微藻(Chlorella sorokiniana)的转录组分析:油脂生物合成相关的途径解析和基因挖掘  

De novotranscriptomic analysis of Chlorella sorokiniana:Pathway description and gene discovery for lipid production

作　　者：李林[1,2] 王钦宏[2] 杨海麟[1] 王武[1] 

机构地区：[1]江南大学,工业生物技术教育部重点实验室,江苏无锡214122 [2]中国科学院天津工业生物技术研究所,中国科学院系统微生物工程重点实验室,天津300308

出　　处：《微生物学报》2014年第9期1010-1021,共12页Acta Microbiologica Sinica

基　　金：国家"973项目"(微藻能源规模化制备的科学基础;2011CB200900);高等学校博士学科点专项科研基金(海洋微藻油脂合成氮源调控机制及信号应答网络研究;20100093120002)~~

摘　　要：【目的】为了构建和改造产油微藻(Chloralla sorokiniana),需要从基因组水平解析脂肪酸、三酰甘油、淀粉等生物大分子的合成和降解途径,并分析和确定其中的重要基因。【方法】本研究选取了初始氮浓度分别为KNO3∶8g/L和KNO3∶2 g/L的培养条件培养微藻C.sorokiniana,培养至84h收集藻细胞进行Illumina Hiseq2000双端测序,利用Trinity进行de novo拼接,得到的转录本通过Nr数据库、UniProtKB/Swiss-Prot数据库、COG数据库进行功能注释及分类,通过KEGG数据库进行相关代谢途径注释。最后利用RSEM计算每个转录本的RPKM值,并对相关代谢途径中的转录本的表达差异进行了初步分析。【结果】Illumina Hiseq2000双端测序共获得49M 100 bp的raw reads,de novo拼接得到49885个转录本,其长度范围在300bp到14100bp之间,N50为1941 bp。其中26479个转录本成功注释到功能,2357个转录本注释到了EC编号,利用这些转录本注释到207条代谢途径。在此基础上构建了C.sorokiniana的脂肪酸、三酰甘油、淀粉等生物大分子的合成及降解途径。并初步确定了代谢途径中基因的上调及下调水平。【结论】通过RNA-seq分析实现了对非模式藻株C.sorokiniana的基因组解析,在此基础上构建了脂肪酸、三酰甘油、淀粉的生物合成和降解途径及重要基因,所构建的代谢途径与模式藻株莱氏衣藻是一致的,并比较了相关途径中的基因的表达差异,这些信息有助于将来对微藻进行遗传改造提高其产油能力。[ Objective] The paucity of genomic information limits the metabolic engineering of non-model microalgae Chlorella sorokiniana. Our study aimed to elucidate the fatty acid, triacylglycerol and starch biosynthetic pathways in the microalgae C. sorokiniana based on de novo transcriptomic analysis. [ Methods ] We cultured C. sorokiniana with different nitrogen concentrations （KNO3： 8g/L and 2g/L） , then sequenced the transcriptomeusing Illumina Hiseq2000 platform. We used Trinity to de novo assemble the reads so as to obtain transcripts, aligned all the transcripts with Nr database, UniProtKB/Swiss-Prot database and COG database to annotate the function and classify using BLASTx algorithm, and assigned the transcript with metabolic pathway by aligning with KEGG database. Then we used RSEM to calculate FPKM value, and used it for preliminary analysis of different gene expression in the related pathways. [ Results] Over 49M high quality raw reads were produced with the length of 100bp, We used Trinity to assembled these reads into 49885 transcripts with an N50 of 1941bp, ranging from 300bp to 14100bp. 26479 transcripts were annotated through BLASTx similarity search, 2357 transcripts were assigned with EC number, and 207 metabolic pathways were assigned in total. Based on these analyses, we reconstructed the fatty acids, triacylglycerol and starch biosynthetic pathways in C. sorokiniana. We also identified preliminarily different geneexpression in the pathways. [ Conclusion] Using RNA-seq technology, we reconstructed the metabolic pathways involving in the fatty acid, triacylglycerol and starch biosynthesis in non-model microalgae C. sorokiniana without genomic data, which is consistent with those in model microalgae Chlamydomonas reinhardtii, and compared the gene expression level under different conditions. These information is very useful for the metabolic engineering of C. sorokiniana and other microalgae to enhance the production of lipids.

关 键 词：微藻 CHLORELLA sorokiniana 三酰甘油 淀粉 RNA-SEQ 

分 类 号：Q78[生物学—分子生物学]