Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated(-73 0 generations,-two days each)to adapt freshwater BGll medium.A number of freshwater medium adapted colonyderived strains were obtained.The strains were verified phylogenetically to be N.oceanica LAMB0001 based on the 18 S ribosomal RNA gene.Freshwater-medium adapted strain(FA1)grew faster in the BG11 medium prepared with freshwater than wild-type N.oceanica grew in f/2 medium prepared with seawater.We assumed that(1)the expression patterns of the genes that expressed differentially between FA1 and the wild-type N.oceanica exposing to the BG11 medium(WT-F)have been reprogrammed;(2)the physiological processes in which these genes involved have been modified;and(3)a Gene Ontology(GO)term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up-or down-regulated if it was enriched simultaneously by up-or down-regulated DEGs between FA1 and WT-F,respectively.Under these assumptions,we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion,membrane and membrane integrity,material transportation,cell movement,and cellular signaling network.These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium.It is feasible to domesticate marine microalgae to a freshwater habitat,which may aid to modify their cultivation performances.     

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.

     

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

1 Introduction Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and pro-gressively smaller polymers composed of glucose units(Windish et al., 1965; Pandey et al., 2000; Chi et al.2001). Amylase is a kind of very important enzyme andconstitutes a class of industrial enzymes sharing approximately 25% of the enzyme market (Sindhu et al.1997; Rao et al., 1998). Amylases are universally distributed throughout the animal, plant and microbial kingdoms…     

Digging out molecular markers associated with low salinity tolerance of Nannochloropsis oceanica through bulked mutant analysis

Journal of Oceanology and Limnology - Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance. It grows fast, contains rich oils, reproduces...     

Antiviral Effects of Stichopus japonicus Acid Mucopolysaccharide on Hepatitis B Virus Transgenic Mice

Hepatitis B virus(HBV) is a significant global pathogen and efficient cure for HBV patients is still a challenging goal. We previously reported that acidic mucopolysaccharide from stichopus japonicus selenka(SJAMP) could inhibit HBs Ag and HBe Ag expression in vitro. However, the potential anti-HBV effects of SJAMP in vivo have not yet been explored. In this study, we show that SJAMP exhibits potent anti-HBV activity in HBV transgenic mice in a dose-dependent manner. Specifically, sixty HBV transgenic male BALB/c mice were randomly selected to receive the treatment of PBS, low dose SJAMP(30 mg kg~(-1)), middle dose SJAMP(40 mg kg~(-1)), high dose SJAMP(50 mg kg~(-1)) and IFN(45 IU kg~(-1)) for 30 d. SJAMP treatment suppressed serum HBV-DNA, and liver HBs Ag and HBc Ag levels in HBV-transgenic mice. The present study highlights the potential application of SJAMP in HBV therapy.     

Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

A Gram negative bacterium Ar/W/b/75°25＇N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25＇N, and longitude 162°25＇W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30(℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0. 5 % to 10 % sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source.About 75.7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.     

Efficient expression of green fluorescent protein （GFP） mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii,a high efficient expression vector was constructed.Green fluorescent protein(GFP) was expressed in C.reinhardtii under the control of promoters:RBCS2 and HSP70A-RBCS2.Efficiency of transformation and expression were compared between two transgenic algae:RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ.Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2,and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ.In addition,a threefold increase of GFP in Tran-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.     

一株H_5N_1亚型禽流感病毒株NA基因的克隆与序列分析

用RT-PCR方法从1个H5N1亚型禽流感病毒分离株A/Chicken/Guangdong/DH/1997扩增NA基因cDNA片段,将其克隆至pMD18-T载体,获得重组质粒pMD-NA,并对其核苷酸序列进行测定和分析。结果表明,该毒株的NA基因长度为1350bp,编码449个氨基酸,与其它H5N1亚型AIV分离株的核苷酸序列同源性为97.0%～99.4%,氨基酸序列同源性为97.7%～99.1%,提示禽流感病毒NA基因保守性较高。NA基因氨基酸序列的聚类分析表明该毒株与来自香港的A/Pheasant/HK/FY155/01和A/Ch/HK/FY150/01两个分离株处于同一进化枝,亲缘关系较近。     

