【作者】 张东；

【导师】 隋正红；

【作者基本信息】 中国海洋大学 ， 遗传学， 2010， 硕士

【摘要】 2007年春季和秋季对北黄海水域(120°34’E-124°03’E,32°00’ N-39°23’ N)微型浮游生物丰度进行调查,其中春季78个站位,秋季80个站位,对微型浮游生物丰度在水平分布,垂昼夜变化和种类组成进行了分析。春季北黄海水域微型浮游生物丰度变化范围为(5.01cells/ml～91.87cells/ml)平均值为29.50 cells/ml.秋季变化范围为(4.70 cells/ml～151.73 cells/ml),平均值为34.15 cells/ml;在水平分布上,山东半岛,辽东半岛的近岸和北黄海暖流经过的海域丰度较高,达到90cells/ml。种类组成上,两个季节主要为硅藻甲藻组成,也有少数的蓝藻、绿藻出现。在昼夜变化上,春季和秋季表现出相似的趋势,表层和10米层在中午12：00-14：00出现峰值,30米层和底层水深的丰度分布相对较均匀,变化不大,没有明显的峰值出现。利用多元相关分析可知,春季微型浮游生物丰度与环境参数有相关性但并不显著,而秋季微型浮游生物丰度与温度呈显著的正相关,与水深呈显著的负相关,与盐度相关性不显著。采用96孔板有限稀释分离培养的方法,将采自青岛太平角的1株微型藻类纯化,对其进行光镜和电镜观察,与纤细角毛藻(Chaetoceros gracilis)在光镜下无法区分,电镜下只有角毛结构稍有差异,故从形态上初步鉴定为角毛藻属(Chaetoceros sp.qd)藻类。角毛每隔0.5μm左右有一互生的刺状突起,顶端呈锥状,其网状骨架主干呈螺旋状,延伸至顶端,骨架之间有横隔相连,形成小孔结构。扩增其18S核糖体基因(18SrDNA),由测序结果得知C.sp.qd的18S序列部分长度为1623bp,经BLAST比对后得到C.sp.qd与Chaetoceros gracilis strain UTEXLB 2375的18SrDNA相似值最高,达到99%,确定为角毛藻属的微藻。然后扩增5.8S核糖体基因(5.8SrDNA)和转录单元内间隔区(Internal Transcribed Spacer, ITS)序列,5.8SrDNA-ITS的序列总长度为837bp。比对后可知此种5.8S区与角毛藻属相似度很高,达到99%,但ITS区差别较大。系统进化树以及遗传距离分析显示此藻种与其他角毛藻种有显著差异,在进化树中单独分支,结合形态学分析将C.sp.qd鉴定为一角毛藻新种.采用96孔板有限稀释分离培养的方法,将采自青岛太平角的1株微型藻类纯化,对其进行光镜和电镜观察,与艾伦海链藻(Thalassiosira allenii)和具毛海链藻(Thalassiosira hispida)在光镜下无法区分,在电镜下与艾伦海链藻壳缘支持突数量上不同,与具毛海链藻在孔纹数量上也有差异。故从形态上初步鉴定为海链藻属(Thalassiosira sp.qd)藻类。扩增其18S核糖体基因(18SrDNA),由测序结果得知T.sp.qd的18S序列部分长度为1640bp,使用硅藻门13个不同属的18S与本种构建的系统发育树可知,本种与海链藻属的藻类聚群,节点支持率为98,故确定为海链藻属的微藻。然后扩增5.8S核糖体基因(5.8SrDNA)和转录单元内间隔区(Internal Transcribed Spacer, ITS)序列,5.8SrDNA-ITS的序列总长度为762bp。比对后可知此种5.8S区与海链藻属相似度很高,达到99%,但ITS区差别较大。在属内种间的系统进化树以及遗传距离分析显示此藻种与其他海链藻种有显著差异,在进化树中单独分支,显示此藻种可能为一新种,由于缺乏与之相近的两种藻的5.8S-ITS序列,无法从分子生物学方面验证我们的推断。但本文提供此藻种较详细的形态学和分子生物学资料,以期为将来准确鉴定此藻种奠定基础。更多还原

