大白菜细胞质雄性不育的分子鉴定及序列分析

为了获得3种大白菜细胞质雄性不育系Ogu CMS,Pol CMS,CMS96和保持系间的多态性以及定位大白菜CMS96不育系所属的不育类型,利用设计的atp6,orf222单一和混合引物PCR扩增3组11份同核异质大白菜细胞质雄性不育系和保持系mtDNA。结果表明,atp6引物在大白菜Ogu CMS不育系扩增的200 bp片段为其特异带;orf222引物仅在大白菜Pol CMS和CMS96不育系有扩增产物,但二者有3点完全不同:大白菜Pol CMS不育系扩增产物为675bp,CMS96不育系扩增产物为669 bp,二者相差6个核苷酸,后者定名为大白菜CMS96-orf222。大白菜CMS96-orf222与甘蓝型油菜Nap CMS的nad5c基因和Nap-orf222基因同源性均为99%,E值为0.0;大白菜Pol CMS的675 bp序列具有ORF224开放阅读框,没有保守结构域,而大白菜CMS96的669 bp序列具有ORF222开放阅读框和保守结构域YMF19。另外,atp6和orf222混合引物多重PCR扩增产物存在明显多态性:800 bp为大白菜保持系的差异带型;2 300 bp和1 500 bp为大白菜Pol CMS不育系特异带型;200 bp为大白菜Ogu CMS不育系特异带型;690 bp为大白菜CMS96不育系特异带型。该方法仅用一次PCR反应快速地将3种大白菜细胞质雄性不育系和保持系一次性全部区分开,为大白菜分子育种和常规育种更好地相结合提供了简单、快速、准确和重复性好的方法和手段。     

The effect of three anaesthetic protocols on the stress response in cane toads (Rhinella marina)

ObjectiveThree anaesthetics (MS222, clove oil and a mixture of ketamine/diazepam) were administered to cane toads to determine their effect on the hypothalamic-pituitary-adrenal (HPA) axis. Time to induction and recovery and any adverse events were also evaluated.Study designProspective randomized experimental trial.AnimalsThirty adult male cane toads (Rhinella marina) with body mass ranging between 130 and 250 g were captured from the field.MethodsThree groups of 10 toads were anaesthetized with ketamine (200 mg kg^?1) and diazepam (0.2 mg kg^?1) by intramuscular injection, MS222 (3 g L^?1) or clove-oil (0.3 mL L^?1) both by immersion. Blood samples were collected to determine plasma corticosterone concentrations. Induction and recovery time were recorded in each treatment. After full recovery animals were euthanized and a complete post-mortem examination was performed.ResultsSignificant differences were found in the activation of the HPA axis and in the times of induction and recovery between treatments (p < 0.001). Animals anaesthetized with clove-oil had the highest levels of corticosterone in plasma (42.5 ± 21.6 ng mL^?1). No differences were found between ketamine/diazepam (15.0 ± 13.3 ng mL^?1) and MS222 (22.0 ± 13.6 ng mL^?1) groups. The mean ± SD induction (minutes) and recovery (hours) times respectively were; ketamine/diazepam 66.5 ± 11 and 8 ± 3, clove oil 39 ± 12 and 7.6 ± 3, and MS222 42.5 ± 11 and 1.5 ± 0.5. Clove oil exposure had 30% mortality. Death followed a period of respiratory distress with changes consistent with non-cardiogenic oedema observed at post-mortem examination.Conclusions and Clinical relevanceBased on shorter induction and recovery times and minimal activation of HPA, MS222 is the anaesthetic of choice in cane toads. If it is not possible to use immersion methods of anaesthesia, ketamine/diazepam can be used but induction and recovery times are prolonged. Clove oil had unacceptable mortality in this study and should be used with extreme caution.     

Physiologic and biochemical measurements and response to noxious stimulation at various concentrations of MS‐222 in Koi (Cyprinus carpio)

ObjectiveTo evaluate the physiological effect and response to noxious stimulation at five concentrations of MS-222 in koi (Cyprinus carpio).Study designProspective experimental study.AnimalsTwenty-one healthy adult unknown sex koi fish weighing mean 450 ± SD 120 g.MethodsEach fish was exposed to five different concentrations of MS-222 (50, 70, 110, 150 and 190 mg L^?1) in a random sequence during the same anaesthetic event. For each concentration of MS-222, vital functions such as heart rate (HR) (via Doppler) and opercular rate (OpR) were recorded after a standardized induction period. Response to two noxious stimuli in the form of haemostat clamp pressure applied on the tail and the lip was evaluated, and blood was drawn to measure biochemical and blood gas values.ResultsDecrease in response to noxious stimulation with an increase of MS-222 concentration both for the lip (p = 0.0027) and the tail (p < 0.0001) stimulus was observed. Biochemical values were unaffected by the concentration of MS-222 with the exception of lactate concentration which was weakly correlated with the duration of anaesthesia (r = 0.31, p < 0.001) and the number of times the fish was clamped or bled prior to sampling (r = 0.23, p < 0.001). Opercular rate decreased with the increase in anaesthetic concentration, and HR was not affected.Conclusions and clinical relevanceOur results indicated a decrease in response to stimulus and a decrease in OpR that were associated with increased concentrations of MS-222. This may assist in establishing anaesthetic protocols using MS-222 in fish and supports the use of supramaximal pressure stimuli to teleost fish under variable MS-222 concentrations as a model for future studies.     

