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1.
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. 相似文献
2.
Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901”
strain, genetic study on variation was conducted to its parents and offsprings of F 6, F 7, F 8, and F 9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp
were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA
(unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could
be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212–0.4767, and the maximum
was between F 7 and F 8 (0.4767). Identity analysis showed that F 6 generation was more close to the female parent (0.6593), and F 7 generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification,
191 loci ranging from 230–2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations
were male parent (0.2239), female parent (0.1072), F 6 (0.2164), F 7(0.2286), F 8(0.2296) and F 9(0.3172). The nearest genetic distance was 0.083 5(F 8, F 9). Total heterozygosity (H T) of F 6, F 7, F 8 and F 9 generations was 0.3186, the average heterozygosity (H S) for F 6, F 7, F 8 and F 9 generations was 0.2480, and deduced coefficient of population differentiation (G st) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed
to be verified in detail as they are significant for molecular marker assistance in breeding and selecting Laminaria. 相似文献
3.
Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901”
strain, genetic study on variation was conducted to its parents and offsprings of F 6, F 7, F 8, and F 9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp
were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA
(unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could
be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212–0.4767, and the maximum
was between F 7 and F 8 (0.4767). Identity analysis showed that F 6 generation was more close to the female parent (0.6593), and F 7 generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification,
191 loci ranging from 230–2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations
were male parent (0.2239), female parent (0.1072), F 6 (0.2164), F 7(0.2286), F 8(0.2296) and F 9(0.3172). The nearest genetic distance was 0.083 5(F 8, F 9). Total heterozygosity (H T) of F 6, F 7, F 8 and F 9 generations was 0.3186, the average heterozygosity (H S) for F 6, F 7, F 8 and F 9 generations was 0.2480, and deduced coefficient of population differentiation (G st) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed
to be verified in detail as they are significant for molecular marker assistance in breeding and selecting Laminaria.
Financially supported by 863 program of China (No.2001AA621090) 相似文献
4.
This study on the effects of ultrasonic treatment on female gametophytes ofLaminaria japonica showed that:
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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.
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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.
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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.
相似文献
5.
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. 相似文献
6.
When cultured under certain environmental conditions (25°C, light intensity 80 μmol/m 2·s, LD 12/12, in enriched seawater medium with 7×10 −4 mol/L NO 3−N, 1.56×10 −4 mol/L, PO 4−P and supplements of other elements like Mn, Fe, I, etc.), male and female gametophytes of U. pinnatifida kept growing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. The empirical formula G
m= G
o·3 m was established to estimate the output of vegetative gametophytes. Vigorous vegetative gametophyte cells began to form reproductive
structures (oogonium and spermatangium, when the temperature was lower than 25°C and other environmental factors were kept
optimal. The sufficient supply of gametophyte cells provided enough seeds for raising Undaria sporelings on production scale. Controlled cross-breeding experiments using selected male and female gametophyte clones which
increase their cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes from the cross-breeding
had almost the same length and width increase rates as those of the control. The fact that vegetative gametophytes can be
purposely selected, propagated quickly, cross-bred, with the resulting sporophytes having almost the same characteristics
leads to a new way to select desired Undaria strains for long time use without losing the desired economic characteristics.
Contribution No. 2696 from the Institute of Oceanology, Chinese Academy of Sciences.
Supported by National Natural Science Foundation of China. 相似文献
7.
Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers
selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance
among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic
(UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively.
It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship
if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship
among the species of Laminaria.
Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences.
This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090). 相似文献
8.
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. 相似文献
9.
A genetic transformation model for the seaweedLaminaria japonica mainly includes the following aspects:
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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
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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
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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.
相似文献
10.
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 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 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 BL 1 (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.
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). 相似文献
11.
Unicellular gametophytes of Undaria pinnatifida (Harv.) Sur. were isolated in Qingdao, P. R. China in April 1993 and in Tokushima, southern Japan in March 1995. Different
intraspecific crossings by using unicellular male and female gametophytes were successfully undertaken in Sept. of 1995 in
Qingdao. Sporophytes were transplanted to two different locations for open sea cultivation. One was at Zhanqiao (ZQ) Bay where
the water current was slower than that of another location—Taipingjiao(TPJ). A total of 218 adult sporophytes were harvested
on January 12 and 18 from TPJ and ZQ respectively. For eacn combination, 10 sporophytes were cultivated. Analysis of the morphological
characteristics of adult sporophytes indicated that the longest length between two bases of the serration of pinnate blades
(W2) is a morphological characteristic that can be transferred from the parent plant to the next generations regardless of
environmental variations. There was evidence that W2 was apparently determined by sex-linked factors, i.e., by male parental
gametophyte. Sporophytes from certain crossing combinations showed more vigorous growth than those from other crossing combinations.
