REAPPRAISAL OF PHYSIOLOGICAL ATTRIBUTES OF NINE STRAINS OF DUNALIELLA (CHLOROPHYCEAE): GROWTH AND PIGMENT CONTENT ACROSS A SALINITY GRADIENT

Among the few taxonomic characters used to circumscribe sections within the subgenus Dunaliella are the physiological response to changes in salt concentration, which define a specific range for optimal growth, and the carotenogenic ability of the vegetative cells, responsible for the change in cell color from green to orange or red, under suboptimal culture conditions. Previous work based on molecular data from seven taxa of different sections of the subgenus showed no correlation between the genetic relationship inferred from the internal transcribed spacer RFLP data and the morphophysiological attributes in use in taxonomy. The present work was performed to experimentally reevaluate the physiological attributes in the same seven previous taxa ( D. tertiolecta Butcher UTEX 999 and CCMP 1320, D. parva Lerche UTEX 1983 and CCMP 362, D. salina Teodoresco UTEX 200, D. viridis Teodoresco CONC 002, and D. peircei Nicolai et Baas Becking UTEX 2192) adding D. parva CCAP 19/9 and D. pseudosalina Massyuk et Radchenko CONC 010. Growth responses and pigment content in a wide range of NaCl concentrations were assessed. The results revealed that two strains of D. parva , two strains of D. tertiolecta , and one strain of D. peircei showed similarity in their growth responses to the whole range of salt concentrations. Dunaliella parva UTEX 1983, on the other hand, showed a growth rate pattern very different from those of their conspecifics and similar to those of D. viridis CONC 002 and D. salina UTEX 200. In relation to the pigment content, none of the strains turned orange or red in color under the whole range of salt concentrations assayed, and the total carotenoids to chlorophyll ratios were always lower or equal to 1.0. These results reaffirm the validity of the physiological attributes used to discriminate sections within the subgenus Dunaliella and show correlation with the previous molecular data, but stress the need for relocating some strains into different sections.     

PHYLOGENETIC RELATIONSHIP AMONG VARIOUS STRAINS OF DUNALIELLA (CHLOROPHYCEAE) BASED ON NUCLEAR ITS rDNA SEQUENCES

Phylogenetic analyses of 15 strains representing 8 taxa of Dunaliella (D. salina, D. bardawil, D. pseudosalina, D. tertiolecta, D. parva, D. viridis, D. peircei, and D. lateralis) belonging to both subgenera and all sections of the genus were carried out using the sequences of the nuclear rDNA spacers (internal transcribed spacer [ITS-1 + ITS-2]). The ITS data agreed with the traditional data in that D. lateralis (from subgenus Pascheria) is only distantly related to the seven taxa of the subgenus Dunaliella. The ITS data also supported the monophyly of the subgenus Dunaliella. Within the subgenus Dunaliella, sequence data resolved five phylogenetic groups; some isolates of D. parva and D. salina separated into different clades containing other species. For example, D. parva UTEX 1983 (section Dunaliella) grouped with D. viridis CONC 002 (section Virides); the former has more nucleotides in common with D. viridis (93.2% similarity) than to its conspecifics (85.6% similarity). Likewise, the strains of D. parva CCMP 362 and CCAP 19 / 9 (section Dunaliella), the three strains of D. tertiolecta (section Tertiolectae), and the one strain of D. peircei (section Peirceinae) formed a strong phylogenetic clade (99%–100% support). Dunaliella salina UTEX 200 is more closely related to D. pseudosalina CONC 010 than to its conspecifics (95% similarity), even though the two taxa differ markedly physiologically. The results revealed that D. parva UTEX 1983 has been misidentified and should be renamed as D. viridis. Similarly, the strains of D. parva CCAP 19 / 9 and CCMP 362 and the strain UTEX 2192 of D. peircei should be renamed as D. tertiolecta. More physiological and molecular work needs to be done to elucidate the correct taxonomic position of D. salina UTEX 200 and D. pseudosalina CONC 010. Finally, the high ITS sequence variability found among the various strains of D. salina underlines the importance of further work to elucidate the species status in this complex taxon.     

