Carrot, a biennial herb of the Apiaceae family, is among the most important vegetable crops in the world. In this study, nine candidate reference genes (GAPDH, ACTIN, eIF-4α, PP2A, SAND, TIP41, UBQ, EF-1α, and TUB) were cloned from carrot. Carrot plants were subjected to abiotic stresses (heat, cold, salt, and drought) and hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid). The expression profiles of the candidate reference genes were evaluated in three technical and biological replicates. Real-time qPCR data analyses were performed using three commonly used Excel-based applets namely, BestKeeper, geNorm, and NormFinder. ACTIN and TUB were the most stable genes identified among all sample groups, but individual analysis revealed changes in their expression profiles. GAPDH displayed the maximum stability for most of single stresses. To further validate the suitability of the reference genes identified in this study, the expression profile of DcDREB-A1 gene (homolog of AtDREB-A1 gene of Arabidophsis) was studied in carrot. The appropriate reference genes were selected that showed stable expression under the different experimental conditions.

Controllable manipulation of fluid flow is crucial for efficient particle separation, which is associated with plenty of biomedical and industrial applications. Microfluidic technologies have achieved promising progress in particle positioning depending on inertial force with or without the help of the Dean effect. Herein, we describe an inertial microfluidic system containing a spiral microchannel for various highly efficient particle separations. We demonstrated that Dean-like secondary flow can be regulated by geometric confinement in the microchannel. On the introduction of a library of micro-obstacles into the spiral microchannels, the resulting linear acceleration of secondary flow can be applied to remarkably enhance particle focusing in time and space. Further, multiple separating and sorting manipulations of particles including polymeric particles, circulating tumor cells, and blood cells, can be successfully accomplished in the dimension-confined spiral channels in a sheathless, high-throughput (typically 3 ml min), long-term (at least 4 h), and highly-efficient (up to 99.8% focusing) manner. The methodological achievement pointing to ease-of-use, effective, and high-throughput particle manipulations is useful for both laboratory and commercial developments of microfluidic systems in life and material sciences.

Tea plant (Camellia sinensis) leaf is an important non-alcoholic beverage resource. The application of quantitative real time polymerase chain reaction (qRT-PCR) has a profound significance for the gene expression studies of tea plant, especially when applied to tea leaf development and metabolism. In this study, nine candidate reference genes (i.e., CsACT7, CsEF-1α, CseIF-4α, CsGAPDH, CsPP2A, CsSAND, CsTBP, CsTIP41, and CsTUB) of C. sinensis were cloned. The quantitative expression data of these genes were investigated in five tea leaf developmental stages (i.e., 1st, 2nd, 3rd, 4th, and older leaves) and normal growth tea leaves subjected to five hormonal stimuli (i.e., ABA, GA, IAA, MeJA, and SA), and gene expression stability was calculated using three common statistical algorithms, namely, geNorm, NormFinder, and Bestkeeper. Results indicated that CsTBP and CsTIP41 were the most stable genes in tea leaf development and CsTBP was the best gene under hormonal stimuli; by contrast, CsGAPDH and CsTUB genes showed the least stability. The gene expression profile of CsNAM gene was analyzed to confirm the validity of the reference genes in this study. Our data provide basis for the selection of reference genes for future biological research in the leaf development and hormonal stimuli of C. sinensis.

WRKY transcription factors belong to one of the largest transcription factor families. These factors possess functions in plant growth and development, signal transduction, and stress response. Here, we identified 95 DcWRKY genes in carrot based on the carrot genomic and transcriptomic data, and divided them into three groups. Phylogenetic analysis of WRKY proteins from carrot and Arabidopsis divided these proteins into seven subgroups. To elucidate the evolution and distribution of WRKY transcription factors in different species, we constructed a schematic of the phylogenetic tree and compared the WRKY family factors among 22 species, which including plants, slime mold and protozoan. An in-depth study was performed to clarify the homologous factor groups of nine divergent taxa in lower and higher plants. Based on the orthologous factors between carrot and Arabidopsis, 38 DcWRKY proteins were calculated to interact with other proteins in the carrot genome. Yeast two-hybrid assay showed that DcWRKY20 can interact with DcMAPK1 and DcMAPK4. The expression patterns of the selected DcWRKY genes based on transcriptome data and qRT-PCR suggested that those selected DcWRKY genes are involved in root development, biotic and abiotic stress response. This comprehensive analysis provides a basis for investigating the evolution and function of WRKY genes.

A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

To achieve effective symbiosis with legume, rhizobia should fine-tune their background regulation network in addition to activating key genes involved in nodulation (nod) and nitrogen fixation (nif). Here, we report that an ancestral zinc finger regulator, MucR1, other than its paralog, MucR2, carrying a frameshift mutation, is essential for supporting nitrogen fixation of Sinorhizobium fredii CCBAU45436 within soybean nodules. In contrast to the chromosomal mucR1, mucR2 is located on symbiosis plasmid, indicating its horizontal transfer potential. A MucR2 homolog lacking the frameshift mutation, such as the one from S. fredii NGR234, can complement phenotypic defects of the mucR1 mutant of CCBAU45436. RNA-seq analysis revealed that the MucR1 regulon of CCBAU45436 within nodules exhibits significant difference compared with that of free-living cells. MucR1 is required for active expression of transporters for phosphate, zinc, and elements essential for nitrogenase activity (iron, molybdenum, and sulfur) in nodules but is dispensable for transcription of key genes (nif/fix) involved in nitrogen fixation. Further reverse genetics suggests that S. fredii uses high-affinity transporters to meet the demand for zinc and phosphate within nodules. These findings, together with the horizontal transfer potential of the mucR homolog, imply an intriguing evolutionary role of this ancestral regulator in supporting nitrogen fixation.

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA-DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean.

A new polydiacetylene liposome-based sensor was successfully constructed for the colorimetric and fluorometric detection of Pb2+.