东海原甲藻基因表达特征研究
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摘要
尽管东海原甲藻(Prorocentrum donghaiense)的命名仍存在分歧,但它是我国特有的赤潮藻种,最近几年已在长江口和舟山群岛产生大规模的赤潮,造成了重大的经济损失和给生态带来了重大的影响,由此引起人们极大的关注。
进年来,有关它的生活史,生态,分类,形态等研究很多,但其分子生物学研究却非常少。为了在东海原甲藻中开展相关的分子遗传学研究,我们构建了人工培养状态下的东海原甲藻对数生长期细胞的cDNA文库。从随机选取的700个克隆获得了565条可用的序列。本论文报道了从这些序列中鉴定的功能基因,尤其是那些与细胞程序化死亡相关的基因。另外,我们还对线粒体细胞色素b基因及细胞色素氧化酶亚基I基因进行了克隆与分析。
在东海原甲藻的cDNA文库构建中,使用UNIQ-10 Trizol Total RNA Preparation Kit来进行总RNA的提取,将cDNA Synthesis Kit和PCR cDNA Library Kit组合使用进行cDNA的扩增,最后,将cDNA与pMD 18-T载体连接后转化到大肠杆菌JM109中。
鉴定功能基因的一个比较恰当的途径是表达序列标签分析。实际上,这样的分析已经在许多真核生物中进行,被认为在某中程度上发挥与全基因组测序相同的作用。表达序列标签就是一段cDNA序列,能定义这些cDNA,但因为是单方向测序产物,不仅节约成本,也能获得与全长基因相似的信息。它们除了作为基因标签,也能用来比较转录物集合,做DNA芯片,尤其是鉴定功能基因。我们从东海原甲藻的cDNA文库中随机挑取克隆进行测序,得到565条EST这些序列可分为272个单一序列和36个重叠群(308个EST组)。在蛋白数据库中比较发现23组与已知功能蛋白相似的克隆,其中的大部分与能量代谢、转录/翻译及光合作用有关。
有9组与甲藻中已知蛋白序列相匹配。另有14组与其它生物体中的已知蛋白序列相匹配。特别重要的是有两组EST分别与两个细胞程序化死亡的关键蛋白同缘,它们分别是半胱氨酰天冬氨酸特异蛋白酶(cysteine proteases)和分裂细胞
Although its scientific name is in debate, Prorocentrum donghaiense, one of dinoflagellates, has caused most frequent large-scale red tides in Changjiang River Estuary and Zhoushan Mountain Archipelago in recent years, bringing us tremendous economic losses and local environments serious ecological impacts.
Studies of dinoflagellate are of importance for both their biology and our understanding of red tide process. Currently, very rich publications concerning the life history, ecology, taxonomy and morphology of P. donghaiense are available. In contrast, molecular biological studies of P. donghaiense are very scarce.
In order to initiate the molecular biological research of P. donghaiense, a cDNA library was constructed for the artificial culture of this species at exponential growth phase. The first batch of 565 usable sequencing reads was generated from 700 clones randomly selected and sequenced. Here our trial identification of functional genes of P. donghaiense from these reads with emphasis on those involved in cell proliferation and death were reported. In addition, mitochondrial cob and cox1 gene fragments of P. donghaiense were cloned and sequenced.
In the cDNA library construction, total RNA was extracted by UNIQ-10 Trizol RNA Preparation Kit, and cDNAs were synthesized and amplified by cDNA Synthesis Kit and PCR cDNA Library Kit. Finally, the ligated cDNAs with pMD 18-T vector were transducted into E.coli JM 109. The constructed cDNA library was used to the latter analysis of EST.
