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。

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.

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.

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.

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.

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.

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.

Population structure of Haliotis rubra from South Australia inferred from nuclear and mtDNA analyses

Microsatellite loci and mtDNA-RFLPs were surveyed in four spatially separated populations of Haliotis rubra and two populations of putative Haliotis conicorpora from South Australia. A high level of microsatellite genetic diversity was observed in all populations although several loci were characterized by homozygote excesses, probably due to null alleles. MtDNA variation was also moderate with an average of 80% haplotype variation across the six populations. Despite the high levels of genetic variation, significant pair-wise spatial differentiation is not detected among the populations. Hierarchical AMOVA analysis revealed very low levels of genetic partitioning on either a spatial or putative species level. Both molecular techniques revealed little genetic differentiation across the six populations, suggesting a panmictic population model for these South Australian abalone populations. Furthermore, no molecular evidence suggests that the putative H. conicorpora individuals sampled from South Australia belong to a separate species.

Phylogenetic diversity of bacterial biofilms covering the settlement substrates of nona-porous abalones (Haliotis diversicolor supertexta)

The settlement substrates of nona-porous abalones （Haliotis diversicolor supertexta） are covered with biofilms in which several types of microorganisms coexist and interact. These microorganisms are usually important causes of juvenile abalone disease as well as organisms useful in promoting abalones’ adhesion. The bacterial community structure of the biofilms remains unclear. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Total DNA of bacteria in biofilms was extracted, and 16S rRNA gene clone library was constructed using the primers specific for the domain bacteria. Subsequently, 30 randomly selected positive clones were screened by PCR-restriction fragment length polymorphism （PCR-RFLP） analysis, and resulted in 15 different RFLP patterns. Sequences analysis of representatives from each unique RFLP type revealed high genetic diversity in the bacterial populations. These sequences fell into nine major lineages of the bacterial domains： α-, β-, γ-and δ-subdivisions of the Proteobacteria; Planctomycete, Actinobacteria, Firmicutes, V errucomicrobium spp., and CytophagaFlexibacter-Bacteroides spp. Phylogenetic analysis indicated that the dominant phylotypes were most closely related to environmental and clinical Burkholderia cepacia of the β-Proteobacteria, and Roseobacteria of the α-Proteobacteria.

Genetic variation of Chinese and Japanese wild Pacific abalone(Haliotis discus hannai)measured by microsatellite DNA markers

Population differentiation and relationships among three wild populations of the Pacific abalone Haliotis discus hannai collected from coastal seas around China and Japan were estimated using microsatellite DNA analysis. The results obtained with six microsatellite loci showed a high genetic diversity for China and Japan populations. The mean number of alleles per locus ranged from 11.7 to 23.0, and the average of observed and expected heterozygosity ranged from 0. 656 to 0. 721, and from 0. 721 to 0. 793, respectively. The observed genotype frequencies at each locus were mostly in agreement with Hardy - Weinberg expectations with five exceptions. Significant differences were detected between Chinese and Japanese H. discus hannai populations [ Weir and Cocker-ham＇ s fixation index（ Fst ） range ： 0. 020～ 0.023 ; Slatkin＇ s fixation index （Rst） range： 0. 016～ 0. 044 ], and no obvious difference was detected between the samples of Japanese H. discus hannm populations （ Fst = 0. 002 ; Rst = 0. 007）. The level of differentiation among populations is further evidenced by the nNeighbor-joining tree topology on which the Japanese samples were closely clustered, and the Chinese population formed a separate cluster. These results suggest that care should be taken in future management of different populations.

Primary Study on Experiment of Tetraploid Induction in Haliotis discus hannai with PEG

The experiment was performed on induction tetraploid of Haliotis discus hannai at two-cell stage through cell fusion with PEG treatment. In this paper, the orthogonal experiment of three factors and three levels [L9 （3^4）] was used. Three factors and three levels were molecular weight of PEG： 8 000, 6 000, 4 000 mol ·g^-1; PEG concentration： 45%, 50%, 55%; treatment duration time： 1, 2, 3 min, respectively. The results showed that the optimal pattern of three factors and three levels on inducing tetraploid of Haliotis discus hannai at two-cell stage through using PEG treatment were： molecular weight 4 000, concentration 55%, treatment duration time： 2 min. The highest tetraploid induction rate was 10.8% at embryo period. The three factors treatment sequence was treatment duration time→concentration→molecular weight.

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.

Comparative transcriptome analysis of abalone Haliotis discus hannai with green and gray egg colors

Haliotis discus hannai is an important marine economic species in China.Its egg color was found to be associated with economic traits,which provides a new idea for breeding.However,the molecular mechanism of the egg-color formation has not been reported.Thus,the pigment composition and comparative transcriptome analyses of H.discus hannai with green and gray egg color were conducted using high-performance liquid chromatography(HPLC)and RNA-Seq methods.Results show that individuals with green and gray eggs both possess the fucoxanthin.Lutein existed in gray-egged individuals,but not in green-egged individuals.In transcriptome analysis,272310 unigenes were received from 461162 transcripts with a mean length of 985 bp and N50 of 1524 bp,respectively.A total of 185 unigenes were identifi ed as diff erentially expressed genes(DEGs).The DEGs involved in“fl avin-containing compound metabolic process”,“melanosome”,“glutathione metabolism”,and“cytochrome b6f complex”were likely related to the formation of the egg color.Our results provide foundational information for the functional analysis of egg-color related genes and are benefi cial to the selective breeding of H.discus hannai.

