Genetic variation of Laminaria japonica (Phaeophyta) populations in China as revealed by RAPD markers

For the population genetics analysis of the naturally grown brown seaweed Laminaria japonica （Laminariales,Phaeophyta） sampled from Dalian,Yantai,Weihai,Rongcheng and Qingdao in China,ten primers were employed to produce 88 bands as revealed by randomly amplified polymorphic DNA （RAPD） markers,and all these bands were polymorphic.According to these band patterns,there were 94 distinct phenotypes occurred in 100 samples indicating the high heterozygosity of this kelp.Dalian population samples showed the highest percentage of polymorphism （71.67%）,and also the higher diversity estimated on the basis of the Shannon’s index （8.498）,suggesting that this population could be chosen as the best resource for genetic breeding.The highest diversity of Yantai population possibly resulted from the introduction of L.longissima used for interspecific cross breeding with L.japonica cultivated in China.From Dalian southwards to Qingdao,the genetic variation of the five populations became less with a decrease in latitude,possibly due to the natural selection especially of high temperature.The genetic distance （Φ ST values） of the five populations was a little significantly correlated with the geographical distance （r=0.496） at P =0.05 by Mantel’s test.Weihai,Rongcheng and Yantai populations were closely grouped genetically together by Neighbor-joining cluster analysis probably in that the dispersal of the kelp by propagules more easily occurring in the range of relatively short distance.The analysis of molecular variance （AMOVA） also demonstrated that the relatively higher variation occurred among populations （71.49%） at an extremely significant level （P 0.000 1）.All these evidence showed that there was a relatively distinct genetic differentiation among the sampled kelp populations,and L.japonica grown in China was also rather heterozygous in heredity.

海带(Laminaria japonica)幼孢子体生长和光合作用的N需求

根据室内和围隔实验中海带幼孢子体在不同硝态氮浓度下的生长情况和光合作用速率(Pmax),分析了幼孢子体的N需求,得到其最大生长率(μm)为0.12d-1,维持生存的最低组织N含量(NQ)为16.8μg/mgDW,以最大生长率生长所必需组织N的临界值(NC)为20.4μg/mgDW,每天以最大速率生长的N需求(Nreq)为2.45μg/mgDWd-1。同时,不同处理组的初始NO3-N浓度越高,海带幼孢子体吸收速率和组织N的累加速率越高,且呈明显的线性相关关系(R2分别为0.8393和0.7793,P<0.05)。现场围隔实验中,叶绿素a含量(R2=0.7907,P<0.05)和组织N含量(R2=0.9147,P<0.01)与Pmax也呈明显的线性相关关系。同时,根据海带幼孢子体N的需求和营养吸收状况,分析认为,海带幼孢子体存在受到N限制的风险,但凭其营养吸收能力有适应N限制的能力。还根据海带的这种生理特征,探讨了大型海藻的养殖对富营养化海水的生态调控。

海带(Laminaria japonica)丝状体遗传多样性的比较研究

采用垂直板式聚丙烯酰胺凝胶电泳技术对分别来自中国青岛与日本北海道海带（Ｌａｍｉｎａｒｉａｊａｐｏｎｉｃａ）丝状体的６种酶系统（乙醇脱氢酶、谷氨酸脱氢酶、异柠檬酸脱氢酶、乳酸脱氢酶、山梨醇脱氢酶和超氧化物歧化酶）进行了检测，发现存在２２个基因位点，分析了不同来源海带丝状体的遗传多样性与遗传分化程度。结果表明：日本北海道海带丝状体的预期杂合度、每个位点有效等位基因数等遗传参数均大于中国青岛海带丝状体。对日本北海道海带丝状体与中国青岛海带丝状体的遗传相似系数和遗传距离进行了计算分析，显示两者发生了遗传分化。日本北海道海带丝状体的遗传多样性高于中国青岛海带丝状体。研究结果还发现在中国青岛海带丝状体的Ｉｄｈ、Ｌｄｈ和Ｓｄｈ中，以及日本北海道海带丝状体的Ａｄｈ、Ｇｄｈ、Ｉｄｈ、Ｌｄｈ、Ｓｄｈ和Ｓｏｄ中存在着不同迁移率的不等距双带酶，可能由特殊基因控制，并集中在第３个基因位点上。

