DNA folding and melting observed in real time redefine the energy landscape

We report real-time observations of the folding and melting of DNA by probing two active sites of a hairpin structure, the bases and the stem end, and using an ultrafast T-jump. Studies at different initial temperatures (before, during, and after melting) provide the time scale of water heating (<20 ps), single-strand destacking (700 ps to 2 ns), and hairpin destacking (microseconds and longer) in solutions of various ionic strengths and pH values. The behavior of transient changes gives direct evidence to the existence of intermediate collapsed structures, labile in destacking but compact in nature, and indicates that melting is not a two-state process. We propose a landscape that is defined by these nucleation structures and destacking for efficient folding and melting

Characterization of Sucrose transporter alleles and their association with seed yield-related traits in Brassica napus L

<p>Abstract</p> <p>Background</p> <p>Sucrose is the primary photosynthesis product and the principal translocating form within higher plants. <it>Sucrose transporters </it>(<it>SUC/SUT</it>) play a critical role in phloem loading and unloading. Photoassimilate transport is a major limiting factor for seed yield. Our previous research demonstrated that <it>SUT </it>co-localizes with yield-related quantitative trait loci. This paper reports the isolation of <it>BnA7.SUT1 </it>alleles and their promoters and their association with yield-related traits.</p> <p>Results</p> <p>Two novel <it>BnA7.SUT1 </it>genes were isolated from <it>B. napus </it>lines 'Eagle' and 'S-1300' and designated as <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b</it>, respectively. The BnA7.SUT1 protein exhibited typical SUT features and showed high amino acid homology with related species. Promoters of <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b </it>were also isolated and classified as <it>pBnA7.SUT1.a </it>and <it>pBnA7.SUT1.b</it>, respectively. Four dominant sequence-characterized amplified region markers were developed to distinguish <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b</it>. The two genes were estimated as alleles with two segregating populations (F₂and BC₁) obtained by crossing '3715'×'3769'. <it>BnA7.SUT1 </it>was mapped to the A7 linkage group of the TN doubled haploid population. <it>In silico </it>analysis of 55 segmental <it>BnA7.SUT1 </it>alleles resulted three <it>BnA7.SUT1 </it>clusters: <it>pBnA7.SUT1.a- BnA7.SUT1.a </it>(type I), <it>pBnA7.SUT1.b- BnA7.SUT1.a </it>(type II), and <it>pBnA7.SUT1.b- BnA7.SUT1.b </it>(type III). Association analysis with a diverse panel of 55 rapeseed lines identified single nucleotide polymorphisms (SNPs) in promoter and coding domain sequences of <it>BnA7.SUT1 </it>that were significantly associated with one of three yield-related traits: number of effective first branches (EFB), siliques per plant (SP), and seed weight (n = 1000) (TSW) across all four environments examined. SNPs at other <it>BnA7.SUT1 </it>sites were also significantly associated with at least one of six yield-related traits: EFB, SP, number of seeds per silique, seed yield per plant, block yield, and TSW. Expression levels varied over various tissue/organs at the seed-filling stage, and <it>BnA7.SUT1 </it>expression positively correlated with EFB and TSW.</p> <p>Conclusions</p> <p>Sequence, mapping, association, and expression analyses collectively showed significant diversity between the two <it>BnA7.SUT1 </it>alleles, which control some of the phenotypic variation for branch number and seed weight in <it>B. napus </it>consistent with expression levels. The associations between allelic variation and yield-related traits may facilitate selection of better genotypes in breeding.</p

Development and Validation of an Effective CRISPR/Cas9 Vector for Efficiently Isolating Positive Transformants and Transgene-Free Mutants in a Wide Range of Plant Species

The CRISPR/Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Previously, we succeeded in effectively generating mutations in Brassica napus using an available CRISPR/Cas9 vector pKSE401, while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive 35S promoter into pKSE401 to facilitate a visual screen of mutants. This modified vector was named pKSE401G and tested in several dicot plant species, including Arabidopsis, B. napus, Fragaria vesca (strawberry), and Glycine max (soybean). Consequently, GFP-positive plants were readily identified through fluorescence screening in all of these species. Among these GFP-positive plants, the average mutation frequency ranged from 20.4 to 52.5% in Arabidopsis and B. napus with stable transformation, and was 90.0% in strawberry and 75.0% in soybean with transient transformation, indicating that the editing efficiency resembles that of the original vector. Moreover, transgene-free mutants were sufficiently identified in Arabidopsis in the T2 generation and B. napus in the T1 generation based on the absence of GFP fluorescence, and these mutants were stably transmissible to next generation without newly induced mutations. Collectively, pKSE401G provides us an effective tool to readily identify positive primary transformants and transgene-free mutants in later generations in a wide range of dicot plant species

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea

BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus

7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus

Ultrafast T-Jump in Water: Studies of Conformation and Reaction Dynamics at the Thermal Limit

We report the development of ultrafast T-jump with time resolution reaching the fundamental time scale of water thermalization time (∼5 ps). The T-jump heats the bulk water up to 20 °C via the overtone absorption of the OH− stretch at 1.5 μm. The example given here shows the application of the methodology, for the first time, and the results demonstrate distinct time scales for solvation, conformational change, and thermal reaction

Design and implementation of the fuel supply for the high-temperature combustion system

This article describes the configuration and working principle of the high-temperature combustion system; according to the control requirements which have a wide range and high precision for fuel flow-rate of the high-temperature combustion system, a set of fuel supply system is designed based on the frequency conversion hydraulic technology and electro-hydraulic proportional technique. An automatic control system with the function of field and remote control is carried out to achieve the precise supply of the fuel. The transfer function which describes the dynamic characteristic of the fuel supply system is given and the dynamic matrix control algorithm is employed to realize the high-quality control of fuel flow-rate. The experimental results show that the response time of flow-rate is about 12 s, almost no overshoot, and control accuracy within 1%. Therefore, the designed fuel supply system can meet the requirements of the high-temperature combustion system, and the designed control system has good control performance