High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Quantifying the concentration and purity of a target protein is essential for high‐throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time‐consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p‐coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co‐expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ～1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high‐throughput protein expression screening.     

Using independent nest survey data to validate changes in reporting rates of Martial Eagles between the Southern African Bird Atlas Project 1 and 2

Repeat monitoring is vital to measure biodiversity change. However, monitoring protocols may change, as survey techniques improve or different questions are asked. Such modifications may cause difficulties when examining changes in wildlife populations. The Southern African Bird Atlas Project (SABAP) 1 and 2 are repeat national bird surveys undertaken 20 years apart. These surveys therefore offer unrivalled potential to examine bird population changes in an African context. However, changes in protocols, both spatially and temporally, between the two surveys have raised concerns over using these data to infer population changes. In this study we use independ- ent nest survey data to test whether changes in reporting rates of Martial Eagles in the Kalahari Gemsbok National Park between the two SABAP surveys were reflected in real change in numbers of nesting pairs. From 11 quarter degree squares (QDS), covering c. 8 000 km2, both SABAP and nest surveys suggested a near identical 44% decline. Levels of agreement were weaker at the individual QDS scale, although in 67% of cases the direction of change was the same using both surveys. These results suggest that comparisons in the reporting rates between SABAP 1 and SABAP 2 accurately reflect changes in the breeding population size of this species.     

The quality of flower-based ecosystem services in field margins and road verges from human and insect pollinator perspectives

We contrasted traditionally used indicators of service provision quality, such as overall species richness and growth form composition, to three more specific functional properties: functional diversity, functional intensity, and functional stability. We defined flower colour as a functional trait perceived differently by humans and insect pollinators, and used user specific colour richness, flower size, and species richness within colour group as indicators of these three properties. We asked (1) do field margins and road verges provide flower-based ecosystem services with the quality of permanent grasslands; and (2) do traditional and detailed functional indicators of service provision quality agree on the service quality ranking of habitats?In an agricultural landscape of central and south-eastern Estonia (115 ÿ 95 km area, centroid 26°49⿲43⿳ and 58°54⿲49⿳) we sampled 87 field margins and 111 road verges as linear grassland-substitution habitats, and 84 permanent grasslands to scale their service quality.Linear habitats generally provided service of lower quality than permanent grasslands, but detailed indicators showed less evident contrast among habitat types than the overall species richness and stronger contrast than the proportion of forbs. Detailed indices, however, had strong seasonal dynamics to take into account. Vegetation in the first year field margins had greater colour richness (functional diversity) and species richness within colour groups (functional stability), but the smallest flower size (functional intensity), in contrast to road verges. By the third year of succession, field margins had become more similar to road verges. Indication of service provision quality differed between humans and pollinators, but their estimates were correlated across habitats.We showed that (1) combinations of specific service quality indicators provide more adequate information than generalized richness or growth form system, and (2) single grassland surrogate habitat type is an insufficient service providing substitute for permanent grasslands, although a mosaic of these habitats might be more efficient. Therefore, remnant fragments of semi-natural grasslands should receive top priority attention for conservation and restoration, particularly in agriculture dominated landscapes.     

MiR-137 affects melanin synthesis in mouse melanocyte by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway

Previously, we created miR-137 overexpressing transgenic mice that produced lighten color phenotypes including gray mice phenotype. However, the miR-137 functional role in coat color regulation is still not well understood. In this study, the quantity of melanin granule and the relative expression of TYRP2 in gray miR-137 overexpression transgenic mouse skin were significantly lower than that in C57BL/6J black mouse skin. The mRNA and protein expression level of c-Kit and c-Kit downstream gene Tyrp2 in miR-137 expression plasmid-transfected melanocytes were significantly down-regulated comparing with that of the control melanocytes. In melanocytes, miR-137 overexpression could decrease the enhanced expression of c-Kit and Tyrp2 and the increased melanin production caused by UV treatment. The target relationship of miR-137 and c-Kit was identified by luciferase assay. The results suggest that miR-137 could inhibit melanogenesis in mouse skin melanocytes by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway.     

