条斑紫菜叶状体细胞对3种抗性筛选剂敏感性的研究

为探讨条斑紫菜叶状体细胞对3种抗生素的敏感性,以确定条斑紫菜遗传转化后代适宜的抗性选择质量浓度,将叶状体用海螺酶解离获得原生质体,在培养皿中20℃培养3d后,在培养液中分别加入G418(0、10、50、100、200、300mg/L)、卡那霉素(0、50、100、300、500、800mg/L)或氯霉素(0、5、15、35、65、100mg/L),观察细胞分裂与死亡率、小紫菜比率,进行显著性检验。试验结果表明,条斑紫菜叶状体细胞对3种抗生素的敏感性存在显著差异,对抗生素的敏感性为氯霉素卡那霉素G418。在条斑紫菜叶状体遗传转化用G418、卡那霉素或氯霉素作抗性筛选时,筛选质量浓度分别为300、50mg/L及15~35mg/L。     

条斑紫菜优良品系(LC-14)的筛选与特性分析

从条斑紫菜(Pyropia yezoensis)绿色突变体和红色突变体种内杂交产生的F_1叶状体中,分离出新品系LC-14。该品系的F_1叶状体在生长速度和品质等方面均显著优于条斑紫菜野生型栽培品系(WT)。日龄70 d的叶状体平均长度和湿重,LC-14品系分别为106.53 cm和3.07 g,分别是WT品系的4.39和10.27倍。培养至第55天,LC-14品系的3种主要光合色素(叶绿素a、藻红蛋白和藻蓝蛋白)含量分别为9.85 mg/g、52.09 mg/g和23.26 mg/g,分别比WT品系提高了65%、65%和49%,而其藻体的平均厚度(20.89μm)反而降低了32%。此外,LC-14品系的壳孢子放散总量为761.32×10~4 ind/壳,是WT品系的1.28倍。上述结果表明,LC-14品系具有生长快、品质优、壳孢子放散量大的优点,藻体颜色与野生色(棕褐色)相近,是条斑紫菜的优良品系,有望在生产中应用。     

条斑紫菜南移福建养殖的研究

条斑紫菜系我国长江以北的养殖品种,1986年和1987年南移福建连江进行了丝状体育苗和叶状体养殖,均获成功。叶状体产量折合亩产干品67.9公斤。条斑紫菜南移存在的主要问题是:丝状体育苗时间长,温度高;叶状体则是生长期短,产量低。实行坛紫菜和条斑紫菜轮养,可提高紫菜的产量和经济效益。     

条斑紫菜两个耐高温品系的耐低盐特性

以条斑紫菜两个耐高温品系(YZ-4和TM-18)为对象,以野生型品系(WT)做对照,分析其叶状体在高温、低盐胁迫下的生长和光系统Ⅱ最大光量子产量(F_v/F_m)的数值,为筛选既耐高温又耐低盐的条斑紫菜抗逆品系提供依据。结果显示,在适宜培养条件(温度18°C和盐度26)下,YZ-4和TM-18叶状体在50～85 d的生长速率快于WT,F_v/F_m和主要光合色素含量均高于WT,各品系的绝对生长率和F_v/F_m之间均具有较高的正相关性。另外,WT叶状体在70 d左右开始形成精子囊时,绝对生长率和F_v/F_m明显下降。在高温胁迫下培养,各品系叶状体的F_v/F_m和绝对生长率均呈下降趋势,且胁迫的温度越高或时间越长,其降幅越大。在24°C高温胁迫35 d后,WT、YZ-4和TM-18的F_v/F_m分别下降了56.7%、43.2%和28.7%。在25°C高温胁迫15 d时,WT的生长完全停滞,而YZ-4和TM-18则分别保持0.51和0.84 cm/d的绝对生长率,说明二者确实是耐高温的。低盐胁迫对叶状体的影响与高温胁迫的类似,在盐度为9的低盐下培养35 d后,WT、YZ-4和TM-18的F_v/F_m降幅分别为46.2%、42.0%和32.0%,三者的绝对生长率分别为0.12、0.10和0.90 cm/d。另外,WT和YZ-4在盐度为5时生长受到明显影响,叶状体在15 d时颜色加深、卷曲变硬并腐烂,而TM-18在相同盐度下培养25 d也未腐烂,F_v/F_m仍高达初始值的72.8%。这表明TM-18在高温或低盐胁迫下的耐受性比WT和YZ-4的都强,其内在原因是叶状体F_v/F_m的降幅较小,间接说明F_v/F_m可作为条斑紫菜抗逆品系选育的新指标。     

