Sex identification and genetic variation of Saccharina (Phaeophyta) gametophytes as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) markers were utilized to investigate the genetic variation between male and female gametophyte populations of strains Rongfu and 901 of Saccharina. In total, 11 ISSR primers were able to generate 135 satisfactory and reproducible loci, of which 134 were polymorphic with 99.26 % polymorphism. The percentages of polymorphism of female gametophyte populations (60 and 62 % for their respective strains) were higher than those of the males (53 %), and the Nei’s genetic diversity and Shannon’s information index showed a similar tendency. The clustering of gametophytes of the same sex from each strain was well resolved by both an unweighted paired group method using the arithmetic average and a principal component analysis, suggesting that any male/female gametophyte pair could represent each strain. However, a single pair was not adequate for germplasm maintenance because the genetic variance among individuals within a population accounted for 57.45 % of the total (P?<?0.0001), as shown by the analysis of molecular variance. The gametophyte sex could be identified by amplification with primer UBC809 because of a differential band present in the females. According to the sequence of this band, a pair of ISSR-derived sequence-characterized amplified region (SCAR) primers was designed. With the primers, one female-specific fragment was detected using PCR and Southern blot hybridization. This converted SCAR marker was localized on one unique chromosome of the female gametophytes of these two strains by use of fluorescence in situ hybridization, confirming that it was a female chromosome-specific marker.     

Roseomonas alkaliterrae sp. nov., isolated from an alkali geothermal soil sample in Tengchong,Yunnan, south-west China

An alkalitolerant, thermotolerant and Gram-stain negative bacterium, designated strain YIM 78007^T, was isolated from an alkaline geothermal soil sample from Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of strain YIM 78007^T were observed to be aerobic and short rod-shaped. The colonies were observed to be orange-red, convex and circular. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 78007^T clustered with members of the genus Roseomonas (with similarities from 97.2 to 92.2 %). Optimal growth of strain YIM 78007 occurs at 40–50 °C and pH 8.0–10.0. The predominant ubiquinone was identified as Q-10 and the major fatty acids were identified as C_18:1 ω7c and C_16:0. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified aminolipids and one unknown phospholipid. The G + C content of the genomic DNA was determined to be 63 mol %. The levels of DNA–DNA hybridization relatedness between strain YIM 78007^T and its closet neighbours (Roseomonas lacus JCM 13283^T and Roseomonas terrae JCM 14592^T) were well below the threshold required for the proposal of a novel species. The results of physiological and biochemical characteristics, the phylogenetic analysis, as well as low DNA–DNA hybridization values, allowed the phenotypic and genotypic differentiation of strain YIM 78007^T from its closest phylogenetic neighbours. Therefore, strain YIM 78007^T is considered to represent a novel species of the genus Roseomonas, for which the name Roseomonas alkaliterrae sp. nov. is proposed. The type strain is YIM 78007^T (=BCRC 80644^T = JCM 19656^T).     

Multichannel,Line-Monitoring Sensing Approach Based on Long-Range Surface Plasmons

The refractive index resolution of a surface plasmon resonance (SPR) sensor has been significantly improved these years; however, higher sensing performance is always desired. In this work, we propose a line-monitoring, long-range SPR sensor whose resolution is much better than conventional SPR sensors. Also, in contrast to mono-channel detection, multichannel detection, using line-monitoring technique, can detect multiple channels concurrently. In this way, this system achieves a refractive index resolution of 4.0?×?10^??7 refractive index units and can monitor multiple molecular interactions simultaneously. Finally, a model experiment detecting the Escherichia coli bacteria has demonstrated the potential for biomedical applications of this system.     

