Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

水稻种子贮藏10年后的活力相关基因定位

以2004年构建并保存在种质库10年的186个单株组成的湘743/Katy F2:3群体为材料,在发芽的第5天和第9天统计亲本和各株系的发芽率和成苗率,应用由129个标记组成的连锁图谱检测与种子活力相关的QTL,一共检测到12个QTLs,共分布在6条染色体的6个区间,单个QTLs对群体性状表型变异的贡献率为5.73%~47.53%,联合贡献率都是50%。其中,在第8染色体RM152~RM310区间检测到一个主效的QTL,对第5天发芽率和第9天发芽率和第9天成苗率的贡献率分别为12.02%、47.53%、38.64%,来自于湘743的基因增加发芽率和成苗率;在第9染色体RM444~RM219区间检测到一个稳定表达的QTL,对第5天发芽率和第9天发芽率和第9天成苗率的贡献率分别为8.85%、7.49%、10.36%,来自于Katy的基因增加发芽率和成苗率;此外,没有检测到显著的上位性互作。     

RNA在离体小鼠肝脏组织中的稳定性

采取肝脏等临床样本时,常因不能及时保存造成核酸降解,从而影响后续实验的进行.本研究旨在通过对室温条件下放置不同时间的小鼠肝脏组织的RNA的完整性进行评价,为肝脏临床样本的采集与保存提供依据.将离体小鼠肝脏组织在室温下放置0～180min后,提取总RNA,采用电泳、RT-PCR和芯片生物分析仪检测RNA的完整性.电泳结果显示,将小鼠肝脏置于室温180min后,RNA尚未发生降解.对β肌动蛋白,GAPDH和Trp53的RT-PCR的分析表明,室温下保存180min的RNA样品均未降解.生物分析仪检测的RNA完整性系数(RIN)都大于7.9,样品可用于后续研究.因此,离体后的小鼠肝脏置于室温下3h以内,RNA仍能保持其完整性.     

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down‐regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9‐hydroperoxyoctadecadienoic acid (9‐HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild‐type seeds by measuring the ultraviolet absorption of 9‐HPOD at 234 nm and by high‐performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS‐91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.     

Ecological longevity of Polaskia chende (Cactaceae) seeds in the soil seed bank,seedling emergence and survival

• Soil seed banks are essential elements of plant population dynamics, enabling species to maintain genetic variability, withstand periods of adversity and persist over time, including for cactus species. However knowledge of the soil seed bank in cacti is scanty. In this study, over a 5‐year period we studied the seed bank dynamics, seedling emergence and nurse plant facilitation of Polaskia chende, an endemic columnar cactus of central Mexico.
• P. chende seeds were collected for a wild population in Puebla, Mexico. Freshly collected seeds were sown at 25 °C and 12‐h photoperiod under white light, far‐red light and darkness. The collected seeds were divided in two lots, the first was stored in the laboratory and the second was use to bury seeds in open areas and beneath a shrub canopy. Seeds were exhumed periodically over 5 years. At the same time seeds were sown in open areas and beneath shrub canopies; seedling emergence and survival were recorded over different periods of time for 5 years.
• The species forms long‐term persistent soil seed banks. The timing of seedling emergence via germination in the field was regulated by interaction between light, temperature and soil moisture. Seeds entered secondary dormancy at specific times according to the expression of environmental factors, demonstrating irregular dormancy cycling.
• Seedling survival of P. chende was improved under Acacia constricta nurse plants. Finally, plant facilitation affected the soil seed bank dynamics as it promoted the formation of a soil seed bank, but not its persistence.