杜氏盐藻叶绿体atpA基因片段的克隆及序列分析

根据莱因衣藻、卵形肾藻、普通小球藻等10种藻类的atpA全基因氨基酸高度保守序列,设计简并引物,利用PCR方法从盐藻叶绿体DNA中扩增出约400bp的片段,将该片段连接到T-vector上进行序列测定。结果表明,核苷酸长度为405bp,编码135个氨基酸。推导的氨基酸序列与莱因衣藻的同源性为92%,普通小球藻88%,Mesostigmaviride87%,卵形肾藻86%,Cyanidioschyzonmerolae85%。以所克隆的DNA片段为探针,与盐藻叶绿体基因组进行SouthernBlot杂交结果有明显的杂交信号。据此可推断本实验中所克隆的序列为杜氏盐藻叶绿体atpA基因片段。该基因序列已被GenBank收录,接受号为AY435096。     

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.     

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of nuclear ribosomal DNA in the Phaseolus-Vigna complex.

Molecular phylogeny based on internal transcribed spacer (ITS) sequences was studied to resolve the taxonomic contradiction in Vigna and its relation to Phaseolus. The ITS region of the 18S-26S nuclear ribosomal DNA repeat was sequenced for 29 Vigna species, selected from five of the nine subgenera, and 9 species of Phaseolus. The length of ITS-1 ranged from 187 to 243 bp and 217 to 290 bp, and that of ITS-2 from 187 to 219 bp and 225 to 243 bp, within Vigna and Phaseolus species, respectively. Phylogenies derived from ITS sequences based on maximum-parsimony and neighbor-joining methods gave trees essentially of similar topology. The ITS phylogeny was generally congruent with recent classifications based largely on morphological, biochemical, cytogenetical, and palynological features, except that subgenus Plectotropis of Neotropical origin was revealed to be closely related to subgenus Vigna instead of forming a link between African (subgenus Vigna) and Asiatic (subgenus Ceratotropis) vignas, and subgenus Sigmoidotropis, featuring morphological characters of both Vigna and Phaseolus, was placed as the sister group to the Phaseolus taxa. The ITS sequences were shown to be useful for identifying wild progenitors of V. mungo, V. radiata, V. umbellata, and V. unguiculata and for clarifying taxonomy-related problems in many previously controversial cases. This study also affirms that V. umbellata and V. angularis are the diploid progenitors of the only tetraploid species (V. glabrescens) known in the genus.     

杜氏盐藻psaB基因cDNA克隆及系统进化分析

根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii及Chlorella vulgaris等光系统Ⅰ反应中心蛋白psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻(Dunaliella salina)细胞的总RNA,通过RT-PCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序以及测序结果推导成氨基酸序列进行同源性比较,表明所克隆的1815bp序列为杜氏盐藻光系统Ⅰ反应中心psaB基因的cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB的核苷酸序列推导成氨基酸序列与一些已知物种的psaB氨基酸序列相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydomonas moewusii 91%,Chlorella vulgaris 86%,Mesostigma viride 85%,Phy -scomitrella patenssubsp.Patens 85%,Nephroselmis olivacea 84%。此外,psaB密码子偏爱性分析表明:杜氏盐藻psaB基因第三位密码子A和T的组成分别为35.7%和39.17%,而G和C分别为7.27%和17.85%,即杜氏盐藻psaB基因密码子的组成大多为NNA和NNT。根据psaB基因的特征,作者对绿藻门的50个物种的psaB基因作了进化分析,结果表明:杜氏盐藻与Haematococcaceae中的大多数种类进化地位最为接近,这为更进一步弄清杜氏盐藻的遗传背景提供了理论依据。     

杜氏盐藻psaB基因cDNA的克隆与序列分析

根据真核生物莱茵衣藻(Chlamydornonas reinhardtii)、Chlamydomonas moewusii、Chlorella vulgaris以及Mesostigma viride的psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻(Dunaliella salina)细胞的总RNA,通过RTPCR,得到的一段长为1．8kb左右的cDNA片段。PCR产物经T-A克隆并测序分析以及测序结果推导成氨基酸序列进行同源性比较．表明所克隆的1815bp序列为杜氏盐藻psaB cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB序列推导成氨基酸序列与一些已知物种的psaB基因相比较,同源性分别为Chlamydomonas reinhardtii 92％,Chlamydornonas moewusii 91％,Chlorella vulgaris 86％,Mesostigma viride 85％,Physcomitrella patens subsp．Patens 85％,Nephroselmis olivacea 84％。据此可推断本实验中所克隆的序列为杜氏盐藻psaB cDNA序列.     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

