Multi-omics analyses provide insights into the evolutionary history and the synthesis of medicinal components of the Chinese wingnut.

Chinese wingnut (Pterocarya stenoptera) is a medicinally and economically important tree species within the family Juglandaceae. However, the lack of high-quality reference genome has hindered its in-depth research. In this study, we successfully assembled its chromosome-level genome and performed multi-omics analyses to address its evolutionary history and synthesis of medicinal components. A thorough examination of genomes has uncovered a significant expansion in the Lateral Organ Boundaries Domain gene family among the winged group in Juglandaceae. This notable increase may be attributed to their frequent exposure to flood-prone environments. After further differentiation between Chinese wingnut and Cyclocarya paliurus, significant positive selection occurred on the genes of NADH dehydrogenase related to mitochondrial aerobic respiration in Chinese wingnut, enhancing its ability to cope with waterlogging stress. Comparative genomic analysis revealed Chinese wingnut evolved more unique genes related to arginine synthesis, potentially endowing it with a higher capacity to purify nutrient-rich water bodies. Expansion of terpene synthase families enables the production of increased quantities of terpenoid volatiles, potentially serving as an evolved defense mechanism against herbivorous insects. Through combined transcriptomic and metabolomic analysis, we identified the candidate genes involved in the synthesis of terpenoid volatiles. Our study offers essential genetic resources for Chinese wingnut, unveiling its evolutionary history and identifying key genes linked to the production of terpenoid volatiles.

Decoy engineering of the receptor-like cytoplasmic kinase StPBS1 to defend against virus infection in potato.

Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1-RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1NIa . StPBS1NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1NIa .

Association of CYP24A1 gene polymorphism with colorectal cancer in the Jiamusi population.

BACKGROUND: The population in Jiamusi has been reported to have the highest prevalence of colorectal cancer (CRC) in China. The genetic causal-effect for this occurrence among the residents remains unclear. Given the long cold seasons with people wearing more clothes and reduced UV exposure, we aimed to study the association between the vitamin D metabolism-related gene CYP24A1 polymorphism and CRC susceptibility. METHOD: A case-control study was conducted that included 168 patients with CRC and 710 age-matched healthy individuals as the control group. Plausible susceptible variations were sought and clinical phenotypic-genotype association analysis was performed. RESULTS: Overall, two CYP24A1 polymorphisms, rs6013905 AX (P = 0.02, OR = 1.89, 95%CI: 1.09-3.29) and rs2762939 GX (P = 0.02, OR = 1.52, 95%CI: 1.08-2.13) were significantly associated with CRC in the Jiamusi population. In the female group, three CYP24A1 polymorphisms, rs6013905 AX (P = 0.04, OR = 2.59, 95%CI: 1.03-6.49), rs2762939 GX (P = 0.01, OR = 2.35, 95%CI: 1.25-4.42), and rs6068816 GG (P = 0.05, OR = 1.89, 95%CI: 0.99-3.59) carriers were significantly associated with CRC. In clinical phenotypic-genotype analysis, rs6013905 GG (P = 0.05, OR = 4.00, 95%CI: 0.92-17.48) and rs2762939 GX (P = 0.03, OR = 4.87, 95%CI: 1.00-23.69) carriers were significantly associated with poorly differentiated CRC, while CYP24A1 rs6068816 AX was significantly associated with the tumor type (P = 0.02, OR = 2.08, 95%CI: 1.10-3.96) and location (P = 0.04, OR = 2.24, 95%CI: 1.05-4.77). CONCLUSION: CYP24A1 gene polymorphism may be a genetic risk factor attributable to the highest prevalence of CRC in Jiamusi people. Individuals with CYP24A1 gene polymorphism may have an increased barrier for vitamin D absorption, thus contributing to the risk of CRC development.

MicroRNA-124 suppresses the invasion and proliferation of breast cancer cells by targeting TFAP4.

