Candidate genes association study to identify allele-specific SNP marker of ω-3 fatty acid in Brassica napus.

Due to the rapid decline in oceanic fish stock, ω-3 fatty acid (C18:3) has attracted serious attention and, hence, the identification of genotypes with high ω-3 content has become the main objective of Brassica napus (rapeseed) breeding. A candidate genes association study permitted us to delineate a genomic region linked to ω-3 content, offering a detailed understanding of the complex genetic mechanism of fatty acid biosynthesis in B. napus. Herein, the candidate genes association study, conducted on 324 genetically diverse rapeseed accessions, detected 114 single nucleotide polymorphisms (SNPs) associated with ω-3 fatty acid. Furthermore, these loci were functionally characterized in Saccharomyces cerevisiae. These associated loci were selected based on their contribution to a high C18:3 ratio, and the selected candidate loci were validated using allele-specific SNPs markers in an inbred population through polymerase chain reaction (PCR). These findings may contribute to improving the fatty acid composition by marker-based breeding and will facilitate the development of rapeseed varieties with high ω-3 content.

Candidate genes-association study to identify loci related to oleic acid in Brassica napus using SNP markers and their heterologous expression in yeast.

Brassica napus (rapeseed) serves as a main source of edible oil, and the oil's quality is mainly determined by the relative proportions of fatty acids. A high oleic acid concentration in B. napus oil increases its shelf life and oxidative stability. Therefore, attaining a high oleic acid concentration is necessary to enhance the nutritional quality of rapeseed oil. Here, an association study of candidate genes was conducted using a population of 324 genetically diverse rapeseed accessions, and several loci related to oleic acid content were identified. Furthermore, these loci were functionally characterized in Saccharomyces cerevisiae to assess their functions, and the promising candidate loci were validated using single nucleotide polymorphic markers in an independent inbred population. The results increased our understanding of fatty acid metabolism in B. napus. Moreover, these findings may assist in marker-based breeding efforts to improve the fatty acid composition and quality of B. napus oil.

Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed (Brassica napus L.). To date, the genetic mechanisms of rapeseed' interactions with S. sclerotiorum are not fully understood, and molecular-based breeding is still the most effective control strategy for this disease. Here, Arabidopsis thaliana GDSL1 was characterized as an extracellular GDSL lipase gene functioning in Sclerotinia resistance. Loss of AtGDSL1 function resulted in enhanced susceptibility to S. sclerotiorum. Conversely, overexpression of AtGDSL1 in B. napus enhanced resistance, which was associated with increased reactive oxygen species (ROS) and salicylic acid (SA) levels, and reduced jasmonic acid levels. In addition, AtGDSL1 can cause an increase in lipid precursor phosphatidic acid levels, which may lead to the activation of downstream ROS/SA defence-related pathways. However, the rapeseed BnGDSL1 with highest sequence similarity to AtGDSL1 had no effect on SSR resistance. A candidate gene association study revealed that only one AtGDSL1 homolog from rapeseed, BnaC07g35650D (BnGLIP1), significantly contributed to resistance traits in a natural B. napus population, and the resistance function was also confirmed by a transient expression assay in tobacco leaves. Moreover, genomic analyses revealed that BnGLIP1 locus was embedded in a selected region associated with SSR resistance during the breeding process, and its elite allele type belonged to a minor allele in the population. Thus, BnGLIP1 is the functional equivalent of AtGDSL1 and has a broad application in rapeseed S. sclerotiorum-resistance breeding.

BnaMPK3 Is a Key Regulator of Defense Responses to the Devastating Plant Pathogen Sclerotinia sclerotiorum in Oilseed Rape.

