A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Overexpression of a Cinnamyl Alcohol Dehydrogenase-Coding Gene, GsCAD1, from Wild Soybean Enhances Resistance to Soybean Mosaic Virus.

Soybean mosaic virus (SMV) is the most prevalent soybean viral disease in the world. As a critical enzyme in the secondary metabolism of plants, especially in lignin synthesis, cinnamyl alcohol dehydrogenase (CAD) is widely involved in plant growth and development, and in defense against pathogen infestation. Here, we performed RNAseq-based transcriptome analyses of a highly SMV-resistant accession (BYO-15) of wild soybean (Glycine soja) and a SMV-susceptible soybean cultivar (Williams 82), also sequenced together with a resistant plant and a susceptible plant of their hybrid descendants at the F3 generation at 7 and 14 days post-inoculation with SMV. We found that the expression of GsCAD1 (from G. soja) was significantly up-regulated in the wild soybean and the resistant F3 plant, while the GmCAD1 from the cultivated soybean (G. max) did not show a significant and persistent induction in the soybean cultivar and the susceptible F3 plant, suggesting that GsCAD1 might play an important role in SMV resistance. We cloned GsCAD1 and overexpressed it in the SMV-susceptible cultivar Williams 82, and we found that two independent GsCAD1-overexpression (OE) lines showed significantly enhanced SMV resistance compared with the non-transformed wild-type (WT) control. Intriguingly, the lignin contents in both OE lines were higher than the WT control. Further liquid chromatography (HPLC) analysis showed that the contents of salicylic acid (SA) were significantly more improved in the OE lines than that of the wild-type (WT), coinciding with the up-regulated expression of an SA marker gene. Finally, we observed that GsCAD1-overexpression affected the accumulation of SMV in leaves. Collectively, our results suggest that GsCAD1 enhances resistance to SMV in soybeans, most likely by affecting the contents of lignin and SA.

Previous School Bullying-Associated Depression in Chinese College Students: The Mediation of Personality.

Previous school bullying was associated with increased risk of depression in students. However, little was known about the role of the Big Five personality traits in this association. The purpose of this study was to investigate the possible mediation by the Big Five personality traits in this association in a large group of Chinese college students, and to provide help for educators to prevent students from serious psychological and mental diseases caused by school bullying. Random stratified cluster sampling was used to survey 2152 college students ranging from freshmen to seniors at three universities in Qiqihar city, Heilongjiang Province, China. The risk factors for previous school bullying included gender, living expenses per month, caregivers, parents often quarreling, and divorced parents. Males were more likely to be bullied at school than females. The influencing factors of depression include gender, caregivers, living expenses per month, frequent parents quarreling, and parental divorce. Females were more prone to depression than males. Depression was significantly correlated with all dimensions of school bullying and the Big Five personality traits (p < 0.05). The Big Five personality traits were found to play a significant mediating role between depression and school bullying in up to 45% of cases involving depression. Our major findings highlighted the promising role of personality-based intervention measures in reducing the risk of depression associated with school bullying in Chinese students.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

Functional activation of a novel R2R3-MYB protein gene, GmMYB68, confers salt-alkali resistance in soybean (Glycine max L.).

Soil salinity significantly reduces soybean (Glycine max L.) production worldwide. Plants resistance to stress conditions is a complex characteristic regulated by multiple signaling pathways. The v-Myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor (TF) plays a crucial role in plant development, secondary metabolism, and abiotic stress responses. GmMYB68-overexpression and RNA interference (RNAi) lines were established for examining the function of G. max GmMYB68 in plant responses to abiotic stresses. The predicted amino acid sequence of GmMYB68 was similar to that of R2R3-MYB proteins. Quantitative real-time PCR analysis revealed that GmMYB68 expression varied in response to abiotic stresses. GmMYB68-overexpression lines showed enhanced resistance to salt and alkali stresses and their osmotic adjustment and photosynthetic rates were also stronger than that of GmMYB68-RNAi and wild type plants. Although wild type and transgenic plants showed no significant differences in agronomic traits under normal conditions, the overexpression of GmMYB68 increased grain number and 100-grain weights under salt stress. Our study identified a valuable TF associated with stress response in soybean, as its overexpression might help improve salt and alkali tolerance in soybean and other crops.

