杜氏盐藻外源基因稳定表达系统的构建

建立了一种单细胞海水绿藻--杜氏盐藻(Dunaliella salina Teod.)的外源基因稳定表达系统.通过电激法将携带乙肝病毒表面抗原基因(HBsAg)和氯霉素乙酰转移酶基因(CAT)的质粒转入盐藻细胞内,CAT基因为筛选基因.PCR和Southern杂交结果显示,HBsAg基因已经整合到盐藻基因组中.Northern杂交结果表明,转化成功细胞内的该基因已转录成mRNA.HBsAgELISA和Western杂交检测证明,HBsAg蛋白在转化的盐藻细胞内稳定地表达.同时,PCR和Southern杂交显示,CAT基因也已整合到盐藻基因组中.且CATELISA检测证明,CAT蛋白在转化体中也已稳定地表达.进一步对转化盐藻进行无氯霉素筛选培养,60代后,HBsAg基因依然稳定地存在并表达.本实验第一次报道了外源基因在杜氏盐藻细胞内的稳定表达.     

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.     

杜氏盐藻外源基因稳定表达系统的构建(英文)

A stable transformation system for the expression of foreign genes in the unicellular greenmarine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed witha plasmid containing the hepatitis B surface antigen (HBsAg） gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAEgene wasintegrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressedat the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAgenzyme-linked immunosorbent assay (HBsAg EUSA) and Western blotting analysis. Also, PCR and Southernblotting analyses showed that the CA Tgene was integrated into the D, salina genome, and CAT EUSAindicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAgprotein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. salina.     

Intron-exon structure and gene copy number of a gene encoding for a membrane-intrinsic light-harvesting polypeptide of the red alga Galdieria sulphuraria

Genes for light-harvesting proteins (lhc genes) of higher plants are well examined. However, little is known about the corresponding genes of algae, although this knowledge might give valuable information about the evolution of photosynthetic antennae. In the case of rhodophytes only two cDNA sequences from a single organism, Porphyridium cruentum, have been published. Here we describe an additional sequence from another species, the thermo-acidophilic red alga Galdieria sulphuraria. For the first time also a genomic sequence for a red algal lhc gene is presented. From a cDNA library of G. sulphuraria we isolated a clone containing an open reading frame for a protein of 302 amino acids with a deduced molecular mass of 33.86kDa. It shares major structural features with eukaryotic light-harvesting polypeptides. A proposed cleavage site between transit peptide and mature protein gives rise to a transit peptide of 119 amino acids and a mature protein of 183 residues. Hydropathy analysis suggests that the mature protein consists of three transmembrane helices. Several amino acid residues supposed to bind chlorophyll a and chlorophyll b in higher plants are conserved. The protein shows up to 69% identity and 81% similarity to the Porphyridium polypeptides in the transmembrane helices 1 and 3. Using oligonucleotides annealing in the regions of the start and stop codons of the gene as primers, a DNA sequence was amplified from nuclear G. sulphuraria DNA by PCR. Compared with the cDNA clone, this sequence contains five additional intervening DNA strings of 50-74bp length. Four of them show typical features of spliceosomal introns with GT-AG borders, and the fifth differs by starting with GC. Three of the supposed introns are located in similar positions as introns of higher plant light-harvesting proteins. Southern blotting and hybridization experiments indicate that G. sulphuraria contains at least three copies of this gene.     

Cloning and expression of a gene coding for the major light-harvesting chlorophyll a/b protein of photosystem II in the green alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied. However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa. The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative PCR, and indicated that this gene expresses different mRNA levels under different light conditions.     

Photoreactivation of (6-4) photolyase in Dunaliella salina.

Dunaliella salina is a unicellular green alga and possesses two types of photolyase: Class II cyclobutane pyrimidine dimers (CPD) photolyase and (6-4) photolyase. The gene of D. salina (6-4) photolyase is the first one found in unicellular organisms. CPD photolyases have been extensively studied but (6-4) photolyases are less understood. Because of the data observed in this study, D. salina (6-4) photolyase is insensitive to high salinity; whether it can tolerate a higher level of salinity than other (6-4) photolyases needs to be studied further. However, evidence is provided that (6-4) photolyases might be highly conserved among different species, not only in the sequence identity but also in the photorepair mechanism.     

Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms

The light-harvesting complexes (LHCs) of land plants and green algae have essential roles in light capture and photoprotection. Though the functional diversity of the individual LHC proteins are well described in many land plants, the extent of this family in the majority of green algal groups is unknown. To examine the evolution of the chlorophyll a/b antennae system and to infer its ancestral state, we initiated several expressed sequence tag projects from a taxonomically broad range of chlorophyll a/b-containing protists. This included representatives from the Ulvophyceae (Acetabularia acetabulum), the Mesostigmatophyceae (Mesostigma viride), and the Prasinophyceae (Micromonas sp.), as well as one representative from each of the Euglenozoa (Euglena gracilis) and Chlorarachniophyta (Bigelowiella natans), whose plastids evolved secondarily from a green alga. It is clear that the core antenna system was well developed prior to green algal diversification and likely consisted of the CP29 (Lhcb4) and CP26 (Lhcb5) proteins associated with photosystem II plus a photosystem I antenna composed of proteins encoded by at least Lhca3 and two green algal-specific proteins encoded by the Lhca2 and 9 genes. In organisms containing secondary plastids, we found no evidence for orthologs to the plant/algal antennae with the exception of CP29. We also identified PsbS homologs in the Ulvophyceae and the Prasinophyceae, indicating that this distinctive protein appeared prior to green algal diversification. This analysis provides a snapshot of the antenna systems in diverse green algae, and allows us to infer the changing complexity of the antenna system during green algal evolution.     

The small subunit of ribulose-1,5-bisphosphate carboxylase is plastid-encoded in the chlorophyll c-containing alga Cryptomonas Φ

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) is located in the large single-copy region of the plastid genome of the chlorophyll c-containing alga Cryptomonas . The coding sequence is 417 base pairs long, encoding a protein of 139 amino acids, considerably longer than most other small subunit proteins. It is found 83 base pairs downstream from the gene for the large subunit and is cotranscribed with it. An 18 base pair perfect inverted repeat is located 8 base pairs beyond the termination codon. Sequence analysis shows the gene to be more closely related to cyanobacterial and cyanelle small-subunit genes than to those of green algae or land plants. This is the first reported sequence of a Rubisco small-subunit gene which is plastid-encoded and it exhibits a number of unique features. The derived amino acid sequence shows extensive similarity to a partial amino acid sequence from a brown alga, indicating that this gene will be of major interest as a probe for the small subunit genes in other algae and for determining possible evolutionary ancestors of algal plastids.     

Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina

Although interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) are implicated in various nuclear functions, the understanding of the regulatory mechanism of MARs is still poor. A few MAR-binding proteins (MARBP) have been isolated from some plants and animals, but not from the unicellular algae. Here, we identify a novel MAR-binding protein, namely DMBP-1, from the halotolerant alga Dunaliella salina. The cDNA of DMBP-1 is 2322-bp long and contains a 1626 bp of an open reading frame encoding a polypeptide of 542 amino acids (59 kDa). The DMBP-1 expressed in Escherichia coli specifically binds A/T-rich MAR DNA. The DMBP-1 fused to green fluorescent protein appears only inside the nuclei of Chinese hamster ovarian cells transfected with the pEGFP-MBP, indicating that the protein is located in the nuclei. The findings mentioned above may contribute to better understanding of the nuclear matrix-MAR interactions.     

