Genome-wide survey and expression analysis of Chlamydomonas reinhardtii U-box E3 ubiquitin ligases (CrPUBs) reveal a functional lipid metabolism module.
Genome-wide survey and expression analysis of Chlamydomonas reinhardtii U-box E3 ubiquitin ligases (CrPUBs) reveal a functional lipid metabolism module.
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DOI：
10.1371/journal.pone.0122600
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Deng X
中科院分区：
文献类型：
--
作者：
Luo Q;Li Y;Wang W;Fei X;Deng X
E3泛素连接酶决定了泛素化的底物特异性。植物U-box(PUB)E3连接酶具有典型的70个氨基酸的U-box结构域，参与植物的发育过程和环境响应。到目前为止，已在拟南芥中鉴定出PUB蛋白，在水稻中已鉴定出77个PUB蛋白。然而，对模式微藻莱茵衣藻中U-box基因的详细研究还缺乏。在这里，我们提出了一个全面的分析编码U-box家族蛋白的基因。经BLASTP分析，在莱茵梭菌基因组序列中鉴定出30个全长U-box基因。对CrPUB基因进行生物信息学分析，以确定每个成员的系统发育关系、染色体位置和基因结构。已鉴定的30个CrPUB蛋白分为3个不同的亚家族，这些蛋白的基因在14条染色体上分布不均。此外，对氮饥饿条件下30个CrPUB基因丰度的实时定量RT-PCR或半定量RT-PCR分析表明，18个CrPUB基因在N饥饿条件下被诱导，7个基因在贫氮环境中受到抑制。我们选择了5个在无氮条件下表现出明显表达变化的CrPUB基因进行RNAi实验，并检测了这些基因沉默的转基因菌株的含油量。在N饥饿下表达下调的CrPUB5和CrPUB14的沉默分别诱导了9.8%-45.0%和14.4%-61.8%的脂肪积累。相反，在无氮条件下显著上调的CrPUB11、CrPUB23和CrPUB28的沉默使脂肪含量分别降低了5.5%-27.8%、8.1%-27.3%和6.6%-27.9%。这些结果为该基因家族的鉴定和功能分析提供了有用的参考，也为微藻脂代谢研究提供了基础信息。
E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.
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