Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达

【目的】了解海洋细菌Shewanella haliotis B P-1中海藻酸裂解酶降解海藻酸钠的生物活性。【方法】应用基因克隆和大肠杆菌异源表达技术,过量表达海藻酸裂解酶,将粗酶液通过 DEAE Sepharose FF柱分离纯化后检测其酶活性。【结果】从S.haliotis BP-1菌株的基因组DNA中克隆得到一个大小为2157 bp的海藻酸裂解酶基因Alg17S ,该基因编码的海藻酸裂解酶 Alg17S属于PL17家族的蛋白,大小为79726 Da,其中包括N端26个氨基酸的信号肽,与Saccharophagus degradans 2-40菌株产生的海藻酸裂解酶 Alg17C 具有高度同源性,相似性为52%。经纯化后获得的重组酶 Alg17S和△snAlg17S（N端不含26个氨基酸的信号肽）均具有降解海藻酸钠的活性,但△snAlg17S对海藻酸钠的催化活性比 Alg17S高,其酶比活力高达9635 U/mg。【结论】重组海藻酸裂解酶△snAlg17S兼具高表达水平及高酶活性,是进一步研究海藻酸盐糖化和生物燃料生产的潜在的优势酶。

Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai

From 2007 to 2009, large-scale blooms of green algae (the so-called "green tides") occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.

Genetic parameter estimates for growth traits at early stage of Pacific abalone,Haliotis discus hannai Ino

Heritability and genetic and phenotypic correlations were estimated for juvenile growth traits of Pacific abalone Haliotis discus hannai Ino. The estimates were calculated from shell length and shell width measurements on progeny resulting from 12 half-sib families and 36 full-sib families obtained using artificial fertilization of mating three females to each male. The measurements were taken at 10, 20 and 30 d after fertilization. It was found that heritability estimates based on sire component ranged from 0.23 to 0.36 for shell length and 0.21 to 0.32 for shell width. Heritability estimates from dam component were larger than those from sire component at three ages, indicating presence of maternal effects, non-additive genetic effects and common environmental effects. Phenotypic correlations were significant at three ages （P 〈 0.05）, with values of 0.92, 0.93 and 0.92, respectively. Genetic correlations from the paternal half-sib correlation analysis were highly positive at three ages, with values of 0.50, 0.78 and 0.81, respectively. The results suggest that selective breeding is an effective approach to improving growth traits of Pacific abalone stocks.

Heterosis and combining ability:a diallel cross of three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino

We conducted a complete diallel cross among three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino to determine the heterosis and the combining ability of growth traits at the spat stage.The three populations were collected from Qingdao(Q) and Dalian(D) in China,and Miyagi(M) in Japan.We measured the shell length,shell width,and total weight.The magnitude of the general combining ability(GCA) variance was more pronounced than the specific combining ability(SCA) variance,which is evidenced by both the ratio of the genetic component in total variation and the GCA/SCA values.The component variances of GCA and SCA were significant for all three traits(P<0.05),indicating the importance of additive and non-additive genetic effects in determining the expression of these traits.The reciprocal maternal effects(RE) were also significant for these traits(P<0.05).Our results suggest that population D was the best general combiner in breeding programs to improve growth traits.The DM cross had the highest heterosis values for all three traits.

INDUCTION OF LARVAL SETTLEMENT AND METAMORPHOSIS OF HALIOTIS DISCUS HANNAI INO(GASTROPODA,MOLLUSCA)

Conspecific foot mucus,excessive[K^+] and gamma-aminobutyric acid (GABA) showed different metamorphosis-inductive effect on the veliger of Haliotis discus hannai.The inductive effect of excessive [K^+]and GABA was developmental stage-dependent and dose-dependent,while that of conspecific foot mu-cus was only developmental stage-dependent.At 20℃ the veliger larvae became competent within 4 daysafter fertilization H.discus hannai larvae showed gregarious settlement pattern on the conspecific foot mu-ous under the conditions of either presence or absence of KCI or GABA.The present studies showed that the effect of conspecific foot mucus on abalone larvae metarnorphosis could be dose-independent.