MicroRNA (miRNA/miR)-124 is widely accepted as the suppressor of different tumors. The present study aimed to improve understanding of the potential role of miR-124 in breast cancer. The gene expression profile change derived from the overexpression of miR-124 was investigated using RNA sequencing and bioinformatics analysis of the breast cancer cell line SKBR3. The results demonstrated that the gene expression profile of SKBR3 cells significantly changed. In addition, the transcription factor activating enhancer-binding protein 4 (TFAP4) gene was identified among the top 10 differentially expressed genes, and was identified as a novel target gene of miR-124 using a dual-luciferase reporter assay. TFAP4 knockdown in notably impaired SKBR3 cell migration and proliferation, which was consistent with decreasing migration and proliferation ability following overexpression of miR-124. Taken together, these results suggest that overexpression of miR-124 can suppress the migration and proliferation of SKBR3 cells by tarsgeting TFAP4. Thus, TFAP4 may act as a novel therapeutic target of breast cancer.

Improvement of host-induced gene silencing efficiency via polycistronic-tRNA-amiR expression for multiple target genes and characterization of RNAi mechanism in Mythimna separata.

Host-induced gene silencing (HIGS) emerged as a new strategy for pest control. However, RNAi efficiency is reported to be low in Lepidoptera, which are composed of many important crop pests. To address this, we generated transgenic plants to develop HIGS effects in a maize pest, Mythimna separata (Lepidoptera, Noctuidae), by targeting chitinase encoding genes. More importantly, we developed an artificial microRNA (amiR) based PTA (polycistronic-tRNA-amiR) system for silencing multiple target genes. Compared with hpRNA (hairpin RNA), transgenic expression of a PTA cassette including an amiR for the gut-specific dsRNA nuclease gene MsREase, resulted in improved knockdown efficiency and caused more pronounced developmental abnormalities in recipient insects. When target gene siRNAs were analysed after HIGS and direct dsRNA/siRNA feeding, common features such as sense polarity and siRNA hotspot regions were observed, however, they differed in siRNA transitivity and major 20-24nt siRNA species. Core RNAi genes were identified in M. separata, and biochemical activities of MsAGO2, MsSID1 and MsDcr2 were confirmed by EMSA (electrophoretic mobility shift assay) and dsRNA cleavage assays, respectively. Taken together, we provide compelling evidence for the existence of the RNAi mechanism in M. separata by analysis of both siRNA signatures and RNAi machinery components, and the PTA system could potentially be useful for future RNAi control of lepidopteran pests.

Synthesis of Full-Length cDNA Infectious Clones of Soybean Mosaic Virus and Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro.

Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2-with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif-attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host-SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system.

Long non-coding RNA PRNCR1 has an oncogenic role in breast cancer.

Long non-coding RNAs (lncRNAs) have important roles in the development and progression of various types of human cancer. However, the expression and function of the lncRNA prostate cancer-associated non-coding RNA 1 (PRNCR1) in breast cancer remains unclear. Reverse transcription-quantitative PCR was performed to measure the levels of mRNA expression. Cell counting kit-8, flow cytometry, wound healing and Transwell assays were also performed to study cell proliferation, cell cycle, migration and invasion, respectively. The results of the present study revealed that PRNCR1 expression levels were higher in breast cancer tissues compared with adjacent normal tissues in a patient study. It was also determined that high expression of PRNCR1 was significantly associated with advanced clinical stage, positive metastasis and poor prognosis for patients with breast cancer. In vitro experiments determined that PRNCR1 was significantly upregulated in the breast cancer cell lines BT-549, MCF-7, SK-BR-3 and MDA-MB-231 compared with the normal human breast cell line, MCF-10A. Silencing of PRNCR1 significantly inhibited the proliferation, colony formation, cell cycle progression, migration and invasion of SK-BR-3 and BT-549 cells, while cell apoptosis was induced. In addition, knockdown of PRNCR1 suppressed epithelial-mesenchymal transition in SK-BR-3 and BT-549 cells. In summary, the present results demonstrated that lncRNA PRNCR1 was significantly upregulated in breast cancer and was associated with cancer progression and poor patient prognosis. In vitro experiments determined that knockdown of PRNCR1 inhibited the malignant phenotypes of breast cancer cells. Taken together, the results indicated that PRNCR1 may be used as a potential therapeutic target for patients with breast cancer.