     

Comparative longevity of Australian orchid (Orchidaceae) seeds under experimental and low temperature storage conditions

Seeds from ten terrestrial orchid species, nine from the south‐west Australian biodiversity hotspot (Caladenia arenicola, Caladenia flava, Caladenia huegelii, Diuris laxiflora, Microtis media ssp. media, Pterostylis recurva, Pterostylis sanguinea, Thelymitra crinita and Thelymitra macrophylla) and one from south‐east Australia (Diuris fragrantissima), were placed into experimental storage to assess their relative longevity and likely optimal conditions for long‐term conservation seed banking. Seeds from all species were desiccation tolerant, germinating after drying at 23% relative humidity (C. arenicola, C. huegelii, P. sanguinea and T. macrophylla) or 5% relative humidity (C. flava, D. laxiflora, M. media ssp. media, P. recurva and T. crinita) at 23 °C. From automatedly determined moisture adsorption and desorption isotherms at 23 °C, these equate to tolerance of drying to 0.03–0.06 g water g^?1 dry weight or 0.013–0.028 g water g^?1 dry weight, respectively. Results of storage experiments at a range of moisture contents and temperatures suggest conventional seed bank storage at ?18 °C after equilibration at c. 23% relative humidity (at 23 °C) may be suitable for most of the species, although there was higher germination of P. recurva seeds stored at ?80 °C and of M. media ssp. media seeds equilibrated at 75% relative humidity. However, there was considerable variation in germination of seeds sampled after different storage periods, making it difficult to identify optimal storage conditions definitively. Results of comparative longevity storage experiments at 60% relative humidity and 40 °C suggest seeds from these orchid species are short‐lived compared with non‐orchid species, and with Australian species in particular. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 26–41.     

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)⁺RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)⁺RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)⁺RNA content but the initial decrease and subsequent increase in poly(A)⁺RNA levels are less marked. On imbibition at 10°C, the poly(A)⁺RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)⁺RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.Abbreviations Poly(A) polyadenylic acid - poly(U) polyuridylic acid - poly(A)⁺RNA polyadenylated RNA     

Inflow of seeds through the forest edge: evidence from seed bank and vegetation patterns

To determine the influence of the proximity of a forest edge on seed bank composition and diversity, we performed a seed bank sampling at ancient deciduous forests bordering intensive arable fields. Also vegetation patterns were taken into account. We hypothised that forest edges may facilitate the entrance of diaspores of invasive species into the forest and the successive incorporation of these species in the forest seed bank. We noticed a substantial influence of the proximity of an edge on seed bank composition at as well the forested side of the edge as the field side. The forest edge zone was limited to 3 m into the forest and the field edge zone extended 3m into the field. The seed bank samples of field and forest edge are characterised by a higher species diversity and seed density and a higher similarity between seed bank and vegetation, compared to field or forest samples. The forest edges contains fewer pioneer species in comparison with the forest interior and more competitive species and species of edges and clearings compared with field and forest samples. The seed longevity index increases towards the forest interior. We can conclude from our data that the forest and edge seed bank are composed by both seeds from recent dispersal processes and local seed set and by seeds originating from past vegetation on the site. Near the edge, actual seed input seems of primal importance. Further towards the forest interior seed input decreases and long-living seeds of past vegetation become more important. Ancient forest edges thus act as a barrier for seeds of species of the surrounding arable field.     

Double-stranded RNA and male sterility in rice

Summary Double-stranded RNA (dsRNA) was isolated from rice Oryza sativa ssp. japonica, but not from other subspecies. The dsRNA has been found in all of the examined cytoplasmic male-sterile (CMS) lines of BT (Chinsurah Boro II)-type rice, but was not detected in their companionate maintainer lines. It is uniquely and positivley correlated with the CMS trait in BT-type rice. Recently, the dsRNA was also found in a nuclear malesterile (NMS) rice, Nongken 58s, but was not found in its normal Nongken 58. The molecular weight of this dsRNA was estimated to be about 18 kb. Electron microscopic analysis reveals that it is linear snapped. The double strandedness of the RNA molecules was characterized by CF-11 cellulose column chromatography and nuclease treatments. It bound to CF-11 cellulose in the presence of 15% ethanol. It was sensitive to RNase A at low salt concentrations, but insensitive to DNase I, SI nuclease, and RNase A at high salt concentrations. The dsRNA was detected in both mitochondrial and cytoplasmic fractions. Dot-blot hybridization reveals that there is no sequence homology between this dsRNA and mtDNA, but there is homology between this dsRNA and nuclear genomic DNA. We have not been able to transmit this dsRNA to fertile rice.     