抗生素抑制农杆菌的效果及对条斑紫菜生长发育的影响

用头孢霉素和卡那霉素抑制农杆菌(LBA4404和EHA105)生长,同时测定在液体培养条件下条斑紫菜组织块对2种抗生素的敏感性,以期找到适合用于农杆菌介导条斑紫菜遗传转化的脱菌剂及农杆菌转化用于条斑紫菜的可行性.试验结果表明,2种抗生素对2株农杆菌菌株有较强的抑制作用,对条斑紫菜叶状体组织块的生长作用效果不同,头孢霉素≥300 mg/L抑制叶状体组织块长度的增加,而卡那霉素≥400 mg/L对组织块的增长有一定的促进作用.头孢霉素对条斑紫菜的发育抑制作用较卡那霉素更大.在培养液中添加头孢霉素≥300 mg/L或卡那霉素质量浓度≥600 mg/L组织块不能发育形成丝状体.头孢霉素和卡那霉素可作为农杆菌介导条斑紫菜遗传转化的抑菌剂,使用较适宜的质量浓度分别为200 mg/L和400 mg/L.     

条斑紫菜品系评价方法的探讨

总结并分析了“国家级紫菜种质库”中有关紫菜种质材料的原始数据和资料,调查研究了条斑紫菜不同品系的形态结构等生物学特性及环境适应性表现;通过对条斑紫菜不同品系经济性状的定量测评、丝状体生长发育特征的测评、品系产品生化成分的分析评价以及分子标记技术在条斑紫菜种质评价分析中的应用等研究,初步得到了条斑紫菜品系的评价方法;筛选...     

坛紫菜与Pyropia radi种间杂交重组优良品系的选育与特性分析

为获得具明显杂交重组优势的紫菜优良品系,实验以印度产紫菜Pyropia radi野生型品系(Pr-WT01,特点:藻体厚、生长慢、韧性好、壳孢子放散量多)为父本,坛紫菜诱变品系(Ph-HMC5,特点:藻体薄、生长快、韧性差、壳孢子放散量少)为母本进行种间杂交,从杂合丝状体产生的F1叶状体中选出4个综合性状优良的杂交重组品系,HR-6品系为其中的最优良品系。父本品系叶状体的绝对生长率最大值为0.39 cm/d,母本品系为5.24 cm/d,而HR-6品系高达9.27 cm/d,在日龄30~50 d期间,它的平均绝对生长率比母本品系的最大生长率还大。日龄60 d的叶状体平均体长父本品系为13.18 cm,母本品系为85.67 cm,而HR-6品系已达218.57 cm,分别是父、母本品系的16.58倍和2.55倍。日龄35 d的HR-6品系叶状体的叶绿素a含量高达9.43 mg/g,比父、母本品系分别增加了50%和20%;另外,它的总藻胆蛋白含量高达99.62 mg/g,分别是父、母本品系的2.18倍和1.74倍。日龄35 d的HR-6品系的叶状体平均厚度为26.22μm,比父本品系降低了37%,比母本品系增加了30%,叶状体的韧性明显增加。HR-6品系的壳孢子放散总量达167.72万个/贝,分别为父、母本品系的1.13和2.65倍。上述研究结果表明,HR-6品系具有生长快、品质好、壳孢子放散量大等优良性状,表现出良好的生产适用性。     

条斑紫菜6个品系的SRAP分析

为鉴别条斑紫菜不同品系的种质,使用相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)标记对条斑紫菜的5个选育品系和1个野生品系进行遗传分析,结果从35对引物组合中筛选出可扩增出稳定清晰条带的组合11对,共获得131个扩增位点,其中多态性位点125个,多态性比例高达95.42%。6个品系 间的遗传距离为0.364 3～0.867 9,平均为0.593 0。用UPGMA法进行聚类分析,结果将6个品系分为2个群,所反映的亲缘关系与各品系的来源基本一致,说明SRAP 标记技术可以成为条斑紫菜品系间遗传分析的有效工具。在131个多态性位点中,选择扩增出的4个位点构建了6个品系的指纹图谱。另外,通过ME1/EM6引物组合 扩增得到耐高温品系TM-18的特异性条带,经回收测序和重新设计引物,该条带在其丝状体和叶状体DNA中均能稳定地被扩增出来,可用于该品系的种质鉴别 。     

主推品种紫菜

[品系来源]自然野生或养殖性状良好的紫菜.上海水产大学利用自然的野生坛紫菜叶状体,通过人工诱变、体细胞克隆和单陛生殖等技术,获得坛紫菜的变异细胞,将变异细胞单个分离并培养成完整叶状体,选育出性状稳定的优良坛紫菜新品系,如"申福1号"、"申福2号"等.     

