山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用

【目的】快速骨骼肌肌钙蛋白T（fast skeletal troponin T3,TNNT3）作为肌钙蛋白(troponin, Tn)家族成员,调节横纹肌收缩、参与骨骼肌的生长发育并影响家畜肉质性状。通过获得山羊TNNT3基因的可变剪切体,分析山羊TNNT3基因可变剪切的表达模式及其在肌细胞分化中的作用,深入解析TNNT3基因在山羊骨骼肌生长发育过程中的作用机制。【方法】基于NCBI已公布山羊TNNT3基因（NM_001314210.1）和牛TNNT3基因（XM_010821200）mRNA序列,使用软件Primer Premier 6.0设计引物,以简州大耳羊胚胎期和出生后7个阶段骨骼肌为试验材料,克隆测序获得山羊TNNT3基因的CDS区可变剪切体,利用软件ORF Finder、EditSeq、DNAMAN、ClustalW和MEGA_X_10.1.8等对序列进行生物信息学分析;进一步设计实时荧光定量（real-time PCR,RT-qPCR）及半定量引物,研究TNNT3基因剪切体在7个不同组织（背最长肌（longissimus dorsi muscle,LD）、半膜肌（semimembranosus muscle,SM）、心、肝、脾、肺、肾）和7个发育阶段（胚胎期E75、E90、E105和出生后B3、B45、B150、B300）肌肉组织（背最长肌和半膜肌）中表达模式;此外,对转录本TNNT3_3进行体外编码能力检测确定其具有编码蛋白的能力,并在山羊骨骼肌卫星细胞（skeletal muscle satellite cells,MuSCs）中过表达,观察细胞形态变化以及检测标志基因的表达变化,研究其对山羊MuSCs分化的作用。【结果】①TNNT3（NM_001314210.1）CDS区全序列主要含有18个外显子,其中外显子16/17相互排斥,转录后单一表达。克隆发现山羊TNNT3基因 5个新转录本（TNNT3_1—5）,其外显子数分别是15、15、20、16、14。②生物信息学分析结果显示山羊TNNT3基因核苷酸序列和氨基酸序列与绵羊、牛、猪等哺乳动物具有很高的一致性,而与鱼类和爬行类动物的一致性较低,说明TNNT3基因序列在哺乳动物高度保守。③TNNT3 mRNA在背最长肌、半膜肌、心、肝、脾、肺、肾7个组织中都有表达,其中在骨骼肌中高度富集(P < 0.01),心脏及肺次之,其余组织中较低;TNNT3 mRNA在背最长肌和半膜肌中的表达始终处于一个动态变化中,胚胎期TNNT3在半膜肌的表达量高于背最长肌（P<0.05）;出生后则背最长肌中高于半膜肌（P<0.05）。④山羊TNNT3基因转录本TNNT3_3重复出现保守的外显子9—11（138bp）,体外翻译实验显示其可编码蛋白且蛋白大小与预期基本相符（37 kD）;相较于对照组,在山羊MuSCs中过表达该转录本使肌分化标志基因Myomaker、MyoG和MyH4 mRNA极显著升高(P < 0.01)。【结论】获得了山羊TNNT3基因具有完整CDS区5个新可变剪切体,TNNT3主要在肌肉组织（背最长肌和半膜肌）中高表达,在哺乳动物中高度保守且促进成肌分化。初步表明TNNT3基因在动物肌肉生长发育中具有重要的生物学功能。     

吉林省西部地区滴灌条件下宜机械粒收玉米品种筛选

确定宜机械粒收的玉米品种是构建吉林省西部旱作补灌区玉米丰产增效技术模式的重要内容之一。2018年和2019年在吉林省西部地区开展滴灌条件下宜机械粒收玉米品种的筛选试验,测定38个玉米品种的收获子粒含水率和产量,并按玉米子粒含水率和产量水平采用双向平均法作图进行品种分类。结果表明,本研究初步筛选出5个滴灌条件下宜机械粒收玉米品种,分别是迪卡159、福莱77,稷秾108、吉农大889和优迪919。根据品种综合性状分析,吉农大889较优,其次为迪卡159,可以作为适宜吉林省西部滴灌条件下丰产高效栽培的推荐品种。     

西方语境的中国古典园林审美认知历史溯源

中国古典园林审美不仅仅在中国文化体系中进行讨论，随着全球化进程，西方语境学者也逐渐涉及相关的内容。尝试寻找一种跨文化的“他者”视角，梳理近3个世纪以来西方语境的中国古典园林审美认知经历的3个主要阶段。以18世纪为开端，经过19—20世纪借助西译艺术著作和海外园林建设实现对审美理解从印象到真实的转变，到进入21世纪后诞生的跨学科的诸多新观，这个过程中展现出以中国传统园林审美为代表的打破自身线性历史的闭关自守，回归全球语境的未来中国古典园林研究趋势。     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

Kernel vitreousness and protein content: Relationship,interaction and synergistic effects on durum wheat quality

Four sets of durum samples were used in this study to further understand the interrelationships among hard vitreous kernels (HVK), protein content, and pigment concentration, with a focus on the interaction and synergistic effects of protein content and vitreousness on durum quality. HVK level increases with higher protein content in the range of 9.5–12.5%, but this relationship is less evident in durum samples with high protein content (12.5–14.5%). Both protein content and kernel vitreousness can significantly affect durum milling quality. White starchy kernels (WSK) in low protein durum have a very detrimental impact on milling and pasta processing quality, but high protein content can mitigate the adverse impact of WSK on durum quality. Although protein content plays a dominant role, higher HVK might contribute positively to pasta firmness. There was no significant difference in yellow pigment content between HVK and WSK. However, pigment loss from semolina to dough was higher for WSK than HVK. Despite the difference in protein content, HVK and WSK have little difference in gluten strength. The monomeric protein was preferentially accumulated in HVK. The glutenin proteins of HVK and WSK were similar in the ratios of 1Bx/1By and HMW/LMW-GS.     