Increases in the longevity of desiccation-phase developing rice seeds: response to high-temperature drying depends on harvest moisture content

Background and Aims Previous studies have suggested that the drying conditions routinely used by genebanks may not be optimal for subsequent seed longevity. The aim of this study was to compare the effect of hot-air drying and low-temperature drying on subsequent seed longevity for 20 diverse rice accessions and to consider how factors related to seed production history might influence the results.Methods Seeds of rice, Oryza sativa, were produced according to normal regeneration procedures at IRRI. They were harvested at different times [harvest date and days after anthesis (DAA), once for each accession] and dried either in a drying room (DR; 15 % relative humidity, 15 °C) or in a flat-bed heated-air batch dryer (BD; 45 °C, 8 h d^–1) for up to six daily cycles followed by drying in the DR. Relative longevity was assessed by storage at 10·9 % moisture content and 45 °C.Key Results Initial drying in the BD resulted in significantly greater longevity compared with the DR for 14 accessions (seed lots): the period of time for viability to fall to 50 % for seeds dried in the BD as a percentage of that for seeds dried throughout in the DR varied between 1.3 and 372·2 % for these accessions. The seed lots that responded the most were those that were harvested earlier in the season and at higher moisture content. Drying in the BD did not reduce subsequent longevity compared with DR drying for any of the remaining accessions.Conclusions Seeds harvested at a moisture content where, according to the moisture desorption isotherm, they could still be metabolically active (>16·2 %) may be in the first stage of the post-mass maturity, desiccation phase of seed development and thus able to increase longevity in response to hot-air drying. The genebank standards regarding seed drying for rice and, perhaps, for other tropical species should therefore be reconsidered.     

Relationship between seed bank expression, adult longevity and aridity in species of Chaetanthera (Asteraceae) in central Chile

BACKGROUND AND AIMS: Broad surveys have detected inverse relationships between seed and adult longevity and between seed size and adult longevity. However, low and unpredictable precipitation is also associated with seed bank (SB) expression in semi-arid and arid areas. The relationship between adult longevity, SB formation, seed mass and aridity is examined in annual and perennial herbs of Chaetanthera (Asteraceae) from the Chilean Mediterranean-type climate and winter-rainfall desert areas over a precipitation range of one order of magnitude. METHODS: Seeds of 18 species and subtaxa (32 populations) were buried in field locations, and exhumed after two successive germination periods. Seeds not germinating in the field were tested in a growth chamber, and remnant intact seed tested for viability. Seed banks were classed as transient or persistent. The effect of life form, species, population and burial time on persistent SB size was assessed with factorial ANOVA. Persistent seed bank size was compared with the Martonne aridity index (shown to be a surrogate for inter-annual variation in precipitation) and seed size using linear regression. ANCOVA assessed the effect of life-form on SB size with aridity as covariate. KEY RESULTS: Three species had a transient SB and 15 a persistent SB. ANOVA revealed a significant effect of life-form on SB size with annuals having larger SB size and greater capacity to form a persistent SB than perennials. Significant inter-population variation in SB size was found in 64% of cases. Seed mass was negatively correlated with persistent SB size. Persistent seed bank size was significantly correlated with the Martonne aridity index in the perennial and annual species, with species from more arid areas having larger persistent SBs. However, when aridity was considered as a covariate, ANCOVA revealed no significant differences between the annual and perennial herbs. CONCLUSIONS: Persistent seed bank size in Chaetanthera appears to reflect environmental selection rather than any trade-off with adult longevity.     

Proteomic analysis of RNA-binding proteins in dry seeds of rice after fractionation by ssDNA affinity column chromatography

In proteomic analysis, one of the major limitations is the detection of low-abundance proteins. To detect low-abundance RNA-binding proteins in mature dry seeds of rice, fractionation by single stranded DNA (ssDNA) affinity column chromatography was carried out before analysis by two-dimensional gel electrophoresis (2-DE). Proteomic analysis of the ssDNA-binding fraction revealed the existence of three types of RNA-binding proteins, including a K homology (KH) domain containing protein, a putative RNA-binding protein and a glycine-rich RNA-binding protein, in mature seeds. In addition, decreases in the putative RNA-binding protein and glycine-rich RNA-binding protein after absorbing water in seeds appear to be associated with seed germination.     

