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当前位置: 首页 > 文章 > 缘管浒苔rbcL全长cDNA克隆与序列分析 水产学报 2010,34 (5) 141-150
Position: Home > Articles > Cloning and sequence analysis of the full length cDNA of rbcL from Ulva linza(Chlorophyceae,Chlorophycophyta) Journal of Fisheries of China 2010,34 (5) 141-150

缘管浒苔rbcL全长cDNA克隆与序列分析

作  者：
应成琦;尹顺吉;林森杰;沈轶;何培民
单  位：
Department;上海海洋大学农业部水产种质资源与养殖生态重点开放实验室
关键词：
缘管浒苔;核酮糖1,5-二磷酸羧化酶/加氧酶大亚基基因(rbcL基因);前导序列;cDNA未端快速扩增;cDNA
摘  要：
对缘管浒苔光合作用第一关键酶Rubisco大亚基基因(rbcL)全长cDNA序列进行了克隆分离。首先应用RT-PCR方法获得了rbcL大片段cDNA序列,序列长度为1101bp,并进行了测序分析。然后分别应用5′RACE方法和3′RACE方法获得2个克隆序列,其序列长度分别为371bp和579bp,并进行了测序分析。在此基础上,将3个片段序列进行拼接,获得了Rubisco大亚基全长cDNA序列(1472bp),其中包括1425bp的编码区序列及47bp的前导序列(NCBI登录号:DQ813496),并推断出蛋白质序列(474个氨基酸)。对序列进行了相关生物信息分析和同源性比对,结果显示与已克隆出全长rbcL基因的7种绿藻比较具有较高同源性,核苷酸序列和蛋白质序列同源性分别达到了82.07%～85.78%和88.00%～94.11%,其中与蛋白核小球藻蛋白质序列同源性最高,为94.11%。在此基础上,使用生物信息学软件对缘管浒苔rbcL氨基酸序列与其它植物或藻类进行了多序列比对,并且进一步应用在线软件程序进行了蛋白质的二级结构分析和三级结构预测。
译  名：
Cloning and sequence analysis of the full length cDNA of rbcL from Ulva linza(Chlorophyceae,Chlorophycophyta)
作  者：
YING Cheng-qi1,2,YIN Shun-ji1,LIN Sen-jie3,SHEN Yi1,HE Pei-min1* (1. Key Lab of Aquatic Genetic Resources & Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Ocean University,Shanghai 201306,China; 2. East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Shanghai 200090,China; 3. Department of Marine Sciences,University of Connecticut,Groton,CT 06340,USA)
关键词：
rbcL; Ulva linza; leader sequence; rapid amplification of cDNA ends(RACE); cDNA
摘  要：
Rubisco (Ribulose 1,5-bisphosphate Carboxylase / Oxygenase) is the first key enzyme in the photosynthesis pathway. In this study,the full length cDNA,encoding Rubisco large subunit (RbsL) was cloned from Ulva linza for the first time. Partial rbcL cDNA sequence of 1 101 bp was first cloned by RT-PCR from total RNA,then,both sequences at 3′ and 5′ ends were amplified using 3′ -RACE and 5′ -RACE technologies that yielded 579 bp and 371 bp DNA fragments respectively. A full length cDNA sequence of 1472 bp was obtained from these cloned sequences and was assigned to a new NCBI accession number DQ813496. It contained the 1 425 bp full coding sequence for Rubisco large subunit (RbsL) and a leader sequence of 47 bp,and it encoded a protein of 474 amino acids. Through bioinformatic analysis and BLAST search,the full cDNA sequence in Ulva linza showed high homologies to known full length cDNAs of the same gene from other green algae where rbcL cDNAs were previously cloned. The identity ranged from 82. 07% to 85. 78% ,and 88. 00% to 94. 11% ,respectively,at DNA and amino acid levels. The highest homology of the protein was obtained between Ulva linza and Chlorella pyrenoidosa. Based on the newly identified cDNA sequence,multiple alignment was performed by using bioinformatic software among amino acid sequences of Ulva linza and other two higher plants and one green alga. Then,the secondary and three-dimensional structures for Rubisco large subunit in Ulva linza were also predicted and analyzed with online programs.

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