The disease caused by Sclerotinia sclerotiorum has traditionally been difficult to control, resulting in tremendous economic losses in oilseed rape (Brassica napus). Identification of important genes in the defense responses is critical for molecular breeding, an important strategy for controlling the disease. Here, we report that a B. napus mitogen-activated protein kinase gene, BnaMPK3, plays an important role in the defense against S. sclerotiorum in oilseed rape. BnaMPK3 is highly expressed in the stems, flowers and leaves, and its product is localized in the nucleus. Furthermore, BnaMPK3 is highly responsive to infection by S. sclerotiorum and treatment with jasmonic acid (JA) or the biosynthesis precursor of ethylene (ET), but not to treatment with salicylic acid (SA) or abscisic acid. Moreover, overexpression (OE) of BnaMPK3 in B. napus and Nicotiana benthamiana results in significantly enhanced resistance to S. sclerotiorum, whereas resistance is diminished in RNAi transgenic plants. After S. sclerotiorum infection, defense responses associated with ET, JA, and SA signaling are intensified in the BnaMPK3-OE plants but weakened in the BnaMPK3-RNAi plants when compared to those in the wild type plants; by contrast the level of both H2O2 accumulation and cell death exhibits a reverse pattern. The candidate gene association analyses show that the BnaMPK3-encoding BnaA06g18440D locus is a cause of variation in the resistance to S. sclerotiorum in natural B. napus population. These results suggest that BnaMPK3 is a key regulator of multiple defense responses to S. sclerotiorum, which may guide the resistance improvement of oilseed rape and related economic crops.

Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus.

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down-regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down-regulated in B. napus by over expressed of AtDA1R358K , which is a functional deficiency of DA1 with an arginine-to-lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.

A Meliorated Multi-Frequency Band Pyroelectric Sensor.

This article proposes a meliorated multi-frequency band pyroelectric sensor for detecting subjects with various velocities, namely extending the sensing frequency under good performance from electrical signals. A tactic, gradually increasing thickness of the ZnO layers, is used for redeeming drawbacks of a thicker pyroelectric layer with a tardy response at a high-frequency band and a thinner pyroelectric layer with low voltage responsivity at a low-frequency band. The proposed sensor is built on a silicon substrate with a thermal isolation layer of a silicon nitride film, consisting of four pyroelectric layers with various thicknesses deposited by a sputtering or aerosol deposition (AD) method and top and bottom electrodes. The thinnest ZnO layer is deposited by sputtering, with a low thermal capacity and a rapid response shoulders a high-frequency sensing task, while the thicker ZnO layers are deposited by AD with a large thermal capacity and a tardy response shoulders a low-frequency sensing task. The fabricated device is effective in the range of 1 KHz~10 KHz with a rapid response and high voltage responsivity, while the ZnO layers with thicknesses of about 0.8 µm, 6 µm, 10 µm and 16 µm are used for fabricating the meliorated multi-frequency band pyroelectric sensor. The proposed sensor is successfully designed, analyzed, and fabricated in the present study, and can indeed extend the sensing range of the multi-frequency band.

Multi-frequency band pyroelectric sensors.

A methodology is proposed for designing a multi-frequency band pyroelectric sensor which can detect subjects with various frequencies or velocities. A structure with dual pyroelectric layers, consisting of a thinner sputtered ZnO layer and a thicker aerosol ZnO layer, proved helpful in the development of the proposed sensor. The thinner sputtered ZnO layer with a small thermal capacity and a rapid response accomplishes a high-frequency sensing task, while the thicker aerosol ZnO layer with a large thermal capacity and a tardy response is responsible for low-frequency sensing tasks. A multi-frequency band pyroelectric sensor is successfully designed, analyzed and fabricated in the present study. The range of the multi-frequency sensing can be estimated by means of the proposed design and analysis to match the thicknesses of the sputtered and the aerosol ZnO layers. The fabricated multi-frequency band pyroelectric sensor with a 1 µm thick sputtered ZnO layer and a 20 µm thick aerosol ZnO layer can sense a frequency band from 4000 to 40,000 Hz without tardy response and low voltage responsivity.

[Transposon expression and potential effects on gene regulation of Brassica rapa and B. oleracea genomes].