A new species of Lysimachia (Myrsinaceae) from Dabieshan Mountain,China

A new species of Myrsinaceae, Lysimachia jinzhaiensis S.B. Zhou & K. Liu, is described and illustrated from Dabieshan mountain, Anhui, China. It is similar to L. christiniae in the prostrate stem, opposite leaves, yellow flowers born singly in leaf axils, but differs by being glabrous throughout or glandular on young parts, and having quadrangular stem, corolla with densely transparent glandular, orange-red corolla base with lobes being significantly overlapping and contorting to left in bud. Moreover, L. jinzhaiensis have a different karyotype, formulated as 2n = 2m + 2sm + 10st (2SAT) + 10t.     

新城疫病毒非编码序列的遗传演化趋势

【目的】探讨新城疫病毒全基因序列中非编码序列的分子演化规律。【方法】结合本研究室2012年自产蛋下降鸭群中分离测序的一株鸭源新城疫病毒全序列,从GenBank下载35株不同基因型新城疫病毒全长cDNA序列,获取非编码序列,分别绘制引导序列、尾随序列、F-HN及HN-L基因间隔序列(IGS)的遗传进化树,比较编码基因内5’及3’UTR序列核苷酸序列替代特点。【结果】非编码序列的长度及位置高度保守,而其核苷酸基因序列在不断发生变异,且变异趋势与编码基因序列相一致。【结论】新城疫病毒在整个基因组上编码和非编码序列同步发生变异。     

Photoacclimation characteristics of Sargassum thunbergii germlings under different light intensities

The photosynthesis and growth responses of Sargassum thunbergii germlings to different light intensities (10, 60, and 300 μmol photons m^?2 s^?1) were investigated. Maximum photochemical efficiency (F _v/F _m), rapid light curves (RLCs), and photochemical and non-photochemical quenching (qP and NPQ) were estimated by a pulse amplitude-modulated fluorometer. The photosynthesis of S. thunbergii germlings exhibited different properties to optimize light capture and utilization. The excitation pressure (1???qP) was rapidly increased to approximately 0.27 showing that germlings responded to high light by chronic photoinhibition with an accumulation of closed reaction centers, which ultimately resulted in a slow growth. This was accompanied by a reduced F _v/F _m with time and a development of high capacity for NPQ. Although F _v/F _m in moderate-light germlings did not fully recover overnight, germlings demonstrated a less severe chronic photoinhibition considering the reduced degree of excitation pressure accumulation of approximately 0.15. The relative stability of photosynthetic capacity (rETR_max, E _k, and α) could endow germlings with the highest relative growth rate (RGR) of approximately 9.3 % day^?1 in moderate light. By contrast, low-light germlings demonstrated high F _v/F _m and F _o, corresponding high α collectively suggested greater efficiency of light absorption and energy transformation. Sustained increases in electron transport capacity (rETR_max and E _k) occurred in low-light germlings, which resulted in a stable RGR of over 8.2 % day^?1. Consequently, S. thunbergii germlings are considered to prefer low light regimes and have a relative capacity of moderate and high light tolerance. However, the light acclimation to oversaturating conditions is at the cost of slow growth to maintain survival.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

丙型肝炎病毒核心蛋白稳定表达肝癌细胞株的构建及生物学研究

旨在构建稳定表达HCV核心蛋白的稳定细胞系Huh7-Core并进行初步的生物学功能研究.利用PCR技术扩增HCV核心蛋白基因,通过酶切连接反应将目的基因克隆至载体pSEB-3Flag中,将重组质粒pSEB-3F-Core和辅助质粒pAmpho共转染Huh7细胞,经过Blasticidine抗性筛选,建立稳定表达HCV核心蛋白的肝癌细胞系Huh7-Core.采用RT-PCR、Western blot鉴定Huh7-Core细胞株中核心蛋白的稳定表达并采用MTS、结晶紫试验观察Huh7-Core稳定细胞株的增殖情况.结果显示,成功构建了表达HCV核心蛋白的稳定细胞株Huh7-Core.结晶紫、MTS试验证实Huh7-Core细胞较Huh7-3Flag细胞增殖速度增快,表达HCV核心蛋白的Huh7-Core稳定细胞株构建成功,Core稳定表达后可促进Huh7细胞生长速度.