大刺鳅促黄体生成素基因克隆及其组织表达分析

[目的]克隆大刺鳅(Mastacembelus armatus)促黄体生成素(LH)基因及明确其表达规律,为进一步阐明LH基因在大刺鳅性腺发育过程中的生理功能及揭示大刺鳅的生殖调控机理提供参考依据.[方法]采用RACE克隆大刺鳅LH基因cDNA全长序列,从GenBank中选择硬骨鱼类、两栖动物、鸟类和哺乳动物等物种的LH氨基酸序列,通过ClustalX 2.1进行氨基酸序列比对,以MEGA 6.0中的邻接法(NJ)构建系统发育进化树,并应用实时荧光定量PCR检测分析大刺鳅LH基因在不同组织及不同性腺发育期的表达情况.[结果]大刺鳅LH基因cDNA序列全长799 bp,包括224 bp的5'非编码区(5'-UTR)、131 bp的3'非编码区(3'-UTR)和444 bp的开放阅读框(ORF),共编码147个氨基酸残基,第1~22位氨基酸残基组成其信号肽区域.大刺鳅LH氨基酸序列C-末端区域高度保守,但N-末端区域与其他硬骨鱼类、两栖动物、鸟类和哺乳动物存在明显差异.大刺鳅LH氨基酸序列与其他鱼类的LH氨基酸序列同源性较高,其中与黄鳝(Monopterus albus)的亲缘关系最近.大刺鳅LH基因在其脑组织中的相对表达量最高,显著高于在其他组织中的相对表达量(P<0.05);在卵巢、心脏、肝脏和精巢中的相对表达量次之;大刺鳅LH基因在不同发育期卵巢和精巢中的表达变化趋势一致,从II期开始其相对表达量随之增加,至V期时达峰值.[结论]大刺鳅LH氨基酸序列具有高度保守区域,其基因在各组织中广泛表达,尤其在脑组织和性腺中具有较高表达量,提示LH的靶基因可能在脑—垂体—性腺轴上,参与大刺鳅的性腺发育调控,主要促进卵母细胞和精子成熟并刺激排卵或排精.     

Increasing pre‐acclimation temperature reduces the freezing tolerance of winter‐type faba bean (Vicia faba L.)

Autumn‐sown winter‐type faba bean (Vicia faba L.) has been shown to have a yield advantage over spring sowing. Still, adoption of this overwintered pulse crop remains limited in temperate locations, due to inadequate winter hardiness. This research sought to understand how the prevailing temperature during emergence and seedling development, that is pre‐acclimation, influences freezing tolerance. Seedlings grown under a controlled “warm” 17/12°C (day/night) pre‐acclimation environment were initially less freezing tolerant than those grown under a “cold” 12/5°C temperature treatment. Stem and particularly root tissues were primarily responsible for slower cold acclimation, and there was a genotype specific response of above‐ground tissues to pre‐acclimation treatment. Both above and below‐ground tissues should be tested across a range of pre‐acclimation temperatures when screening faba bean germplasm for freezing tolerance.     

Spraying exogenous synthetic cytokinin 6‐benzyladenine following the waterlogging improves grain growth of waterlogged maize in the field

6‐Benzyladenine (6‐BA, a synthetic cytokinin) could improve the ability to resist adversity. However, very little attention has been given to its role in alleviating waterlogging damages on grain growth. A field experiment was performed to investigate effects of exogenous 6‐BA after waterlogging for 6 days at the third leaf stage (V3‐WL + 6‐BA) or the sixth leaf stage (V6‐WL + 6‐BA) on grain filling, and endogenous hormone of summer maize hybrids DengHai605 (DH605) and ZhengDan958 (ZD958), which were planted popularly in China. Exogenous 6‐BA alleviated waterlogging damages on grain filling by improving grain weight and volume, which was beneficial to yield increase. Grain yield of DH605 under V3‐WL + 6‐BA and V6‐WL + 6‐BA increased by 16% and 12%, respectively, compared with that of V3‐WL (waterlogging at the third leaf stage) and V6‐WL (waterlogging at the sixth leaf stage), while the corresponding values for ZD958 increased by 20% and 15%, respectively. Moreover, exogenous 6‐BA alleviated waterlogging damages on grain endogenous hormone content by increasing levels of endogenous indole‐3‐acetic acid, zeatin riboside, and gibberellic acid, and decreasing level of abscisic acid in grain of waterlogged summer maize during grain‐filling periods. Clearly, exogenous 6‐BA improved grain‐filling characteristics, and endogenous hormone, resulting in a significant yield increase of waterlogged summer maize.     

