长石莼(缘管浒苔)(Ulva linza)rbcL全长基因的克隆与序列分析

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。     

胶州湾浮游植物遗传多样性及其季节变化研究

分别于春、夏、秋三季提取了胶州湾浮游生物总DNA,用PCR方法扩增了浮游植物核酮糖1,5-二磷酸羧化/氧化酶大亚基(rbcL)基因片段,构建了三季节rbcL基因片段质粒文库.随机从各文库中选择约50个克隆,测定了这些克隆的序列.以氨基酸序列相同为标准,共获得59个分类操作单元(OTUs),其中春季特有的21个,夏季特有的13个,秋季特有的23个.有两个OTUs为春、夏季表层海水共有.基于OTU丰度计算的香农氏指数能反映遗传多样性水平.胶州湾春、夏、秋三季的香农氏指数分别为2.69,2.44和2.76,表明秋季胶州湾浮游植物遗传多样性最丰富.春、夏、秋三季浮游植物群落组成差异显著.除夏秋季有两个OTUs相同外,其他OTUs在季节间完全不同.表层海水浮游植物群落结构是动态的,随时间、空间的不同而变化.     

长菱形藻(Nitzschia longissima)和新月细柱藻(Cylindrotheca closterium)分子分类研究

本研究自胶州湾分离了两种底栖硅藻,形态学初步鉴定为长菱形藻和新月细柱藻。对其18S rDNA和rbcL基因进行了测序并用邻接法构建了系统树。结果表明,两藻在18S rD-NA和rbcL基因序列上均存在较大的差异,基于DNA序列的分类结果与形态学分类结果一致。在依据形态指标难以确定藻类分类地位的情况下,18S rDNA和rbcL基因序列是有用的鉴定工具。     

红翎菜科4种海藻ITS和rbcL序列的分子系统学研究

通过PCR扩增基因片段的方法测定了红翎菜科琼枝属(Betaphycus)、卡帕藻属(Kappaphycus)和麒麟菜属(Eucheuma)4种海藻共19株个体的核糖体基因转录间隔区(ITS)(含5.8SrDNA)和核酮糖-1,5-二磷酸羧化酶/加氧酶基因大亚基(rbcL)全长基因序列。其中rbcL全长序列为首次测定,为从蛋白质水平探讨红翎菜科分子系统进化提供了可靠的保证。琼枝属、卡帕藻属、麒麟菜属ITS序列长度分别为1 024、629~669、1 001bp,GC含量在45.6%~52%之间;rbcL序列长度均为1 467bp,GC含量在37.1%~37.6%之间。结合GenBank中现有的红翎菜科海藻ITS和rbcL序列进行系统进化分析,2个片段聚类结果均明显的将所有样品分为琼枝属、卡帕藻属和麒麟菜属3大分支,表现出明显的属间差异。本研究的11株长心卡帕藻根据ITS序列差异分成明显2种类型,推测这2种类型长心卡帕藻的ITS序列差异与其地理环境、无性繁殖时间(代数)和藻体颜色无关。     

Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

1 IntroductionRibulose-1,5-bisphosphatecarboxylase/oxygenase(EC 4.1.1.39;Rubisco) plays an important role in photo-synthetic carbon fixation by controlling the initial stageof the Calvin cycle and possessing the functions of bothCO2 fixation and ribulose-1, 5-bisphosphate (RuBP)oxygenation. Rubisco is composed of eight large andeight small subunits in higher plants and green algae,and the large subunit is encoded by chloroplast genomeand the small subunit by nuclear genome. The gene en-cod…     

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.     

Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbc L and 18S rDNA sequence data

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer (ITS) regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-1 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbcL genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan’ao Island were identical. The ITS, 18S rDNA and rbcL genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U. flexuosa had the least evolutionary distance from U. californica in both the ITS regions (0.009) and the 18S rDNA (0.002). These data verified that Ulva and Enteromorpha are not separate genera.     

Phylogenetic diversity of ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

Phylogenetic diversity of Form Ⅰ and Form Ⅱ ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer sets were designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries were constructed by amplification and cloning of approximately 640-800 bp sequences of bacterioplankton populations. The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The results show that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while that of Form II is higher at the inshore station where salinity is lower and productivity is higher. Several clusters of sequences obtained are deeply rooted and show low similarity (60%-78%) to the known rbcL in existing databases. The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity, pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for genetic diversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.     

海南岛海洋绿藻新记录种未氏仙掌藻(Halimeda velasquezii)的形态学与分子系统学分析

2019年4月在海南岛冯家湾潮间带的野外调查过程中采集到一种仙掌藻, 利用传统形态分类学与分子系统分类学结合的方法, 将该种鉴定为未氏仙掌藻Halimeda velasquezii W.R. Taylor。该种的形态特征为藻体呈绿色钙化, 直立生长, 固着器单一较小, 由横卵形或肾形节片连接形成, 节片表面观形态为近圆形, 内部为管状髓丝, 节点处髓丝多发生侧面成对融合, 皮层下髓丝末端无囊状膨大。1,5-二磷酸核酮羧化酶/氧化酶大亚基基因(ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, rbcL)的序列分析结果显示, 在系统发育树上, 本研究样本与印度洋-太平洋地区产的未氏仙掌藻处于同一分支, 且自展支持值为100, 碱基基因差异为9bp (0.69%)。该种在海南岛海域首次被发现, 为新记录种。本研究同时首次提取并分析了中国种群的rbcL基因序列, 丰富了该种的中国种群的生态学知识, 扩展了其地理分布范围, 对了解海南岛海域海藻资源的生物多样性及其分布特征具有重要意义。