用微卫星标记分析皱纹盘鲍群体的遗传变异

利用微卫星标记技术,对皱纹盘鲍山东长岛和辽宁獐子岛的两个野生群体以及山东崆峒岛一个养殖群体的遗传变异进行了分析。对6个微卫星基因座的多态性进行了评估,各基因座的多态信息含量(PIC)值均大于0.5,适合对鲍群体遗传结构的分析。结果表明,这6个基因座在3个皱纹盘鲍群体中共获得57个等位基因,等位基因数(A)平均为9.50,有效等位基因数(Ne)平均为5.8572,平均杂合度观测值(Ho)和期望值(He)分别为0.6925和0.7966;两个野生群体的杂合度观测值(Ho)和期望值(He)均高于养殖群体。上述结果为保护和利用皱纹盘鲍的遗传多样性提供了依据。

养殖杂色鲍暴发病超微病理学研究

从1999年至2002年在中国南方沿海许多养殖场的杂色鲍(Haliotis diversicolor Reeve)出现了严重的疾病,该病可感染各种规格的鲍,死亡很快,死亡率高。病鲍的外观症状表现为外套膜收缩,腹足表面变黑,肌肉变得僵硬。通过电镜观察,在肝胰腺、外套膜、肾、鳃及肠等组织中发现了一种球状病毒。病毒粒子大小为100～130nm。该病毒一般存在于间质细胞的细胞质中,为双层质膜所包裹。染病鲍细胞器如滑面内质网扩张、线粒体崩解,糖原粒、核糖体减少,核膜松疏、溶解、消失,核质边缘化。病毒的病原性通过对健康鲍的感染试验得到证实。

饲料中维生素B_6对皱纹盘鲍幼鲍蛋白质代谢的影响

以皱纹盘鲍(Haliotis discus hannaiIno)幼鲍为研究对象,探讨维生素B6对其蛋白质代谢的影响。设计了3种维生素B6不同含量(0,40,4 000 mg/kg)的半精制饲料,在循环水养殖系统中养殖幼鲍240 d。结果表明,皱纹盘鲍内脏团和肌肉中磷酸吡哆醛(PLP)的含量随饲料中维生素B6的添加而显著升高(P<0.05)。饲料中不同维生素B6水平对肌肉组织中蛋白含量没有显著影响(P>0.05),而显著升高内脏团中蛋白质的含量(P<0.05)。内脏团和肌肉中的谷丙转氨酶(ALT)和谷草转氨酶(AST)活力随着饲料中维生素B6添加量的升高而显著增强,分别在维生素B6的添加量为4 000 mg/kg时取得最大值(P<0.05)。内脏团中所检测的各种氨基酸的含量与饲料中维生素B6添加量没有显著影响(P>0.05)。然而,肌肉中的天冬氨酸(Asp)、苏氨酸(Thr)、丝氨酸(Ser)、牛磺酸(Tau)的含量随着饲料中维生素B6添加量的升高而显著增加,并且分别在维生素B6添加量为4 000 mg/kg时取得最大值(P<0.05)。肌肉中甘氨酸(Gly)、丙氨酸(Ala)、缬氨酸(Val)、蛋氨酸(Met)的含量在两个维生素B6添加组(40 mg/kg和4 000 mg/kg)之间没有显著差异(P>0.05),但均显著高于维生素B6缺乏组(0 mg/kg)的值(P<0.05)。

皱纹盘鲍野生与养殖群体遗传多样性的研究

用随机扩增多态DNA(RAPD)技术,对山东长岛(CW)、辽宁大连(DW)两个野生群体和山东崆峒岛(KC)一个养殖群体的皱纹盘鲍遗传多样性进行了分析。结果表明,CW和DW群体间的遗传距离为0.1703,CW和KC群体间的遗传距离为0.3673,DW和KC群体间的遗传距离为0.3477,显示野生群体与养殖群体之间的亲缘关系已发生了变化。CW,DW和KC三个群体的多态位点比例分别为49.22%、50.78%和43.75%;平均杂合度分别为0.3506,0.3408和0.3079。与野生群体相比,养殖群体的多态位点比例和杂合度都有不同程度的降低。

皱纹盘鲍基因型与环境互作的初步研究

以11个皱纹盘鲍(Haliotis discus hannaiIno)家系为材料分别在12,16和20℃养殖40 d,方差分析的结果表明温度和家系的相互作用对壳长增长率存在显著影响(P<0.05)。采用线性回归模式对皱纹盘鲍基因型与环境温度互作数据进行分析,得出了不同基因型的壳长增长率对环境温度的响应模式和对环境温度的灵敏度程度上所表现的序关系。

一种消除九孔鲍苗细菌性病原的无公害绿色生物方法的研究

从海洋环境中分离出4株蛭弧菌(Bdellovibriosp.),并对34株九孔鲍(Haliotisdiversicolor)苗细菌性病原进行了裂解试验。结果表明,Bh04-4、Bh04-41a、Bh04-A+和Bh04-1f等4株蛭弧菌分别可裂解11株、13株、22株和28株病原菌,裂解率为32.4%、38.2%、64.7%和87.5%;4株共同作用,则可裂解32株病原菌,裂解率高达94.1%。研究结果展示了应用蛭弧菌控制九孔鲍苗细菌性病原的可行性。