激光显微切割条斑紫菜叶状体的参数优化  

Establishment of Laser Microdissection on Pyropia yezoensis Thallus

作　　者：曹逸飞 李小姣 曲伟华 茅云翔[1,2] 杜国英 CAO Yi-Fei;LI Xiao-Jiao;QU Wei-Hua;MAO Yun-Xiang;DU Guo-Ying(College of Marine Life Sciences,Ocean University of China,Qingdao 266033,China;College of Fisheries and Life Science,Hainan Tropical Ocean University,Sanya 572022,China)

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]海南热带海洋学院水产与生命学院,海南三亚572022

出　　处：《中国海洋大学学报（自然科学版）》2021年第1期51-58,共8页Periodical of Ocean University of China

基　　金：山东省海洋科学技术基金项目(2018SDKJ0302-4);国家重点研究发展计划项目(2018YFC1406704);农业部现代农业人才支持项目资助。

摘　　要：本研究以经济红藻条斑紫菜(Pyropia yezoensis)为研究对象,应用激光显微切割技术(Laser Microdissection,LMD)分离不同时期、不同部位、不同组织紫菜叶状体细胞团。统计细胞团中成活细胞比例,测定成活率,并应用叶绿素荧光成像法测定成活细胞光合活性,建立适合不同类型细胞的激光显微切割流程。结果表明:(1)整个切割流程需要在30 min内完成,不同材料成活率由高到低为:精子囊、成熟紫菜顶部、紫菜幼苗基部、成熟紫菜基部、幼苗顶基部,同株紫菜相同区域的中央与边缘细胞切割后成活率相近;(2)显微切割获得的紫菜细胞团的各项叶绿素荧光参数中,最大光量子传递效率Fv/Fm、实际光量子传递效率Y(Ⅱ)、光化学淬灭qP下降、非光化学淬灭qN上升,其中Fv/Fm、Y(Ⅱ)变动均高于其他参数,可作为指示切割细胞团光合能力的敏感指标;(3)不同时期紫菜适用的切割技术不同:成熟条斑紫菜在10、20、63倍镜下切割的直径100~300μm细胞团,幼苗在6.3、10、20倍镜下切割的直径150~300μm细胞团,成活率高且具有较高的光合活性。本研究针对条斑紫菜不同材料摸索形成适宜的切割技术参数,最少可从100μm直径细胞团(约20细胞)中获取1~6个成活细胞,为细胞的精确分离提供了新的技术手段。This study employed laser microdissection(LMD)technology to dissect different type cells of Pyropia yezoensis thallus.To establish specific LMD protocol of certain cell type,we calculated the survival rates of cell clusters and measure the chlorophyll flourescence of cell clusters to assess photosynthetic activity.The results showed that(1)The whole LMD process needed to be completed within 30 min;the survival rate of dissected cells from high to low followed the sequence of spermatangium cells,mature top cells,seedling base cells,mature base cells,and seedling base cells;the central and marginal cells had similar survival rates;(2)All tested chlorophyll fluorescence parameters of P.yezoensis cell cluster,Fv/Fm,Y(Ⅱ),qN and qP,changed after cutting by LMD.Among these parameters,Fv/Fm and Y(Ⅱ)varied more largely than other parameters,which can be used as a sensitive indicator to indicate the photosynthetic capacity of cell cluster;(3)Different cutting parameters were suitable for different stages of P.yezoensis the diameter of mature cells in 10,20 and 63 lens was 100~200μm,and the diameter of seedlings in 6.3 and 10 lens was 300μm.This study provides a feasible dissection protocol,at least 1~6 living cells can be dissection from 100μm cluster(about 20 cells).It provides a new method for the precise separation of P.yezoensis and would be benefit for follow-up research.

关 键 词：条斑紫菜 激光显微切割 细胞分离 叶绿素荧光成像技术 光合活性 

分 类 号：Q949.29[生物学—植物学]