不同植物激素对条斑紫菜体细胞生长发育的影响

用添加不同浓度植物激素(IBA和6一BA)的PES培养液对条斑紫菜体细胞进行培养，观察其对条斑紫菜体细胞生长发育的影响。结果表明：酶解的条斑紫菜近基部体细胞离体培养可发育成细胞团和叶状体，低浓度的IBA有利于体细胞向叶状体方向发育，而低浓度的6一BA有利于体细胞发育成细胞团。     

不同贝龄栉孔扇贝数量性状的相关性和通径分析

为深入了解栉孔扇贝形态性状与湿重之间的关系,为栉孔扇贝选育工作中亲贝的挑选提供策略,本研究使用来自同一家系的栉孔扇贝子代324只一龄个体以及生长到二龄的230只个体的性状数据进行通径分析。性状数据包括壳长(x_1/cm),壳高(x_2/cm),壳宽(x_3/cm),湿重(y/g)。结果显示,所有形态性状和湿重之间的相关系数均达到极显著水平。其中与一龄贝湿重相关系数最大的是壳长,为0.939,与二龄贝湿重相关系数最大的是壳高,为0.808。通径分析结果显示,壳长对一龄贝湿重的直接影响最大(0.532),壳高对一龄贝湿重的直接影响最小(0.163)。壳高对二龄贝湿重的直接影响最大(0.451),壳长对二龄贝湿重的直接影响最小(0.191),决定系数与以上通径分析结果的变化趋势一致。利用多元回归的方法构建了一龄贝和二龄贝形态性状与湿重间的回归方程,一龄贝:y=–10.527+0.287x_1+0.087x_2+0.409x_3,R~2=0.926;二龄贝:y=–68.609+0.254x_1+0.719x_2+2.008x_3,R~2=0.830。本研究结果为栉孔扇贝种贝的挑选提供了理论依据。     

池塘养殖三疣梭子蟹的生长规律

为探讨池塘养殖三疣梭子蟹的生长规律和雌雄差异，本研究对1～6月龄池塘养殖三疣梭子蟹各生长指标进行了比较分析，并采用Logistic模型分别拟合三疣梭子蟹的体重(BW)、体长(BL)、体高(BH)、全甲宽(FCW)、甲宽(CW)、大螯长节长(MLC)、大螯不动指长(FFLC)、第一步足长节长(MLFP)等8个形态性状的生长特征。结果显示，三疣梭子蟹雌性和雄性的生长存在差异，早期雄性生长较快，后期雌性生长较快，而雌雄混合分析介于二者之间。三疣梭子蟹各生长性状的Logistic模型拟合结果显示，除雌性MLC和MLFP以及雌雄混合分析的MLFP之外，其余性状R²均达到0.990以上；雌性和雄性体质量的极限生长值分别为290.27和195.91g，快速生长区间分别为2.74～5.10月龄和2.33～4.14月龄，拐点分别为3.92和3.24月龄。研究表明，三疣梭子蟹的生长过程均符合“慢-快-慢”的特征，雄性比雌性更早进入快速生长期，但是快速生长期的持续时间不及雌性。本研究对三疣梭子蟹不同生长指标的规律特征，以及各指标在混合养殖条件下雌性和雄性的优势阶段进行了研究，为实现三疣...     

Dietary histidine affects lens protein turnover and synthesis of N-acetylhistidine in Atlantic salmon (Salmo salar L.) undergoing parr–smolt transformation

