sikFBA4基因启动子的克隆及低温表达模式分析

天山雪莲生长于常年积雪覆盖高海拔处的高山区域,具有很强的极端低温生境适应能力,属于研究植物低温适应的良好模式植物。前期的研究表明,sikFBA4基因可以显著提高番茄的抗寒能力。为进一步研究sikFBA4基因的耐寒响应模式,以天山雪莲为材料,采用实时荧光定量PCR分析sikFBA4在低温胁迫下的表达模式;利用高效热不对称PCR法(Hi-Tail PCR)克隆sikFBA4启动子序列并进行生物信息学分析。为分析PsikFBA4序列克隆的完整性及转录表达特性,将PsikFBA4与GUS基因融合在烟草中进行瞬时表达。结果表明,sikFBA4在低温胁迫条件下的表达发生瞬时显著上调,并在1 h达到峰值,而后表达下调。通过启动子克隆分析,在PsikFBA4序列-1648 bp位置处具有冷响应元件LTRE;进一步的GUS染色和酶活力测定结果表明,低温能够显著提高GUS基因的表达活性,说明sikFBA4属于低温诱导型基因。SikFBA4基因启动子的克隆、序列分析以及表达分析,为进一步探究雪莲果糖-1,6-二磷酸醛缩酶(sikFBAs)基因的表达与调控机制奠定了基础。     

炭化条件对竹原纤维炭孔隙结构的影响分析

为研究炭化竹原纤维的特性,进一步提升竹原纤维的使用性能,拓宽竹原纤维的应用领域,采用可控电炉制备了不同炭化温度和不同保温时间条件下的炭化竹原纤维,利用全自动比表面积及孔隙度分析仪测试了炭化竹原纤维的比表面积、比孔容及平均孔径,探讨了炭化条件对其性能的影响。结果表明：随炭化温度的升高和保温时间的延长,炭化竹原纤维的比表面积、比孔容和孔径分布先增大后减小,在较优的工艺条件下,炭化竹原纤维的比表面积和比孔容最大值分别可达819.35m2/g和0.7358cm3/g,平均孔径最小可达2.0836nm。     

菊花ClERF1基因的克隆与表达分析

以甘菊叶片为试验材料,采用RT-PCR和Tail-PCR以及生物信息学的方法,研究了甘菊ClERF1及其启动子序列特征、表达模式,以及甘菊ClERF1在低温胁迫下的响应。以期为进一步研究甘菊ClERF1功能提供参考依据。结果表明:ClERF1基因序列全长1 242 bp,最大开放阅读框(ORF)618 bp,可编码206个氨基酸。经理化性质分析,ClERF1分子量23 631.35 Da,理论等电点5.19,不稳定系数53.84,脂肪指数62.04,平均亲水性-0.786,亚细胞定位在细胞核内。系统进化树表明,ClERF1与拟南芥At3g23240亲缘关系最近,属于ERF亚家族。qRT-PCR分析表明ClERF1在叶片中表达最高,其次在茎段中。该研究克隆了ClERF1启动子序列1 534 bp,主要包括低温反应和乙烯响应元件、生长素和茉莉酸甲酯反应元件等。甘菊低温处理幼苗ClERF1表达量显著高于未低温处理的幼苗,其中5℃时ClERF1相对表达量最高。     

荔枝FKBP16-2基因启动子的克隆与瞬时表达分析

前期研究中发现了一个果肉低表达而叶片中高表达的荔枝基因FKBP16-2。本文克隆了该基因1 578 bp的启动子片段并对其功能进行了初步分析,结果表明:荔枝FKBP16-2基因启动子序列中含有大量的TATA-box和CAAT-box保守元件,以及TCA-element,ARE,HSE,GCN4_motif,O2-site等各种转录调控相关的顺式作用元件。该启动子能驱动GUS基因在荔枝的花、叶、根、果皮以及种子中表达而在果肉中不表达,表达具有组织特异性。     

鸡脂肪组织TCF21基因启动子区DNA甲基化与其表达的关系

旨在研究鸡脂肪组织中TCF21基因启动子区DNA甲基化水平与其表达的关系。以东北农业大学高、低腹脂双向选择品系（简称高、低脂系）第24世代7周龄肉鸡为试验材料，利用RT-qPCR检测高、低脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平；利用生物信息学和双荧光素酶报告系统分析TCF21基因启动子的结构与功能；利用Sequenom MassARRAY飞行质谱检测高、低脂系肉鸡腹部脂肪组织中TCF21基因启动子区CpG位点的甲基化水平；利用CpG甲基转移酶处理TCF21启动子报告基因质粒，分析DNA甲基化对TCF21基因启动子活性的影响。结果显示，高脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平极显著高于低脂系（P<0.001）；TCF21基因的启动子区存在40个CpG位点，且在启动子的近端和远端均有分布，但不存在CpG岛；将TCF21基因的启动子划分为5个功能区域，分别为R1区域（-2 000~-1 500 bp）、R2区域（-1 500~-1 000 bp）、R3区域（-1 000~-500 bp）、R4区域（-500~-200 bp）和Core区域（-200~-100 bp）；高脂系R2、R3和R2+R3区域的DNA甲基化水平显著或极显著高于低脂系（P<0.05或P<0.001）；R2、R3、R2+R3区域的DNA甲基化水平与TCF21基因mRNA表达水平呈显著正相关（R2区域：r=0.438，P<0.05；R3区域：r=0.371，P<0.05；R2+R3区域：r=0.489，P<0.05）；R2区域的DNA甲基化显著抑制其转录活性（P<0.05）。综上所述，TCF21基因在高、低脂系肉鸡腹部脂肪组织中的表达水平主要与其启动子R2区域的DNA甲基化水平有关。     

Humanized mice in infectious diseases

The pathogenesis of infectious agents with human tropism can only be properly studied in an in vivo model featuring human cells or tissue. Humanized mice represent a small animal model featuring human cells or tissue that can be infected by human-specific viruses, bacteria, and parasites and also providing a functional human immune system. This makes the analysis of a human immune response to infection possible and allows for preclinical testing of new vaccines and therapeutic agents. Results of various studies using humanized mice to investigate pathogens with human tropism are presented in this review. In addition, the limitations of humanized mice and methods to improve this valuable animal model are discussed.     