Circular RNA circMAN2B2 facilitates lung cancer cell proliferation and invasion via miR-1275/FOXK1 axis.

Lung cancer remains a leading cause of cancer-related deaths worldwide. In the past years, increasing reports indicate that circular RNAs (circRNAs) exert a great important role in human cancers, including lung cancer. However, the knowledge about circRNA in lung cancer remains very little so far. In the present study, we screened out a highly expressed novel circRNA named circMAN2B2 in lung cancer tissues. We investigated the function of circMAN2B2 and found that circMAN2B2 knockdown significantly inhibited cell proliferation and invasion in both H1299 and A549 lung cancer cells. Mechanistically, we found that circMAN2B2 could sponge miR-1275 to inhibit its level. Through a series of functional experiments, we dissected the role of miR-1275 in lung cancer and proved the anti-tumor role of miR-1275. Furthermore, we found that miR-1275 exerted its role in lung cancer by regulating FOXK1 expression. In addition, we demonstrated that restoration of FOXK1 could rescue circMAN2B2 knockdown-induced repression of cell proliferation and invasion. Taken together, our study demonstrated that circMAN2B2 acts as an oncogenic role in lung cancer through promoting FOXK1 expression by sponging miR-1275.

Down-regulation of genes coding for core RNAi components and disease resistance proteins via corresponding microRNAs might be correlated with successful Soybean mosaic virus infection in soybean.

Plants protect themselves from virus infections by several different defence mechanisms. RNA interference (RNAi) is one prominent antiviral mechanism, which requires the participation of AGO (Argonaute) and Dicer/DCL (Dicer-like) proteins. Effector-triggered immunity (ETI) is an antiviral mechanism mediated by resistance (R) genes, most of which encode nucleotide-binding site-leucine-rich repeat (NBS-LRR) family proteins. MicroRNAs (miRNAs) play important regulatory roles in plants, including the regulation of host defences. Soybean mosaic virus (SMV) is the most common virus in soybean and, in this work, we identified dozens of SMV-responsive miRNAs by microarray analysis in an SMV-susceptible soybean line. Amongst the up-regulated miRNAs, miR168a, miR403a, miR162b and miR1515a predictively regulate the expression of AGO1, AGO2, DCL1 and DCL2, respectively, and miR1507a, miR1507c and miR482a putatively regulate the expression of several NBS-LRR family disease resistance genes. The regulation of target gene expression by these seven miRNAs was validated by both transient expression assays and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) experiments. Transcript levels for AGO1, DCL1, DCL2 and five NBS-LRR family genes were repressed at different time points after SMV infection, whereas the corresponding miRNA levels were up-regulated at these same time points. Furthermore, inhibition of miR1507a, miR1507c, miR482a, miR168a and miR1515a by short tandem target mimic (STTM) technology compromised SMV infection efficiency in soybean. Our results imply that SMV can counteract soybean defence responses by the down-regulation of several RNAi pathway genes and NBS-LRR family resistance genes via the induction of the accumulation of their corresponding miRNA levels.

Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors.

BACKGROUND: RNAi (RNA interference) is a technology for silencing of target genes via sequence-specific manner. RNAi technology has been used for development of anti-pathogenic crops. In 2007, development of transgenic plants resistant to insect herbivore using RNAi technology was first reported, leading to a burst of efforts aimed at exploitation of RNAi mechanism and control strategy against variety of insect species based on this technique. Mythimna separata belongs to noctuidae family of lepidoptera and is posing threat to crops of economic importance. Recently, outbreaks of M. separata severely threatens corn production in Northern China, calling for new control approaches. RESULTS: Chitinase genes were chosen as the target genes as they were expressed predominantly in the gut tissue and were reported to be ideal silencing targets in several insect species. Interfering sequences against the target genes were cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3) which was defective in dsRNA degradation activity, so preserving the dsRNA from degradation by cellular machinery. The bacteria were mixed with artificial diet and were fed to M. separata. We showed that oral delivery of bacterially expressed dsRNA would lead to RNAi effects in the recipient insect. Quantitative real-time PCR results showed that expression level of target MseChi1 and MseChi2 genes in gut tissue of M. separata were down-regulated after oral delivery of engineered bacteria expressing the corresponding dsRNA. Sequence-specific siRNA (small interfering RNA) was detected in recipient insects, supporting the existence of siRNA-mediated silencing effects in M. separata. Furthermore, knockdown of MseChi1 and MseChi2 resulted in increased mortality and reduced body weight of the feeding larvae. CONCLUSION: We reported a simple and low cost experimental procedure to silence M. separata endogenous gene expression. Our research provides both an experimental foundation for using RNAi technology to control M. separata and also a useful research tool for loss-of-function study of important developmental and regulatory genes in this insect species.