Abundance, biomass and composition of spring ice algal and phytoplankton communities of the Laptev Sea （Arctic）

Abundance,biomass and composition of the ice algal and phytoplank-ton communities were investigated in the southeastern Laptev Sea in spring 1999.Diatoms dominated the algal communities and pennate diatoms dominated the dia-tom population.12 dominant algal species occurred within sea ice and underlyingwater column,including Fragilariopsis oceanica,F.cylindrus,Nitzschiafrigida,N.promare,Achnanthes taeniata,Nitzschia neofrigida,Naviculapelagica,N.vanhoef fenii,N.septentrionalis,Melosira arctica,Clindrothecaclosterium and Pyrarnimonas sp.The algal abundance of bottom 10 cm sea icevaried between 14.6 and 1562.2×10~4 ceils l~(-1)with an average of 639.0×10~4cells l~(-1),and the algal biomass ranged from 7.89 to 2093.5μg C l~(-1)with an av-erage of 886.9μg C l~(-1),which were generally one order of magnitude higherthan those of sub-bottom ice and two orders of magnitude higher than those ofunderlying surface water.The integrated algal abundance and biomass of lower-most 20 cm ice column were averagely 7.7 and 12.2 times as those of upper 20 mwater column,respectively,suggesting that the ice algae might play an importantrole in maintaining the coastal marine ecosystem before the thawing of sea ice.Icealgae influenced the phytoplankton community of the underlying water column.However,the“seeding”of ice algae for phytoplankton bloom was negligible be-cause of the iow phytoplankton biomass within the underlying water column.     

Denitrification potential evaluation of a newly indigenous aerobic denitrifier isolated from largemouth bass Micropterus salmoides culture pond

This work evaluates the application potential of a new indigenous aerobic denitrifi er, strain Pseudomonas CW-2, isolated from a largemouth bass culture pond. The rate of ammonium-N removal by strain CW-2 was approximately 97% at a DO concentration of 5.2 mg/L. Furthermore, when nitrate and ammonia coexisted, the strain gave priority to assimilating ammonia, and thereafter to denitrifi cation. Under optimal cultivation conditions, citrate and acetate were the carbon resources, C/N was 8, dissolved oxygen was 5.2 mg/L, and pH was 7; the removal rate of ammonium reached nearly 90%. The changing patterns of different bacteria in strain CW-2-treated and the control pond water were also compared. Lower levels of ammonia, nitrite, and phosphates were observed in the treated water as compared with the controls. Meanwhile, phylum-level distributions of the bacterial OTUs revealed that P roteobacteria, Bacteroidetes, Planctomycetes, and N itrospirae continuously changed their relative abundances in relation to carbon and the addition of strain CW-2; this finding implies that the conventional denitrifi cation process was weakened under the ef fects of carbon or the presence of strain CW-2. We propose that strain CW-2 is a promising organism for the removal of ammonium in intensive fish culture systems, according to our evaluations of its denitrifi cation performance.     

Amylase production by the marine yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 °C, aeration rate 6 Lmin⁻¹ and agitation speed 250 rmin⁻¹. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.     

一株海水氨氧化细菌的系统发育分析

应用PCR技术扩增16S rRNA基因和amoA(ammonia monooxygenase subunit A)的基因片段,并测定其序列,对一株源于海水养殖水体的高效氨氧化细菌(ammonia-oxidizing bacteria,AOB)进行了系统发育分析。结果表明,经PCR扩增得到了1 098 bp的16S rRNA基因片段和491 bp的amoA基因片段,将其序列用NCBI-Blast软件在GenBank数据库中进行同源性检索后发现,该菌株的16S rRNA基因序列和amoA基因序列分别与亚硝化单胞菌Nitrosomonas sp.NS20的相对应基因片段相似性分别为98.4%和96.7%。在此基础上构建了系统发育树,表明用该菌株的16S rRNA基因片段和amoA基因片段构建的系统发育树均与亚硝化单胞菌属类聚在一起,结合该菌株形态和生理生化特性,鉴定该株氨氧化细菌属亚硝化单胞菌。     

Preliminarily study on the maximum handling size,prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have lesser environmental impacts than non-transgenic carp.