Ribosomal RNA integrity and rate of seed germination

The integrity of ribosomal RNA (the percentage of complete, un-nicked molecules) in seeds was studied by electrophoresis under denaturing conditions. Two batches of carrot seed, harvested at different stages of maturity, and four batches ofNicotiana seed stored for various times were used. Within each species, there was a correlation between the integrity of the rRNA of the dry seed and the rate of germination of that seed. In carrot seed, there was extensive degradation of existing rRNA in both the embryo and endosperm during the first two days of imbibition.     

Identification of specific proteins from seed embryos by two-dimensional gel electrophoresis for the discrimination between indica and japonica rice

Summary Proteins extracted from seed embryos of 29 different cultivated rice (Oryza sativa L.) and one wild rice (O. rufipogon Griff.) were compared by two-dimensional gel electrophoresis analysis. Among more than 300 protein spots on the gel we found some interesting variations in ten spots which were individually designated as proteins A-J. Protein E was observed in all indica cultivars but was not found in those of the subspecies japonica. In contrast, protein F was only detected in japonica cultivars. Protein A existed in all japonica cultivars but, with the exception of IR-36, could not be found in other indica cultivars. Therefore, proteins A, E and F can be used as markers for the identification of indica and japonica. Some so-called Javanica cultivars showed the characteristics of japonica subspecies with regard to proteins A and F, while one other cultivar of Javanica expressed a type intermediate between indica and japonica interms of proteins A and E. One feature discriminating between Javanica and japonica cultivars was found in the D, G, and J proteins which were expressed strongly in Javanica cultivars but were scarcely expressed in those of japonica. Expression of subspecies-specific proteins E and F in f₁ hybrids was also investigated.     

水稻籽实中砷的结合形态特征及其稳定性

利用凝胶层析方法对水稻籽实中As的结合形态特征及其稳定性进行了研究,结果表明,水稻籽实中存在的As主要与表观分子量为54.5KD和5.5KD的蛋白质形成结合体,分子量为54.5KD的蛋白质-As结合体不太稳定,并且在蒸煮加热和体外消化酶的作用下容易分解,生成相对稳定的、小分子的结合体形式。     

Effects of photooxidation on membrane integrity in Salix nigra seeds

Background and Aims

Salix nigra seeds are desiccation-tolerant, as are orthodox seeds, although in contrast to other orthodox seeds they lose viability in a few weeks at room temperature. They also differ in that the chloroplasts of the embryo tissues conserve their chlorophyll and endomembranes. The aim of this paper was to investigate the role of chlorophyll in seed deterioration.

Methods

Seeds were aged at different light intensities and atmospheric conditions. Mean germination time and normal and total germination were evaluated. The formation of free radicals was assessed using electronic spin resonance spectroscopy, and changes in the fatty acid composition from phospholipids, galactolipids and triglycerides using gas–liquid chromatography. Membrane integrity was studied with electronic spin resonance spin probe techniques, electrolyte leakage and transmission electron microscopy.

Key Results

Light and oxygen played an important role in free-radical generation, causing a decrease in normal germination and an increase in mean germination time. Both indices were associated with a decrease in polyunsaturated fatty acids derived from membrane lipids as phospholipids and galactolipids. The detection of damage in thylakoid membranes and an increase in plasmalemma permeability were consistent with the decrease in both types of lipids. Triglycerides remained unchanged. Light-induced damage began in outermost tissues and spread inwards, decreasing normal germination.

Conclusions

Salix nigra seeds were very susceptible to photooxidation. The thylakoid membranes appeared to be the first target of the photooxidative process since there were large decreases in galactolipids and both these lipids and the activated chlorophyll are contiguous in the structure of that membrane. Changes in normal germination and mean germination time could be explained by the deteriorative effects of oxidation.