Genome-wide analysis of the SCAMPs gene family of soybean and functional identification of GmSCAMP5 in salt tolerance.

The effect of salt damage on plants is mainly caused by the toxic effect of Na+. Studies showed that the secretory carrier membrane proteins were associated with the Na+ transport. However, the salt tolerance mechanism of secretory carrier protein (SCAMP) in soybean was yet to be defined. In this study, ten potential SCAMP genes distributed in seven soybean chromosomes were identified in the soybean genome. The phylogenetic tree of SCAMP domain sequences of several plants can divide SCAMPs into two groups. Most GmSCAMPs genes contained multiple Box4, MYB and MYC cis-elements indicated they may respond to abiotic stresses. We found that GmSCAMP1, GmSCAMP2 and GmSCAMP4 expressed in several tissues and GmSCAMP5 was significantly induced by salt stress. GmSCAMP5 showed the same expression patterns under NaCl treatment in salt-tolerant and salt-sensitive soybean varieties, but the induced time of GmSCAMP5 in salt-tolerant variety was earlier than that of salt-sensitive variety. To further study the effect of GmSCAMP5 on the salt tolerance of soybean plants, compared to GmSCAMP5-RNAi and EV-Control plants, GmSCAMP5-OE had less wilted leave and higher SPAD value. Compared to empty vector control, less trypan blue staining was observed in GmSCAMP5-OE leaves while more staining in GmSCAMP5-RNAi leaves. The Na+ of GmSCAMP5-RNAi plants leaves under NaCl stress were significantly higher than that in EV-Control plants, while significantly lower Na+ in GmSCAMP5-OE plants than in that EV-Control plants. The contents of leaves K+ of GmSCAMP5-RNAi, EV-Control, and GmSCAMP5-OE plants under NaCl stress were opposite to that of leaves Na+ content. Finally, salt stress-related genes NHX1, CLC1, TIP1, SOD1, and SOS1 in transformed hairy root changed significantly compared with the empty control. The research will provide novel information for study the molecular regulation mechanism of soybean salt tolerance.

A Natural Glucan from Black Bean Inhibits Cancer Cell Proliferation via PI3K-Akt and MAPK Pathway.

A natural α-1,6-glucan named BBWPW was identified from black beans. Cell viability assay showed that BBWPW inhibited the proliferation of different cancer cells, especially HeLa cells. Flow cytometry analysis indicated that BBWPW suppressed the HeLa cell cycle in the G2/M phase. Consistently, RT-PCR experiments displayed that BBWPW significantly impacts the expression of four marker genes related to the G2/M phase, including p21, CDK1, Cyclin B1, and Survivin. To explore the molecular mechanism of BBWPW to induce cell cycle arrest, a transcriptome-based target inference approach was utilized to predict the potential upstream pathways of BBWPW and it was found that the PI3K-Akt and MAPK signal pathways had the potential to mediate the effects of BBWPW on the cell cycle. Further experimental tests confirmed that BBWPW increased the expression of BAD and AKT and decreased the expression of mTOR and MKK3. These results suggested that BBWPW could regulate the PI3K-Akt and MAPK pathways to induce cell cycle arrest and ultimately inhibit the proliferation of HeLa cells, providing the potential of the black bean glucan to be a natural anticancer drug.

WGCNA Reveals Hub Genes and Key Gene Regulatory Pathways of the Response of Soybean to Infection by Soybean mosaic virus.

