蓖麻和线虫的两种不同结构脂肪酸去饱和酶的原核表达

目的:通过在原核表达系统中表达蓖麻的可溶性脂肪酸去饱和酶基因和线虫的fat-1脂肪酸去饱和酶基因,为脂肪酸去饱和酶序列结构与功能的研究奠定基础。方法:将蓖麻RCD△9脂肪酸去饱和酶和线虫fat-1脂肪酸去饱和酶基因亚克隆到大肠杆菌BL21表达载体pET32a+中,获得重组表达载体pET32a+-R9,pET32a+- F1,并通过SDS-PAGE和Western Blotting鉴定蛋白的表达情况。结果:经PCR和测序鉴定,证实两个重组质粒含有目的基因片段;SDS-PAGE和Western Blotting证实两种蛋白在大肠杆菌中获得表达,但表达量具有明显的不同;Anthepro软件对蛋白跨膜结构的分析,验证蓖麻△9脂肪酸去饱和酶和线虫fat-1脂肪酸去饱和酶在结构上的不同。结论:蓖麻的RCD脂肪酸去饱和酶和线虫的fat-1脂肪酸去饱和酶都得到了表达,但线虫fat-1脂肪酸去饱和酶表达量偏低;这可能与fat-1脂肪酸去饱和酶是一类跨膜蛋白的性质直接相关。因此,对于线虫fat-1脂肪酸去饱和酶的基于蛋白纯化的结构分析有待进一步的研究。

牛胎儿成纤维细胞的分离培养及转染线虫ω-3脂肪酸去饱和酶基因

为利用核移植法获取转基因克隆牛提供供体细胞,体外分离培养牛胎儿成纤维细胞并进行了携带线虫ω-3脂肪酸去饱和酶cDNA的表达载体转染。首先采用组织块贴壁法分离培养牛胎儿成纤维细胞,线虫ω-3脂肪酸去饱和酶基因表达载体以脂质法介导转染牛胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆,PCR鉴定外源基因在细胞基因组中的整合,对阳性克隆进行传代2～3次后利用RT-PCR检测外源基因的转录。结果表明,组织块贴壁后7天可获取原代成纤维细胞,基因转染后利用G418筛选9天即可获得转基因细胞,对转基因细胞进行传代,PCR检测显示CMV启动子和ω-3脂肪酸去饱和酶cDNA整合到细胞基因组中,RT-PCR检测显示ω-3脂肪酸去饱和酶mRNA在转基因传代细胞中得到有效转录,为下一步通过核移植方法获得转基因肉牛提供了基础。

牛耳皮肤成纤维细胞的分离培养及转染线虫ω-3脂肪酸去饱和酶基因研究

为了核移植法制作转线虫ω-3脂肪酸去饱和酶基因克隆牛提供供体细胞,体外分离培养牛耳成纤维细胞并进行了线虫-ω3脂肪酸去饱和酶基因。采用胰蛋白酶消化法分离培养牛耳皮肤成纤维细胞,线虫ω-3脂肪酸去饱和酶基因表达载体以脂质法介导转染细胞,通过G418筛选9 d获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合,对阳性克隆进行传代2~3次后,利用RT-PCR检测外源基因的转录。结果表明：细胞贴壁后9 d可获取原代皮肤成纤维细胞,基因转染后利用G418筛选9 d即可获得转基因细胞,对转基因细胞进行传代,PCR检测显示CMV启动子和ω-3脂肪酸去饱和酶基因整合到细胞基因组中,RT-PCR检测显示ω-3脂肪酸去饱和酶基因在转基因传代细胞中得到有效转录。本研究为下一步通过核移植方法获得转基因肉牛提供了条件。

骨骼肌特异表达线虫ω-3脂肪酸去饱和酶基因载体的构建与验证

根据猪α-actin基因已知DNA序列设计合成了两个特异性引物,以猪基因组DNA为模板,通过PCR扩增得到α-actin 5'调控序列,然后与线虫ω-3脂肪酸去饱和酶基因cDNA、去除CMV启动子的表达载体pcDNA3.1连接构成肌肉特异性表达载体pcDNA3.1-AF,小鼠股四头肌注射该重组载体,RT-PCR检测证明其表达有效性。PCR扩增出1 815 bp的特异性片段,DNA测序结果证明该克隆片段与α-actin 5'端调控区序列相比具有很高的一致性,含有肌肉特异性元件;小鼠股四头肌注射重组载体,RT-PCR显示肌肉内ω-3脂肪酸去饱和酶基因得到有效转录。

为获得大自然拥有的缤纷色彩

赤橙黄绿青蓝紫，谁持彩练当空舞？人类是怎样模拟大自然，用人工的方法获得大自然拥有的那些缤纷色彩，制造出与彩虹比美赛艳的染料的呢？

Effect of n-3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream (Sparus macrocephalus)

Requirement for dietary n-3 HUFA (n-3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers and Artemia at various levels of n-3 HUFA. Five treatments with rotifers and Artemia differing in n-3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n-3 HUFA significantly influence fish n-3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n-3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n-3 HUFA fatty acid ethyl esters from ethyl-esterified oil.