The objective of this study was to determine the genetic relatedness among the Cercospora and Pseudocercospora species closely related to Cercospora apii by using a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the internal transcribed spacer (ITS) region. A single PCR fragment (about 550 bp) was obtained from all Cercospora species categorized as the C. apii-group, Pseudocercospora purpurea, Pseudocercospora conyzae, and Pseudocercospora cavarae. Cercospora caricis yielded a 680 bp PCR fragment. The similarity in the PCR fragment size and RFLP profiles among the C. apii-group isolates, including Pseudocercospora purpurea, and Pseudocercospora conyzae strongly suggests that these species are conspecific. Synonymy with C. apii (lectotype) at a subspecific rank has been proposed. Amplified ITS regions of genomic DNA extracted from spinach leaves showing 12 and 233% leaf spot disease symptoms caused by Cercospora beticola yielded two PCR fragments (i.e., one from the fungus and one from the host plant) and were resolved by electrophoresis of the PCR product in 3% LMP agarose. Digestion of the total PCR product with HinfI restriction enzyme yielded RFLP profiles similar to those obtained from amplified DNA from the causative agent, C. beticola. The method described in this preliminary study offers rapid detection and diagnosis of fungal infections in plants for disease prediction and management and screening of plant materials for quarantine purposes.     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

作物数量遗传学基础 二、遗传力及其估算

两栖类染色体的研究,过去多使用以生殖 细胞和端蚌尾部上皮细胞为材料的水低渗压片 法^[6]。然而,生殖细胞和峪蚌组织受季节的限 制颇大,所得的中期分裂相亦不甚多。随着 低等脊稚动物组织培养技术^[1,2]与血液培养技 术^[3]的发展,近年来已更多的使用离体培养的 细胞来进行研究。     

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Phylogenetic position of Dunaliella acidophila (Chlorophyceae) based on ITS and rbcL sequences

Dunaliella acidophila is one of the most extreme acidophiles on earth and is able to survive in highly acidic habitats. This characteristic has made this organism the universal model for the study of abiotic stress. Although D. acidophila is currently circumscribed to the subgenus Pascheria within Dunaliella Teodoresco (Chlorophyceae), its taxonomic position has stirred controversy. The comparison of D. acidophila CCAP19/35 internal transcribed spacers (including ITS2 secondary structure analysis) and RuBisCo large subunit (rbcL) sequences with other Dunaliella species confirms that D. acidophila should maintain its phylogenetic position within the genus Dunaliella, suggesting its inclusion within the subgenus Dunaliella. Furthermore, the ITS1 and ITS2 data revealed that D. acidophila was highly divergent from the other freshwater species assessed, D. lateralis, with which it barely shares a 56.8% similarity.     

Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately 16-24 microm long and 10-15 microm wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of beta-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of beta-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate beta- carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.     

14 Phylogeny of chlamydomonads isolated from saline habitats: inferences from 18s and 26s rDNA data

Although the vast majority of chlamydomonadalean organisms are found in freshwater habitats, several prominent lineages (e.g., Dunaliella, Brachiomonas and some species of Chlamydomonas ) are found in brackish, marine or brine environments. Unknown chlamydomonads from Salt Plains National Wildlife Refuge (SPMO; OK USA) and from the Culture Collection of Marine Phytoplankton (CCMP; Bigelow Labs, ME USA) were included in a broad phylogenetic analysis using both 18S and 26S rDNA data. Results indicate that several halotolerant/halophilic lineages can be identified among the Chlamydomonadales. One lineage, which includes Chlamydomonas parkeae, Chl. hedleyi (symbiont of foraminifer) and five CCMP isolates, is allied in the Tetracystis clade with Chl. moewusii and Chl. noctigama . A second group, which includes Chl. reginae , Chl. uva-maris and CCMP 233, is allied with Chl. monadina . Brachiomonas , which is sister to Haematococcus lacustris , has no close halotolerant/halophilic allies. The three SPMO isolates plus CCMP 220 and CCMP 1641 are resolved as close allies of various Dunaliella taxa. Two of the SPMO isolates are distinctive from a typical Dunaliella cell in that they regularly form non-flagellated cellular aggregations in normal saline media. In summary, these observations collectively suggest that the ability to live in a saline habitat has arisen, independently, in at least four chlamydomonadalean lineages. In addition, substantial taxonomic revision is required to accommodate virtually all of the unidentified isolates included in this investigation. Supported by NSF DEB 9726588, DEB 0129030, MCB 0132083 and MCB 0132097.     

