Selective Fractionation of Maize Sperm Cells by RCA I Sepharose 6MB Affinity Chromatography

RCA I-Sepharose 6MB was employed for affinity chromatography of maize ( Zea mays L. ) sperm cells. The result indicates that the loading capacity of 0.1 mL RCA I-6MB is about 1.5 × 10 . If the amount of sperm cells is less than this capacity, about 70% of the sperm cells specifically bind to RCA I-6MB. 42.1% of the absorbed sperm cells can be released from RCA I-6MB in 0.2 mol/L galactose and recovered with less impurities and with some viability, which will facilitate further study on plant sperm cells. It remains to be elucidated whether there are two types of maize sperm cells different from each other in monosaccharide residues on their plasma membranes.     

The glycoprotein hGC-1 binds to cadherin and lectins

Human granulocyte colony stimulating factor stimulated clone-1 (hGC-1, also known as GW112, OLM4, and hOlfD) is an olfactomedin-related glycoprotein of unknown function. We performed a series of biochemical studies to characterize its function. Using hGC-1 purified from baculovirus Sf9 cells we demonstrated that hGC-1 is a secreted glycoprotein containing N-linked carbohydrate chains and forms disulfide-bonded multimers. It binds to cell surfaces and to the locutions ricinus communis agglutinin I, concanavalin A and wheat germ agglutinin. Purified hGC-1 enhanced NIH3T3 and 293T/17 cell spreading and attachment, as did hGC-1-enriched culture supernatants of 293T/17 cells transfected with an hGC-1 expression vector. Coimmunoprecipitation studies demonstrated that hGC-1 interacts with cadherin in 293T/17 cells. This interaction depends on the C-terminal olfactomedin domain, but does not require the five well-conserved cysteine residues. However, cysteine residues at 83, 85, 246 and 437 are essential for secretion, and cysteine 226 is critical for hGC-1 multimer formation. Our studies demonstrated that hGC-1, an extracellular matrix glycoprotein, facilitates cell adhesion. Its potential interaction with endogenous cell surface lectins and cadherin may mediate this function.     

稻瘟病抗病基因Pi15的精细定位

稻瘟病抗病基因Pi15曾被作者鉴定为与已知抗病基因Pii具有连锁关系,但是,Pii基因究竟位于染色体6还是9上存在争议.为了确定Pi15基因的染色体位置,利用分子标记在由15个抗病个体和141个感病个体组成的F2群体中,通过混合群体分离法(BSA)与隐性群体分析法(RCA)相结合的手段,对目标基因进行了连锁分析.首先,从染色体6和9分别选择10个微卫星标记进行了分析,结果表明,只有位于染色体9的RM316与目标基因连锁,重组率为(19.1±3.7)%.为了进一步确定这种连锁关系,从染色体9选择了4个序列标定位点(STS)标记进行分析,结果表明,只有G103与目标基因连锁,重组率为(5.7±2.1)%.为了获得与目标基因更加紧密连锁的分子标记,对目标基因进行了RAPD)分析.在筛选、分析了1 000个随机引物之后,从中获得了3个目标基因紧密连锁的分子标记BAPi15486、BAPi15782、BAPi15844.它们与目标基因的重组率分别为0.35%、0.35%和1.1%.这些紧密连锁的分子标记可作为分子标记辅助基因聚合和克隆的出发点.     

Helper virus-free gutless adenovirus (HF-GLAd): a new platform for gene therapy

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replication-competent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases.     

Root cortical aerenchyma,characteristics and nutrient use efficiency of different maize hybrids during drought and rehydration process

The importance of cortical aerenchyma in flood and drought resistance is well established. However, effects of the combination of the two factors remain unclear. We tested the hypothesis that under conditions of substantial water alteration, enhanced root cortical aerenchymas (RCAs) could not only exert a positive effect on root functions under drought periods but also influence nitrogen capture, soil exploration and compensation growth after rehydration. Three maize varieties, Yurui2 (YR2), Yunrui6 (YR6) and Yunrui167 (YR167), were grown under rainfed field conditions, and a subset was grown initially under well-watered and extreme-drought conditions, followed by the same simulated rainfed field conditions, in order to compare the effects on the RCAs during the growing season. Root morphology and function parameters were measured, and variation in the RCAs was induced by water stress. Under extreme drought, we found substantial variation in the RCAs (from 0% to 37%) and drought tolerance performance. During the rehydration stage, different RCA components exerted different effects on maize, recovery parameters and plant N use efficiency. YR6 showed resilient growth, whereas recovery was inhibited in YR167 and YR2, respectively, which possessed the lowest and highest RCA area percentages. The results support the hypothesis that RCA area percentage under drought periods are important for maize recovery after post-drought rehydration.     

Gluten,a lectin with oligomannosyl specificity and the causative agent of gluten-sensitive enteropathy

The pathogenesis of gluten-sensitive enteropathy or coeliac disease is as yet unknown. We can demonstrate by laser nephelometric measurements that gluten has lectin-like properties. Gluten binds ‘high-mannose type’ glycoproteins and the complex formation is inhibitable by mannan. As known for other lectins the reaction is absolutely Ca-dependent. Glycoproteins from the immature crypt cells from the small intestine are highly more reactive than glycoproteins from the mature villous zone. The possibility of a genetically determined deficiency of the growth-dependent N-acetyl-glucosaminyltransferase-1 as the pathogenic factor of the gluten-sensitive enteropathy is discussed.     

利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年，RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中，鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性，进一步提高家族miRNA鉴别的灵敏度，滚环扩增的程度用相对荧光强度来表示。研究结果显示，T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率，从而提高RCA的检测特异性。本文利用优化的RCA技术，实现对let 7高度同源的家族miRNAs高灵敏度的鉴别，灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550，而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol，与本研究相比，灵敏度低了近1 000倍。本研究的结果表明，利用RCA技术鉴别高度同源性miRNAs是高效灵敏的，此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。