Effect of heavy metals (Cd, Cu) on the gametophytes of Laminaria japonica Aresch

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminaria japonica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal (Cd) distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu tip to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning (the first day) of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd- and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods. All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.

Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

A new method of Laminaria japonica strain selection and sporeling raising by the use of gametophyte clones

One-celled female and male gametophytes of three Laminaria japonica strains were isolated, cultured and gametophyte clones were formed. A technique combining strain selection with sporeling raising by the use of these female and male gametophyte clones was studied. Experiments on 9 different crossing combinations was conducted in November of 1997 in Qingdao, P. R. China. The main economic characteristics, frond length and fresh weight, of sporophytes of different crossing combinations were measured. F-1 sporophytes of No. 2 showed a higher fresh weight and longer length, therefore, No. 2 (Wh860 + x Lid) was selected as a good combination. Its parental female and male gametophyte clones are being mass cultured for sporeling production. By this method, the time needed for strain selection was shortened from 5-6 to 2 years. As compared with the routine method of sporeling raising by the collection of zoospores, the time of sporeling raising of this method decrease by 50%, and the production cost is also reduced by 50%. It is believed that this method will be labour and time saving and a more economic way for strain selection and sporeling raising in L. japonica cultivation industry.

Review of genetic engineering of Laminaria japonica (Laminariales, Phaeophyta) in China

Progress has been made in establishing a genetic transformation model for Laminaria japonica (Phaeophyta, Laminariales). The model includes introduction of foreign genes by biolistic bombardment, use of promoter SV40 to drive gene expression, algal regeneration by parthenogenesis and selection by chloramphenicol or hygromycin.

Purification of High-quality Plastid DNA from the Brown Alga Laminaria japonica Sporophytes

Extracting DNA from a variety of algae is rather difficult because of high levels of polysaccharides, tannins, and phenolics as these interfere with DNA isolation and downstream applications. High-quality plastid DNA (ptDNA) purification is particularly difficult because of its small proportion in total genomic DNA. This report describes an improved protocol for ptDNA purification that efficiently produces high-quality ptDNA from sporophytes of Laminaria japonica and several other algae. This improved protocol simplifies procedures for ptDNA purification and improves yield to 150-200 mu g of ptDNA per 100 g of frozen algal tissue. Polymerase chain reaction (PCR) amplification of conserved sequences has been used to verify purity of the ptDNA product.

Temperature tolerance of young sporophytes from two populations of Laminaria japonica revealed by chlorophyll fluorescence measurements and short-term growth and survival performances in tank culture

The cold-water subtidal brown alga Laminaria japonica has been commercially fanned in the Far East and has been on top of all marine-fanned species in terms of farming area and annual output worldwide. The successful trials of transplantation of young sporophytes from the north to the south in winter along the Chinese coast in the 1950s led to the spreading of cultivation activities down to a latitude of 25-26 degrees N. Up to today, nearly 50% of the annual output of this farmed alga, as a cold-water species, comes from the sub-tropical south in China. The demand to have high-temperature-tolerant strains/ecotypes in farming area calls for a practical method to judge and select the desired parental plants for breeding programs and for seedling production. In this paper, we report our results on using chlorophyll fluorescence measurement and short-term growth performance in tank culture to estimate the temperature tolerance of offspring from two populations, Fujian Farmed Population (FFP) sampled from Fujian province (latitude: 25-26 degrees N) in subtropical area and Qingdao Wild Population (QWP) sampled from Qingdao (latitude: 36 degrees N). Contrary to what has been usually thought, the results revealed that offspring from Qingdao wild population in the north showed better performance both in short-term growth and survival rates and in optimal quantum efficiency (F-v/F-m) when exposed to higher temperature (20-25 degrees C). This result was further confirmed by fluorescence quenching analysis. QWP distributed along the southern distribution limit at a latitude of 36 degrees N in the Pacific west coast is thus taken as a more ideal one than the fanned population in subtropical region as a source of parental plants for breeding high-temperature-tolerant varieties. (c) 2006 Elsevier B.V. All rights reserved.

