丛粒藻细胞的透射电镜观察和rbcL、18SrRNA序列分析

摘要

采用透射电镜对丛粒藻（ Botryococcus braunii）藻株AGB-Bb02和AGB-Bb03进行了亚显微结构观察，以rbcL和18S rRNA序列为目标基因进行克隆，结合研究从GenBank获取的黄藻纲、绿藻纲和共球藻纲13株相关微藻的rbcL、18S rRNA基因序列，运用Clustal X 1.8和MEGA4.0软件进行分析，用邻接法构建了系统发育树，并计算bootstrap值以评估其可靠性。结果显示，丛粒藻细胞由多层细胞壁包被，细胞内壁和外壁的腔隙中含有烃类物质，独特而发达的内质网膜系统与细胞质膜部分相连，叶绿体基质类囊体平行紧贴、无基粒结构，脂滴的分布与内质网和叶绿体相关； AGB-Bb02和AGB-Bb03的rbcL和18 S rRNA序列存在差异，系统发育树显示丛粒藻藻株与共球藻纲的微藻聚成一个单系群，并获得bootstrap值的高度支持。研究表明，丛粒藻烃类主要储存于细胞外壁中，内质网的产物可经由膜连接直接输送至邻近的细胞质膜，再分泌到细胞壁中存储；丛粒藻的分类学地位应归属共球藻纲。%The ultrathin sections of Botryococcus braunii AGB-Bb02 and AGB-Bb03 were observed by transmission electron microscope.The target rbcL and 18S rRNA genes were cloned respectively by PCR from total DNA and sequenced bidirec-tionally.Based on the rbcL and 18S rRNA genes, evolutionary relation among AGB-Bb02, AGB-Bb03 and other 13 rela-tive samples containing members of the Xanthophyceae , Chlorophyceae and Trebouxiophyceae obtained from GenBank was studied.Clustal X 1.8 and MEGA4.0 software were used to align and analyze those sequences .A neighbor-joining tree was constructed from this alignment for phylogenetic relationship and taxonomic placement analyses of B.branu ii, and boot-strap analysis was carried out to evaluate the statistical reliability .The results showed that each cell of B.braunii was sur-rounded by multi-layer cell wall filled with hydrocarbons in the compartments .The unique and well-developed endoplasmic reticulum ( ER) system was partially connected with the plasma membrane .Stroma thylakoids parallel and closely arranged in the chloroplast with no grana .The distribution of lipid droplets in the cell were associated with envelope of the ER and chloroplast.The rbcL, 18S rRNA sequences varied between AGB-Bb02 and AGB-Bb03.The phylogenetic tree revealed that the two isolates of B.braunii were clustered into a monophyletic clade within the Trebouxiophyceae with high bootstrap support .The research indicated that hydrocarbons were primarily stored in the extracellular cell wall , and products synthe-sized in the ER could be transported to the adjacent plasma membrane and then secreted into the cell wall for accumulation . 18S rRNA phylogenetic analysis sustained the findings of rbcL analysis .The analysis of the molecular study strongly led to the conclusion that B.braunii should belong to the Trebouxiophyceae class .