It is therefore possible to select gametophyte strains which can be used as parental gametophytes for the seedling production
of sporophytes with more vigorous growth within shorter cultivation period. The morphology of hybrids from a Qingdao strain
and a Tokushima cultivated strain resembled that of both parental plants in frond features (wrinkled or smooth) and W2. Sporophyll
formation also varied with strains. The fact that adult sporophytes resulting from the same crossing combinations have identical
morphological characteristics under the same environmental conditions indicates the possibility of a new way to select strains
which are expected to be ideal for commerical production by purposely selecting, propagating, and seeding unicellular gametophytes
for sporeling production through freeliving techniques of gametophytes.
Project 39400102 supported by NSFC, also supported by National Climbing Plan B (PD-B642); and Bioengineering Center, SSTC. 相似文献
12.
Polysiphonia urceolata is one type of potential commercial red seaweeds used for breeding and cultivation, because of its significant biochemical
and biomedical application. However, the information of breeding and seedling incubation for cultivation is limited, especially
the early development. In this study, tetrasporohyte and gametophyte of P. urceolata were taken as the study materials in Huiquan Bay, Qingdao, China. The cleaned and sterilized tetrasporophytes and gametophytes
were pre-cultured in sterilized seawater, then nurtured at 18°C, 25 μmol photons m −2 s −1 in 12:12 h (light:dark) photoperiod. Continuous observation under microscope showed that the early development consists of
bipolar division stage and seedling stage. In the division stage, tetraspores germinate into bipolar sporelings that further
differentiate into a colorless rhizoidal portion and a lightly pigmented upright shoot. The lightly pigmented rhizoidal cell
develops to a rhizoid and the larger pigmented cell transforms to an erect axis. In the seedling stage, several quasi-protuberances
appear on the erect axis and form juvenile seedlings. The results demonstrate the culture of P. urceolata from tetraspores under laboratory conditions.
Supported by National Key Technology Support Program, Development Program of China (No.2006AA09Z21), National Natural Science
Foundation of China (No. 40618001 and N_CUHK438/06) and Shandong Agricultural Seed Stock Breeding Project 相似文献
13.
The annual life cycle of the brown seaweed Undaria pinnatifi da(Harvey)Suringer comprises a macroscopic diploid sporophyte stage and a microscopic haploid gametophyte stage.In 2011,an unusual zoospore-derived monoecious gametophyte isolate(designated as line 10-5-3)of U.pinnatifi da was observed.To understand this phenomenon,a comprehensive screening of eighty-two previously identifi ed male gametophyte cultures,isolated from three randomly selected cultivars(lines 10,7,and 5)was performed.Thirty-six of the isolates developed both antheridia and oogonia on the same fi lamentous fragment in a standard gametogenesis test(SGT: 18°C,60 μmol photons/(m 2·s)).Selfi ng of the monoecious gametophyte or crossing it with a normal male gametophyte both gave rise to morphologically normal sporophytic offspring.However,crossing resulted in a much higher fertilization rate(89.7%).The hybrid and selfed sporophytic offspring were grown to maturity in fl ow tanks at an ambient temperature of 10–18°C over a period of 69 days.Active zoospores were released from both types of mature sporophylls.The majority of these developed into male gametophytes,while 15%–20% developed into the observed monoecious structures on the same fi lament.Using PCR amplifi cation it was found that all the monoecious gametophyte isolates and the sporophytic offspring resulting from the selfi ng and crossing lacked the femalelinked microsatellite sequence(a part of the locus Up-AC-2A8,GenBank accession No.AY738602.1),indicating their male nature.U.pinnatifi da is an invasive species in some regions and the implications of the above fi ndings for this species in nature are briefl y discussed. 相似文献
14.
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. 相似文献
15.