Effects of Calanoid Copepod Schmackeria poplesia as a Live Food on the Growth, Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder, Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.

Nutrient effects of broodstocks on the larvae in Patinopecten yessoensis

Patinopecten yessoensis is a commercial valuable species. This study deals with the effect of nutrient effects of the broodstock(mainly ovaries) on the larvae. Concentrations of total carbohydrate, total protein and total lipid in the gonads of P. yessoensis from three Hatcheries(Hatchery 1, Hatchery 2, and Hatchery 3) were determined before and after spawning. The relationship between the nutrient concentration in ovaries before spawning(BC) and that of larvae(LC) was assessed as well as the change in nutrient levels in ovaries after spawning(DC). Results indicate that the BC of total carbohydrate(7.66%) and total lipid(14.48%) in ovaries were significantly higher than in testes(5.20%, 5.20% respectively), whereas the BC of total protein in the ovaries was lower(61.76%) than in the testes(81.67%). The different gonadal composition suggests the different nutrient demands between male and female broodstocks in breeding season. P atinopecten yessoensis gonads contained a higher proportion of lipids, in comparison to other bivalves, which might be a response to the low ambient water temperatures. Further analysis of fatty acids showed that the concentrations of n-3PUFA, EPA and DHA in larvae(LC) were positively correlated with BC and DC, indicating the significant nutrient influence of broodstocks on the larvae. As these fatty acids are important in metabolism, and have been demonstrated to be influential to the viability of the larvae, larval growth and the settlement, spat growth, and juvenile survival in many bivalves, they could possibly be used as indexes to evaluate, and predict condition of broodstocks and larvae.

Amino Acid and Fatty Acid Profile in Epidermal Mucus of Bluestreak Cleaner Wrasse （Labroides dimidiatus）： Possible Role as Defense Mechanism against Pathogens

Labroides dimidiatus has been proven to remove ectoparasites and monogeneans from client fishes and studies showed that they were not infected with the parasite. Due to this, there is a possibility that a defense mechanism against pathogen and parasitic invasion exist in the epidermal mucus which serves as a mechanical as well as biochemical barrier. The study was performed to identify the amino acid and fatty acid components using GC （gas chromatography） and HPLC （high performance liquid chromatography） in epidermal mucus ofL. dimidiatus. The present study revealed 16 components of amino acid and 22 types of fatty acid in epidermal mucus ofL. dimidiatzts. Linoleic acid （C18：2n6c） was the most prominent PUFA （polyunsaturated fatty acid） which contributed approximately 11.69% of total fatty acids. The other major fatty acids are palmitic acid （C 16：0）, oleic acid （C 18： l n9c）, linoledaidic acid （C18：2n6t）, arachidic acid （C20：0）, Gamma-Linoleic acid （C18：3n6） and gadoleic acid （C20：1） which contained reasonable amounts of 9.52%, 8.06%, 6.26%, 8.33%, 6.21% and 9.05% of total fatty acids, respectively. This present study also demonstrated the presence of various amino acids in skin extract. Glycine, glutamine, arginine, asparagin and alanine were found at high concentration of 8.09%, 6.95%, 5.73%, 4.74%, 4.58% respectively. The most abundance percentage of linoleic acid （C18：2n6c） was found to be the metabolic precursor of arachidonic acid （AA） which inducing platelet aggregation, facilitate the blood clotting process and adhesion in endothelial cells during wound healing and might be responsible for rapid tissue growth in L. dimidiatus. It can be concluded that the amino acid and fatty acid profile from the epidermal mucus ofL. dimidiatus contains most of the essential components required to play a possible role in its defense mechanism. Understanding the biochemical properties of L. dimidiatus epidermal mucus in defense mechanism would enable to determine how this fish protect itself from parasitic infection.

The New Pest Scatella tenuicosta Collin （Diptera： Ephydridae） for Spirulina Ponds

The goal of this study was to discover Scatella tenuicosta Collin （Diptera： Ephydridae） for the first time as a pest on blue-green algae Spirulina platensis. This algae has been recognized as a very important commercial product in the world today and as one of the most promising ones among the other microscopic algae due to its richness in polyunsaturated fatty acids, as well as in gamma-linolenic acid （GLA）, and in pigments including phycocyanin, myxoxanthophyll and zeaxanthin. Both immature and adult development stages ofS. tenuicosta have been studied under the temperature of 15±2℃. The development times for eggs, larval stages, including first, second, and third one, and for pupae as well as for adults were found to be 1.4, 1.0, 1.1, 1.1, 3.0 and 8.7 respectively. Some pest control practices ofS. tenuicosta on S. platensis have been also examined in this study.