Generation and Identification of the Number of Copies of Exogenous Genes and the T-DNA Insertion Site in SCN-Resistance Transformation Event ZHs1-2.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) causes an estimated economic loss of about USD 3 billion each year in soybean (Glycine max L.) production worldwide. Overexpression of resistance genes against SCN provides a powerful approach to develop SCN resistance cultivars in soybean. The clarification of molecular characterization in transformation events is a prerequisite for ecological risk assessment, food safety, and commercial release of genetically modified crops. Here, we generated transgenic events harboring the BCN (beet cyst nematode) resistance Hs1pro-1 gene using the Agrobacterium-mediated method in soybean, evaluated their resistance to SCN infection, and clarified the molecular characterization of one of the transformation events. Five independent and stable inheritable transformation events were generated by an Agrobacterium-mediated transformation method. SCN resistance tests showed the average number of developed females per plant and female index (FI) in T4 ZHs1-1, ZHs1-2, ZHs1-3, ZHs1-4, and ZHs1-5 transformation events were significantly lower than that in the nontransgenic control. Among these, the ZHs1-2 transformation event had the lowest number of developed females per plant and FI. Southern hybridization showed the exogenous target Hs1pro-1 gene was inserted in one copy and the Bar gene was inserted two copies in the ZHs1-2 transformation event. The exogenous T-DNA fragment was integrated in the reverse position of Chr02: 5351566-5231578 (mainly the Bar gene expression cassette) and in the forward position of Chr03: 17083358-17083400 (intact T-DNA, including Hs1pro-1 and Bar gene expression cassette) using a whole genome sequencing method (WGS). The results of WGS method and Southern hybridization were consistent. All the functional elements of exogenous T-DNA fragments were verified by PCR using specific primer pairs in the T5 and T6 ZHs1-2 transformation events. These results demonstrated that the overexpression of Hs1pro-1 gene enhanced SCN resistance, and provide an important reference for the biosafety assessment and the labeling detection in transformation event ZHs1-2.

Wheat grain protein accumulation and polymerization mechanisms driven by nitrogen fertilization.

In wheat (Triticum aestivum) grain yield and grain protein content are negatively correlated, making the simultaneous increase of the two traits challenging. Apart from genetic approaches, modification of nitrogen fertilization offers a feasible option to achieve this aim. In this study, a range of traits related to nitrogen-use efficiency in six Australian bread wheat varieties were investigated under different nitrogen treatments using 3-year multisite field trials. Changes in the individual storage protein composition were detected by high-performance liquid chromatography. Our results indicated that wheat grain yield and grain protein content reacted similarly to nitrogen availability, with grain yield being slightly more sensitive than grain protein content, and that genotype is a vital determinant of grain protein yield. Measurement of the glutamine synthetase activity of flag leaves and developing grains revealed that high nitrogen availability prompted the participation of glutamine in biological processes. In addition, a more significant accumulation of gluten macropolymer was observed under the high-nitrogen treatment from 21 days post-anthesis, and the underlying mechanism was elucidated by a comparative proteomics study. A yeast two-hybrid experiment confirmed this mechanism. The results of this study revealed that peptidyl-prolyl cis-trans isomerase (PPIase) was SUMOylated with the assistance of small ubiquitin-related modifier 1 and that high nitrogen availability facilitated this connection for the subsequent protein polymerization. Additionally, luminal-binding protein 2 in the endoplasmic reticulum played a similar role to PPIase in the aggregation of protein under high-nitrogen conditions.

BvcZR3 and BvHs1pro-1 Genes Pyramiding Enhanced Beet Cyst Nematode (Heterodera schachtii Schm.) Resistance in Oilseed Rape (Brassica napus L.).