This study was conducted to investigate protein synthesis rates and metabolism of histidine (His)‐derivatives in lenses of Atlantic salmon (Salmo salar L.) of different dietary His background during parr–smolt transformation. Two populations of Atlantic salmon parr of equal origin were established in freshwater (FW), 3 months prior to transfer to seawater (SW). The populations were fed either a control diet (CD) containing 8.9 g kg^?1 His or the same diet added crystalline His to a total level of 14.2 g kg^?1 (HD). On the basis of these two populations, ¹⁴C His force‐feeding studies were performed; in FW 3 weeks prior to sea transfer and in SW 6 weeks after transfer. The studies were conducted by force‐feeding the respective diets enriched with ¹⁴C labelled His, with subsequent measurements of incorporation of ¹⁴C His into lens free amino acid pool, as well as into lens proteins and other free His pool fractions. The latter included the major lens imidazole N‐acetylhistidine (NAH). Lens concentrations of His and NAH were clearly influenced by dietary His history, both in parr and smolt. The lens His and NAH concentrations in the CD population were considerably lower in SW than in FW, while in the HD group the His level was equal and the NAH level 50% higher in SW than in FW. Fractional synthesis rate for NAH, K_{S (NAH)}, in FW was 8.2 and 4.2 μmol g^?1 day^?1 for fish in the CD and HD populations, respectively. The corresponding K_{S (NAH)} values in SW were 5.1 and 33.0 μmol g^?1 day^?1. Our data show that free His is rapidly converted to NAH in the lens, and that NAH seems to have a very high turnover, especially in salmon reared in SW. Fractional synthesis rate for lens proteins, K_{S (PROTEIN)}, ranged between 1.8 and 17.3% day^?1 (182 and 2791 μg g^?1 day^?1, respectively), and was generally higher in SW than in FW (P < 0.01). In SW, K_{S (PROTEIN)} was highest in fish in the HD population (P < 0.05), whereas lens protein retention in the HD group was significantly lower than the CD group (P = 0.01). In a second model assuming that His from lens NAH is available for protein synthesis, calculated values of K_{S (PROTEIN)} ranged between 0.17% day^?1 (17.6 μg g^?1 day^?1) and 0.48% day^?1 (70.2 μg g^?1 day^?1). Cataract scores recorded in the His populations at a later point (day 204), showed that the CD fish had significantly higher mean cataract scores than individuals in the HD population (P < 0.01), confirming that low levels of lens His and NAH are associated with cataract development.     

Long-term separate and combined effects of environmental hypercapnia and hyperoxia in Atlantic salmon (Salmo salar L.) smolts

Atlantic salmon (Salmo salar L.) parr (mean start weight 50 g) were reared in freshwater (FW) and exposed to three levels of oxygen saturation measured in effluent water; control group (93% O₂, LO₂), medium (111% O₂, MO₂) and high (123% O₂, HO₂). Further three groups were exposed to similar water oxygen levels in combination with elevated carbon dioxide levels (17–18 mg L^– 1 CO₂), named LO₂–CO₂, MO₂–CO₂ and HO₂–CO₂, respectively. The experiment was run in duplicate tanks for 42 days, and the fish were subsequently transferred to the same seawater (SW) regime for 45 days for an assessment of post-smolt growth. As a consequence of the CO₂ addition, tank pH levels in the FW period were reduced from 6.7 to 5.9 for the hypercapnia groups compared to for the normcapnia groups. Water temperature in FW ranged between 6.4 and 9.0 °C. Citrate was added to the water to complex labile aluminium.In the CO₂ groups observed ventilation frequencies were significantly increased compared to the control (p < 0.05). This difference declined towards the end of the FW period, suggesting acclimation to elevated CO₂. The degree of oxygenation appeared to contribute to the acclimation as the lowest mean ventilation frequency on day 36 was found in the HO₂–CO₂ group and the highest in the LO₂–CO₂ group. Lower plasma chloride and sodium levels were observed in the CO₂ groups relative to the respective oxygenation groups during the FW period, while plasma chloride and sodium levels were normalised to equal levels for all groups after 44 days in SW. No significant differences were found among treatments for blood concentrations of red blood cells, haemoglobin, potassium and glucose during the experiment.By termination of the FW period, the HO₂ group had significantly higher body weight than all other groups (p < 0.05), with specific growth rate significantly higher than the CO₂ groups (p < 0.05). Further, the condition factor was significantly lower in all the CO₂ groups at the end of the FW period compared to the control and normcapnia groups (p < 0.05). Although variable among replicates, occurrence of nephrocalcinosis was 10 times higher in the hypercapnia groups than in the control and normcapnia groups. Mortality was negligible (< 2.0%) during the trial, and most of the mortality occurred following SW transfer.     

Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller's dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 ± 0.5 °C, 32.2 ± 0.4‰) with juvenile L. vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg^− 1 CP; 8.03 g kg^− 1 lipid; 2.01 kcal g^− 1) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 °C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx²), n = 26. Best in vitro APD predictions were obtained at 25 °C, 4 U: 80 mg CP both for ingredients (R² = 0.86; P = 0.001) and test diets (R² = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD − determined APD) for corn gluten meal, feather meal, poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD > 80% showed higher prediction precision (R² = 0.98; P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R² = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information.     