【Abstract】 To study the distribution and daily variation of nanoplankton in the northern Yellow Sea, nanoplankton abundance in spring and autumn was determined Nano-plankton Direct Count in the northern Yellow Sea was in the range between 5.01 cells/ml and 91.87 cells/ml at the 78 sampling stations, with a mean of 29.50 cells/ml in spring. Direct Count in the northern Yellow Sea was in the range between 4.70 cells/ml and 151.73 cells/ml at the 80 sampling stations, with a mean of 34.15 cells/ml in autumn. Higher nanoplankton abundance was found at the 10m,20m layers in offshore waters of the Liaodong Peninsular,Shandong Peninsular and the way of Bohai warm current than those in other waters in both seasons. The results show that diatoms and dinoflagellates are the major components of phytoplankton community in the survey region, with cynobacteria, green algae being also common found in some stations.The data of both season showed the similar trend that the diurnal variation of nanoplankton abundance of the surface and the 10m layer was more significant than those of the 30 m and the bottom, while the variation of the abundance of the bottom layer was small. According to multicorrelation analysis, nanoplankton abundance in spring had correlation with the environmental parameters but not significant, while in autumn the abundance had significant positive correlation with the temperature, significant nagitive correlation with the depth, no significant correlation with the salinity. A nanoplanktic strain was isolated from Taiping Corner of Qingdao through limited dilution method on 96 multiwell cell culture plate. It was impossible to distinguish it from Chaetoceros gracilis in light microscope and there were few differences in the structure of flagellum in electronic microscope, so it was prelimin-ary identified as a Chaetoceros species (Chaetoceros sp.qd) based on morphological features.There was a alternative protuberance every 0.5μm on the flagellum whose top liked a wimble, reticular framework presented as helix structure which extended tothe top,there was diaphragm plate that linked the framework and shaped the hole structure. The 18SrDNA sequence was amplified, sequenced to be 1623bp long and had highest level similarity (99%) with strain UTEX LB 2375 of Chaetoceros gracilis retrived from GenBank database. The strain was ascribed to the Chaetoceros genus. The 5.8S rDNA and ITS sequence was further amplified which were 837bp, the BLAST analy-sis showed that Chaetoceros sp.qd had high level similarity with Chaetoceros at 99% in 5.8S region, but there were remarkable differences in ITS region. Phylogenic tree construction and genetic distance analysis indicated that the isolated Chaetoceros sp.qd diverged with other Chaetoceros species, formed a separate branch in the phylo-genic tree. Combined with morphological results, Chaetoceros sp.qd is suggested to be a new species of Chaetoceros.A nanoplanktic strain was isolated from Taiping Corner of Qingdao through limited dilution method on 96 multiwell cell culture plate. It was impossible to distinguish it from Thalassiosira allenii and Thalassiosira hispida in light microscope, in electronic microscope, there are many differences in the quantity of support enation between Thalassiosira sp.qd and Thalassiosira allenii, it also has many discrepancy in the quantity of pore texture between Thalassiosira sp.qd and Thalassiosira hispida, so it was preliminary identified as a Thalassiosira species (Thalassiosira sp.qd) based on morphological features. The 18SrDNA sequence was amplified, sequenced to be 1640bp long and had highest level similarity (98%) with strain UTEX LB 2375 of Thalassiosira angulata strain BEN02-35 retrived from GenBank database. The strain was ascribed to the Thalassiosira genus. The 5.8SrDNA and ITS sequence was further amplified which were 762bp, the BLAST analysis showed that Chaetoceros sp.qd had high level similarity with Thalassiosira at 99% in 5.8S region, but there were remarkable differences in ITS region. Phylogenic tree construction and genetic distance analysis indicated that the isolated Thalassiosira sp.qd diverged with other Thalassiosira species, formed a separate branch in the phylogenic tree, This suggested that Thalassiosira sp.qd was a new specie. However we can’t verified the deduction in molecular biology because lack of the 5.8S-ITS sequence. But we provide the detailed data in morphology and molecular biology, so it established the foundation to identificate the nanoplankton precisely in future.更多还原