基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。     

Relationship between body condition score loss and mRNA of genes related to fatty acid metabolism and the endocannabinoid system in adipose tissue of periparturient cows

The endocannabinoid system (ECS) controls feed intake and energy balance in nonruminants. Recent studies suggested that dietary management alters the expression of members of the ECS in the liver and endometrium of dairy cows. The aim of this study was to determine the relationship between body condition score (BCS) loss and the mRNA abundance of genes related to fatty acid metabolism and the ECS in the subcutaneous adipose tissue (AT) of dairy cows. The BCS was determined in multiparous (3.2 ± 0.5 lactations) Holstein cows at −21 and 42 days relative to calving (designated as d = 0). Cows were grouped into three categories according to BCS loss between both assessments as follows: (1) lost ≤0.25 unit (n = 8, low BCS loss (LBL)), (2) lost between 0.5 and 0.75 units (n = 8, moderate BCS loss (MBL)) and (3) lost ≥1 unit (n = 8, high BCS loss (HBL)). Concentrations of haptoglobin and non-esterified fatty acids (NEFAs) were determined in plasma. Real-time PCR was used to determine mRNA abundance of key genes related to fatty acid metabolism, inflammation and ECS in AT. Milk yield (kg/day) between week 2 and 6 post-calving was greater in the LBL group (49.4 ± 0.75) compared to MBL (47.9 ± 0.56) and HBL (47.4 ± 0.62) groups (P < 0.05). The overall mean plasma haptoglobin and NEFA concentrations were greater in MBL and HBL groups compared with the LBL group (P < 0.05). The mRNA abundance of TNF-α, Interleukin-6 (IL-6) and IL-1β was greatest at 21 and 42 days post-calving in HBL, intermediate in MBL and lowest in LBL groups, respectively. Cows in the HBL group had the greatest AT gene expression for carnitine palmitoyltransferase 1A, hormone sensitive lipase and adipose triglyceride lipase at 21 and 42 days post-calving (P < 0.05). Overall, mRNA abundance for very long chain acyl-CoA dehydrogenase and peroxisome proliferator-activated receptor gamma, which are related to NEFA oxidation, were greater in MBL and HBL groups compared to the LBL group at 42 days post-calving. However, mRNA abundance of fatty acid amide hydrolase was lower at 21 and 42 days post-calving in HBL cows than in LBL cows (P < 0.05). In summary, results showed a positive association between increased degree of BCS loss, inflammation and activation of the ECS network in AT of dairy cows. Findings suggest that the ECS might play an important role in fatty acid metabolism, development of inflammation and cow’s adaptation to onset of lactation.     

军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

耐除草剂玉米G1105E-823C定性检测方法

转基因耐除草剂玉米G1105E-823C是经过改造的转mG2-aroA基因耐草甘膦玉米新品系,具有更高的草甘膦耐受性,目前已完成生产性试验,具有重要的产业化应用前景。但目前尚无针对G1105E-823C的转化体特异性检测方法的相关报道,这十分不利于对该品系的检测及监管。基于此,以G1105E-823C转化体特异性序列为靶标,建立了转基因耐除草剂玉米G1105E-823C的普通PCR和实时荧光PCR定性检测方法。结果表明,2种方法均能检测出转基因耐除草剂玉米G1105E-823C转化体成分,且具有较高的特异性。普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。研究建立的2种定性检测方法为转基因耐除草剂玉米G1105E-823C的精准检测提供了新的技术手段,可为农业转基因监管提供技术支撑。