Family × Environment interactions in the growth and survival of Arctic charr (Salvelinus alpinus) grown in brackish and fresh water

We investigated whether Fraser strain Arctic charr Salvelinus alpinus L. reared in fresh water (FW) differed in survival and growth from full siblings transferred to brackish water (BW) and whether family performance was consistent throughout 1 year of commercial growth. Fish were grown communally (n = 3600) in either FW or BW from May 2008 to May 2009. Mean family survival in BW was moderately correlated with mean family survival in FW (r = 0.342; P = 0.032). Fish which survived until the conclusion of the experiment were significantly heavier in body weight at the baseline assessment than their full‐sibs which died. Family by treatment interactions were detected for body weight, condition factor and specific growth across all measurement dates (May 2008, October 2008 and May 2009) and growth intervals. Despite the varied response of families to treatment, mean family body weight in FW was correlated phenotypically with BW body weight in October 2008 (r = 0.633, P < 0.0001) and marginally correlated in May 2009 (r = 0.289, P = 0.061). These data suggest that body weight in FW and body weight in BW should be analysed as separate, but correlated traits in Arctic charr breeding programs.     

四种抗生素对共培养的萱藻丝状体和膨胀色球藻的影响

为抑制或消除萱藻丝状体扩增阶段出现的膨胀色球藻,本研究探讨了头孢噻肟钠、阿莫西林、四环素和红霉素4种抗生素对共培养的萱藻丝状体和膨胀色球藻产生的影响。结果显示,浓度为50和100 mg/L的头孢噻肟钠均显著抑制膨胀色球藻的生长,进而保证萱藻丝状体正常生长与发育;浓度为50~1 000 mg/L的阿莫西林对膨胀色球藻和萱藻丝状体均无抑制作用,阿莫西林不适于去除共培养体系中的膨胀色球藻;浓度为100和200 mg/L的四环素对膨胀色球藻有显著的抑制作用,但此浓度下萱藻丝状体细胞质萎缩,生长状况较差;浓度为0.10~1.00 mg/L的红霉素对膨胀色球藻的生长均有显著的抑制作用,但当其浓度超过0.50 mg/L时,萱藻丝状体的生长亦受到抑制,甚至死亡。     

不同月龄缢蛏新品种数量性状的相关与通径分析

为了检测缢蛏新品种"申浙1号"不同月龄数量性状间的相关性。随机选取4、9、11月龄缢蛏个体60个以上,测量其壳长(SL)、壳高(SH)、壳宽(SW)、活体质量(W),采用相关与通径分析方法分析壳形态性状对活体质量的作用效果。结果显示,各数量性状之间的相关关系均达到极显著水平,其中与4、11月龄质量相关系数最大的是壳长,分别为0.911、0.631,与9月龄活体质量相关系数最大的是壳高,为0.653。通径分析结果显示,对4、9、11月龄活体质量的直接影响最大的分别是壳长(0.562)、壳宽(0.433)、壳高(0.394),决定系数与以上通径分析结果的变化趋势一致。利用逐步回归的方法分别建立4、9、11月龄缢蛏活体质量(W)的最优回归方程:W4=–0.756+0.039SL+0.112SW,R2=0.849;W9=–10.622+0.095SL+0.404SH+0.644SW,R2=0.631;W11=–23.772+0.207SL+0.805SH+0.587SW,R2=0.909。研究结果为缢蛏人工选育过程中的亲贝选择和产量预测等提供了基础数据。     

Effect of chloride concentration on the acute toxicity of nitrite to common carp, Cyprinus carpio L., fry

Abstract. Duplicate static bioassays were conducted for 168 h each to determine the median lelhal concentration (LC50) of nitrite (NO^?₂) for common carp, Cyprinus carpio L., fry at five different chloride (Cl^?) levels. The acute toxicity of nilrite ceased towards the end of 96 h at all levels of chloride concentration. There was a highly significant positive correlation between the chloride concentration tested and the 96-h LC50. The 96-h LC50 values are 2·55, 5·77, 14·41 27·26 and 48·70 mgl^?1 NO^?₂-N at chloride concentration of 1·0. 5·0, 10·5, 27·3 and 45·0 mgl^?1 Cl^? respectively. The linear relationship between chloride concentration and 96-h LC50 is best described by the equation: y= 1·03x+ 1·49 (r=+ 0·996; d.f. = 3;P <0·001), where y= 96 h LC50 of NO^?₂-N and x= concentration of Cl^?. A NO^?₂-N to Cl^? ratio of about 1:1·5–3·0 prevented complete mortality over the 168-h experimental period. A NO^?₂-N to Cl^? ratio of 1:5 is recommended for protection of carp fry against nilrite mortality in fish farms.