Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

According to the known sequence of iron stress-induced gene (isiAB operon), we cloned its 1.5 kb fragment by PCR, and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment, a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system, we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS),rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene, as the marker gene, was also subcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment and several expression elements, such asgroESL promoter, MCS,rbcS polyA terminator and kanamycin resistance gene, were all included.

After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942, as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942, the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10⁻⁶. By southern blot analysis, it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942, located on the site of theisiAB gene, and can be replicated with the chromosomal DNA.

     

Proteocephalus sinocyclocheili n. sp. (Cestoda: Proteocephalidae) in a fishSinocyclocheilus grahami tingi Fang from Yunnan, China

A new species ofProteocephalus from the fishSinocyclocheilus grahami tingi, a subspecies endemic to Fuxian Lake in Southwest China,is described. This is the first report of cestode from this host. The new species differs fromP. exiguus, P. Longicollis, P. parasiluri andP. torulosus, the only other species ofProteocephalus currently reported from China, by lacking an apical sucker, fromP. exiguus by having 45–108 rather than 24–54 testes per proglottis, and a maximum of 15 rather than 28 uterine diverticula; fromP.longicollis by having an average of 45–108 rather than 75–115 testes per proglottis and testes being in a single layer instead of 2 layers; and fromP. torulosus by having 45–108 instead of 150 testes per proglottis and testes being in 3–4 layers; and fromP. parasiluri by having 45–108 rather than 180 testes per proglottis. It resemblesP. percae, P. pollanicola, andP. thymalli in number of testes and uterine diverticula, but differs by lacking an apical sucker.Proteocephalus thymalli, like the new species, has a vagina that opens dorsally to the cirrus sac, butP. thymalli slao has postovarian testes, which the new species lacks. The possible significance of parasitological examinations of the other two subspecies ofS. grahami in two lakes close to Fuxian Lake is discussed in relation to host speciation and coevolution of the host-parasite system.     

Study on transient expression of gus gene inChlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

Study on the transient expression of GUS gene at different growing stage ofChlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed thatC. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.

     

A transformation model forLaminaria Japonica (Phaeophyta,Laminariales)

A genetic transformation model for the seaweedLaminaria japonica mainly includes the following aspects:

1. 1.

   The method to introduce foreign genes into the kelp,L. japonica

   Biolistic bombardment has been proved to be an effective method to bombard foreign DNA through cell walls into intact cells of both sporophytes and gametophytes. The expression ofcat andlacZ was detected in regenerated sporophytes, which suggests that this method could induce random integration of foreign genes.

   Promoters to drive gene expression

2. 2.

   The CaMV35S promoter was first used by us to induce the expression of GUS gene in brown algae. But results of further studies suggested that CaMV35S could be a tissue-specific promoter. Our use of SV40 promoter resulted in both transient and stable expression oflacZ andcat in sporophytes or gametophytes. No GUS or LacZ background was found in either sporophytes or gametophytes.The regeneration route of transgenic kelp

   The regeneration efficiency of explants is still very low. By using female gametophytes as gene hosts and parthenogenesis as regeneration route, CAT activity and LacZ activity were detected in regenerated sporophytes of parthenogenetic kelp. li]4.|The way to select transgenic kelp

3. 1.

   Results of sensitivity tests showed that kelp was only sensitive to chloramphenicol and hygromycin among many antibiotics. The regenerated sporophytes by parthenogenesis were more sensitive to hygromycin than to chloramphenicol. Resistant kelp was created by transforming female gametophytes with pSV40-CAT and stimulating parthenogenesis followed by selection in medium with lethal concentration of chloramphenicol.

   Safety consideration of transgenic kelp

   L. japonica was originally introduced from Japan. In China it is a cultured population. The possibility of its negative impact on natural populations is very low. 2) The vectors and target genes used for transformation should be restricted in order to avoid any negative impacts on human health and environment. 3) Specially devised containers (3.6 L, made of 200 μm membrane) were used to ensure that the kelp cannot escape or be eaten by marine animals. 4) To avoid the release of spores, it is very necessary to harvest the kelp at suitable age before the sporangium forms.

