Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig

To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples.     

In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding β‐galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.     

鹅细小病毒NS2基因以及VP1与VP3非重叠序列基因的真核表达

采用pBlueBacHis2A系统筛选鹅细小病毒（GPV）的检测抗原，利用PCR扩增和双酶切方法分别构建了含有NS2基因和VPl与VP3非重复序列基因的重组质粒pBlueBacHis2A-GPV-NS2、pBlueBa-cHis2A-GPV-VP（1-3），分别转染sf9细胞，得到重组病毒，分别表达出了54ku的NS2融合蛋白和24ku的VP（1-3）融合蛋白。Western-blotting和Dot-ELISA检测结果证实，表达产物具有特异性；间接免疫荧光试验证实，重组蛋白在细胞中获得了表达。     

Selection on multiple QTL with control of gene diversity and inbreeding for long-term benefit

The purpose of this study was to develop and investigate selection strategies that aim at maximizing long-term genetic response while conserving gene diversity and controlling inbreeding in populations of limited effective size, assuming complete knowledge of all genes affecting a quantitative trait. Three selection strategies were proposed to select on 100 quantitative trait loci (QTL) and compared with truncation selection on breeding value. Alternative selection strategies aimed at maximizing the average breeding value of parents with a penalty on (1) the number of unfavourable QTL genotypes among parents (OS-I), (2) the negative of the logarithm of the frequency of the favourable allele at each QTL among parents (OS-II), and (3) the average pedigree relationship among parents (OS-III). When all QTL and their effects were known, the strategies examined were able to obtain extra long-term responses, conserve QTL diversity and reduce inbreeding, compared with truncation selection. Strategy OS-II was the most effective in conserving QTL diversity and OS-III in reducing inbreeding. By changing the magnitude of the penalties applied, the impact on long-term response, inbreeding and diversity can be controlled. Extra long-term responses over truncation selection of OS-I and OS-II were even greater when effects of QTL were estimated rather than assumed known, indicating the applicability of results to practical strategies for marker-assisted selection. Extra responses are expected to be reduced for larger population sizes.     

杧果PEPC基因家族全基因组鉴定及表达分析

PEPC家族蛋白在植物光合碳同化过程中起到重要作用,同时具有多种非光合生物学功能,目前杧果中尚未报道。本研究对杧果MiPEPCs进行了全基因组鉴定,并分析了它们在不同组织（包括成熟叶片、成熟树皮、种子、根、花、果肉和果皮）和两个品种（高糖品种‘台农1号’和低糖品种‘热农1号’）中的表达情况。结果获得4个杧果MiPEPC基因家族成员,其理化特征较为相似,且所有MiPEPCs蛋白亚细胞定位预测均存在与细胞质中。基因组定位发现杧果MiPEPCs分布于4条不同的染色体。联合拟南芥,水稻,菠萝的PEPCs进行系统进化分析显示可分为2个亚组,MiPEPCs分布在第I（PTPC）和II亚族（BTPC）。杧果植物型MiPEPCs和细菌型MiPEPCs分离时间早于单子叶植物和双子叶植物的分离时间,两者蛋白序列差异大,但仍具有一定的保守性。顺式作用元件分析显示,MiPEPCs含有数量较多的光响应元件和激素响应元件。转录组表达分析结合qPCR实验验证发现,MiPEPCs在不同杧果组织中的表达具有偏好性,可能参与杧果的多个生物学过程（包含光合和非光合过程）,并初步推测MiPEPC1、MiPEPC3可能与杧果果实成熟过程。本研究为进一步探究MiPEPC家族在杧果中的生物学功能奠定了基础。     

大肠杆菌O157:H7 rfbE基因和fliC基因的克隆与序列分析

根据已发表的E．coli O157：H7 EDL933株的rfbE基因和fliC基因，设计了两对引物，分别对两个O157：H7，菌株的rfbE基因和一个菌株的fliC基因进行PCR，并将PCR产物克隆于pMD18-T栽体质粒。测序结果表明，获得到了与理论相符的582bp rfbE基因片段和802bp fliC基因片段。一株菌的rfbE基因存在无意义突变（两个）。而另一株菌fliC基因有一个有意义突变（aac→gac）     

绵羊FecB基因打靶载体构建

利用peGFP-C1,pRET.IL.PGK-TK和pMD19-T Simple等基本质粒,构建绵羊FecB基因打靶载体。以克隆载体pMD19-T Simple为载体骨架,插入一个正选择标记eGFP基因、一个负选择标记基因HSV-tk基因,并在eGFP基因两侧各添加一个同源长臂和同源短臂,从而构建绵羊FecB基因打靶载体。结果:经多个限制性内切核酸酶酶切鉴定和测序证实,所构建的基因打靶载体符合设计要求,表明成功构建了绵羊FecB基因打靶载体。     