Transposons or transposable elements (TEs) are ubiquitous and most abundant DNA components in higher eukaryotes. Recent sequencing of the Brassica rapa and B. oleracea genomes revealed that the amplification of TEs is one of the main factors inducing the difference in genome size. However, the expressions of TEs and the TE effects on gene regulation and functions of these two Brassica diploid species were unclear. Here, we analyzed the RNA sequencing data of leaves, roots, and stems from B. rapa and B. oleracea. Our data showed that overall TEs in either genome expressed at very low levels, and the expression levels of different TE categories and families varied among different organs. Moreover, even for the same TE category or family, the expression activities were distinct between the two Brassica diploids. Forty-one and nine LTR retrotransposons with the transcripts that read into their adjacent sequences have the distances shorter than 2 kb and 100 bp compared to the downstream genes. These LTR retrotransposon readout transcriptions may produce sense or antisense transcripts of nearby genes, with the effects on activating or silencing corresponding genes. Meanwhile, intact LTRs were detected at stronger readout activities than solo LTRs. Of the TEs inserted into genes, the frequencies were ob-served at a higher level in B. rapa than in B. oleracea. In addition, DNA transposons were prone to insert or retain in the intronic regions of genes in either Brassica genomes. These results revealed that the TEs may have potential effects on regulating protein coding genes.

Simultaneous analysis of multiple endogenous plant hormones in leaf tissue of oilseed rape by solid-phase extraction coupled with high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry.

INTRODUCTION: Plant hormones are important signalling molecules that act at lower concentrations to regulate numerous plant physiological and developmental processes. In order to study the functions of plant hormones, it is necessary to develop a high-throughput and highly selective and sensitive method for determination of plant hormones. OBJECTIVE: Based on SPE-HPLC-ESI-MS/MS method, a highly selective and sensitive method for determination of six plant hormones in leaf tissue of oilseed rape was developed. METHODOLOGY: The extraction was performed with C18 solid-phase extraction cartridge and the sample was subsequently analysed by HPLC-ESI-MS/MS. Methanol and H2O with 0.05% formic acid was selected as the mobile phase for HPLC, using a gradient of increasing methanol content. The plant hormones were quantified in MRM mode and identified in IDA mode by the hybrid triple quadrupole/linear ion trap mass spectrometer with high sensitivity and selectivity. RESULTS: Under the optimal conditions, good linearities were obtained for six plant hormones with the correlation coefficients above 0.9924. The detection limits of the target compounds were in the range of 0.005-0.2 ng/mL. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations. The extraction recovery yields of plant hormones under SPE conditions ranged from 67.03 to 119.83%. Compared with previous methods, sample preparation time and amount of sample required for analysis of plant hormones were reduced, and more classes of hormones were quantitatively measured.

A visual method for direct selection of high-producing Pichia pastoris clones.

BACKGROUND: The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs. RESULTS: A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology. CONCLUSIONS: A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.

Electrocatalytic oxidation of phytohormone salicylic acid at copper nanoparticles-modified gold electrode and its detection in oilseed rape infected with fungal pathogen Sclerotinia sclerotiorum.

Salicylic acid (SA) is a biological substance that acts as a phytohormone and plays an important role in signal transduction in plants. It is important to accurately and sensitively detect SA levels. A gold electrode modified with copper nanoparticles was used to assay the electrocatalytic oxidation of salicylic acid. It was found that the electrochemical behavior of salicylic acid was greatly improved at copper nanoparticles, indicating that anodic oxidation could be catalyzed at copper nanoparticles. And the pH had remarkable effect on the electrochemical process, a very well-defined oxidation peak appeared at pH 13.3 (0.2M NaOH). The kinetics parameters of this process were calculated and the heterogeneous electron transfer rate constant (k) was determined to be 1.34x10(-3)cms(-1), and (1-alpha)n(alpha) was 1.22. The gold electrode modified with copper nanoparticles could detect SA at a higher sensitivity than common electrodes. The electrode was used to detect the SA levels in oilseed rape infected with the fungal pathogen Sclerotinia sclerotiorum. The results showed that the SA concentration reached a maximum during the 10th-25th hours after infection. This result was very similar to that determined by HPLC, indicating that the gold electrodes modified with copper nanoparticles could be used as salicylic acid sensors.

A robust sampling approach for identification and quantification of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signaling in Sclerotinia sclerotiorum resistance.

