链状亚历山大藻生长衰亡相关基因的筛选  

Screening of growth decline related genes from Alexandrium catenella

作　　者：仲洁[1] 隋正红[1] 王春燕 丁弘叶[1] 周伟[1] 张杨 

机构地区：[1]中国海洋大学海洋生物遗传育种教育部重点实验室,青岛266003 [2]山东嫁禾生化(沂南)有限公司,沂南276300 [3]青岛海底世界,青岛266003

出　　处：《生态学报》2012年第2期431-437,共7页Acta Ecologica Sinica

基　　金：高校博士点基金资助课题(20100132110007)

摘　　要：采用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术构建了衰亡期的链状亚历山大藻(Alexandriumcatenella)特异表达的cDNA文库。共获得800个克隆,利用巢式引物进行PCR筛选,最终确定阳性克隆556个。利用斑点杂交技术对这些阳性克隆进行差异筛选,获得差异表达克隆160个。测序后得到片段125个,经过归类,获得21种序列,其中6种在NCBI中经Blast,获得功能基因与其匹配,这些功能基因分别是CHK1类似检测点蛋白(checkpoint-like protein)、核酸外切酶复合体、谷氧还蛋白、Na+/K+ATPase、叶绿体中的一个开放阅读框和pG1蛋白,其他blast无同源序列,可能为新基因。推测链状亚历山大藻的衰亡过程可能涉及到DNA损伤、mRNA降解过程、氧化还原状态的变化、离子动态平衡的变化以及叶绿体一些生理状况的变化等过程。With worldwide industrialization, red tide events have been reported more frequently and their harm to humans has become increasingly severe. Although great efforts have been made to elucidate the ecology and biology of red tide events, studies of their molecular mechanisms are still limited, including those of the mechanisms involved in the decline phase of red tide. Alexandrium catenella is an important causative dinoflagellate associated with harmful algal blooms and paralytic shellfish poisoning. In this study, a growth decline specific eDNA library of A. catenella was constructed using suppression subtractive hybridization （SSH） technology to identify genes expressed in response to decreased growth of red tide algal cells as a primary step in elucidating the red tide process. Samples collected during the decline phase of growth were analyzed and compared with those collected during other phases of growth, which were considered to be controls. A total of 800 clones were obtained from the SSH library, and 556 positive clones were identified by nested PCR. Dot blot hybridization was conducted to screen differentially expressed genes from the positive clones, and a total of 160 differentially expressed clones were identified. After sequencing and classification, 21 sequences were obtained, six of which were found to be homologous with functional genes in the NCBI database. Specifically, these genes were homologous with CHK1 checkpoint-like protein, exosomal 3＇-5＇ exoribonuclease complex, glutaredoxin, Na^＋/ K^＋ ATPase, chloroplast gene and pG1 protein. The remaining sequences did not match any present functional genes in the database and were thereforeassumed to be novel. Checkpoint is a biochemical process that detects DNA damage or problems during DNA replication and restricts problematic cells by maintaining them in certain cell phases or inducing apoptosis. Chkl is a serine/threonine kinase that functions as an important signal transducer in the DNA redox balance and resist oxidative damage damag

关 键 词：链状亚历山大藻 衰亡 抑制性消减杂交 

分 类 号：Q943[生物学—植物学] X173[环境科学与工程—环境科学]