Dmrt7基因重组腺病毒载体的构建及鉴定

为了研究Dmrt7基因与GFP基因复制缺陷型腺病毒载体的构建,试验将购得的pReceiver-M01-Dmrt7质粒酶切、纯化后,与IRES-EGFP片段共同插到pShuttle-CMV载体中,经测序验证后,利用Adeasy-1系统细菌内同源重组原理构建Dmrt7基因的复制缺陷型腺病毒载体Ad-Dmrt7,再经PmeⅠ酶切线性化,转染HEK293细胞,观察GFP表达并检测重组腺病毒的效价。结果表明:重组腺病毒载体Ad-Dmrt7经PacⅠ酶切得到23 kb和2.9 kb 2个特异性片段,表明同源重组成功;将此种重组腺病毒经脂质体介导转染293细胞24 h后,在荧光显微镜下可观察到绿色荧光,表明Dmrt7重组腺病毒包装成功,效价为0.95×108pfu/mL。     

Potential sources of neck and back pain in clinical conditions of dogs and cats: a review

Pathological neck and back pain occurs in many medical conditions of dogs and cats. Pain may arise from a variety of structures including the intervertebral discs, facet joint capsules, dorsal root ganglia, vertebral ligaments, the vertebral periosteum, and the meninges. The source of this pain is dependent upon the type of disease process and its location within or surrounding the spinal column. Diseases can directly or indirectly stimulate pain sensors (nociceptors). Inflammatory diseases may hypersensitize these receptors or nociceptive pathways with inflammatory mediating substances such as serotonin, histamine and potassium. Diseases resulting in mechanical compression of nociceptors or nociceptive pathways may also result in neck or back pain. A thorough understanding of spinal pain occurring in dogs and cats will lead to more accurate diagnoses and treatments and may provide information regarding prognoses for various diseases. Evidence pointing to sources of spinal pain taken from scientific and clinical studies of a variety of species including humans is provided. Suspected or known sources of neck and back pain occurring in several clinical conditions of dogs and cats are discussed.     

猪流感病毒H1N1亚型济南株HA、NA、NP基因的克隆与序列分析

根据GenBank中流感病毒H1N1亚型HA、NA、NP基因序列,分别设计扩增HA、NA、NP基因的特异性引物,用RT-PCR方法扩增猪流感病毒(SIV)H1N1济南株(SW/SD/JT/07)HA、NA、NP基因,分别将RT-PCR产物克隆入pMD18-T载体,进行测序分析。结果显示,SW/SD/JT/07(H1N1)HA基因长为1 701bp,编码566个氨基酸;HA蛋白切割位点为IPSIQSR↓G,属于非高致病性毒株;HA蛋白有8个糖基化位点,其中有6个在HA1蛋白上,有2个在HA2蛋白上,与个别参考毒株糖基化位点个数不同;HA蛋白上的受体结合位点与多数参考毒株一致。SW/SD/JT/07(H1N1)NA基因长为1 410bp,编码469个氨基酸;NA基因颈部未发现氨基酸缺失现象;NA蛋白的头部有5个可能的抗原位点Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ分别由5个区段组成,在340、346、366位分别为P、I、N,其余参考毒株在相应位置的氨基酸分别为S、V、S;NA蛋白共有8个糖基化位点,与个别参考毒株的糖基化个数不同。SW/SD/JT/07(H1N1)NP基因长为1 565bp,编码498个氨基酸,根据HA、NA、NP基...     

FOXL2基因重组腺病毒载体的构建及鉴定

为了构建叉头框L2(Forkhead box L2,FOXL2)基因与绿色荧光蛋白(GFP)基因复制缺陷型腺病毒载体,试验将克隆的FOXL2基因与IRES-GFP片段通过酶切、纯化等方法共同连接到pShuttle-CMV载体中,再与pAdEasy-1腺病毒质粒在BJ5183大肠杆菌中进行同源重组,得到复制缺陷型AD-FOXL2腺病毒载体,再用PacⅠ酶线性化后转染HEK293细胞,观察绿色荧光表达,并测量病毒效价。结果表明:将AD-FOXL2腺病毒载体用PacⅠ酶线性化后,得到1个大于23 kb的大片段和1个4.5 kb的特异性小片段,证明同源重组成功;将所得的腺病毒载体转染HEK293细胞,可以观察到GFP的表达,证明包装成功,并测得病毒效价为1×10-8.61/0.1 mLTCID50。     