     

RAPD study on some common species ofPorphyra in China

Electroporation, PEC, PEG plus electroporation and Biolistics methods were tested in gene transformation ofP. yezoensis. The exogenousgus was from plasmid of pBI121 and pCAMBIA1301, both contain the CaMV35S promoter. The receptors included the protoplasts, tissues and free-living conchocelis filaments ofP. yezoensis. Several factors, for example, the voltage, capacitance and bivalent cations, etc., were studied. Results show that these four methods are all efficient for gene transformation inP. yezoensis; and that PEG is the best one, with transformation efficiency of up to 4×10⁻⁵. GUS activity was detected 26 days after transformation by using PEG method.

     

Cell biotechnology of rhodophytes in China

Cell biotechnology of rhodophytes is important not only in theoretical research but also in cultural practice and for exploitation of genetic resources. In this paper, cell biotechnology ofPorphyra is reviewed. Tissue culture and protoplast studies on other rhodophytes in China are discussed.

     

Comparative studies on the larval development of the penaeid shrimps,Penaeus Chinensis,P. merguiensis andP. penicillatus

The morphology of larval and 1st postlarval stages ofPenaeus penicillatus are described. Results from comparative studies on larval development ofP. penicillatus, P. merguiensis andP. chinensis are as follows: These three species could not be identified during their naupliar stages. In the 1st protozoea, the antennule L1/L2 value is 1.7–2.0 inP. merguiensis, but less than 1.7 inP. chinensis andP. penicillatus; in the 2nd protozoea, the supra-orbital spine inP. chinensis is not bifurcated, while those ofP. merguiensis andP. penicillatus are bifurcated; in the 3rd protozoea, there is a minute (or no) dorso-median spine on the posterior margin of the 1st and 2nd abdominal somite inP. chinensis, but they are prominent inP. merguiensis andP. penicillatus. In the mysis and 1st postlarval stages,P. chinensis differs fromP. merguiensis andP. penicillatus in having 9 (8 in the other 2 species) long setae on the exopod of pereopods 1–3; additionally, one dorsal tooth appears on the rostrum ofP. chinensis in the 2nd mysis and that of the other 2 species in the 3rd mysis;P. chinensis has 2 (mostly) or 1 dorsal tooth on the rostrum in the 3rd mysis and 2–3 in the 1st postlarva, whileP. penicillatus andP. merguiensis have only 1 in the 3rd mysis and 1st postlarva. Comparative studies on larval development showedP. penicillatus has closer affinity withP. merguiensis than withP. chinensis. Contribution No. 2218 from the Institute of Oceanology, Chinese Academy of Sciences     

Isolation,purification and characterization of the hydrogen evolution promoting factor of hydrogenase ofSpirulina platensis

A component (s-factor) with obvious promoting effect on hydrogen evolution of hydrogenase has been isolated and extracted from a cell-free preparation ofSpirulina platensis. The effect of the s-factor in the reaction system is similar to that of Na₂S₂O₄, but is coupled with light. The s-factor has the maximum absorption peak at 620 nm in the oxidized state, at 590 nm in the reduced state. The partially purified s-factor showed two bands by SDS-PAGE and is distinctly different from phycocyanin, which has no change of oxidized state and reduced state absorption spectra, and also has no promoting effect on hydrogenase ofSpirulina platensis under the light. The research supported by the NSFC.     

Spirulina cultivation in China

This paper reviews and discusses the development and many problems ofSpirulina cultivation in China, points out the advantages and disadvantages of open photobioreactor system, and predicts that seawaterSpirulina cultivation will be a new trend to be strengthened and emphasized due to its special physiological characteristics, easier management, lower fertilizer cost, and higher resistance to contaminants and rare pollution of chemicals.