Toxic effects of several phthalate esters on the embryos and larvae of abalone Haliotis diversicolor supertexta

As the most widely used plasticizers in the world, phthalate esters （PAEs） are potential endocrine disruption compounds （EDCs）. In the present study, the toxicity of dimethyl phthalate （DMP）, diethyl phthalate （DEP）, dibutyl phthalate （DBP）, di （2-ethylhexyl） phthalate （DEHP） on embryogenesis and larvae development of the marine univalve Haliotis diversicolor supertexta was examined in laboratory. The results show that the malformation of embryos appeared during the experiment, such as embryos died or lysed, small transparent flocculent rings studded on the periphery of the embryo, and the larvae could failed to hatch. In embryo toxic test, embryos incubated at the highest concentration of DMR DEP and DBP solutions showed significantly high abnormal rate compared with the control, while DEHP solutions displayed no significant difference. In larval toxic test, in all concentrations of DMP, DEP and DBP solutions, larval settlement rates were low significantly than that of the control. Similarly, DEHP solutions show nearly no effect on the larval settlement. The order of toxicity on embryos and larvae is DBP〉DEP〉DMP〉DEHE Being a simple and easy stimulation to indoor spawn, sensitive to environmental factors, and short culture time, the embryos of H. diversicolor supertexta can be used to indicate toxicity of the PAEs.

Cloning and Characterization of an Abalone(Haliotis discus hannai)Actin Gene

An actin encoding gene was cloned by using RT-PCR, 3’ RACE and 5’ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3’ untranslated region of 307 base pairs and 79 base pairs of 5’ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the mi-crosatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (< 20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 （Hsp70） is one important member of heat shock protein （Hsp） family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone （Haliotis discus hannai Ino）. For the chronic exposure, abalones were maintained at 8, 12, 20, and 30∩ for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30~C-acclimated group, followed by the 8~C-acclimated group and then the 12~C- and 20~C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8~C and 38~C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

Effects of dietary menadione on the activity of antioxidant enzymes in abalone, Haliotis discus hannai Ino

A 240-day growth experiment in a re-circulating water system was conducted to investigate the effects of dietary menadione on the growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 1.19 ± 0.01 g; shell length: 19.23 ± 0.01 mm) were fed to satiation with 3 semi-purified diets containing 0, 10, and 1 000 mg menadione sodium bisulfite (MSB)/kg, respectively. Results show that there were no significant differences in the rate of weight gain or in the daily increment in shell length of abalone among different treatments. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) in viscera were significantly decreased with dietary menadione. However, activities of these enzymes except for GPX in muscle were increased. Therefore, antioxidant responses of abalone were increased in muscle and decreased in viscera by dietary menadione.

南海岛礁羊鲍(Haliotis ovina)资源及其栖息地环境研究

鲍是重要的海珍品,羊鲍(Haliotis ovina)是主要生活在热带岩岸潮间带大型经济鲍类底栖动物。为了研究南海岛礁羊鲍资源及其栖息环境,我们在宣德群岛的永兴岛和七连屿,永乐群岛的羚羊礁、甘泉岛和晋卿岛进行了羊鲍资源分布及其栖息水环境研究。按照《海岛调查技术规程》中潮间带岩岸大型底栖生物调查方法,对羊鲍进行取样调查。按照《海洋调查规范第4部分:海水化学要素调查》(GB T 12763.4-2007),对羊鲍栖息地水环境的总无机氮、活性磷酸盐、悬浮物和叶绿素a等海水化学要素进行分析。结果表明,羊鲍在永乐群岛和宣德群岛都有分布,这些岛礁按永兴岛、七连屿、羚羊礁、甘泉岛和晋卿岛的顺序,分布密度依次增加,个头大小依次增大,总体来说羊鲍资源量小,有的岛礁海区几乎绝迹,亟需开展生态增殖和资源养护研究工作。水质调查表明,除永兴岛站位的悬浮物指标因填海作业未达标外,各站位的水质指标均达一类水标准且具有一定稳定性。因此,尽管南海岛礁受人类活动的影响,但该海域仍适合羊鲍栖息,是羊鲍资源养护、底播生态增殖和生态养护的适宜场所,符合建立海洋保护区的国家水质标准和环境条件。