奶山羊BLG基因敲除载体的构建

根据已知的BLG序列设计引物,通过PCR技术克隆同源臂序列,5′端同源臂2 264bp,包括外显子1,3′端同源臂4 461bp,包括全部的外显子3、4、5、6、7,分别连入克隆载体pMD18-T Simple载体中并测序。然后以含有正负筛选标记基因的ploxpⅡ载体为基础,将5′端和3′端同源臂片段先后连入其中,进行酶切、PCR鉴定。将构建好的基因敲除载体转化组成型表达Cre重组酶的大肠杆菌BM25.8,验证Loxp位点的活性。结果表明,构建了奶山羊BLG基因第2外显子缺失的基因敲除载体pBLG2T,且pBLG2T载体中的正选neo基因可以被Cre重组酶去除。为获得BLG 1条等位基因缺失型细胞株以及培育高产优质奶山羊新品种奠定基础。     

CRISPR/Cas9系统:基因组定点编辑技术及其在植物基因功能研究中的应用

CRISPR/Cas系统是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御系统,可用来对抗入侵的病毒及外源DNA。相比锌指核酸酶(ZFNs)和TALE核酸酶(TALENs),基于RNA指导的Ⅱ型CRISPR/Cas9系统为基因组定点编辑开辟了一条新的道路,在基因功能研究中具有效率高、成本低、易于操作等显著优点。本文从CRISPR/Cas9系统的基本结构、作用机制及其在植物基因功能研究和作物遗传育种中的应用等方面进行了简述,最后对该系统的发展前景进行了展望。     

军牧1号白猪肉质主效基因的多态性检测分析

猪的肉质属于数量性状,受多个主效基因和候选基因的控制。为检测军牧1号白猪肉质主效基因多态性,本试验采用RFLP技术检测了400头军牧1号白猪氟烷基因(RYR1)、心脏脂肪酸结合蛋白基因(H-FABP)和酸肉基因(RN)3个重要肉质基因的多态性。群体遗传结构分析表明,军牧1号白猪群体中RYR1基因的N基因频率为0.908 3,n基因频率为0.091 7;心脏脂肪酸结合蛋白基因的5′区的HinfⅠ位点的H基因频率为0.688 8,h基因频率为0.311 2;H-FABP基因内含子-2HaeⅢ位点D基因频率为0.478 8,d基因频率为0.521 2;H-FABP基因内含子-2MSPⅠ位点不存在多态性;RN基因也不存在多态性。     

猫细小病毒NS部分基因的克隆及序列分析

将疫苗用猫细小病毒(FPV)株提取基因组DNA,针对NS特定片段设计引物,利用PCR扩增出了部分基因并将该基因克隆到pMD18-T Vector中测序.结果表明,该基因长613bp,编码204个氨基酸.分离株基因与犬的细小病毒(CPV)、貂的细小病毒(MEV)毒株核苷酸同源性均为99%.氨基酸同源性达到97%、98%以上,与犬、貂的肠炎病毒有密切的亲缘关系.     

Radiography of the distal extremity of the manus in the donkey foal: Normal images and quantitative characterization from birth to 2 years of age: A pilot study

This study describes a radiographic survey of the anatomical development of the distal extremity of the manus in the donkey from 0 to 2 years of age. The right distal limb of 10 donkey foals, born in the spring of 2012, underwent radiographs every month for the first 6 months of age and every 3 months during the following 18 months. Latero‐medial radiographs with and without barium marker at the coronary band and dorso‐palmar radiographs with both front feet in weight bearing were obtained. The distal physis of the third metacarpal bone and the proximal physis of the proximal phalanx (phalanx proximalis) were closed at the mean age of 18.6 months. The distal physis of the proximal phalanx appeared as a clear radiolucent line at 2 weeks of age and was still subtly visible in some donkeys at 24 months. The proximal physis of the middle phalanx (phalanx media) was closed at the mean age of 16.7 months. The distal physis of this phalanx was visible at birth, but closed at 4 days. The distal phalanx (phalanx distalis) was triangular at birth. At the age of 20–21 months, the palmar processes (processus palmares) were both developed. The navicular bone (os sesamoideum distalis) was developed at the mean age of 9 months. The proximal sesamoid bones (ossa sesamoidea proximalia) were seen in continuously development during the 24 months. It seems that the physes in the distal extremity of the manus in the donkey close at an older age than the physes in the horse.