     

Molecular identification of Prorocentrum (Dinophyceae) species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains ofProrocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo I, Hae III, and RSA I) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except forP. arcuatum (ME 131), which had the same pattern toP. micans (ME160 and 04). The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthicProrocentrum species were different from each other, and also from other cultured ones examined in this study.P arcuatum (ME132) could not be differentiated fromP. micans (ME160 and 04), it was probably mis-identified, since they are quite different morphologically.P. redfieldii (ME138) could also not be distinguished formP. triestinium (ME132), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found inP. micans, compared with previous sequence data. Project supported by National Basic Research Priorities Program (2001CB409701, 2001CB409710) and supported by NSFC (40376040, 40025614)     

High efficiency induction of callus and regeneration of sporophytes ofLaminaria japonica (Phaeophyta)

This study on the effects of ultrasonic treatment on female gametophytes ofLaminaria japonica showed that:

1. 1.

   Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certain period of time, the average length of filamentous female gametophytes was shortened.

2. 2.

   Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with time of treatment. Ultrasonic treatment had harmful effect on cells.

3. 3.

   Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, output of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regeneration of female gametophytes into sporophytes was effected. Female ganetophytes could not recover after too long period of treatment.

     

Iron ions increase the thermostability of phycocyanin ofSpirulina maxima

A spectral method to investigate the effect of Fe³⁺, Fe²⁺ on the thermostability of phycocyanin (PC) ofSpirulina maxima showed that iron ions provent decrease of visible light absorbance and fluorescence intensity of PC. Increase in denaturation temperature caused by Fe³⁺ was observed by the micro-differential scanning calorimetric method. All results showed iron ions maintain the aggregation stability of the PC. The absorption spectrum of phycocyanobilin (PCB, a prosthetic group of PC) with Fe³⁺ in chloroform was quite different from that of free PCB.

     

Hydrogen photoproduction ofA. Variabilis incorporated in a photobioreactor

H₂ photoproduction and nitrogenase activities in two strains ofAnabaena variabilis marked wild type ATCC 29413 and mutant PK84 exposed to thermal stress (temperature higher than the normal incubation temperature of 30°C) were studied. Cultures of both strains collected from any interval of logarithmic growth phase exhibited high H₂ photoproduction and nitrogenase activities when exposed to limited time heat shock during the assay process. In contrast, the algal H₂ photoproduction rate of both strains fluctuated with long term thermal stress caused by increasing the growth temperature from 30°C to 36°C.

The changes of nitrogenase (the key H₂ photobiosynthetic enzyme) activities in the mutant PK84 showed variation tendency similar to that of H₂ photoproduction during exposure to thermal stress, indicating that fluctuation of H₂ photoproduction in the mutant was mainly due to the variation of nitrogenase activities. A temporary maximal H₂ photoproduction in the mutant PK84 (wild type ATCC29413) was observed when cells grew at 36°C for 14 (6) days. However, the responses of nitrogenase activities in the wild type to thermal stress were not completely similar to those in the mutant in spite of similar variations of H₂ photoproduction in both strains. The data obtained in these studies suggested that the activities of other enzymes (in the wild strain) involved in H₂ photoproduction were affected by thermal stress since H₂ photoproduction maximized or dropped to 0 without variation tendency similar to that of nitrogenase activities.

Furthermore, an enhancement of H₂ photoproduction speed of the mutant strain cultured in a 4.4 L laboratory photobioreactor was also observed when it was subjected to short time continuous charge of argon, and temperature rise.

All these results indicated that high temperature plays an important role in the photo-autotrophic H₂ photoproduction, and that long term thermal stress is unfavourable for net H₂ photoproduction in both strains ofA. variabilis though short-time heat shock is conducive to H₂ photoproduction.

     

Effect of germanium dioxide on growth of Spirulina platensis

This study on the effect of different concentrations of germanium dioxide (GeO₂) on the specific growth rate (SGR), pigment contents, protein content and amino acid composition ofSpirulina platensis showed that Ge was not the essential element of this alga; that GeO₂ could speed up growth and raise protein content ofS. platensis, and could possibly influence the photosynthesis system. The concentration range of GeO₂ beneficial to growth ofS. platensis is from 5–100mg/l. GeO₂ is proposed to be utilized to remove contamination byChlorella spp. usually occurring in the cultivation ofSpirulina.     

