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.

     

Application of RAPD in genetic diversity study onGracilaria lemaneiformis III. Phase and sex related markers

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 random primers (P-1 to P-10) inGracilaria lemaneiformis. Phase and sex specific bands were amplified by primers P-2, P-6, P-7 and P-8: for P-2 a 1.4 kb band was found in female gametophytes and tetrasporophytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.76 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in female gametophytes and tetrasporophytes; for P-8, a 0.73 kb band only appeared in male gametophytes.

     

Identification and characterization of a DnaJ gene from red alga <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> (Bangiales,Rhodophyta)

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis (PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

Early development of Costaria costata (C. Agardh) Saunders and cultivation trials

Costaria costata (C. Agardh) Saunders is one of common kelps distributed in many coastal areas worldwide; however, in China, no reports have been made on cultivation of the genus. To investigate potential cultivation of the species in the northern part of China, trials on isolation and preservation of the gametophytes were conducted using C. costata from Korea; growth and development of the gametophytes were observed. We showed that at 10±1°C, 60 μmol m⁻²s⁻¹ and 12:12 h (L:D), freshly released zoospores settled down within 1 hour, and then developed into the primary cell during the following 2 days. After a vegetative growth phase lasting 6–8 days, female gametophytes became 3–4 times larger in diameter than that of the primary cell, but still remained at a unicellular stage, while male gametophytes divided into 4–10 cells with only a slight change in size. Fertilization occurred within 10 days after the zoospores were released from the sporangia, and the apical and basal tissues of the juvenile sporophyte divided and differentiated into the blade and stipe. Temperature and irradiance influenced gametophytic vegetative growth and developmental patterns. Generally, low irradiance (15 μmol m⁻²s⁻¹ and 30 μmol m⁻²s⁻¹) was unfavorable to the induction of fertility, but it enhanced female gametophyte division. The optimal conditions for vegetative growth were 15°C and 30 μmol m⁻²s⁻¹. After transplantation of the juvenile seedlings and after eight months cultivation, the harvested mature blade reached 194 cm in length and 32.7 cm in width. Our study proves that it is feasible to implement propagation and large scale cultivation of C. costata in northern China.     

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.

     

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis （Bory） is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplified fragment length polymorphism （AFLP） technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplification primers were used in this study. The primer combination E-TG/M-CCA detected a specific band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplified region （SCAR） primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identification of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae...     

Effects of blue light on gametophyte development of Laminaria japonica (Laminariales, Phaeophyta)

Laminaria gametophyte was greatly influenced by light in its growth and development. Using light-emitting diodes (LED) as blue and red light sources, we analyzed the light effect on gametophytes development ofLaminaria japonica Aresch. The gametophytes were obtained from zoospores collected in April, May, July, 2003 and September, 2004. We found that the growth of gametophytes was stimulated by increasing intensity of blue light (BL) and red light (RL) illumination, of which BL was obviously stronger than that of RL. The fertilization of gametophytes depended largely on BL, and only sufficient BL illumination could take the reproductive effect. In addition, we noticed that there was a significant difference in light responses for gametophytes developed from zoospore collected in different times. For zoospores released in April, under BL₁ (73.90 μmol photons/m·s), the unicellular female gametophytes and multi-cellular male gametophytes produced eggs and sperms respectively, and further developed towards sporophytes. However, for gametophytes developed in May, July or September, they became multi-cellular and never formed oogonia or antheridia. It is believed that theLaminaria sporangium maturation stage could affect the gametophytes reaction to BL under laboratory culture conditions. Therefore, cryptochrome- or phototropin-like BL photoreceptors is probably involved in BL-induced development ofLaminaria gametophytes. This research was supported by the NSFC (No.40376049) and Japan Society for the Promotion of Science (The Invitation Fellowship for Research in Japan 2002).     

Cloning and expression analysis of a ubiquitin gene (Ub L40 ) in the haemocytes of Crassostrea hongkongensis under bacterial challenge

Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5’ untranslated region (UTR) of 34 bp, a 3’-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge.     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and stone flounder <Emphasis Type="Italic">Kareius bicoloratus</Emphasis>

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)     

Cryopreservation of Gametophytes of Laminaria japonica （Phaeophyta） Using Encapsulation-Dehydration with Two-Step Cooling Method

Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m-2 s-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molL-1 sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for ＞24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃seawater and re-hydrated in 0.05 molL-1 citrate sodium prepared using 30‰ NaC1 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.     

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.

     

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales,Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments,and consequently may have highly effi cient ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)activity for carbon assimilation.In our study,we cloned the full-length Rubisco gene from S.japonica(SJ-rbc).It contained an open reading frame for a large subunit gene(SJ-rbcL)of 1 467 bp,a small subunit gene(SJ-rbcS)of 420 bp,and a SJ-rbcL /S intergenic spacer of 269 bp.The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa,5.81 and 15.84 kDa,4.71,respectively.After induction with 1 mmol/L isopropyl-β-Dthiogalactopyranoside for 5 h and purifi cation by Ni 2+ affi nity chromatography,electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein.Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night.This observation implied that Rubisco played a role in normal gametophytic growth and development.In juvenile sporophytes,mRNA levels of SJ-rbcL,carbonic anhydrase,Calvin-BensonBassham cycle-related enzyme,and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance.Similarly,expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m 2·s).Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Characterization of defensin gene from abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals＇ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides （including six Cys）, molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar （70% in common） to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin （hd-def）, a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.     

Induction and cultivation of cloned filaments of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

A filamentous clone of Polysiphonia urceolata was regenerated from segments cut from the fronds of gametophytes. Unlike wild thalli with short virgate branchlets, the clone was filamentous with few branches. Many transparent trichoblasts arose from pericentral cells during the induction culture, but these were seldom observed during normal growth. The trichoblasts were uniseriate, often colorless, and formed lobed rhizoids rapidly when they came into contact with solid substrates. In addition to morphological characteristics, the photosynthetic properties and growth conditions of the clone differed from those of the mother plant. Cross-gradient light and temperature culture experiments revealed that the most favorable conditions for culture of the filamentous clone were 22°C and 95-120 μE/(m2-s) light intensity. The photosynthetic light saturation value for filaments was approx. 100 μE/(m2-s), which is far lower than that of wild thalli. These results could be used to develop techniques for mass cultures of P. urceolata in photobioreactors for production of seed stock or bioactive products.