Experimental evaluation of heterobeltiosis and heterosis between two populations of Pacific abalone Haliotis discus hannai Ino

A2 × 2 factorial cross between two populations of Pacific abalone Haliotis discus hannai Ino, collected separately from Dalian （D） in China and Miyagi （M） in Japan, was conducted to compare performances in fertilization rate, hatching rate, metamorphosis rate and growth at days 20, 43, 160 and 330 between purebreds （DD and MM） and crossbreds （DM and MD） and investigate the magnitude of heterobeltiosis （better parent） and heterosis （mid-parent）. Heterobeltiosis and heterosis for all the traits analyzed were evidently different between crossbreds DM and MD. Heterobeltiosis in the crossbred DM varied among traits, with values of 2.5% for the fertilization rate, 2.2% for the hatching rate, - 1.9% for the metamorphosis rate and 7.4% for the growth at the day 330. The crossbred DM displayed positive heterotic values for fertilization rate （5.4%）, hatching rate （7.4%）, metamorphosis rate （7.6%） and growth （ 12.0% ） at the day 330. However, both heterobeltiosis and heterosis for all the traits in the crossbred MD were negative except those for the growth at days 20 and 43. The results indicate the importance of selecting superior hybrid varieties if the exploitation of hybrid vigor is considered in the Pacific abalone breeding program.

An AFLP Genetic Linkage Map of Pacific Abalone(Haliotis discus hannai)

A genetic linkage map of Pacific abalone(Haliotis discus hannai) was constructed using AFLP markers based on a two-way pseudo-testcross strategy in a full-sib family. With 33 primer combinations,a total of 455 markers(225 from the female parent and 230 from the male parent) segregated in a 1:1 ratio,corresponding to DNA polymorphism:heterozygous in one parent and null in the other. The female framework map consisted of 174 markers distributed in 18 linkage groups,equivalent to the H. discus hannai haploid chromosome number,and spanning a total length of 2031.4 cM,with an average interval of 13.0 cM between adjacent markers. The male framework map consisted of 195 markers mapped on 19 linkage groups,spanning a total length of 2273.4 cM,with an average spacing of 12.9 cM between adjacent markers. The estimated coverage for the framework linkage maps was 81.2% for the female and 82.1% for the male,on the basis of two estimates of genome length. Fifty-two markers(11.4%) remained unlinked. The level of segregation distortion observed in this cross was 20.4%. These linkage maps will serve as a starting point for linkage studies in the Pacific abalone with potential application for marker-assisted selection in breeding programs.

Effects of chemical cues on larval survival,settlement and metamorphosis of abalone Haliotis asinina

Low larval survival,poor settlement,and abnormal metamorphosis are major problems in seed production of donkey-ear abalone Haliotis asinina.We examined the effects of chemical cues including epinephrine,nor-epinephrine,and serotonin on larval survival,settlement,and metamorphosis in order to determine the possibility of using these chemicals to induce the problems.The results show that epinephrine could enhance metamorphosis rate at 10-6 mol/L only but higher concentrations(10-3-10-4 mol/L);and nor-epinephrine could inhibit the performance significantly,and serotonin could increase significantly the performance at a wide-range concentration(10-3-10-6 mol/L).Treatment with serotonin at 10-5 mol/L for 72 hours resulted in the highest settlement rate(42.2%) and survival rate(49.3%),while at 10-4 mol/L for 72 hours resulted in the highest metamorphosis rate(38.8%).Therefore,serotonin may be used as a fast metamorphosis inducer in abalone culture.

The hydrogen peroxide impact on larval settlement and metamorphosis of abalone Haliotis diversicolor supertexta

Abalone Haliotis diversicolor supertexta is an important economic mollusk.The settlement and metamorphosis are two critical stages during its development period,which has direct influence on abalone survival and production.The influence of reactive oxygen species(hydrogen peroxide) on abalone embryo and juvenile development were examined in this study.Larvae of Haliotis diversicolor supertexta were induced to settlement and metamorphose by exposure to seawater supplemented with hydrogen peroxide.They had the best performance at 800 μmol/L.The concentration of 1 000 μmol/L or higher was toxic to the larvae,as the larvae could settle down only at benthic diatom plates without complete metamorphosis.In addition,H2O2 adding time was critical to the larval performance.24h after two-day post-fertilization was proved to be the optimal adding time.In this paper,two action mechanisms of hydrogen peroxide are discussed:(1) hydrogen peroxide has direct toxicity to ciliated cells,thus cause apoptosis;(2) hydrogen peroxide,as a product from catecholamines' autoxidation process in vivo,can reverse this process to produce neuro-transmitters to induce abalone metamorphosis.