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Complete mitochondrial genome of the Asian paddle crab Charybdis japonica (Crustacea: Decapoda: Portunidae): gene rearrangement of the marine brachyurans and phylogenetic considerations of the decapods

Given the commercial and ecological importance of the Asian paddle crab, Charybdis japonica, there is a clearly need for genetic and molecular research on this species. Here, we present the complete mitochondrial genome sequence of C. japonica, determined by the long-polymerase chain reaction and primer walking sequencing method. The entire genome is 15,738 bp in length, encoding a standard set of 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes, plus the putative control region, which is typical for metazoans. The total A+T content of the genome is 69.2%, lower than the other brachyuran crabs except for Callinectes sapidus. The gene order is identical to the published marine brachyurans and differs from the ancestral pancrustacean order by only the position of the tRNA (His) gene. Phylogenetic analyses using the concatenated nucleotide and amino acid sequences of 13 protein-coding genes strongly support the monophyly of Dendrobranchiata and Pleocyemata, which is consistent with the previous taxonomic classification. However, the systematic status of Charybdis within subfamily Thalamitinae of family Portunidae is not supported. C. japonica, as the first species of Charybdis with complete mitochondrial genome available, will provide important information on both genomics and molecular ecology of the group.

Rapid optimization of process conditions for cultivation of transgenic Laminaria japonica gametophyte cells in a stirred-tank bioreactor

Batch cultivation for transgenic kelp gametophyte cells was investigated in an online controlled 5 L stirred-tank photo-bioreactor to rapidly optimize the process conditions by monitoring the rate of increase of pH. The transgenic kelp gametophytes with heterologous gene encoding hepatitis B surface antigen (HBsAg) could rapidly grow in the bioreactor. Optimal temperature and agitation rate for bioreactor cultivation of gametophytes were 15 degrees C and 200 rpm. Optimal incident light intensities depended on the initial cell densities. (c) 2006 Elsevier B.V. All fights reserved.

QTL Mapping for Frond Length and Width in Laminaria japonica Aresch (Laminarales, Phaeophyta) Using AFLP and SSR Markers

In Laminaria japonica Aresch breeding practice, two quantitative traits, frond length (FL) and frond width (FW), are the most important phenotypic selection index. In order to increase the breeding efficiency by integrating phenotypic selection and marker-assisted selection, the first set of QTL controlling the two traits were determined in F-2 family using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Two prominent L. japonicas inbred lines, one with "broad and thin blade" characteristics and another with "long and narrow blade" characteristics, were applied in the hybridization to yield the F-2 mapping population with 92 individuals. A total of 287 AFLP markers and 11 SSR markers were used to construct a L. japonica genetic map. The yielded map was consisted of 28 linkage groups (LG) named LG1 to LG28, spanning 1,811.1 cM with an average interval of 6.7 cM and covering the 82.8% of the estimated genome 2,186.7 cM. While three genome-wide significant QTL were detected on LG1 (two QTL) and LG4 for "FL," explaining in total 42.36% of the phenotypic variance, two QTL were identified on LG3 and LG5 for the trait "FW," accounting for the total of 36.39% of the phenotypic variance. The gene action of these QTL was additive and partially dominant. The yielded linkage map and the detected QTL can provide a tool for further genetic analysis of two traits and be potential for maker-assisted selection in L. japonica breeding.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Metabolism of polychaete Neanthes japonica Izuka: relations to temperature, salinity and body weight

Polychaete Neanthes japonica is a species geographically specific in China and Japan with important scientific implication and commercial value. In this study, the relations of body weight, salinity and temperature to oxygen consumption and ammonia excretion of N. japonica were determined. Three different groups in body weight (large: 2.34 +/- 0.36 g, middle: 1.50 +/- 0.21 g and small: 0.62 +/- 0.12 g) were set for all experiments. Results show that the body weight is negatively related to the rates of oxygen consumption and ammonia excretion; and the relationship is significant. The oxygen consumption and ammonia excretion at 24A degrees C decreased at salinity from 5 to 30 and increased above 30, indicating that both lower and higher salinity are adverse and certain degree of salinity stress is necessary for enhancing the energy demand. At salinity 30, rising temperature from 18A degrees C to 30A degrees C, the oxygen consumption increased before 27A degrees C and then decreased. However, the relation of ammonia excretion and temperature seems more complex. Two-way ANOVA shows that salinity, temperature and body weight all have a significant effect on the oxygen consumption and ammonia excretion of the worm. Moreover, interaction between salinity/temperature and body weight is also significant. O:N (oxygen/nitrogen) ratio varies greatly in this case from 5.97 to 463.22, indicating that N. japonica can regulate the type of metabolic substrate against environment changes.