A new lean no time test baking method with improved discriminating power

In response to customer concerns related to gluten strength in commercial baking, the Canadian Grain Commission assessed whether the Canadian Short Process (CSP) test bake method was generating useful data related to intrinsic strength of wheat varieties. Assessment of CSP loaf volume data for Canadian variety trials spanning 2003 to 2013 showed very little correlation with dough strength parameters as measured by farinograph and extensigraph. A lean no time (LNT) test baking method was developed that can better discriminate genotypes and provide objective indicators of the effect of intrinsic dough strength on baking quality. From early method development, through method validation and verification using diverse sets of samples targeting different Canadian wheat classes and grown in three different crop years, results showed the LNT method to be more discriminating and easily adopted by other laboratories. In 2015, the LNT method was adopted as the method of choice in future Canadian variety registration trials. The LNT method is fast, simple and well-suited to high throughput test baking conditions encountered in the evaluation of large numbers of breeder lines. A new objective parameter, loaf top ratio, was also introduced and found to correlate well with dough strength and dough handling properties.     

广州近代城市公园的中西合璧特征探析

广州近代城市公园设计精巧,风格独特,在岭南造园史上具有重要地位。结合历史图文资料,运用风景园林史学和风景园林美学相结合的研究方法,归纳分析其造园特征。广州近代城市公园的中西合璧特征主要体现在3个方面:因地制宜的空间布局、中西融合的建筑小品、多姿多彩的植物景观。探析其中西合璧特征,有利于丰富岭南近代园林历史与理论研究,推进对广州近代城市公园的保护与造园智慧的传承。     

Secretoglobin and Transferrin Expression in Bronchoalveolar Lavage Fluid of Horses with Chronic Respiratory Disease

Background

Lower expression of secretoglobin and transferrin has been found in the bronchoalveolar lavage fluid (BALF) of a small number of horses with experimentally induced signs of recurrent airway obstruction (RAO) compared to healthy controls.

Hypothesis/Objectives

Secretoglobin and transferrin BALF expression will be similarly decreased in horses with naturally occurring clinical signs of RAO and in horses with experimentally induced clinical signs of RAO as compared to healthy controls and intermediate in horses with inflammatory airway disease (IAD).

Animals

Recurrent airway obstruction‐affected and control horses were subjected to an experimental hay exposure trial to induce signs of RAO. Client‐owned horses with a presumptive diagnosis of RAO and controls from the same stable environments were recruited.

Methods

Pulmonary function and BALF were evaluated from control and RAO‐affected research horses during an experimental hay exposure trial (n = 5 in each group) and from client‐owned horses (RAO‐affected horses, n = 17; IAD‐affected horses, n = 19; healthy controls, n = 5). The concentrations of secretoglobin and transferrin in BALF were assessed using Western blots.

Results

Naturally occurring and experimentally induced RAO horses had similar decreases in BALF transferrin expression, but secretoglobin expression was most decreased in naturally occurring RAO. Secretoglobin and transferrin expression were both lower in BALF of RAO‐affected horses than in IAD‐affected and control horses.

Conclusions and Clinical Importance

Secretoglobin and transferrin expression is decreased in BALF of RAO‐affected horses after both experimental and natural exposure. Secretoglobin and transferrin likely play clinically relevant roles in the pathophysiology of RAO, and may thus be used as biomarkers of the disease.     

Do active‐dispersing insects dominate the invertebrate fauna of rock pools in the wet–dry tropics,Kimberley, Australia?

1. Invertebrate assemblages of tropical freshwater rock pools (FRPs) are poorly known compared with temperate regions, where FRPs contain specialized, endemic taxa of high conservation value. Invertebrates were sampled from pristine FRPs in the Morgan River catchment in the Australian wet–dry tropics in the 2015 and 2016 wet seasons. Taxa were classified as either active or passive dispersers and by functional feeding groups. A range of physical variables were examined to identify associations with invertebrate assemblages.
2. Forty‐seven taxa were identified from FRPs; most were actively dispersing predatory insects, primarily Coleoptera and Hemiptera. Five adult insect species were found in most pools; other taxa were rare and scattered across FRPs. Two previously undescribed species of Spinicaudata were collected, but few crustacean taxa were recorded, and passively dispersing taxa were rare. Species accumulation curves indicated that had more pools been sampled, more taxa would have been collected in 2015, but in 2016 most species were collected.
3. There was no pattern in FRP assemblages related to any physical variable, but the assemblages in pools were correlated between sampling times. An opportunistic and mobile assemblage composed mostly of actively dispersing predatory insects may explain the absence of relationships with measured variables. Low hydrological stability in the wet–dry tropics may be associated with low taxon richness.
4. The dominance of actively dispersing insects is in contrast with FRP assemblages in temperate and arid regions. Studies of a wider range of FRPs from tropical regions are required to determine whether the fauna of these Kimberley FRPs are typical, and to fully describe short‐range endemic species across the Kimberley and elsewhere.
5. The FRPs have conservation value as habitat for endemic, specialist rock‐pool taxa, contributing to regional species pools. At present, a lack of knowledge of endemic FRP specialist species in tropical regions, including the Kimberley, impedes our ability to protect and conserve these species from disturbance.

     

Generation of mouse monoclonal antibodies specific to tilapia immunoglobulin using fish immunoglobulin/BSA complex for monitoring of the immune response in Nile tilapia Oreochromis niloticus

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.