褐藻酸降解菌在海带(Laminaria japonica)幼苗藻体表面数量分布特点及其对海带回染的初步研究

在威海泊于、崮山及荣成 3家海带育苗场 ,采用回染实验方法对采集的海带藻体表面褐藻酸降解菌数量分布及特点进行了初步研究。结果表明 ,采自荣成的病烂海带幼苗上 ,其总异养细菌数量 ( 1 2 2× 1 0 8cfu/g)比威海两家育苗场的正常海带幼苗上总异养细菌数量 (崮山 :1 1 5× 1 0 6cfu/g及泊于 :1 1 1× 1 0 6cfu/g)高 1 0 0多倍 ,其褐藻酸降解菌的数量 ( 4 88× 1 0 7cfu/g)比后两家 (崮山 :8 0× 1 0 4 cfu/g及泊于 :4 3× 1 0 5cfu/g)的高 1 0 0— 5 0 0倍。与正常海带幼苗相比 ,烂苗脱落后的总异养细菌数量较高 ( 2 42× 1 0 7∶1 1 1× 1 0 6) ,而褐藻酸降解菌的数量相对更高 ( 1 5 0× 1 0 7∶4 3× 1 0 5) ;其中 ,脱落烂苗上的褐藻酸降解菌数量约占其总异养细菌数量的61 98%。将分离、纯化的部分褐藻酸降解菌对海带孢子体组织块进行回染实验 ,能引起海带组织产生绿烂现象 ,证明褐藻酸降解菌是海带烂苗的条件致病菌。

我国引种海带(Laminaria japonica)和长海带(L．longissima)配子体克隆的随机扩增多态性DNA分析

运用RAPD技术,对引种我国的海带(Laminaria japonica)和长海带(L.longissima)的22个配子体克隆进行分析。筛选的19条随机引物共获得252个位点.扩增片段大小在200～2000bp之间,多态性位点228个,占90.5%。配子体克隆遗传变异分析表明,长海带和海带的遗传多样性都比较高,两者之间的遗传分化较为明显,且种间变异大于种内变异。长海带和海带雄配子体克隆的遗传变异程度均低于雌配子体克隆。另外,两个种的雌、雄配子体之间均存在一定程度的分化,且长海带雌、雄配子体之间的遗传分化大于海带配子体。

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Larninaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as A lteromonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that： （1）The blades of L.japonica exhibited symptoms of lesion,bleaching and deteriortion when infected by the bacterium. and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species （ROS） generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde （MDA） and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. （2） The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase （SOD） and catalase （CAT） increased greatly when infected by the bacterium, but glutathione peroxidase （Gpx） and ascorbate peroxidase （APX） did not exhibit active responses to the bacterium throughout the experiment. （3） The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

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 molLl 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‰ NaCl 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.

Genetical study on the parthenogenesis in Laminaria japonica

The parthenogenesis and natural doubling of chromosomes in wild type female gametophytes of Laminaria japonica were studied. The results indicate that not all the female gametophytes from the wild type hybrid parent can propagate through parthenogenesis. Most parthenosporophytes can mature, their spores germinate into gametophytes, the latter then developed into female sporophytes. To form these parthenosporophytes the natural doubling of chromosomes occurred mainly at the first and second cell divisions of the spores. It is thus considered that the parthenogenesis of L. japonica is inheritable and the relative genes link closely with the genes controlling the natural doubling of chromosomes and the female determination.