金叶银杏半同胞子代无性系的叶色和色素含量变化及呈色机制分析

对4月份至11月份金叶银杏‘万年金'( Ginkgo biloba ‘Wannianjin')32个半同胞子代无性系与亲本的叶色差异进行比较;比较了不同色系叶片的色素含量和比值及叶色参数(L*、a*和b*)的变化,分析了叶色参数与叶片色素含量的相关性;并观察了不同色系的叶绿体超微结构。结果表明：32个半同胞子代无性系可被分为金黄、浅黄、草绿和蓝绿4个色系。随时间推移,草绿和蓝绿色系叶片的总叶绿素( Chl)、叶绿素a( Chla)、叶绿素b( Chlb)和类胡萝卜素( Car)含量均呈“双峰型”变化趋势,Car/Chl和Car/Chla比值的变幅均较小;而金黄和浅黄色系叶片的上述色素含量呈“升高—降低—升高”变化趋势,Car/Chl和Car/Chla比值总体呈“迅速下降—相对稳定—缓慢升高”的变化趋势。各色系叶片的上述色素含量在夏季均不同程度下降,Car/Chlb比值变化差异较大,且金黄和浅黄色系的各色素含量均低于草绿和蓝绿色系。随时间推移,金黄和浅黄色系叶片的L*、a*和b*值以及草绿和蓝绿色系叶片的L*和b*值均先降低后升高,后2个色系的a*值则先升高后下降;并且,前2个色系的L*和b*值总体上显著高于后2个色系,而a*值则总体上低于后2个色系。金黄色系的Chla和Chl含量与L*和a*值显著负相关,而其Car/Chl和Car/Chla比值则与L*、a*和b*值显著或极显著正相关;浅黄色系的Chlb含量与a*值显著负相关,其Car/Chla比值与L*和b*值以及Car/Chlb比值与a*值均显著正相关;草绿色系的Chla含量与L*值显著负相关,其Car/Chla比值与L*和b*值以及Car/Chlb比值与a*值均显著正相关;这3个色系叶片的其余指标间以及蓝绿色系叶片的各指标间均无显著相关性。观察结果显示：金黄和浅黄色系的叶绿体基粒片层发育不健全,基粒片层可见但排列较疏松,且无明显垛叠,分布范围小而稀疏;蓝绿和草绿色系叶绿体的基粒类囊体垛叠层数均较多,基粒片层发达且排列紧致、整齐,分布范围大而稠密。综合分析结果表明：‘万年金'4个色系半同胞子代无性系叶片的呈色差异和叶色变化由多种因素控制,其中,Car/Chl和Car/Chla比值高且叶绿体基粒片层发育不健全是叶片呈黄色的主要原因。     

3种卫矛属植物叶片秋冬季转色期生理生化特征研究

以疏花卫矛(Euonymus laxiflorus)、丝棉木(E.maackii)和卫矛(E.alatus)3种卫矛属植物为试验材料,测定叶片转色期叶色参数和相关生理生化指标,探讨各项指标的变化规律和内在联系,为优良色叶植物的筛选提供依据。结果表明:(1)转色期,疏花卫矛的明亮度参数L~*和色素参数b~*(黄/蓝)呈上升趋势,色素参数a~*(红/绿)变化不大;丝棉木的L~*和b~*呈先上升后下降的单峰曲线,a~*呈上升趋势;卫矛的L~*和b~*变化不大,a~*呈上升趋势。(2)3种植物的叶绿素含量在转色期呈明显下降趋势;疏花卫矛的花色素苷相对含量、花色素苷/叶绿素的值较为平稳,类胡萝卜素/叶绿素的值呈上升趋势;丝棉木和卫矛的花色素苷相对含量、花色素苷/叶绿素的值均呈上升趋势,类胡萝卜素/叶绿素的值保持平稳。(3)3种植物苯丙氨酸解氨酶(PAL)活性呈下降趋势,查尔酮异构酶(CHI)活性呈先上升后下降的单峰曲线;卫矛的过氧化物酶(POD)和多酚氧化酶(PPO)活性保持平稳,疏花卫矛和丝棉木的POD活性呈上升趋势,疏花卫矛的PPO活性保持平稳,丝棉木的PPO活性呈下降趋势。(4)疏花卫矛和卫矛的可溶性糖和淀粉的质量分数呈先上升后下降的单峰曲线,丝棉木的可溶性糖和淀粉的质量分数呈上升趋势。(5)相关性分析显示,疏花卫矛呈现黄色主要是因为叶绿素的分解,丝棉木和卫矛呈现红色是因为花色素苷的合成,可溶性糖、淀粉、CHI对花色素苷的合成有一定作用,POD对叶片呈现红色有促进作用,PAL和PPO活性对花色素苷的合成无显著影响。