein evolution, which should be widely applicable in related researches such as the prevalence and drug-resistance of evolving pathogens.