An appropriate approach of identifying functional genes of a species is to generate and analyze expressed sequence tags (EST). EST analysis has been implemented in functional genomic researches of a wide range of eukaryotes. EST are short cDNA sequences that serve as not only gene tags, but also the basis of multiple uses such as profiling transcripts, making cDNA arrays and most importantly, identifying functional genes. A cDNA library was constructed for P. donghaiense at exponential growth phase, and 565 usable sequencing reads were obtained from 700 clones selected randomly. Messenger RNA corresponding reads were clustered into 36 contigs and 272 singletons (EST groups). Comparisons using BlastX program
进年来,有关它的生活史,生态,分类,形态等研究很多,但其分子生物学研究却非常少。为了在东海原甲藻中开展相关的分子遗传学研究,我们构建了人工培养状态下的东海原甲藻对数生长期细胞的cDNA文库。从随机选取的700个克隆获得了565条可用的序列。本论文报道了从这些序列中鉴定的功能基因,尤其是那些与细胞程序化死亡相关的基因。另外,我们还对线粒体细胞色素b基因及细胞色素氧化酶亚基I基因进行了克隆与分析。
在东海原甲藻的cDNA文库构建中,使用UNIQ-10 Trizol Total RNA Preparation Kit来进行总RNA的提取,将cDNA Synthesis Kit和PCR cDNA Library Kit组合使用进行cDNA的扩增,最后,将cDNA与pMD 18-T载体连接后转化到大肠杆菌JM109中。
鉴定功能基因的一个比较恰当的途径是表达序列标签分析。实际上,这样的分析已经在许多真核生物中进行,被认为在某中程度上发挥与全基因组测序相同的作用。表达序列标签就是一段cDNA序列,能定义这些cDNA,但因为是单方向测序产物,不仅节约成本,也能获得与全长基因相似的信息。它们除了作为基因标签,也能用来比较转录物集合,做DNA芯片,尤其是鉴定功能基因。我们从东海原甲藻的cDNA文库中随机挑取克隆进行测序,得到565条EST这些序列可分为272个单一序列和36个重叠群(308个EST组)。在蛋白数据库中比较发现23组与已知功能蛋白相似的克隆,其中的大部分与能量代谢、转录/翻译及光合作用有关。
有9组与甲藻中已知蛋白序列相匹配。另有14组与其它生物体中的已知蛋白序列相匹配。特别重要的是有两组EST分别与两个细胞程序化死亡的关键蛋白同缘,它们分别是半胱氨酰天冬氨酸特异蛋白酶(cysteine proteases)和分裂细胞
Although its scientific name is in debate, Prorocentrum donghaiense, one of dinoflagellates, has caused most frequent large-scale red tides in Changjiang River Estuary and Zhoushan Mountain Archipelago in recent years, bringing us tremendous economic losses and local environments serious ecological impacts.
Studies of dinoflagellate are of importance for both their biology and our understanding of red tide process. Currently, very rich publications concerning the life history, ecology, taxonomy and morphology of P. donghaiense are available. In contrast, molecular biological studies of P. donghaiense are very scarce.
In order to initiate the molecular biological research of P. donghaiense, a cDNA library was constructed for the artificial culture of this species at exponential growth phase. The first batch of 565 usable sequencing reads was generated from 700 clones randomly selected and sequenced. Here our trial identification of functional genes of P. donghaiense from these reads with emphasis on those involved in cell proliferation and death were reported. In addition, mitochondrial cob and cox1 gene fragments of P. donghaiense were cloned and sequenced.
In the cDNA library construction, total RNA was extracted by UNIQ-10 Trizol RNA Preparation Kit, and cDNAs were synthesized and amplified by cDNA Synthesis Kit and PCR cDNA Library Kit. Finally, the ligated cDNAs with pMD 18-T vector were transducted into E.coli JM 109. The constructed cDNA library was used to the latter analysis of EST.
An appropriate approach of identifying functional genes of a species is to generate and analyze expressed sequence tags (EST). EST analysis has been implemented in functional genomic researches of a wide range of eukaryotes. EST are short cDNA sequences that serve as not only gene tags, but also the basis of multiple uses such as profiling transcripts, making cDNA arrays and most importantly, identifying functional genes. A cDNA library was constructed for P. donghaiense at exponential growth phase, and 565 usable sequencing reads were obtained from 700 clones selected randomly. Messenger RNA corresponding reads were clustered into 36 contigs and 272 singletons (EST groups). Comparisons using BlastX program
引文
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