Beet cyst nematode (Heterodera schachtii Schm.) is one of the most damaging pests in sugar beet growing areas around the world. The Hs1pro-1 and cZR3 genes confer resistance to the beet cyst nematode, and both were cloned from sugar beet translocation line (A906001). The translocation line carried the locus from B. procumbens chromosome 1 including Hs1pro-1 gene and resistance gene analogs (RGA), which confer resistance to Heterodera schachtii. In this research, BvHs1pro-1 and BvcZR3 genes were transferred into oilseed rape to obtain different transgenic lines by A. tumefaciens mediated transformation method. The cZR3Hs1pro-1 gene was pyramided into the same plants by crossing homozygous cZR3 and Hs1pro-1 plants to identify the function and interaction of cZR3 and Hs1pro-1 genes. In vitro and in vivo cyst nematode resistance tests showed that cZR3 and Hs1pro-1 plants could be infested by beet cyst nematode (BCN) juveniles, however a large fraction of penetrated nematode juveniles was not able to develop normally and stagnated in roots of transgenic plants, consequently resulting in a significant reduction in the number of developed nematode females. A higher efficiency in inhibition of nematode females was observed in plants expressing pyramiding genes than in those only expressing a single gene. Molecular analysis demonstrated that BvHs1pro-1 and BvcZR3 gene expressions in oilseed rape constitutively activated transcription of plant-defense related genes such as NPR1 (non-expresser of PR1), SGT1b (enhanced disease resistance 1) and RAR1 (suppressor of the G2 allele of skp1). Transcript of NPR1 gene in transgenic cZR3 and Hs1pro-1 plants were slightly up-regulated, while its expression was considerably enhanced in cZR3Hs1pro-1 gene pyramiding plants. The expression of EDS1 gene did not change significantly among transgenic cZR3, Hs1pro-1 and cZR3Hs1pro-1 gene pyramiding plants and wild type. The expression of SGT1b gene was slightly up-regulated in transgenic cZR3 and Hs1pro-1 plants compared with the wild type, however, its expression was not changed in cZR3Hs1pro-1 gene pyramiding plant and had no interaction effect. RAR1 gene expression was significantly up-regulated in transgenic cZR3 and cZR3Hs1pro-1 genes pyramiding plants, but almost no expression was found in Hs1pro-1 transgenic plants. These results show that nematode resistance genes from sugar beet were functional in oilseed rape and conferred BCN resistance by activation of a CC-NBS-LRR R gene mediated resistance response. The gene pyramiding had enhanced resistance, thus offering a novel approach for the BCN control by preventing the propagation of BCN in oilseed rape. The transgenic oilseed rape could be used as a trap crop to offer an alternative method for beet cyst nematode control.

Physiological Regulation of Photosynthetic-Related Indices, Antioxidant Defense, and Proline Anabolism on Drought Tolerance of Wild Soybean (Glycine soja L.).

Wild soybean (Glycine soja L.), drought-tolerant cultivar Tiefeng 31 (Glycine max L.), and drought-sensitive cultivar Fendou 93 (Glycine max L.) were used as materials to investigate the drought tolerance mechanism after 72 h 2.5 M PEG 8000 (osmotic potential -0.54 MPa)-simulated drought stress at the seedling stage. The results indicated that the leaves of the G. soja did not wilt under drought stress. However, both the drought-tolerant and drought-sensitive cultivated soybean cultivars experienced varying degrees of leaf wilt. Notably, the drought-sensitive cultivated soybean cultivars exhibited severe leaf wilt after the drought stress. Drought stress was determined to have a significant impact on the dry matter of the above-ground part of the drought-sensitive cultivar Fendou 93, followed by the drought-tolerant cultivar Tiefeng 31, with the lowest reduction observed in G. soja. Furthermore, the presence of drought stress resulted in the closure of leaf stomata. G. soja exhibited the highest proportion of stomatal opening per unit area, followed by the drought-tolerant cultivar Tiefeng 31, while the drought-sensitive cultivar Fendou 93 displayed the lowest percentage. Photosynthesis-related indexes, including photosynthetic rate, intercellular CO2, transpiration rate, and stomatal conductance, decreased in Fendou 93 and Tiefeng 31 after drought stress, but increased in G. soja. In terms of the antioxidant scavenging system, lower accumulation of malondialdehyde (MDA) was observed in G. soja and Tiefeng 31, along with higher activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) to counteract excess reactive oxygen species and maintain cell membrane integrity. In contrast, the drought-sensitive cultivar Fendou 93 had higher MDA content and higher activities of ascorbate peroxidase (APX, EC 1.11.1.11) and peroxidase (POD, 1.11.1.7). G. soja and Tiefeng 31 also exhibited less accumulation of osmolytes, including soluble sugar, soluble protein, and free proline content. The activities of δ-OAT, ProDH, and P5CS, key enzymes in proline anabolism, showed an initial increase under drought stress, followed by a decrease, and then an increase again at the end of drought stress in G. soja. Before drought stress, Tiefeng 31 had higher activities of ProDH and P5CS, which decreased with prolonged drought stress. Fendou 93 experienced an increase in the activities of δ-OAT, ProDH, and P5CS under drought stress. The δ-OAT gene expression levels were up-regulated in all three germplasms. The expression levels of the P5CS gene in Fendou 93 and Tiefeng 31 were down-regulated, while G. soja showed no significant change. The expression of the P5CR gene and ProDH gene was down-regulated in Fendou 93 and Tiefeng 31, but up-regulated in G. soja. This indicates that proline content is regulated at both the transcription and translation levels.