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the ß-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean.

KEY MESSAGE: Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302 bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

Sleep-related factors and work-related injuries among farmers in Heilongjiang Province, People's Republic of China.

The association between sleep and work-related injuries among Chinese farmers has not been well studied. This study examined the impact of lack of sleep on agricultural work-related injuries among farmers in China. Data were from a cross-sectional survey of farm-workers in northeastern China. Information was obtained on injuries that occurred in 12 months prior to the survey, on eight sleep-related variables, and on socio-demographic variables. Logistic regression analyses were conducted to test the hypothesis that lack of sleep significantly increased the risk of work-related injuries after controlling for other injury-related risk- factors. Farmers who slept less than six hours per night were 59% more likely to be injured than those who slept more than eight hours per night (OR = 1.59; 95% CI = 1.04, 2.41). The odds of a work-related injury was 2.46 (1.56-3.89) for farmers who reported going to sleep after midnight at least once a week compared with farmers who reported going to sleep after midnight once a month. Farmers who reported having difficulty falling asleep or waking frequently during the night, who often having nightmares, or who experienced daytime sleepiness were at higher injury risk compared with the reference group after controlling for age, gender and alcohol consumption. Reduced sleep hours and poor sleep quality significantly increased the risk of work-related injuries in Chinese farmers. Sleep hours and sleep quality should be considered when assessing occupational safety among farmers.

Synthesis and characterization of Co-SBA-15 via rapid ultrasonic technique and their catalytic properties.

The rapid preparation of SBA-15 with cobalt (Co) introduction was performed via the ultrasonic irradiation in combination with "pH-adjusting" method. The catalytic properties of the synthesized Co-SBA-15 were investigated by examining the oxidation of styrene with hydrogen peroxide. The effect of pH values on the textural properties were extensively investigated using small-angle X-ray Diffraction (XRD), N2 adsorption-desorption isotherms and Transmission Electron Microscipy (TEM). The characterization results showed that the incorporation of Co by ultrasonic method did not destroy the mesoporous structure of SBA-15. The Co-SBA-15 catalyst with Co/Si (the molar ratio of 0.03) at pH of 7.5 exhibited a well-ordered hexagonal mesoporous structure with higher surface area and pore volume. This catalyst had excellent styrene conversion and selectivity to benzaldehyde of 21.8% and 92.3%, respectively. The physicochemical properties and catalytic activity of the Co-SBA-15 catalysts via ultrasonic technique possessed the comparable characteristics as those prepared via conventional hydrothermal method.

Isolation of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing Isochrysis galbana H29 and its use for the reconstitution of the alternative Δ8 pathway in Saccharomyces cerevisiae.

A novel gene (IgASE2) encoding a C18-Δ9 polyunsaturated fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, Isochrysis galbana H29. The IgASE2 gene was 1,653 bp in length, contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with Δ9 elongase, IgASE1, and possessed a 44 bp 5'-untranslated region (5'-UTR) and a 823 bp 3'-untranslated region (3'-UTR). IgASE2, by its heterologous expression in Saccharomyces cerevisiae, elongated linoleic acid (LA, 18:2n-6) and α-linolenic (ALA, 18:3n-3) to eicosadienoic acid (EDA, 20:2n-6) and eicosatrienoic acid (ETrA, 20:3n-3), respectively. The conversions of LA to EDA and ALA to ETrA were 57.6 and 56.1%, respectively. Co-expression of this elongase with Δ8 desaturase required for the synthesis of C20-polyunsaturated fatty acids resulted in the accumulation of dihomo-γ-linolenic acid (20:3n-6) from LA and eicosatetraenoic acid (20:4n-6) from ALA. These results demonstrated that IgASE2 exhibited C18-Δ9-PUFAs-specific elongase activity and the alternative Δ8 pathway was reconstituted.