Structural studies on agar fractions extracted sequentially from chinese red sea weeds:Gracilaria sjeostedtii,G. textorii andG. salicornia using13C-NMR and IR spectroscopy

The polysaccharides of three species of ChineseGracilaria (Rhodophyta) were extracted by sequential solvent extraction and the chemical structures of the fractions obtained were investigated by using¹³C-NMR and IR spectroscopy. The¹³C-NMR spectra showed that the main extract, cold water fraction, fromG. sjeostedtii consisted of repeating disaccharides of agarobiose and (1→3)-β-D-galactopyranosy 1-(1→4)-α-L-galactopyranose 6-sulfate, the latter being a precursor to agarobiose and converted to agarobiose after alkali modification. The component of the 60% ethanol fraction fromG. textorii was composed mainly of agarose 6-sulfate, 6-O-methyl-agarose and some amount of agarose, whse structures remained unchanged after alkali treatment. The autoclave extract fromG. salicornia revealed a complex structure composed of 6-O-methyl-agarose, 2-O-methyl-agarose, branched 4-O-methyl-L-galactose and agarose 4-sulfate. All the substituents in these agaroses were stable against alkali treatment. The alkali extracts fromG. sjeostedtii andG. textorii showed the distinct spectra of amylopectin-like floridean starch, while that fromG. salicornia showed a mixture of agaroses and floridean starch. From the IR spectra the relative contents of total sulfate, 3,6-anhydro-galactose, 4-sulfate and 6-sulfate in some fractions of polysaccharides of the algae were qualitatively estimated by the ratios of absorption bands of 1250/2920, 930/2920, 850/2920 and 820/2920, respectively. Contribution No. 1489 from Institute of Oceanology, Academia Sinica     

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

1 INTRODUCTION The unicellular Dunaliella salina, is one of the well studied microalgae for mass culture and is of commercial importance as a source of natural beta-carotene (Avron and Ben-Amot, 1992). D. sa- lina has desirable properties of halotolerance…     

Simultaneous estimates ofsynechococcus spp. Growth and grazing mortality rates in the English Channel

The marine chroococooid phycoerythrin-containingSynechococcus spp. cyanobacterium has been implicated as a substantial component of the photosynthetic picoplankton in the ocean. Although its importance as food source for heterotrophic nanoplankton is now recognized, information about the cycling ofSynechococcus biomass and its diel pattern is limited and study methodology varies among authors. The selective metabolic inhibitor method was used to simultaneously estimate growth and grazing disappearance rates ofSynechococcus in the English Channel where growth rates ranged from 0.25 to 0.72/d (mean ±SD=0.51±0.17/d) and grazing mortality rates ranged from 0.19 to 0.64/d (mean ±SD=0.48±0.17/d). Size-fractionated experiments demonstrated that up to 70% ofSynechococcus disappearance could be attributed to grazers going through a 2 μm Nuclepore filter.Synechococcus grazing mortality rates (mean=0.74 ±0.25/d) during the day were always higher than that (mean=0.2±0.20/d) during the night, while growth rates showed no clear diel pattern. A positive correlation was observed between growth rates andin situ temperature ranging from 9 to 17°C, while in contrast grazing was independent of temperature. The close similatiry between average growth and grazing rates suggests a rapid recycling ofSynechococcus biomass in English Channel coastal waters.     

ANALYSIS OF EVOLUFIONARY RELATIONSHIP BETWEEN ASTASIA LONGA AND EUGLENA GRACILIS BY USING RAPD TECHNIQUE AND CLADISTIC ANALYSIS

Although both Astasia longa and Euglena gracdlis belong to different genera, they share many morphological characters except that A. longa has no chloroplast. In the 1940‘ s, on the basis of the finding that in darkness or upon addition of scrne chemicals, E. gmcilis would fade reversibly or irreversibly, scrne scholars hypothesised that A. longa evolved from E. gracilis by losing chloroplast.The authors‘ use of RAPD and cladistic analyses in a study on the evolutionary relationship between A .longa and E. gmcilis showed that the A. longa ‘ s relationship with E. gmcilis was closer than that with other green euglenoids. This proves the hypothesis that A. longa evolved from E. gmcills is reasonable. The results d this study suggest that saprophytic colorless euglenoids were transformed fromgreen euglenoids by losing their choroplasts.