miR-222 enhances HBx-HepG2 cell growth via regulation of BCL2L13 gene

AIM: To investigate the regulation of miR-222 on BCL2L13 gene and its effect on the growth and apoptosis of HBx-HepG2 cells, and to explore the underlying molecular mechanisms. METHODS: The expression level of miR-222 was detected by RT-qPCR. The HBx-HepG2 cell growth was examined by MTT and colony formation assays. The cell cycle and apoptosis were analyzed by flow cytometry. The recombination vector pmirGLO-BCL2L13 was constructed, and dual-luciferase reporter experiment was performed to validate the target of miR-222. RESULTS: The expression level of miR-222 in the HBx-HepG2 cells was significantly higher than that in the L02 cells (P<0.05). Over-expression of miR-222 enhanced HBx-HepG2 cell growth, changed cell cycle, and inhibited apoptosis (P<0.05). Knockdown of miR-222 reduced HBx-HepG2 cell growth, changed cell cycle, and increased cell apoptotic rate (P<0.05). BCL2L13 was down-regulated in the HBx-HepG2 cells as compared with L02 cells (P<0.05), and knockdown of miR-222 in the HBx-HepG2 cells increased the expression level of BCL2L13 (P<0.05). The results of dual-luciferase reporter assay and restore experiment showed that miR-222 negatively regulated the expression of BCL2L13 via targeting 3'UTR of BCL2L13, resulting in the promotion of HBx-HepG2 cell growth. CONCLUSION: miR-222 enhances HBx-HepG2 cell growth via down-regulation of BCL2L13.     

Simultaneous effect of sex and dose on efficacy of clove oil,tricaine methanesulfonate, 2‐phenoxyethanol and propofol as anaesthetics in guppies,Poecilia reticulata (Peters)

We studied the simultaneous effect of sex and dose on anaesthesia efficacy to estimate, if possible, the lowest effective dose (LED) for clove oil, tricaine methanesulphonate (MS‐222), 2‐phenoxyethanol (2‐PE) and propofol in mature guppies. LED is the lowest dose needed to reach A5 stage in a mean time of 3 min, with mean recovery (R5) time of 5 min. We used four doses/anaesthetic: 25, 50, 75 and 100 mg/L for clove oil; 120,140,160 and 180 mg/L for MS‐222; 800, 1,000, 1,200 and 1,400 mg/L for 2‐PE, and 7.50, 8.25, 10.00 and 11.25 mg/L for propofol. Each dose was tested on 10 females and 10 males. Morbidity, mortality and behavioural changes were checked on two control groups (10 males and 10 females/group). Sedation (A3), A5 and R5 times were recorded. Significant interactive effect dose*sex on A5 time was found for each anaesthetic agent (p_dose*sex < .05). Except for MS‐222 (p_dose*sex = .284), significant interactive effect dose * sex on R5 time was found (p_dose*sex < .05). A5 time in females tended to be greater than in males, but, in general, R5 times were longer in males. Body size differences between males and females could explain these differences in MS‐222 on A5 time and for clove oil, 2‐PE and propofol on R5 time. No dose simultaneous meet LED′s conditions relating to both A5 and R5 times; therefore the lowest doses inducing A5 in a mean time of 3 min could be a safe guideline for anaesthetic procedure in both male and females.     

Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status

Responses to anaesthesia with essential oil (EO) of Aloysia triphylla (135 and 180 mg L^?1) and tricaine methanesulfonate (MS222) (150 and 300 mg L^?1) were assessed in silver catfish. Exposure to the anaesthetics elicited a stress response in the species. In the case of MS222, it was displayed as a release of cortisol into bloodstream, elevation in hematocrit and plasma ion loss. The EO presented cortisol‐blocking properties, but increased haematocrit and disturbances of hydromineral balance were observed. Liver antioxidant/oxidant status of EO and MS222‐anaesthetized silver catfish was also estimated. The synthetic anaesthetic induced lipoperoxidation, notwithstanding increased catalase contents, whereas the naturally occurring product was capable of preventing the formation of lipid peroxides, possibly due to combined actions of catalase and glutathione‐S‐transferase. Anaesthetic efficacy was also tested via induction and recovery times. Overall, the promising results obtained for the physiological parameters of the EO‐treated fish counterbalanced the slight prolonged induction time observed for 180 mg L^?1. As for 135 mg L^?1, both induction and recovery times were lengthy; despite that, the EO was able to promote oxidative protection and mitigate stress. None of the MS222 concentrations prompted such responses concomitantly.     

