长石莼(缘管浒苔)(Ulva linza)rbcL全长基因的克隆与序列分析  

CLONING AND SEQUENCE ANALYSIS OF THE FULL-LENGTH rbcL GENE FROM ULVA LINZA

作　　者：应成琦[1,2] 蔡春尔[1] 尹顺吉[1] 徐韧[3] LIN Sen-Jie 周志刚[1] 马家海[1] 何培民[1] 

机构地区：[1]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,上海201306 [2]中国水产科学研究院东海水产研究所,中国水产科学研究院盐碱地渔业工程技术中心,上海200090 [3]国家海洋局东海环境监测中心,上海200137 [4]Department of Marine Sciences,University of Connecticut,Groton,CT 06340,USA

出　　处：《海洋与湖沼》2010年第4期555-562,共8页Oceanologia Et Limnologia Sinica

基　　金：国家自然科学基金项目,30371101号;教育部博士点基金资助项目,2006-2007;上海市优秀学科带头人计划项目,08XD14037号;上海市科委“长三角联合攻关”项目,062358101号;上海市教委优势(重点)学科资助项目,S30701号

摘　　要：克隆分离了长石莼(缘管浒苔)光合作用第一关键酶Rubisco大亚基全长基因(rbcL)。首先应用PCR和RT-PCR方法扩增rbcL大片段DNA序列和大片段cDNA序列,结果表明,获得的2个克隆序列完全相同,该rbcL大片段基因不存在内含子。其次应用基因步移方法分别对rbcL基因的5′上游未知序列和3′下游未知序列进行扩增,在此基础上,将3个片段序列进行拼接,获得了rbcL全长基因序列(NCBI登陆号:DQ813497)。序列全长2124bp,其中编码区序列长1425bp,为单外显子基因,编码474个氨基酸;5′非翻译区序列长225bp,转录起始位点为位于翻译起始密码子ATG上游第47个核苷酸G,在距离转录起始位点-9bp—-48bp的范围内有推测的类似原核生物的启动子序列(-10区:TAAAAT、-35区:TTGAAA);3′非翻译区序列长474bp,在距离转译终止密码子TAA下游54—98bp处有一段较长的回文序列,可形成一个22bp的茎结构。密码子偏好性分析结果表明,长石莼(缘管浒苔)rbcL基因偏向使用第三位核苷酸碱基为A和T的密码子。The full-length DNA sequence of Rubisco,the first key enzyme of photosynthesis in Ulva linza was cloned.Firstly,partial rbcL DNA and cDNA sequence both with 1101bp in size were obtained using PCR and RT-PCR technology,respectively,and both of their PCR products were sequenced and analyzed.The partial rbcL DNA sequence was the same as the rbcL cDNA sequence,indicating there is no intron in the partial rbcL DNA sequence.In addition,gene walking technology was used to clone the 5＇ upstream sequence and the 3＇ downstream sequence.The sizes of the cloned sequences at 5＇ upstream and 3＇ downstream were 474bp and 1053bp,respectively.Based on these three cloned sequences,a full-length DNA sequence of 2124bp was acquired（NCBI accession number：DQ813497）,which includes a coding sequence of 1425bp,encoding 474 amino acids.The 5＇ upstream sequence is 225bp and includes a putative promoter between-56bp—-95bp upstream the ATG codon.The 474bp long downstream sequence contains a long palindrome sequence locating between 54—98bp downstream from the termination codon and being capable of forming a 22bp stem.Codon preference analysis performed with the SMS software indicates that codons with the third base being A or T are in preference in the rbcL gene in Ulva linza.

关 键 词：RBCL 长石莼(缘管浒苔) 全长序列 基因步移 

分 类 号：Q953[生物学—动物学]