Temperature-regulated expression of a glycine-rich RNA-binding protein in the halotolerant alga Dunaliella salina

Acclimation of the halotolerant alga Dunaliella salina to low temperature induced the accumulation of a 12.4 kDa protein (DsGRP-1) and reduction of a 13.1 kDa protein (DsGRP-2). DsGRP-1 and DsGRP-2 are boiling-stable proteins that are localised in the cytoplasm, as revealed by sub-cellular fractionation and by immuno-localisation. The proteins were partially purified and their corresponding genes were cloned. The predicted sequences are homologous to Glycine-Rich RNA-binding Proteins (GRPs) from plants and cyanobacteria. The nucleotide sequences of grp1 and grp2 differ in a short insert encoding 9 amino acids in the glycine-rich domain of DsGRP-2. grp2 contains a single intron at position 179 indicating that DsGRP-1 and DsGRP-2 are not derived from alternative splicing of a common gene. The level of grp mRNA increased at 7 degrees C and was rapidly depressed at 24 degrees C. Analysis of binding to ribonucleotide homopolymers revealed that DsGRP-1 and DsGRP-2 bind preferentially to poly-G and to poly-U indicating that they are RNA-binding proteins. It is proposed that DsGRP-1 and DsGRP-2 are encoded by distinct genes which are differentially regulated by temperature.     

Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina

Two clones have been isolated from a genomic library of the moss Physcomitrella patens and a cDNA library of the halotolerant green alga Dunaliella salina. The isolates contain genes coding for the major light-harvesting chlorophyll-a/b-binding protein (CAB) in the photosystem II (PSII) light-harvesting complex (LHCII). The 2544-bp insert of the moss genomic clone contains the complete CAB-coding region and 5' and 3' flanking sequences. The coding region contains an intron of 359 bp which is spanned by a pair of 9-bp perfect direct repeats. There are two CCAAT boxes and five enhancer-like elements related to (G)TGGTTTAAA(G) (Weiher et al., 1983) residing in the intron. Comparisons of the moss cab gene with sequences of light-inducible genes of higher plants reveal homologous and repeated sequences similar to the enhancer element in the 5' region upstream from the TATA and CCAAT boxes thought to be responsive to light inducibility. The 1256-bp algal cDNA contains the complete CAB-coding sequence, a 170-bp 5'-nontranslated region, and a 264-bp 3'-nontranslated region. While the overall homology in the nontranslated regions is low between the cab gene of the moss and that of the alga, the 3'-nontranslated regions of the two contain some sequences that are conserved among the cab genes in higher plants. The deduced amino acid sequences of these two clones are highly conserved except for the N-terminal region. Their hydropathic plots are very similar and both possess three hydrophobic segments that are likely alpha-helical transmembrane segments. The proposed CAB transit peptide sequence of the alga is divergent from that of the moss or higher plants, suggesting that they may have evolved from different origins. Southern blot analysis shows that the cab genes in the moss and the alga, as in higher plants, are encoded by a number of homologous genes constituting a multigene family.     

杜氏盐藻分子生物学最新进展及展望

杜氏盐藻是一种无细胞壁的单细胞双鞭毛真核藻类,是一种十分重要的藻类资源。过去对杜氏盐藻的研究多集中在形态学、耐盐机理及β-胡萝卜素等方面,近年来,随着藻类基因工程的快速发展,本研究课题组及国内外在杜藻盐藻分子生物学方面做了大量工作,现就杜氏盐藻在这一领域的研究进展进行综述,主要是重要功能基因的克隆与分析、杜氏盐藻调控序列的研究以及杜氏盐藻作为宿主表达外源基因等。     

Identification of N- and C-terminal amino acids of Lhca1 and Lhca4 required for formation of the heterodimeric peripheral photosystem I antenna LHCI-730