Soybean mosaic virus (SMV) is one of the main pathogens that can negatively affect soybean production and quality. To study the gene regulatory network of soybeans in response to SMV SC15, the resistant line X149 and susceptible line X97 were subjected to transcriptome analysis at 0, 2, 8, 12, 24, and 48 h post-inoculation (hpi). Differential expression analysis revealed that 10,190 differentially expressed genes (DEGs) responded to SC15 infection. Weighted gene co-expression network analysis (WGCNA) was performed to identify highly related resistance gene modules; in total, eight modules, including 2256 DEGs, were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 2256 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant-pathogen interaction pathway, mitogen-activated protein kinases (MAPK) signaling pathway, and plant hormone signal transduction pathway. Among these pathways, we found that the flg22, Ca2+, hydrogen peroxide (H2O2), and abscisic acid (ABA) regulatory pathways were fully covered by 36 DEGs. Among the 36 DEGs, the gene Glyma.01G225100 (protein phosphatase 2C, PP2C) in the ABA regulatory pathway, the gene Glyma.16G031900 (WRKY transcription factor 22, WRKY22) in Ca2+ and H2O2 regulatory pathways, and the gene Glyma.04G175300 (calcium-dependent protein kinase, CDPK) in Ca2+ regulatory pathways were highly connected hub genes. These results indicate that the resistance of X149 to SC15 may depend on the positive regulation of flg22, Ca2+, H2O2, and ABA regulatory pathways. Our study further showed that superoxide dismutase (SOD) activity, H2O2 content, and catalase (CAT) and peroxidase (POD) activities were significantly up-regulated in the resistant line X149 compared with those in 0 hpi. This finding indicates that the H2O2 regulatory pathway might be dependent on flg22- and Ca2+-pathway-induced ROS generation. In addition, two hub genes, Glyma.07G190100 (encoding F-box protein) and Glyma.12G185400 (encoding calmodulin-like proteins, CMLs), were also identified and they could positively regulate X149 resistance. This study provides pathways for further investigation of SMV resistance mechanisms in soybean.

3-Hydroxy-3-methylglutaryl coenzyme A reductase genes from Glycine max regulate plant growth and isoprenoid biosynthesis.

Isoprenoids, a large kind of plant natural products, are synthesized by the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. As one of the rate-limiting enzymes in the MVA pathway of soybean (Glycine max), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is encoded by eight isogenes (GmHMGR1-GmHMGR8). To begin, we used lovastatin (LOV), a specific inhibitor of GmHMGR, to investigate their role in soybean development. To further investigate, we overexpressed the GmHMGR4 and GmHMGR6 genes in Arabidopsis thaliana. The growth of soybean seedlings, especially the development of lateral roots, was inhibited after LOV treatment, accompanied by a decrease in sterols content and GmHMGR gene expression. After the overexpression of GmHMGR4 and GmHMGR6 in A. thaliana, the primary root length was higher than the wild type, and total sterol and squalene contents were significantly increased. In addition, we detected a significant increase in the product tocopherol from the MEP pathway. These results further support the fact that GmHMGR1-GmHMGR8 play a key role in soybean development and isoprenoid biosynthesis.

Roles of a CCR4-NOT complex component GmNOT4-1 in regulating soybean nodulation.

Legume-rhizobial symbiotic nitrogen fixation is the most efficient nitrogen assimilation system in the ecosystem. In the special interaction between organ-root nodules, legumes supply rhizobial carbohydrates for their proliferation, while rhizobials provide host plants with absorbable nitrogen. Nodule initiation and formation require a complex molecular dialogue between legumes and rhizobia, which involves the accurate regulation of a series of legume genes. The CCR4-NOT complex is a conserved multi-subunit complex with functions regulating gene expression in many cellular processes. However, the functions of the CCR4-NOT complex in rhizobia-host interactions remain unclear. In this study, we identified seven members of the NOT4 family in soybean and further classified them into three subgroups. Bioinformatic analysis showed that NOT4s shared relatively conserved motifs and gene structures in each subgroup, while there were significant differences between NOT4s in the different subgroups. Expression profile analysis indicated that NOT4s may be involved in nodulation in soybean, as most of them were induced by Rhizobium infection and highly expressed in nodules. We further selected GmNOT4-1 to clarify the biological function of these genes in soybean nodulation. Interestingly, we found that either GmNOT4-1 overexpression or down-regulation of GmNOT4-1 by RNAi or CRISPR/Cas9 gene editing would suppress the number of nodules in soybean. Intriguingly, alterations in the expression of GmNOT4-1 repressed the expression of genes in the Nod factor signaling pathway. This research provides new insight into the function of the CCR4-NOT family in legumes and reveals GmNOT4-1 to be a potent gene for regulating symbiotic nodulation.

[Comparative study on FTIR spectra of garlic from different geographical populations].

