Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.     

羊草种子萌发相关基因的筛选及表达分析

前期实验表明,变温处理(28 ℃ 12 h/16 ℃ 12 h)可显著提高羊草种子的萌发率,且羊草种子萌发中的第1天是接受变温信号的关键时期。以此为研究基础,结合羊草种子变温萌发的转录组测序数据,针对羊草种子萌发初期对变温处理的响应筛选出与种子萌发、休眠及低温相关的基因24个,利用测序结果中这些基因的RPKM值制作基因表达热图并分析其表达差异。以萌发率高、低的两种羊草种质的种子为材料,对24个基因在恒温12 h(28 ℃)和变温1 d(28 ℃ 12 h/16 ℃ 12 h)萌发处理中的表达分别进行了定量分析。结果表明,与恒温对照相比,变温处理12 h后,表达明显上调的基因有SAIN1,PP2C62,EXPB3,EXPB4,GA3ox,EXPA2和EXPA7,而表达明显下调的基因有bHLH49,GID1,ABI8,Chi1,11833,CBF3,NAC2,PP2C72,SAIN2和5423。通过进一步分析相关基因在高、低萌发率两个种质中表达的差异,筛选出其中可能与羊草种子萌发相关的基因有几丁质酶基因Chi1,转录因子基因CBF3,羊草新基因5423,赤霉素合成基因GA3ox,细胞松弛素蛋白基因EXPB4和羊草新基因SAIN1,将为下一步阐明羊草种子萌发的分子作用机理奠定基础。     

人工改造野生大豆GsDREB2基因对植物耐盐和耐渗透胁迫能力的影响

DREB (dehydration responsive element binding protein)转录因子是一个干旱应答元件的结合蛋白,它能特异结合启动子中含有DRE/CRT顺式元件,激活逆境诱导基因的表达,调控植物对干旱、低温、高盐、高温等胁迫的耐逆性。大量研究表明DREB转录因子在信号传导、作用机理及基因表达方面存在复杂性。为了野生大豆来源GsDREB2基因能更有效地发挥功能,人工突变该基因的负向调节结构域(negative regulatory domain, NRD,140~204),经改造命名为GsDREB2-mNRD。在酵母中比较全长基因(FLDREB2)和GsDREB2-mNRD转录激活和与DRE元件结合的能力,并验证GsDREB2-mNRD核定位情况。分别将FLDREB2和GsDREB2-mNRD转化拟南芥,通过拟南芥幼苗期盐胁迫和渗透胁迫试验,比较GsDREB2-mNRD和FLDREB2在提高植物耐盐和渗透胁迫方面的差异。结果表明,GsDREB2基因内部存在着负向调节结构域(NRD),抑制了GsDREB2转录激活功能和DRE元件结合的特性;经改造的GsDREB2基因依然能定位在细胞核;超量表达GsDREB2-mNRD基因的拟南芥耐盐和渗透胁迫能力明显强于非转基因对照,也高于FLDREB2基因超表达的拟南芥;野生大豆来源的GsDREB2基因NRD结构域的缺失可增强该基因在植物耐盐、渗透胁迫等逆境胁迫下的功能。     

低温胁迫对狗牙根生理及基因表达的影响

以耐寒性差异较大的杂交狗牙根品种运动百慕大、天堂419和普通狗牙根品种保定狗牙根为试验材料,分析了昼夜温度为适温(30 ℃/25 ℃)、亚适温(18 ℃/12 ℃)、冷害(8 ℃/4 ℃)和冻害(4 ℃/-4 ℃)4种温度处理下,低温对狗牙根生长速率和草坪质量、MDA含量、叶绿素荧光(F_v/F_m)、光合作用(P_n、G_s、C_i、T_r)、H₂O₂和O2·-含量、抗氧化酶活性(SOD、POD、CAT、APX)、抗氧化酶及耐寒相关基因(CBF1、COR、LEA)表达的影响。结果表明,随着温度的降低,狗牙根草坪质量、生长速率、F_v/F_m和光合作用显著下降,但在耐寒性强的运动百慕大中下降幅度较小;低温下叶片MDA和H₂O₂含量及O2·-产生速率显著升高,其中在耐寒性弱的保定狗牙根中升高程度更大;狗牙根叶片抗氧化酶活性及抗氧化酶基因的表达水平随着处理温度的下降而显著升高,其中在运动百慕大中更为明显;狗牙根CBF1、COR和LEA基因表达量随着温度的下降而显著升高,尤其是在耐寒狗牙根运动百慕大中更为显著。低温诱导的抗氧化酶和耐寒相关基因上调表达,增强了细胞内的抗氧化防御,有助于狗牙根植株维持较高的光合作用和细胞膜稳定性,延缓叶片的衰老,从而提高了狗牙根的抗寒能力。     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

对虾养殖池副溶血弧菌的分离鉴定及其耐药特征、毒力基因分析

为了解对虾养殖池中副溶血弧菌(Vibrio parahaemolyticus)的耐药性和毒力基因的携带情况,2018年从山东4个地区的对虾养殖池收集分离副溶血弧菌,采用Kirby-Bauer纸片法检测其对12种抗生素的耐药性,用PCR方法检测其携带耐热直接溶血素基因(tdh)和耐热相关溶血素基因(trh)的情况。从对虾养殖池共分离副溶血弧菌50株。药敏实验结果显示,副溶血弧菌对庆大霉素、硫酸新霉素和氨苄西林的耐药情况最为严重,耐药率分别高达98%、90%和86%,对氟苯尼考、氯霉素、头孢他啶等敏感性较高,耐药率分别为10%、10%和20%。88%的菌株具有多重耐药性。毒力基因检测结果显示,所有菌株均不携带tdh基因,4%的菌株表现为trh阳性。本研究表明,对虾养殖水环境中的副溶血弧菌对抗生素的耐药性较为严重,应加强副溶血弧菌的病原学监测,在养殖过程中合理使用抗生素,以实现水产养殖业健康发展。