Parentage Analysis of Dongfang No.2, a Hybrid of a Female Gametophyte Clone of Laminaria japonica (Laminariales, Phaeophyta) and a Male Clone of L. longissima

The cultivation of the first filial generation of monoploid gametophyte clones of different Laminaria species (hybrid Laminaria) is an effective way of utilizing heterozygous vigor (heterosis). A female gametophyte clone of L. japonica and a male gametophyte clone of L. longissima were hybridized, generating Dongfang No.2 hybrid Laminaria. The parentage of this hybrid Laminaria was determined using AFLP of total DNA, SNP of the ITS region of ribosomal RNA transcription unit and microsatellite DNA variation at two loci. In addition to 167 AFLP bands shared by Dongfang No.2, L. japonica and L. longissima, Dongfang No.2 hybrid Laminaria shared another 70 and 55 bands with L. japonica and L. longissima, respectively, which were obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No.2 held both ‘T’ and ‘C’ at position 847 of the ITS region, while ‘T’ at this position was specific for L. japonica and ‘C’ for L. longissima, respectively. Dongfang No.2 also held the microsatellite DNA alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No.2 is a true hybrid of L. japonica and L. longissiuma. Unfortunately, the origin of the chloroplast of Dongfang No.2 was not determined based on the variation of RuBisCo spacer. More sequence variants of both ITS region and RuBisCo spacer were identified in Dongfang No.2 and most of them did not exist in either L. japonica or L. longissima. The unexpected variants may be due to the mutation of ga- metophyte clones occurring during their vegetative amplification.

EFFECTS OF ULTRASONIC TREATMENT ON FEMALE GAMETOPHYTES OF LAMINARIA JAPONICA (PHAEOPHYTA)

This study on the effects of ultrasonic treatment on female gametophytes of Laminariajaponica showed that:1. Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certainperiod of time, the average length of filamentous female gametophytes was shortened.2. Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with timeof treatment. Ultrasonic treatment had harmful effect on cells.3. Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, out-put of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regner-ation of female gametophytes into sporophytes was effected. Female gametophytes could not recover aftertoo long period of treatment.

Salicylic acid and heat acclimation pretreatment protects Laminaria japonica sporophyte(Phaeophyceae) from heat stress

Possible mediatory roles of heat acclimation and salicylic acid in protecting the sporophyte of marine macroalga Laminaria japonica(Phaeophyceae) from heat stress were studied.Heat stress resulted in oxidative injury in the kelp blades.Under heat stress significant accumulation of hydrogen peroxide(H_2O_2) and malonaldehyde(MDA),a membrane lipid peroxidation product,and a drastic decrease in chlorophyll a content were recorded.Activity of the enzymatic antioxidant system was drastically affected by heat stress.The activity of superoxide dismutase(SOD) was significantly increased while peroxidase(POD),catalase(CAT) and glutathione peroxidase(GPX) were greatly inhibited and,simultaneously,phenylalanine ammonia-lyase was activated while polyphenol oxidase(PPO) was inhibited.Both heat acclimation pretreatment and exogenous application of salicylic acid alleviated oxidative damage in kelp blades.Blades receiving heat acclimation pretreatment and exogenous salicylic acid prior to heat stress exhibited a reduced increase in H_2O_2 and MDA content,and a lower reduction in chlorophyll a content.Pretreatment with heat acclimation and salicylic acid elevated activities of SOD,POD,CAT,GPX and PPO.Considering these results collectively,we speculate that the inhibition of antioxidant enzymes is a possible cause of the heat-stress-induced oxidative stress in L.japonica,and enhanced thermotolerance may be associated,at least in part,with the elevated activity of the enzymatic antioxidant system.

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 devel- opment of Laminaria 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 inten- sity 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 repro- ductive 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 the Laminaria 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 of Laminaria gametophytes.

Immunosuppressive Activities of Fucoidan from Laminaria japonica

Effects of fucoidan from Laminaria japonica on 2,4 dinitrochlorobenzene induced delayed type hypersensitivity (DTH) reaction and the serum levels of IgG, IgM, complement C3 and C4 were investigated in the present study. Results showed that oral administration of fucoidan at dose of 150 and 300 mg/(kg·d) for 9 days before the hapten challenge significantly inhibited 2,4 dinitrochlorobenzene induced delayed type hypersensitivity reaction; and also inhibited the humoral immunity. Serum C3 and C4 levels were markedly reduced by fucoidan at dose of 150 and 300 mg/kg; and serum IgG and IgM levels were reduced by fucoidan at dose of 300 mg/kg. The inhibitory effects of fucoidan on delayed type hypersensitivity suggested that it may be potential medication for chronic inflammatory diseases in the future.