In the present paper, 25 garlic samples from different geographical populations were studied. FTIR spectra for each sample were obtained by using Fourier transform infrared spectrometer, and the similarity of garlic samples from different geographical populations was compared through "quick comparison" function in software of the spectrometer. The results showed that there are differences among FTIR spectra of garlic samples from different geographical populations. The quick comparison showed that the similarity is from 76.3% to 99.8% and the diversity of differentiation is more obvious. To some extent, the results reflected the effects of populations environment on physical and chemical properties of garlic. The study provided a simple, rapid, non-destructive and new methods for identification and evaluation of garlic germplasm resources.

Analysis of Genetic Regions Related to Field Grain Number per Spike From Chinese Wheat Founder Parent Linfen 5064.

Wheat founder parents have been important in the development of new wheat cultivars. Understanding the effects of specific genome regions on yield-related traits in founder variety derivatives can enable more efficient use of these genetic resources through molecular breeding. In this study, the genetic regions related to field grain number per spike (GNS) from the founder parent Linfen 5064 were analyzed using a doubled haploid (DH) population developed from a cross between Linfen 5064 and Nongda 3338. Quantitative trait loci (QTL) for five spike-related traits over nine experimental locations/years were identified, namely, total spikelet number per spike (TSS), base sterile spikelet number per spike (BSSS), top sterile spikelet number per spike (TSSS), fertile spikelet number per spike (FSS), and GNS. A total of 13 stable QTL explaining 3.91-19.51% of the phenotypic variation were found. The effect of six of these QTL, Qtss.saw-2B.1, Qtss.saw-2B.2, Qtss.saw-3B, Qfss.saw-2B.2, Qbsss.saw-5A.1, and Qgns.saw-1A, were verified by another DH population (Linfen 5064/Jinmai 47), which showed extreme significance (P < 0.05) in more than three environments. No homologs of reported grain number-related from grass species were found in the physical regions of Qtss.saw-2B.1 and Qtss.saw-3B, that indicating both of them are novel QTL, or possess novel-related genes. The positive alleles of Qtss.saw-2B.2 from Linfen 5064 have the larger effect on TSS (3.30%, 0.62) and have 66.89% in Chinese cultivars under long-term artificial selection. This study revealed three key regions for GNS in Linfen 5064 and provides insights into molecular marker-assisted breeding.

The relationship between SARS-COV-2 RNA positive duration and the risk of recurrent positive.

BACKGROUND: The management of discharge COVID-19 patients with recurrent positive SARS-CoV-2 RNA is challenging. However, there are fewer scientific dissertations about the risk of recurrent positive. The aim of this study was to explore the relationship between SARS-COV-2 RNA positive duration (SPD) and the risk of recurrent positive. METHODS: This case-control multi-center study enrolled participants from 8 Chinese hospital including 411 participants (recurrent positive 241). Using unadjusted and multivariate-adjusted logistic regression analyses, generalized additive model with a smooth curve fitting, we evaluated the associations between SPD and risk of recurrent positive. Besides, subgroup analyses were performed to explore the potential interactions. RESULTS: Among recurrent positive patients, there were 121 females (50.2%), median age was 50 years old [interquartile range (IQR): 38-63]. In non-adjusted model and adjusted model, SPD was associated with an increased risk of recurrent positive (fully-adjusted model: OR = 1.05, 95% CI: 1.02-1.08, P = 0.001); the curve fitting was not significant (P = 0.286). Comparing with SPD < 14 days, the risk of recurrent positive in SPD > 28 days was risen substantially (OR = 3.09, 95% CI: 1.44-6.63, P = 0.004). Interaction and stratified analyses showed greater effect estimates of SPD and risk of recurrent positive in the hypertension, low monocyte count and percentage patients (P for interaction = 0.008, 0.002, 0.036, respectively). CONCLUSION: SPD was associated with a higher risk of recurrent positive and especially SPD > 28 day had a two-fold increase in the relative risk of re-positive as compared with SPD < 14 day. What's more, the risk may be higher among those with hypertension and lower monocyte count or percentage.

Favipiravir in the treatment of patients with SARS-CoV-2 RNA recurrent positive after discharge: A multicenter, open-label, randomized trial.