Use of the nitrate‐specific ion electrode for the determination of nitrate nitrogen in surface and ground water

Abstract

The usefulness of the nitrate‐specific ion electrode (NE) for the determination of nitrate nitrogen (NO3‐N) in surface and ground water was determined. The NE method had several advantages that made it superior to the phenoldisulfonic acid (PDS) method in our environmental quality studies. Samples could be analyzed quickly and with a minimum of sample preparation. Sample coloration and soluble salts did not interfere with the performance of the electrode. Range of detection without dilution was much wider than by the PDS method. Also, the values obtained by the NE method, although slightly greater than those obtained by the PDS method, were satisfactory.     

Definition and clinical evaluation of a recruiting airway pressure based on the specific lung elastance in anesthetized dogs

ObjectiveTo determine the specific lung elastance (SE_L) in anesthetized dogs and to evaluate the efficacy of a SE_L-based recruiting airway pressure (RP_aw) at improving global and regional lung aeration.Study designRetrospective and prospective clinical study.AnimalsA total of 28 adult dogs were included in the retrospective study and six adult dogs in the prospective study.MethodsRetrospective study: SE_L and SE_L-based RP_aw were determined using previously published data. In mechanically ventilated dogs undergoing thoracic computed tomography (CT), SE_L was calculated as ΔP_L/(V_T/EELV), where ΔP_L is the driving transpulmonary pressure, V_T is the tidal volume and EELV is the end-expiratory lung volume. The ratio of lung to respiratory system elastance (E_L/E_rs) was determined. SE_L and E_L/E_rs were used to calculate the SE_L-based RP_aw. Prospective study: dogs underwent thoracic CT at end-expiration and at end-inspiration using the SE_L-based RP_aw, and global and regional aeration was determined. For analysis of regional aeration, lungs were divided into cranial, intermediate and caudal regions. Regional compliance was also calculated. A p value <0.05 was considered significant.ResultsThe SE_L and E_L/E_rs were 12.7 ± 3.1 cmH₂O and 0.54 ± 0.07, respectively. The SE_L-based RP_aw was 29.1 ± 7.6 cmH₂O. In the prospective study, the RP_aw was 28.2 ± 1.3 cmH₂O. During RP_aw, hyperinflation increased (p = 0.0003) whereas poorly aerated (p < 0.0001) and nonaerated (p = 0.01) tissue decreased. Normally aerated tissue did not change (p = 0.265). Regional compliance was higher in the intermediate (p = 0.0003) and caudal (p = 0.034) regions compared with the cranial region. Aeration did not differ between regions (p > 0.05).Conclusions and clinical relevanceAn SE_L-based RP_aw reduces poorly and nonaerated lung tissue in anesthetized dogs. In nonsurgical anesthetized dogs, an RP_aw near 30 cmH₂O is effective at improving lung aeration.     

水牛犊牛血浆外泌体的分离与鉴定

研究旨在从水牛犊牛血浆中分离外泌体,并对分离的外泌体进行分子生物学特征分析和鉴定。采用差速离心法分离3头水牛犊牛的血浆外泌体,并在透射电子显微镜下观察其形态,使用纳米粒度及Zeta电位仪对提取的外泌体进行粒径大小分析,应用Western blotting方法检测外泌体标记蛋白钙联蛋白（Calnexin）、肿瘤易感基因101（TSG101）、CD9、CD81在3头水牛犊牛血浆外泌体中的表达情况。结果显示,从水牛犊牛血浆中分离的外泌体形态多为圆形和椭圆形,直径在30~150 nm;Western blotting检测结果表明,在水牛血浆外泌体表面,特异性蛋白Calnexin、TSG101和CD81阳性表达,CD9不表达。本研究从形态学和分子生物学特征等方面证实研究获得的提取物为外泌体,由此说明,差速离心法可以成功分离提取水牛犊牛血浆中的外泌体,为水牛外泌体的后续研究提供了重要技术基础和参考。     

Effects of dietary lysozyme levels on growth performance,intestinal morphology,non‐specific immunity and mRNA expression in weanling piglets

The aim of the present study was to determine the effect of dietary lysozyme levels on growth performance, gut health and non‐specific immunity of weanling piglets. A total of 150 weanling piglets were allocated to six treatments. The piglets were fed the same basel diet supplemented with 0, 30, 60, 90 and 120 mg/kg lysozyme as well as antibiotics for 28 days. From day 14 to day 28 of dietary treatment, piglets fed 90 mg/kg lysozyme had greater average daily gain than piglets fed control diet. During the whole experimental period, piglets fed 120 mg/kg lysozyme tended to have greater average daily gain than piglets fed control diet. Compared with piglets fed control diet, piglets fed diets containing antibiotics and 90 mg/kg lysozyme had greater villus height to crypt depth ratio in duodenum and jejunum. Additionally, dietary supplementation of 60 and 90 mg/kg lysozyme as well as antibiotics enhanced the phagocytic activity of peritoneal macrophages in piglets. In conclusion, dietary lysozyme can accelerate the growth of weanling piglets by improving gut health and non‐specific immunity and supplementing 90 mg/kg lysozyme is as effective as antibiotics (20 mg/kg colistin sulphate + 50 mg/kg kitasamycin) in improving the growth performance of weanling piglets.