STUDY ON THE BLOOD FAT REGULATION OF THE FUCOIDA-GALACTOSAN SULFATE (FGS) FROM LAMINARIA JAPONICA

FGS was isolated and purified from laminaria japonica by enzymolysis, chromatography methods and so on. The high blood fat model was established by feeding mouse on high fat feedstuff. Compared with control group, the concentration of HDL-C, the ratio of HDL-C/TC increased;while the concentration of TC, LDL-C and TG decreased in experimental groups. Conclusion: FGS was an effective serum lipid regulator and can be developed as medicine and health food.

The extraction of pigments from fresh Laminaria japonica

The pigments in Laminaria japonica was extracted with six organic solvents and analyzed in spectroscopy analysis. The extractions conditions were screened by an orthogonal test and the quantity of extracted pigments was determined spectroscopically. The results show that: (1) among the six organic solvents, acetone was the most effective one for the extraction; (2) the optimum extraction conditions were as follows: the ratio of S/M (solvent volume/material weight) was 30 ml/g; fresh seaweed was extracted 2 times in 2h; (3) the average total content of pigments was 1.85 mg/g (calculated with dry L. japonica).

STUDIES ON THE ISOLATION PURIFICATION AND CHARACTERIZATION OF THE FUCOIDAN-GALACTOSAN SULFATE (FGS) FROM LAMINARIA JAPONICA

FGS, isolated from the water solution of enzymolyzed laminaria japonica, is a mixture of acid heteroglycans. Four fractions F1, F2, F3, and F4 were obtained from FGS by ion exchange chromatography. After further purified by gel filtration chromatography on a sepharose 2B and 6B column, we obtained F9, F10, F11, and F12. They showed single band when identified by electrophoresis. The molecular weight of F9, F10, F11, and F12 was estimated to be 216, 120, 138 and 140KD respectively. They containedа-glucosidic bond by IR and 1H-NMR analysis. The typical absorption peaks of these polysaccharides were showed in UV and IR spectra. These polysaccharides contained rha, fuc, man, gal, and uronate when identified by paper chromatography (p.c) and gas chromatography (GC). The molar ratio of these sugars was also assayed.

Molecular Weight Controllable Degradation of Laminaria japonica Polysaccharides and Its Antioxidant Properties

In this study, molecular weight controllable degradation of algal Laminaria japonica polysaccharides(LPS) was investigated by ultrasound combined with hydrogen peroxide. Three main factors, i.e., ultrasonic power(A), ultrasonic time(B), and H_2O_2 concentration(C) were chosen for optimizing parameters by employing three-factors, three-levels BBD. The influence of degradation on structure change and antioxidant activities was also investigated. A second-order polynomial equation including molecular weight(Y) of Laminaria japonica polysaccharides and each variable parameter, i.e., ultrasonic power(A), ultrasonic time(B), and H_2O_2 concentration(C), was established: Y=20718.67-4273.13A-4000.38B-1438.75C+2333.25AB+1511.00AC+873.00BC+2838.29A^2 + 2490.79B^2+873.04C^2. The equation regression coefficient value(R^2 = 0.969) indicated that this equation was valid. The value of the adjusted determination coefficient(adjusted R^2 = 0.914) also confirmed that the model was highly significant. The results of selected experimental degradation conditions matched with the predicted value. FT-IR spectra revealed that the structures of LPS before and after degradation were not significantly changed. Antioxidant activities of LPS revealed that low Mws possessed stronger inhibitory than the original polysaccharides. The scavenging effects on superoxide radicals was the highest when IC50 of crude LPS was 4.92 mg mL^(-1) and IC50 of Mw 18.576 KDa was 1.02 mg mL^(-1), which was fourfold higher than initial polysaccharide.