INTRODUCTION: Methyl jasmonate (MJA), which is a natrual hormonal regulator, is thought to be essential for the regulation of systemic defence responses. The information about MJA levels in plant tissues is helpful for the study of the disease resistance mechanism and genetically engineered cultivars with increased resistance. Therefore, the quantification of MJA levels in plant tissues by means of a sensitive and reliable method is of interest. OBJECTIVE: Development of a film extraction method coupled with GC for determination of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signalling in sclerotinia sclerotiorum resistance. METHODOLOGY: A robust polydimethylsiloxane film was prepared and used for extraction of MJA in leaf tissues. By using in-solution extraction mode, optimum extraction efficiency was achieved with methanol-water (1 : 5, v/v) as extraction medium at 40 degrees C for 60 min. RESULTS: Under the optimal conditions, a detection limit of 0.2 ng/mL was achieved. Excellent reproducibility was found over a linear range of 1-1000 ng/mL. MJA in leaves infected by sclerotinia sclerotiorum was determined, with the results showing that basal levels of MJA (15 ng/g) were present in noninfested controls, but increased to 313 ng/g 10 h after fungal attack. CONCLUSION: The film extraction method is a simple, rapid and inexpensive sampling technique for determination of endogenous MJA in plant tissues that can be applied to most plants.

Real-time monitoring of oxidative burst from single plant protoplasts using microelectrochemical sensors modified by platinum nanoparticles.

Oxidative bursts from plants play significant roles in plant disease defense and signal transduction; however, it has not hitherto been investigated on individual living plant cells. In this article, we fabricated a novel sensitive electrochemical sensor based on electrochemical deposition of Pt nanoparticles on the surface of carbon fiber microdisk electrodes via a nanopores containing polymer matrix, Nafion. The numerous hydrophilic nanochannels in the Nafion clusters coated on the electrode surface served as the molecular template for the deposition and dispersion of Pt, which resulted in the uniform construction of small Pt nanoparticles. The novel sensor displayed a high sensitivity for detection of H(2)O(2) with a detection limit of 5.0 x 10(-9) M. With the use of this microelectrochemical sensor, the oxidative burst from individual living plant protoplasts have been real-time monitored for the first time. The results showed that oxidative burst from single protoplasts triggered by a pathogen analogue were characterized by quanta release with a large number of "transient oxidative microburst" events, and protoplasts from the transgenic plants biologically displayed better disease-resistance and showed a distinguished elevation and longer-lasting oxidative burst.

Microsensor in vivo monitoring of oxidative burst in oilseed rape (Brassica napus L.) leaves infected by Sclerotinia sclerotiorum.

Oxidative burst is the rapid and transient production of large amounts of reactive oxygen species, including superoxide anion, hydrogen peroxide (H(2)O(2)), and hydroxyl radical. A rapid and simple technique was employed for in vivo detection of oxidative burst in oilseed rape (Brassica napus L.) leaves, using a modified electrode. Platinum (Pt) micro-particles were dispersed on a Pt electrode, coated with a poly (o-phenylenediamine) film. This exhibited high sensitivity, selectivity and stability in H(2)O(2) detection. Amperometry was used to obtain satisfactory linear relationships between reductive current intensities and H(2)O(2) concentrations at -0.1 V potential in different electrolytes. This electrode was used in vivo to detect oxidative burst in oilseed rape following fungal infection. Oxidative bursts induced by infection of the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary exhibited notably different mechanisms between a susceptible and a resistant glucose oxidase-transgenic genotype.

In vivo measurements of changes in pH triggered by oxalic acid in leaf tissue of transgenic oilseed rape.

Oxalic acid (OA), a non-host-specific toxin secreted by Sclerotinia sclerotiorum during pathogenesis, has been demonstrated to be a major phytotoxic and pathogenic factor. Oxalate oxidase (OXO) is an enzyme associated with the detoxification of OA, and hence the introduction of an OXO gene into oilseed rape (Brassica napus L.) to break down OA may be an alternative way of increasing the resistance of the plant to Sclerotinia sclerotiorum. In order to investigate the activation of OXO in transgenic oilseed rape, a convenient and accessible method was used to monitor changes in pH in response to stress induced by OA. The pH sensor, a platinum microcylinder electrode modified using polyaniline film, exhibited a linear response within the pH range from 3 to 7, with a Nernst response slope of 70 mV/pH at room temperature. The linear correlation coefficient was 0.9979. Changes induced by OA in the pH values of leaf tissue of different oilseed rape species from Brassica napus L. were monitored in real time in vivo using this electrode. The results clearly showed that the transgenic oilseed rape was more resistant to OA than non-transgenic oilseed rape.