Epitranscriptomic technologies and analyses

RNA can interact with RNA-binding proteins(RBPs), mRNA, or other non-coding RNAs(ncRNAs) to form complex regulatory networks. High-throughput CLIP-seq, degradome-seq, and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions. However, assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs) remains technically challenging. Chemical modifications to mRNA also play important roles in regulating gene expression. Investigation of the functional roles of these modifications relies highly on the detection methods used. RNA structure is also critical at nearly every step of the RNA life cycle. In this review, we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs. We also summarize methods used to detect RNA modifications and to probe RNA structure.     

Constitutive Gαi Coupling Activity of Very Large G Protein-coupled Receptor 1 (VLGR1) and Its Regulation by PDZD7 Protein

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gα_i coupling. Co-expression of the Gα_iq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gα_i protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gα_i coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gα_i signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome.     

Genetic diversity of five Kobresia species along the eastern Qinghai-Tibet plateau in China

Plants of the genus Kobresia are alpine grass species of high ecological and economic importance. Vegetative growth is the dominant means of reproduction for the Kobresia. Studies suggest that substantial vegetative growth can reduce genetic diversity and renders populations less able to buffer changing and extreme conditions. Kobresia are dominant species in the Qinghai-Tibet plateau in China where they face harsh conditions and frequent disturbance. The genetic diversity of five Kobresia species (K. humilis, K. royleana, K. kansuensis , K. tibetica and K. setchwanensis) from the Qinghai-Tibet plateau was assessed. The results reveal high genetic diversity at the population level for all of the species and there does not appear to be a relationship between altitude and genetic diversity. AMOVA analysis shows that most genetic variability resides among individuals within populations, whereas only a minor portion is found among populations. Of the five species, K. royleana and K. kansuensis have the highest levels of gene flow and the lowest genetic differentiation. While K. setchwanensis has the lowest gene flow and the greatest genetic differentiation. The level of gene flow between populations and the mating system play a critical role in the genetic structure of these Kobresia populations. Despite the predominance of vegetative growth enough sexual reproduction occurs to maintain the relatively high genetic diversity in Kobresia populations.     

草莓拟盘多毛孢叶斑病的病原菌

于2013年在北京市房山区发现了一种新的草莓叶部病害,为明确引起该病害的病原菌种类,采用组织分离法对病叶进行了病原菌的分离,经纯化得到一株病原菌CMF4。通过室内人工接种对该菌株的致病性和寄主范围进行测定,发现该菌株可以导致草莓叶片坏死和果实腐烂,可引起有伤供试植物牡丹、海棠、芍药、杏、樱桃、桃和月季发病,但不能无伤侵入,对人工接种发病的植株进行病原菌再分离可得到原接种病原菌。采用rDNA-ITS序列分析方法并结合该病原菌的形态特征进行鉴定,发现引起该病害的病原菌为棒孢拟盘多毛孢Pestalotiopsis clavispora。这是棒孢拟盘多毛孢所致草莓叶斑病在国内的首次报道。     

Energy Budget,Behavior and Leptin in Striped Hamsters Subjected to Food Restriction and Refeeding

Food restriction induces a loss of body mass that is often followed by rapid regaining of the lost weight when the restriction ends, consequently increasing a risk of development of obesity. To determine the physiological and behavioral mechanisms underlining the regaining, striped hamsters were restricted to 85% of initial food intake for 4 weeks and refed ad libitum for another 4 weeks. Changes in body mass, energy budget, activity, body composition and serum leptin level were measured. Body mass, body fat mass and serum leptin level significantly decreased in food-restricted hamsters, and increased when the restriction ended, showing a short “compensatory growth” rather than over-weight or obesity compared with ad libitum controls. During restriction, the time spent on activity increased significantly, which was opposite to the changes in serum leptin level. Food intake increased shortly during refeeding, which perhaps contributed to the rapid regaining of body mass. No correlation was observed between serum leptin and energy intake, while negative correlations were found in hamsters that were refed for 7 and 28 days. Exogenous leptin significantly decreased the time spent on activity during food restriction and attenuated the increase in food intake during refeeding. This suggests that low leptin in restricted animals may function as a starvation signal to induce an increase in activity behavior, and high leptin likely serves as a satiety signal to prevent activity during refeeding. Leptin may play a crucial role in controlling food intake when the restriction ends, and consequently preventing overweight.     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

不同种植行距的大穗型小麦品种'兰考矮早八'中几种与旗叶衰老有关的生理指标变化

在大田高产栽培条件下,检测不同种植行距的大穗型小麦品种‘兰考矮早八’开花后与旗叶衰老相关生理指标以及产量性状的结果表明,相对于传统行距为20cm而言,‘兰考矮早八’种植行距缩至15cm的花后旗叶中叶绿素降解减慢,丙二醛（MDA）含量下降,抗氧化系统酶活性增强,功能叶衰老减缓,穗粒重提高;行距缩至10cm时,尽管灌浆中后期过氧化物歧化酶（SOD）活性增强,但过氧化氢酶（CAT）和过氧化物酶（POD）活性下降,花后旗叶中叶绿素降解加快,MDA含量升高,植株衰老加快,穗粒重下降。