Two filamentous microalgae as feed ingredients improved flesh quality and enhanced antioxidant capacity and immunity of the gibel carp (Carassius auratus gibelio)

A 40‐day experiment was conducted to investigate the effects of two filamentous microalgae as feed ingredients on growth performance, tissue fatty acid profiles, pigmentation and immunity of gibel carp (204.83 ± 2.17 g, mean ± SD). Three diets (control, Oedocladium sp. and Tribonema sp.) were formulated. In the control diet, no microalgal meal was added and into the Oedocladium and Tribonema diets were added 40 g/kg Oedocladium sp. meal and 50 g/kg Tribonema sp. meal, respectively. Compared to the control, the addition of Oedocladium sp. and Tribonema sp. had significant effects on tissue fatty acid profiles, antioxidant capacity and immunity without compromising growth, body composition and pigmentation. The addition of Tribonema sp. and Oedocladium sp. significantly increased the EPA, DHA profiles and the ratio of n‐3/n‐6 polyunsaturated fatty acids, and simultaneously decreased the n‐6 polyunsaturated fatty acids profile in fish muscles. Moreover, addition of Tribonema sp. to the diet significantly increased muscle palmitoleic profile and EPA + DHA contents. Furthermore, the addition of either microalga significantly increased the total superoxide dismutase activity level and the complement 3 and immunoglobulin M contents in the plasma. These results demonstrate that Oedocladium sp. and Tribonema sp. can be used as feed ingredients to improve flesh quality and increase the immunity of fish.     

Formulated diet alters gut microbiota compositions in marine fish Nibea coibor and Nibea diacanthus

Nibea, an economical marine fish, is generally fed on trash fish (the low‐value fish), which can cause high feed costs and waste pollutions in high‐density aquaculture. To assess the effect of formulated diet on the gut microbiota in Nibea coibor and Nibea diacanthus, denaturing gradient gel electrophoresis, clone libraries analysis and Illumina sequencing of the 16S ribosomal RNA gene were used in this study. Two Nibea fishes were both dominated by Gammaproteobacteria (especially Photobacterium) and shared a set of gut microbiota, including Bacilli, Mollicutes, Alphaproteobacteria and Fusobacteriia. Statistical analyses revealed that formulated diet led to lower feed conversion ratio (p < 0.001), lower abundance of Vibrio (p = 0.040) and infectious diseases pathways (p = 0.001), higher abundance of polysaccharide‐degrading bacterium Cellvibrio (p = 0.006) in two Nibea species, with higher weight gain rate (p = 0.023) and microbial diversity (Shannon, p = 0.049 and Simpson, p = 0.044) and more carbohydrate metabolism (p = 0.020) observed in N. coibor. The distribution and correlation network of 17 potential short‐chain fatty acid producing bacteria were obtained and visualized in all treatment groups. The results reveal that formulated diet has beneficial effects on the gut microbial ecology in two Nibea fishes, which suggests the possibility of replacing trash fish diet with formulated diet in Nibea aquaculture.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

急性冰水应激对犬血清甲状腺激素、NO及Na、K、Cl离子含量的影响

通过测定急性冰水应激时犬血清甲状腺激素、NO及Na、K、Cl离子含量的变化,来评价急性冰水应激对犬体内能量代谢、氧化应激状态及电解质平衡的影响。试验选取8只成年杂种犬,保定躯干浸于冰水混合物中(0~4)℃,使其进行冰水浴30 min。分别于处理前0.5 h(-0.5 h)及处理后0、0.5、1、3、7 h进行桡静脉采血2 mL,分离血清后测定其甲状腺激素(T3、T4)、一氧化氮(NO)及Na^+、K^+、Cl^-的含量。结果表明:与-0.5 h(处理前)相比,处理后各时间点犬血清T3含量无显著变化(P>0.05),与-0.5 h及0、3、7 h相比,血清T4含量在0.5 h时显著降低(P<0.05);血清NO含量在处理后各时间点均显著高于处理前0.5 h(P<0.05),且1、3、7 h显著高于0、0.5 h(P<0.05);与-0.5 h相比,处理后3 h血清Na^+含量显著升高(P<0.05),血清K^+含量处理后0 h显著高于1、3、7 h(P<0.05),血清Cl^-含量处理前后差异不显著(P>0.05)。研究表明,急性短期冰水应激会扰乱机体的能量代谢状态、降低T4含量,同时造成氧化应激损伤、使NO含量大量升高;引起血清Na、K离子平衡紊乱、含量升高,提示临床上人或动物发生冰水应激时应及时纠正机体能量代谢、氧化还原平衡和电解质的紊乱。     

11个谷子新品种的主要农艺性状灰色关联度分析与综合评价

以引进的11个谷子新品种为材料，采用渗水地膜穴播技术，对8个主要农艺性状进行了灰色关联度分析，综合评价了品种的优劣性。结果表明，参试品种主要农艺性状对产量的关联度值由大到小依次为穗数、生育期、穗长、穗粒重、穗重、千粒重、株高。通过等权关联度和加权关联度分析，结合生长期的抗病性、抗倒伏等抗逆性指标，筛选出适宜在当地种植的品种为张杂谷13、白良谷和黄金苗。