Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

Chinese tallow (Sapium sebiferum L.) is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB) were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt). TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow

GelMA hydrogel scaffold containing curcumin-loaded solid lipid nanoparticles promotes the regeneration of degenerative discs

Abstract Intervertebral disc degenerative disease (IDD), which usually causes lower back and neck pain, is one of the most widespread musculoskeletal disorders and often causes a low quality of life. However, the surgical and conservative treatments commonly used in clinical practice are not effective. Previous studies have identified curcumin (Cur) as a potential therapeutic agent. However, its development in this regard has been limited due to its low dissolution, instability in water, and rapid metabolism. In this study, we developed a novel anti-inflammatory composite hydrogel scaffold with curcumin encapsulated in solid lipid nanoparticles and mixed it with gelatin methacrylate (GelMA) hydrogel to treat IDD. The hydrogel scaffold, denoted Cur-solid lipid nanoparticles (SLNs)/GelMA, promoted the restoration of Collagen type II (Col II) and aggrecan expression levels in vivo, indicating that the regeneration of the intervertebral discs was effective. Combined in vitro studies showed that Cur-SLNs inhibited the expression of the inflammatory factors TNF-α and IL-6. Additionally, immunofluorescence and western blotting experiments verified that Cur-SLNs regulated the recovery of Col II and aggrecan in an inflammatory environment and promoted the metabolic homeostasis of the extramedullary cell matrix. In conclusion, this study provides a new strategy to promote IDD regeneration, which brings new application prospects

Hollow Mesoporous Microspheres Coating for Super-Hydrophobicity Wood with High Thermostability and Abrasion Performance

Super-hydrophobic phenomena generally exist in nature, and wood can also obtain hydrophobicity by specific processing on the surface, being like the construction of microscale rough surface or decoration with low surface energy materials. In this research, the formation of hydrophobic layers on wood surface was investigated without breaking the wood’s original structure. The core-shell structure particles were prepared by penetrating orthosilicate and polystyrene into the hollow mesoporous microsphere structure with tetrahydrofuran. A wood sample was coated with polydimethylsiloxane (PDMS) resin layer to enhance the adhesion of nano and micron hollow mesoporous microsphere on its surface. According to the surface structure of super-hydrophobic subjects in nature, the nano and micron hollow mesoporous microsphere were sprayed with different ratios several times to form a hydrophobic surface. The water contact angle could reach 150°, revealing that the hydrophobic behavior of the nano and micron hollow mesoporous microsphere coating was achieved. The microstructures of wood samples were examined by the scanning electron microscopy, and the chemical functional groups were investigated by the Fourier transform infrared; both verified that the hydrophobic surface was successfully coated. The thermogravimetric examination revealed the improved thermal stability of the hydrophobic wood. The scratch test was used to measure the abrasion resistance of the nano and micron hollow mesoporous microsphere coatings on wood surface. It was suggested that the nano and micron hollow mesoporous microsphere coating was an effective method to fabricate extremely hydrophobic wood products

Main orthologs involved in abotic stress response.

<p>Main orthologs involved in abotic stress response.</p

Putative pathway of flower development in <i>S. sebiferum</i>.

<p>External (vernalization, photoperiod) and internal (autonomous, age, gibberellins) signals integrate into elaborate genetic network to regulate flowering. The genes components of the genetic network have been discussed in text.</p

Characterization of searching the assembled unigenes against NR, NT and Swiss-Prot protein databases.

<p>a. E-value distribution of BLAST hits for the assembled unigenes in NR database; b.E-value distribution of BLAST hits for the assembled unigenes in NT; c. E-value distribution of BLAST hits for the assembled unigenes in Swiss-Prot database. d. Species distribution of top hits in NT database.</p

Flower Bud Transcriptome Analysis of <i>Sapium sebiferum</i> (Linn.) Roxb. and Primary Investigation of Drought Induced Flowering: Pathway Construction and G-Quadruplex Prediction Based on Transcriptome

<div><p><i>Sapium sebiferum</i> (Linn.) Roxb. (Chinese Tallow Tree) is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on <i>S. sebiferum</i> genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of <i>S. sebiferum</i> flower has been generated by sequencing and <i>de novo</i> assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in <i>S. sebiferum</i> flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis. Besides, the G-Quadruplexes in RNA of <i>S. sebiferum</i> also have been predicted. An interesting finding is that the stress-induced flowering was observed in <i>S. sebiferum</i> for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, <i>GA1</i>, <i>AP2</i> and <i>CRY2</i>, accorded with stress-related genes, such as <i>GRX50435</i> and <i>PRXⅡ39562</i>. This transcriptome provides functional genomic information for further research of <i>S. sebiferum</i>, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other <i>Euphorbiaceae</i> family plants.</p></div

Drought induced flowering (right) and analysis of the expression of related genes by semi-quantitative PCR (left).

<p>Under drought stress, one-year-old seedling (+ in right) began to bloom (red arrow indicating the staminate flowers of <i>S. sebiferum</i>) and normal control (CK at the left) performed no flower or flower bud on the shoot apex (white arrow). Besides the difference of flower, the precocious-flowering seedling had distinct phonotype of senescence, such as dark leaf color and withered leaf tips. The expression of genes related with flowering and drought stress has been analyzed by semi-quantity PCR, and detailed discussion has been shown in text. le, <u>le</u>aves tissue; flo, <u>flo</u>wer tissue.</p