Silencing of Target Chitinase Genes via Oral Delivery of dsRNA Caused Lethal Phenotypic Effects in Mythimna separata (Lepidoptera: Noctuidae).

Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous, migratory corn pest. Outbreak of M. separata has led to severe damage to corn production recently in China. RNAi (RNA interference) is a gene silencing technology applied both in model and non-model organisms, and it is especially useful for the latter in which the reverse genetic research tools are not available. RNAi approach was broadly investigated in many plant pathogens and was used for the generation of anti-pest transgenic plants. We are proposing to use this technology to silence M. separata endogenous genes, thereby, providing a biocontrol method for this insect. Feeding of dsRNAs for target Chitinase genes resulted in substantial decreases of their transcript levels in M. separata. Furthermore, silencing of target Chitinase genes led to phenotypic effects such as reduced body weight and increased mortality. Our study provided both reverse genetic research tool and potential control strategy for this insect species.

Silencing of Mythimna separata chitinase genes via oral delivery of in planta-expressed RNAi effectors from a recombinant plant virus.

OBJECTIVES: To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression. RESULTS: Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %. CONCLUSION: Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.

Induction of autophagy by the MG­132 proteasome inhibitor is associated with endoplasmic reticulum stress in MCF­7 cells.

The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is involved in MG­132­induced autophagy, and to determine the effects of the inhibition of autophagy and ER stress on cell viability following MG­132 treatment. The proteasome inhibitor, MG­132, was used to induce autophagy in MCF­7 cells, and 3­methyladenine (3­MA) and salubrinal were used to inhibit autophagy and ER stress, respectively. An MTT assay was used to analyze cell viability. Apoptosis and the cell cycle were analyzed using flow cytometry. The expression levels of apoptosis­ and ER stress­associated genes were investigated using western blot and reverse transcription­quantitative polymerase chain reaction analyses. MG­132 inhibited cell proliferation, and induced apoptosis and cell cycle arrest at the G2 phase of the cell cycle. Notably, MG­132 increased the autophagy­associated conversion of microtubule­associated protein 1 light chain 3 (LC3)­I to LC3­II, which was partially attenuated by the ER stress inhibitor, salubrinal. In addition, MG­132 inhibited the protein expression of the anti­apoptotic protein, B­cell lymphoma (Bcl)­2, whereas the expression levels of Bcl­2­associated X protein and caspase­3 were upregulated. These effects were enhanced by co­treatment with either 3­MA or salubrinal. Furthermore, the mRNA and protein levels of the ER stress­associated genes, glucose­regulated protein 78, growth arrest and DNA damage induced gene­153, and caspase­12, were upregulated by MG132, and these levels were significantly inhibited by co­treatment of the cells with salubrinal. Taken together, the results of the present study indicated that the induction of autophagy by the proteasome inhibitor was associated with ER stress in the MCF­7 cells, and that the inhibition of autophagy or ER stress enhanced MG­132­induced apoptosis. These findings suggest the potential application of inhibitors of ER stress and autophagy, in combination with proteasomal inhibitors, for the development of combinatorial targeted cancer therapy.

Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy.

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA( P.fluorescens ) indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K ( i ) for glyphosate compared to those of the AroA( E.coli ), while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA ( P.fluorescens ) gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.

Expression, characterization and 2,4,6-trichlorophenol degradation of laccase from Monilinia fructigena.

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V (max) values are 243.9 and 10.55 Um min(-1) mg(-1), respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.