BACKGROUND: The clinical characteristics and treatment of patients who tested positive for COVID-19 after recovery remained elusive. Effective antiviral therapy is important for tackling these patients. We assessed the efficacy and safety of favipiravir for treating these patients. METHODS: This is a multicenter, open-label, randomized controlled trial in SARS-CoV-2 RNA re-positive patients. Patients were randomly assigned in a 2:1 ratio to receive either favipiravir, in addition to standard care, or standard care alone. The primary outcome was time to achieve a consecutive twice (at intervals of more than 24 h) negative RT-PCR result for SARS-CoV-2 RNA in nasopharyngeal swab and sputum sample. RESULTS: Between March 27 and May 9, 2020, 55 patients underwent randomization; 36 were assigned to the favipiravir group and 19 were assigned to the control group. Favipiravir group had a significantly shorter time from start of study treatment to negative nasopharyngeal swab and sputum than control group (median 17 vs. 26 days); hazard ratio 2.1 (95% CI [1.1-4.0], p = 0.038). The proportion of virus shedding in favipiravir group was higher than control group (80.6% [29/36] vs. 52.6% [10/19], p = 0.030, respectively). C-reactive protein decreased significantly after treatment in the favipiravir group (p = 0.016). The adverse events were generally mild and self-limiting. CONCLUSION: Favipiravir was safe and superior to control in shortening the duration of viral shedding in SARS-CoV-2 RNA recurrent positive after discharge. However, a larger scale and randomized, double-blind, placebo-controlled trial is required to confirm our conclusion.

Colorimetric detection of class A soybean saponins by coupling DNAzyme with the gap ligase chain reaction.

Class A saponins are responsible for the taste of soybean products, and the rapid identification of class A saponins from soybean food is essential for both food safety and cultivar screening. In this study, we propose a colorimetric assay based on the coupling of gap ligase chain reaction (Gap-LCR) with DNAzyme to detect the target GmSg-1 genes of class A soybean saponins with the naked eye, without the involvement of expensive instruments. The limits of detection (LODs) for the GmSg-1a and GmSg-1b genes were determined to be 0.1618 and 0.1625 µM, respectively, with a linear range of 0.2-1.2 µM. The DNAzyme-based Gap LCR assay was successfully employed to identify the target genes from different soybean cultivars, providing a simple means for monitoring the quality of soybean products.

Colorimetric Detection of Class A Soybean Saponins by G-Quadruplex-Based Hybridization Chain Reaction.

Soybean saponin is one of the important secondary metabolites in seeds, which has various beneficial physiological functions to human health. GmSg-1 gene is the key enzyme gene for synthesizing class A saponins. It is of great significance to realize the visual and rapid detection of class A saponins at the genetic level. The hybridization chain reaction (HCR) was employed to the visual detection of GmSg-1 gene, which was implemented by changing the length of the target fragment to 92 bp and using the hairpin probes we designed to detect the GmSg-1 a and GmSg-1 b genes. The best condition of HCR reaction is hemin (1.2 µM), Triton X-100 (0.002%), ABTS (3.8 µM), and H2O2 (1.5 mM). It was found that HCR has high specificity for GmSg-1 gene and could be applied to the visual detection of different soybean cultivars containing Aa type, Ab type, and Aa/Ab type saponins, which could provide technical reference and theoretical basis for molecular breeding of soybean and development of functional soybean products.

Detection of quantitative trait loci for yield and drought tolerance traits in soybean using a recombinant inbred line population.

To investigate the genetic basis of drought tolerance in soybean (Glycine max L. Merr.) a recombinant inbred population with 184 F(2:7:11) lines developed from a cross between Kefeng1 (drought tolerant) and Nannong1138-2 (drought sensitive) were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci (QTLs) were identified: 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and D1b, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.

Mapping QTLs for seed yield and drought susceptibility index in soybean (Glycine max L.) across different environments.

Drought stress has long been a major constraint in maintaining yield stability of soybean (Glycine max (L.) Merr.) in rainfed ecosystems. The identification of consistent quantitative trait loci (QTL) involving seed yield per plant (YP) and drought susceptibility index (DSI) in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F(2:7:11) lines from a cross of Kefeng1 and Nannong1138-2 was studied under water-stressed (WS) and well-watered (WW) conditions in field (F) and greenhouse (G) trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identified. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits related to yield and other agronomic traits. One QTL on molecular linkage group (MLG) K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.