Anaesthetic efficacy of clove oil,benzocaine, 2‐phenoxyethanol and tricaine methanesulfonate in juvenile marbled spinefoot (Siganus rivulatus)

Anaesthetics are used in aquaculture and fisheries to facilitate routine procedures, such as capture, handling, transportation, tagging, grading and measurements that can often cause injury or induce physiological stress. Two experiments were performed to assess the efficacies of four anaesthetic agents, clove oil, benzocaine, 2‐phenoxyethanol and MS‐222 on juvenile marbled spinefoot rabbitfish (Siganus rivulatus). In the first experiment we tested the lowest effective doses that produced induction and recovery times in 3 min or less and 5 min or less respectively. Dosages were 70 mg L^?1 for clove oil, 60–70 mg L^?1 for benzocaine, 400 μL L^?1 for 2‐phenoxyethanol and 100–125 mg L^?1 for MS‐222. In the second experiment, we determined optimal concentrations of the four anaesthetics if they were to be used to transport rabbitfish fry. Anaesthetic concentrations suitable for handling and transport were: 10–15 mg L^?1 of MS‐222, 5–10 mg L^?1 of benzocaine, 5 mg L^?1 of clove oil and 50–100 μL L^?1 of 2‐phenoxyethanol. All anaesthetic agents are acceptable for use on S. rivulatus, however, 2‐phenoxyethanol, MS‐222 and clove oil appear to be more suitable than benzocaine. Further studies need to be conducted on effects of high and low doses of anaesthetic agents on physiology of marbled spinefoot.     

香猪睾丸miR-449b和miR-222分子的发育性变化

为了阐明miR-449b和miR-222对贵州从江香猪睾丸发育的调节作用。运用qPCR方法检测香猪睾丸组织中miR-449b和miR-222表达量的发育性变化,通过miRSystem数据库、Kobas数据库对预测的靶基因进行信号转导通路富集分析。经克隆测序,得到香猪miR-222序列,与猪的相似性为100%,而且物种之间miR-449b和miR-222两个分子的保守性都很高。香猪睾丸中miR-449b于1~4月龄期间的表达量逐渐上升,4月龄时表达量达到峰值,之后下降;miR-222的表达量呈现相反的变化趋势,于1~4月龄期间表达量逐渐下降,1月龄时最高,4月龄时最低,之后的表达量有所回升;已知物种的miR-449b和miR-222序列高度保守;经预测miR-449b靶基因有420个,富集于卵母细胞减数分裂、Notch信号通路、Ras信号通路等100个途径(P0.05),miR-222的靶基因有429个,富集于细胞周期、Fox O信号通路、TGF-β信号通路等69个通路(P0.05);miR-449b和miR-222的靶基因共享的通路为Wnt信号通路、MAPK信号通路、雌激素信号通路等42个(P0.05)。     

麻醉剂MS-222对金鱼体内AKP、CAT和ACP活性的影响

在实验室条件下,采用4个浓度0mg/L、30mg/L、60mg/L和90mg/L,分析了麻醉剂MS-222对金鱼鳃、肝脏和肌肉组织中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)和过氧化氢酶(CAT)活性的影响。试验结果表明:ACP明显出现一个先增后降的趋势,呈抛物线状;在低浓度(≤60mg/L)下,酶活性在各组织中呈现一定的稳定状态,而当麻醉剂浓度升高和麻醉时间增长,酶活性逐渐下降。AKP在低浓度麻醉剂溶液中,酶活性有明显的增加,随着浓度的升高(≥60mg/L)和时间的增长,酶活性呈抑制状态。而在肌肉中这种情况并不明显。CAT在一定阶段(30mg/L～60mg/L)呈现稳定的状态、而后抑制逐渐明显,当麻醉剂MS-222浓度达到一定量(≥60mg/L)以后,对鱼体的刺激就不再增加了,酶活性也相对处于较稳定的状态;当麻醉剂随着时间的增长和浓度的增大时,在鱼体内富集的麻醉剂破坏鱼体内的过氧化氢酶的分子构象,使酶体失去活性。     

麻醉剂MS-222对金鱼体内AKP、CAT和ACP活性的影响

在实验室条件下,采用4个浓度0mg/L、30mg/L、60mg/L和90mg/L,分析了麻醉剂MS-222对金鱼鳃、肝脏和肌肉组织中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)和过氧化氢酶(CAT)活性的影响。试验结果表明:ACP明显出现一个先增后降的趋势,呈抛物线状;在低浓度(≤60mg/L)下,酶活性在各组织中呈现一定的稳定状态,而当麻醉剂浓度升高和麻醉时间增长,酶活性逐渐下降。AKP在低浓度麻醉剂溶液中,酶活性有明显的增加,随着浓度的升高(≥60mg/L)和时间的增长,酶活性呈抑制状态。而在肌肉中这种情况并不明显。CAT在一定阶段(30mg/L～60mg/L)呈现稳定的状态、而后抑制逐渐明显,当麻醉剂MS-222浓度达到一定量(≥60mg/L)以后,对鱼体的刺激就不再增加了,酶活性也相对处于较稳定的状态;当麻醉剂随着时间的增长和浓度的增大时,在鱼体内富集的麻醉剂破坏鱼体内的过氧化氢酶的分子构象,使酶体失去活性。