Apoproteins of higher plant light-harvesting complexes (LHC) share considerable amino acid sequence identity/similarity. Despite this fact, they occur in different oligomeric states (i.e., monomeric, dimeric, and trimeric). As a step toward understanding the underlying structure requirements for different oligomerization behavior, we analyzed whether amino acids at the N- and C-termini of Lhca1 and Lhca4 are involved in the formation of the heterodimeric LHCI-730. Using altered proteins produced by deletion or site-directed mutagenesis for reconstitution, we were able to identify amino acids required for the assembly of LHCI-730. At the N-terminus of Lhca1, W4 is involved in heterodimerization. This interaction probably depends on aromatic properties because only replacement of W4 by F resulted in dimer formation. Also, at the C-terminus of Lhca1, W seems to play a crucial role for interaction with Lhca4. A detailed analysis by point mutants revealed the importance of an aromatic residue at position 185. One or more other amino acid(s) located downstream of position 188 may exert additional stabilizing effects, presumably in a cooperative way. The scenario for Lhca4 is different. Dimerization broke down only after the deletion of the entire extrinsic N- or C-terminal region, demonstrating that the termini of Lhca4 are not involved in strong interactions with Lhca1 decisive for dimerization. At the N-terminus, dimerization was abolished after the removal of the same number of amino acids at which monomer formation failed. Site-specific mutagenesis of the amino acid decisive for LHC-formation in a deletion study demonstrated that its character is of no importance for dimerization and, therefore, that abolition of dimer formation may be the consequence of a loss in monomer formation. At the C-terminus of Lhca4, an even higher number of amino acids than required for monomer formation could be removed without the loss of dimerization. The decisive position is I168, located in the third transmembrane region. Because all point mutants of I168 in the full-length protein yielded dimers, failure of dimerization may be caused by either falling below a critical length of the polypeptide chain, resulting in the loss of too many weak interactions, or by too strong an impairment of Lhca4-folding. Interestingly, N- and C-terminal mutants of Lhca4 not able to form stable monomers formed stable dimers, indicating stabilization of labile monomeric complexes by the Lhca1 subunit in dimerization. Finally, the significance for dimer formation of amino acids in other parts of Lhca1 and Lhca4 which may be involved, besides the amino acids identified here in the specific assembly of the heterodimeric LHCI-730, is discussed. Their identification will result in a better understanding of structure characteristics determining the different oligomerization behavior of LHCs.     

小麦TaLhca基因的克隆及表达分析

为了从分子水分研究小麦的光合作用,该研究采用RT-PCR方法,从小麦品种‘百农207’的叶中克隆到1个捕光叶绿素a/b结合蛋白基因,命名为TaLhca。序列分析结果表明,TaLhca的编码区序列(coding DNA sequence,CDS)长810 bp,编码269个氨基酸,推测分子量为29.31 kD,等电点为8.69。TaLhca被定位于叶绿体,无信号肽,存在3个明显的跨膜区域,预测其蛋白结构含有典型的捕光叶绿素a/b结合蛋白功能域(chlorophyll a/b binding domain),为亲水性非分泌蛋白。蛋白序列比对和进化树分析表明,小麦与二穗短柄草(Brachypodium distachyon)和水稻(Oryza sativa)中的Lhca序列相似性最高,亲缘关系最近。启动子顺式作用元件预测表明,启动子区域包含多个光响应元件及逆境响应元件。实时荧光定量PCR分析表明,TaLhca基因在小麦根、茎、叶中均有表达。其中在叶中表达量最高,在根中表达量最低,且受NaCl、干旱、ABA、H2O2和低温胁迫表达增强,受黑暗胁迫表达降低。该研究结果为进一步解析小麦光合作用机理及其相关基因的诱导表达特性提供了依据。     

Identification and expression analysis of cDNA encoding a chloroplast recombination protein REC1, the chloroplast RecA homologue in Chlamydomonas reinhardtii

Chloroplasts of plant cells have their own genome, and a basic recombination protein homologous to the eubacterial RecA was suggested to be involved in the perpetuation of chloroplast DNA. A candidate cDNA sequence encoding the chloroplast RecA protein was identified from the Kazusa EST database for the unicellular green alga, Chlamydomonas reinhardtii (http://www.kazusa.or.jp/en/plant/chlamy/EST/). Analysis of the cDNA sequence identified an open reading frame (ORF) of 414 amino acids encoding a eubacteria-type RecA protein. Thus the corresponding gene was named REC1. The predicted protein contains an N-terminal extension that does not show any similarity with other RecA proteins. Transient expression of a REC1-sGFP (green fluorescent protein) fusion construct in tobacco cells has indicated that this N-terminal sequence functions as a transit peptide for import into chloroplasts. Since DNA-damaging reagents induced the REC1 mRNA, REC1 was suggested to have roles in DNA recombination and repair of the chloroplast DNA in C. reinhardtii.