A TRANSFORMATION MODEL FOR LAMINARIA JAPONICA (PHAEOPHYTA, LAMINARIALES)

A genetic transformation model for the seaweed Laminaria japonica mainly includesthe following aspects:1. The method to introduce foreign genes into the kelp, L. japonicaBiolistic bombardment has been proved to be an effective method to bombard foreign DNA throughcell walls into intact cells of both sporophytes and gametophytes. The expression of cat and lacZ was de-tected in regenerated sporophytes, which suggests that this method could induce the integration of for-eign genes. 2. Promoters to drive gene expressionThe 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 ofSV40 promoter resulted in both transient and stable expression of lacZ and cat in sporophytes or gameto-phytes. No GUS or LacZ background was found in either sporophytes or gametophytes.3. The regeneration route of transgenic kelpThe regeneration efficiency of explants is still very low. By using female gametophytes as gene hostsand parthenogenesis as regeneration route, CAT activity and LacZ activity were detected in regeneratedsporophytes of parthenogenetic kelp.4. The way to select transgenic kelpResults of sensitivity tests showed that kelp was only sensitive to chloramphenicol and hygromycin a-mong many antibiotics. The regenerated sporophytes by parthenogenesis were more sensitive to hygromycinthan 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 chloram-phenicol,.5. Safety consideration of transgenic kelp1) L. japonica was originally introduced from Japan. In China it is a cultured population. The pos-sibility of its negative impact on natural populations is very low. 2) The vectors and target genes used fortransformation should be restricted In order to avoid any negative impacts on human health and environ-ment. 3)Specially devised containers (3. 6 L, made of 200 μm membrane) were used to ensure that thekelp cannot escape or be eaten by marine animals. 4) To avoid the release of spores, it is very necessaryto harvest the kelp at suitable age before the sporangium forms.

HIGH EFFICIENCY INDUCTION OF CALLUS AND REGENERATION OF SPOROPHYTES OF LAMINARIA JAPONICA(PHAEOPHYTA)

Four media (PESI solid, MS liquid, MS solid and ASP-C-I solid medium) were usedto induce callus from excised tissues of the kelp Laminaria japonica. Only PESI solid medium and MSsolid medium produced calli. Modified MS solid medium supplemented with mannitol (3%, W/V), yeastextract (0.1%, W/V), VB2(0. 5 mg/ml), VB12(0.5 mg/ml), kinetin (0. 108 μg/ml) and NAA(1.860μg/ml) showed much better effect on callus induction than non-modified MS solid medium. After24 days of induction 75 .5% of tissues in PESI solid medium showed callus formation. For modified MSsolid medium, after three months of induction 67. 3% of tissues dedifferentiated into calli. No calluscould be found after five months of induction in either MS liquid or ASP-C-I solid medium. When calliwere squashed and cultured in N-P enriched autoclaved seawater, MS liquld medium and ASP12-NTAliquid medium (both modified with kelp extract), differentiation of cells and regeneration of sporophyteswere only observed in ASP12-NTA medium supplemented with kelp extract. Gametophyte-like filamentsformed first, then eggs were released. It was suggested that sporophyte formation could be a process ofparthenogenesis. Sterilization techniques in tissue culture of L. japonica were also tested in this study.

The effects of fucoidans from Laminaria japonica on AAPH mediated oxidation of human low-density lipoprotein

Five fucoidan fractions from Laminaria japonica with different sulfate content and molecular weight were prepared by anion-exchange chromatography and mild acid hydrolysis. Their antioxidant effects on azo radicals 2-2＇-Azobis （2-amidinopropane） dihydrochloride （AAPH） induced oxidation of human low-density lipoprotein （LDL） were evaluated by monitoring cholesteryl ester hydroperoxides （CHL-OOH） formation kinetics through reverse-phase high performance liquid chromatography （RP-HPLC） analysis. Fucoidan F - C with a low molecular mass of 2 000 ～8 000 and a sulfate content of 24.3% had much stronger protective antioxidant effect than other four fucoidan fractions on the hydrophilic radical AAPH induced LDL oxidation. However, the highly sulfated fucoidan fraction L - B with a molecular mass of 20 000 was completely ineffective in protecting LDL from the AAPH induced oxidation. The results suggested that both molecular mass and sulfate content of fucoidan played very important roles in their effects on the AAPH induced LDL oxidation.