Relationships Among Group IV Phytophthora Species Inferred by Restriction Analysis of the ITS2 Region

The polymerase chain reaction (PCR) was used to amplify the ITS2 region of nuclear ribosomal DNA from six Phytophthora species which comprise taxonomic Group IV. Digestion of the ca. 600 bp PCR product with restriction enzymes Alu I, Dra I, Hha I, Hinf I, Msp I, and Taq I revealed variation which allowed relationships among the species to be assessed. P. infestans , P. mirabilis and P. phaseoli were indistinguishable from one another with all enzymes tested. With Alu I and Taq I. P. ilicis , P. colocasiae . and P. hibernalis each showed unique banding patterns different from the common banding pattern shared by P. infestans . P. mirabilis . and P. hibernalis . Dra I allowed differentiation of P. ilicis and P. colocasiae from P. infestans , P. mirabilis , P. phaseoli , and P. hibernalis . all of which shared a common banding pattern. Hha I allowed differentiation of P. colocasiae and P. hibernalis from P. infestans, P. mirabilis, P. phaseoli , and P. ilicis . Hinf I allowed differentiation of P. ilicis and P. hibernalis , (each of which showed a unique banding pattern) from P. infestans, P. mirabilis, P. phaseoli , and P. colocasiae . Msp I allowed differentiation of P. hibernalis from the other five species. Species groupings determined by restriction analysis of ITS2 were consistent with those based on morphological criteria. These results show that restriction analysis of PCR-amplified TS2 regions can be useful as an adjunct to morphological criteria in Phytophthora species identification.     

Differentiation of closely related Carnobacterium food isolates based on 16S-23S ribosomal DNA intergenic spacer region polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763(T) and C. mobile DSM 4849(T) generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766(T) and C. piscicola NCDO 2762(T) were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA(Ile) and tRNA(Ala) genes. The M-ISR included one tRNA(Ala) gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.     

盐藻线粒体GIY-YIG族归巢内切酶基因受到盐胁迫时增强转录

盐藻 (Dunaliellasalina (Chlorophyta) )极强的耐盐能力使之成为研究植物耐盐分子机制的重要模式生物 .在前期分离到的一个盐藻基因片段在盐胁迫时增强表达的基础上 ,通过RACE获得了该基因 5′端cDNA序列及部分 3′端cDNA序列 .序列分析结果表明 ,该基因是一个编码线粒体GIY YIG族归巢内切酶的Ⅰ型内含子基因 .RNase freeDNase处理及Northern杂交结果表明 ,盐藻线粒体DNA在盐处理组中可能发生了扩增 .这些结果结合前人的研究成果说明 ,该内含子基因的增强表达可能并非盐藻对抗盐胁迫的一种手段 ,而是盐藻对抗盐胁迫的副产品 .被该内含子基因插入的基因是盐藻对抗盐胁迫所需要增强表达的 .线粒体DNA的扩增可能是细胞在受到盐胁迫时线粒体数量增加的结果     

杜氏盐藻硝酸盐还原酶基因5′上游序列的克隆与功能分析

目的:克隆杜氏盐藻硝酸盐还原酶（NR）基因5′上游序列序列,并对其功能进行分析。方法: 利用BamHI、EcoRI、HindIII、PstI、SalI、Xbal 6种限制性内切酶分别酶切盐藻基因组DNA,并与接头连接,构建成盐藻基因组步行文库。采用 LA-PCR方法,从上述盐藻步行基因组文库中扩增NR基因5′上游序列序列,测序并进行分析。为检测其表达特性,构建了该片段与GUS 嵌合基因的表达载体pNR-GUS, 通过电击法将所构建的重组表达载体转化盐藻,组织化学染色法观察GUS的表达。结果: 从盐藻基因组步行文库中扩增出约1200bp特异片段,序列分析表明5′上游序列含有启动子的特征性序列。GUS瞬时表达染色结果显示,该DNA 片段具有硝酸盐诱导和铵抑制的启动子活性。结论：所克隆的盐藻的5′上游序列可能是一种具有"开关"活性的可控性启动子。