Diatom diet selectivity by early post-larval abalone Haliotis diversicolor supertexta under hatchery conditions

Benthic diatoms constitute the primary diet of abalone during their early stages of development. To evaluate the dietary preferences of early post-larval abalone, Haliotis diversicolor supertexta, we analyzed the gut contents of post-larvae that settled on diatom films. We compared the abundance and species diversity of diatom assemblages in the gut to those of the epiphytic diatom assemblages on the attachment films, and identified 40 benthic diatom species in the gut contents of post-larvae 12 to 24 d after settlement. The most abtmdant taxa in the gut contents were Navicula spp., Amphora copulate, and Amphora coffeaeformis. Navicula spp. accounted for 64.0% of the cell density. In the attachment films, we identified 110 diatom species belonging to 38 genera. Pennate diatoms were the dominant members including the species Amphiprora alata, Cocconeis placentula var. euglypta, Cylindrotheca closterium, Navicula sp. 2, and A. coffeaeformis. Nano-diatoms （〈20 pm in length） accounted for a considerable proportion of the total species number and cell density of the diatom assemblages in the gut contents and on the films. This suggests that nano-diatoms are important to the efficient production of abalone seed. The difference of the composition and abundance of diatoms between in the guts and on the biofilms suggests that early post-larval grazing was selective. An early post-larval abalone preferred nano-diatoms and the genera Navicula and Amphora during the month after settlement.

Effects of dietary vitamin A on antioxidant responses of abalone Haliotis discus hannai Ino

A 240 d growth experiment was conducted in a re-circulated water system to investigate the effects of dietary vitamin A on growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone [ initial mass was （0.96±0. 02） g, shell length was （17.70±0.06） mm] were fed to satiation one of three send-purified diets containing 0, 1×10^3, 1×10^6 IU vitamin A per kilogram diet, respectively. Results showed that the daily increment in shell length （DISL） of abalone in the treatment with 1 × 10^3 IU vitamin A per kilogram diet was significantly higher than that with 0 or 1 × 10^6 IU vitamin A per kilogram supplementation （P 〈 0.05）. Vitamin A deficiency （0 IU/kg） significantly elevated the activities of superoxide dismutase （SOD）, glutathione peroxidase （GPX） and glutathione reductase （GR） in the viscera of abalone （P 〈 0.05）. In muscle, the effects of vitamin A deficiency on SOD and GPX activities were the same as those in viscera, however, the activity of GR significantly decreased （P 〈 0.05）. Vitamin A deficiency significantly decreased the ratio of CAT to SOD （eatalase/superoxide dismutase） in viscera （P 〈 0.05）. Nevertheless, it significantly decreased the ratio of GR to GPX in muscle （P 〈 0.05）. Compared with the supplement of 1 x 103 IU vitamin A per kilogram, excessive vitamin A （ 1 × 106 IU/kg） had no significant effects on the activities of CAT, SOD and glutathione-S-transferase （GST） （P 〉 0.05）, but significantly elevated GPX and GR activities in viscera （P 〈 0. 05）. In muscle, the activities of CAT, SOD, GPX, GST and GR were significantly decreased by the excessive dietary vitamin A supplement （P 〈 0.05）. Compared with the supplement of 1×10^3 IU vitamin A per kilogram, vitamin A-excessive had no significant effect on the value of ratio of CAT to SOD either in viscera or in muscle （ P 〉0.05 ）. The ratio of GR to GPX was significantly decreased in viscera, but significantly elevated in muscle in the vitamin A-excessive group （P 〈 0.05 ）. In conclusion, optimum supplement of vitamin A （ 1 × 103 IU/kg） was beneficial for abalone to maintain maximum growth and antioxidant system. Hypoor hyper-vitaminosis A would decrease growth and the efficiency of scavenging reactive oxygen species （ROS） of abalone.