Eighteen gametophytes including L.japonica ,L.ochotensis and L.longissima,were verified with random amplified polymorphic DNA (RAPD) technique.Eighteen ten-base primers were chosen from 100 primers selected for final amplification test.Among the total of 205 bands amplified ,181(88.3%) were polymorphic ,The genetic distance among different strains ranged from 0.072 to 0.391,The dendrogram constructed by unweighted pair -group method with arithmetic(UPGMA) method showed that the female and male gamtophytes of the same cell lines could be grouped in pairs respectively,It indicated that RAPD analysis could be used not only to distin-gusih different strains of Laminariz ,but also to distinguish male and female gametophyte within the same cell lines,There is ambiguous systematic relationship if judged mereely by the present data It seems that the use of RAPD marker is limited to elucidstion of the phylogenetic relationship among the species of Laminaria. 相似文献
16.
During the induction of gamete-producing gametangia, induced gametophytes were collected at 4 days intervals (0,4,8,12 d) and total RNAs were isolated by CsCl gradient ultracentrifugation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from different developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned and sequenced. This gene was specifically expressed during 4 days of induction, and had partial homologous sequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene which is related to the IAA regulating function during the development of the gametophytes. 相似文献
18.
During sexual reproduction of seaweeds, spermatozoid (sperm) discharge is triggered by chemical messengers (pheromones) released by the female gametes. The chemotactic ability of the sperm ensures fertilization success. Using unialgal male and female gametophyte material under designated standard gametogenesis testing (SGT) conditions, the potential life-span of the sperm of two seaweeds, Saccharina japonica and Undaria pinnatifida, was assessed by their ability to fertilize eggs. Results show that within 20-30 min after being discharged, sperm of both species could complete fertilization without an apparent decline in fertilization rate. Although fertilization rate 60–120 min after sperm discharge dropped significantly in both species, some sperm were viable enough to fertilize the eggs. In S. japonica, at 12°C, some sperm were able to fertilize eggs up to 12 h after discharge. In both species, egg discharge rates (EDR) in the male and female mixed positive controls were significantly higher than those of all the sperm-testing groups. Doubling the seeded male gametophytes of S. japonica in the SGT tests significantly increased the EDR, further confirming the effect of the presence of the male on the female in terms of facilitating egg discharge from oogonia. 相似文献
19.
Seven compounds (1–7) were identified from the cultivation of the endophytic fungus Exophiala oligosperma (EN-21) that was isolated from the inner tissue of the marine red alga Laurencia similis. Their structures were identified with spectroscopic and chemical methods as 2-phenoxynaphthalene (1), (2S, 3R, 4E, 8E)-1-O-β-D-glucopyranosyl-3-hydroxy-2-[(R)-2′-hydroxyoctadecanoyl] amino-9-methyl-4, 8-octadeca-diene (2), (22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol (3), (22E, 24R)-3β, 5α, 9α-trihydroxy- ergosta-7, ... 相似文献
20.
The present work studies the chemical constituents from marine-derived streptomyces 3320^# and their antitumor activities. The n-BuOH extract of the ferment broth of 3320^# was chromatographed on silica gel, Sephadex LH-20, ODS columns and HPLC to separate the compounds with antitoumor activities. Their structures were identified using IR, UV, NMR, MS spectroscopic techniques and compared with published data. The antitumor activities of the isolates were assayed using SRB method and flow cytometry assay, accompanied with the morphological observation of the cells under light micro- scope against mammalian tsFT210 cells. Ten compounds, cyclo-（Ala-Leu） 1, cyclo-（Ala-Ile） 2, cyclo-（Ala-Val） 3, cyclo- （Phe-Pro） 4, cyclo-（Phe-Gly）5, cyclo-（Leu-Pro）6, 1-methyl-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid 7, N-（4- hydroxyphenethyl） acetamide 8, 4-methyoxy-l-（2-hydroxy） ethylbenzene 9 and uridine 10, were isolated from the ferment broth of streptomyces 3320^# . Among them, compounds 6, 7, 8 and 10 showed potent cytotoxicity against the tsFT210 cell with the IC50 values of 3.6, 7.2, 5.2 and 1.6 mmol L-1, respectively. Compounds 8, 10 also exhibited apoptosis inducing activity under 2.0 mmol L-1. Compounds 6, 7, 8 and 10 are the principle bioactive constituents responsible for the antitumor activities of marine streptomyces 3320^#. Compound 7 was isolated from this species for the first time. 相似文献
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