AVAILABILITY OF PHOSPHORUS FROM SELECTED INORGANIC PHOSPHATES TO JUVENILE ABALONE,HALIOTIS DISCUS HANNAI INO.

The availability of phosphorus to juvenile abalone, Haliotis discus hannai from primary, secondary and tertiary calcium phosphate, primary sodium or potassium phosphate separately or in combination was determined in a 120-day feeding trial. Seven semi-purified diets were formulated to contain relatively constant dietary phosphorus, ranging from 0.78%-0.82%. The concentrations of dietary phosphorus were presumed to be slightly below the requirement for abalone. A brown alga, Laminaria japonica, was used as a control diet. Abalone juveniles of similar size (average weight 1.18 g; average shell length 18.74 mm) were distributed in a single-pass, flow-through system using a completely randomized design with eight treatments and three replicates each treatment. The survival ranged from 71.1% to 81.1%, and was not significantly (P>0.05) related to dietary treatment. However, the weight gain rate (41.72%-65.02%), daily increment in shell length (36.87-55.07 μm) and muscle RNA-DNA ratio (3.44-4.69) were significantly (P<0.05) affected by dietary treatment. Soft body alkaline phosphatase activity (10.9-19.8 U/g wet tissue) and carcass levels of lipid (7.71%-9.33%) and protein (46.68%-49.35%) were significantly (P<0.05) responsive to available phosphorus of the diets. Dietary treatment had significant effect (P<0.05) on concentrations of phosphorus in the whole body (WB) and soft body (SB). Apparent digestibility coefficients (45%-97%) of phosphorus were also significantly (P<0.05) different between dietary treatments. The results indicated that among these inorganic compounds, primary calcium, potassium and sodium phosphates separately or in combination could be utilized effectively by juvenile abalone as dietary phosphorus sources (availability ranging from 87%-97%). However, secondary and tertiary calcium phosphates were found to be low in availability, the values being 45% and 77%, respectively.

Molecular and functional characterization of ferritin in abalone Haliotis diversicolor supertexta

Ferritin is an iron storage protein that plays a key role in the processes of physiology and pathology.In the present study,the authors reported the ferritin gene from abalone Haliotis diversicolor supertexta,which we named hds-ferritin.The full-length of hds-ferritin cDNA consisted of 879 bp with an ORF encoding a 171 amino acids.Amino acid sequence analysis revealed that hds-ferritin shared highly homology with other species.Real time PCR and western blot analysis showed that hds-ferritin was distributed ubiquitously in abalone tissues and had the highest expression level in digestive glands,but its transcripts are not modified remarkably by the stimulation with LPS.The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3),and the titre of anti-ferritin antibody was about 1∶14000.The effects of ROS and RNS on ferritin were analyzed in the present study.The results showed that H2O2 played an important role in decreasing hds-ferritin,however NO cation appeared to have a protecting effect on H2O2-medied reduction of hds-ferritin.

Development and Characterization of Microsatellite Markers for the Pacific Abalone (Haliotis discus) via EST Database Mining

The EST database of the Pacific abalone (Haliotis discus) was mined for developing microsatellite markers. A total of 1476 EST sequences were registered in GenBank when data mining was performed. Fifty sequences (approximately 3.4%) were found to contain one or more microsatellites. Based on the length and GC content of the flanking regions, cluster analysis and BLASTN, 13 microsatellite-containing ESTs were selected for PCR primer design. The results showed that 10 out of 13 primer pairs could amplify scorable PCR products and showed polymorphism. The number of alleles ranged from 2 to 13 and the values of Ho and He varied from 0.1222 to 0.8611 and 0.2449 to 0.9311, respectively. No significant linkage disequilibrium (LD) between any pairs of these loci was found, and 6 of 10 loci conformed to the Hardy-Weinberg equilibrium (HWE). These EST-SSRs are therefore potential tools for studies of intraspecies variation and hybrid identification.