Evaluation of liver function in patients with liver cirrhosis and chronic liver disease using functional liver imaging scores at different acquisition time points.

Purpose: This paper aims to explore whether functional liver imaging score (FLIS) based on Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) images at 5, 10, and 15 min can predict liver function in patients with liver cirrhosis or chronic liver disease and its association with indocyanine green 15-min retention rate (ICG-R15), Child-Pugh (CP) score, albumin-bilirubin (ALBI) score, and model for end-stage liver disease (MELD) score. In addition, it also examines the inter- and intra-observer consistency of FLIS and three FLIS parameters at three different time points. Methods: This study included 110 patients with chronic liver disease (CLD) or liver cirrhosis (LC) (93 men, 17 women; mean ± standard deviation = 56.96 ± 10.16) between July 2019 and May 2022. FLIS was assigned in accordance with the sum of the three hepatobiliary phase characteristics, all of which were scored on the 0-2 ordinal scale, including the biliary excretion, hepatic enhancement and portal vein signal intensity. FLIS was calculated independently by two radiologists using transitional and hepatobiliary phase images at 5, 10, and 15 min after enhancement. The relationship between FLIS and three FLIS quality scores and the degree of liver function were evaluated using Spearman's rank correlation coefficient. The ability of FLIS to predict hepatic function was investigated using receiver operating characteristic (ROC) curves. Results: Intra- and inter-observer intraclass correlation coefficients (ICCs) (ICC = 0.937-0.978, 95% CI = 0.909-0.985) for FLIS at each time point indicated excellent agreement. At each time point, FLIS had a moderate negative association with liver function classification (r = [-0.641]-[-0.428], p < 0.001), and weak to moderate correlation with some other clinical parameters except for creatinine (p > 0.05). FLIS showed moderate discriminatory ability between different liver function levels. The area under the ROC curves (AUCs) of FLIS at 5, 10, and 15 min after enhancement to predict ICG-R15 of 10% or less were 0.838, 0.802, and 0.723, respectively; those for predicting ICG-R15 greater than 20% were 0.793, 0.824, and 0.756, respectively; those for predicting ICG-R15 greater than 40% were 0.728, 0.755, and 0.741, respectively; those for predicting ALBI grade 1 were 0.734, 0.761, and 0.691, respectively; those for predicting CP class A cirrhosis were 0.806, 0.821, and 0.829, respectively; those for predicting MELD score of 10 or less were 0.837, 0.877, and 0.837, respectively. No significant difference was found in the AUC of FLIS at 5, 10 and 15 min (p > 0.05). Conclusion: FLIS presented a moderate negative correlation with the classification system of hepatic function at a delay of 5, 10, and 15 min, and patients with LC or CLD were appropriately stratified based on ICG-R15, ALBI grade, MELD score, and CP classification. In addition, the use of FLIS to evaluate liver function can reduce the observation time of the hepatobiliary period.

CRISPR/Cas9 mediated gene-editing of GmHdz4 transcription factor enhances drought tolerance in soybean (Glycine max [L.] Merr.).

The HD-Zip transcription factors play a crucial role in plant development, secondary metabolism, and abiotic stress responses, but little is known about HD-Zip I genes in soybean. Here, a homeodomain-leucine zipper gene designated GmHdz4 was isolated. Chimeric soybean plants, GmHdz4 overexpressing (GmHdz4-oe), and gene-editing via CRISPR/Cas9 (gmhdz4) in hairy roots, were generated to examine the GmHdz4 gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed GmHdz4 belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as AtHB12, Oshox12, and Gshdz4. The GmHdz4 was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that GmHdz4 expression varied in tissues and was induced by PEG-simulated drought stress. The gmhdz4 showed promoted growth of aboveground parts, and its root system architecture, including the total root length, the root superficial area, and the number of root tips were significantly higher than those of GmHdz4-oe even the non-transgenic line (NT) on root tips number. The better maintenance of turgor pressure by osmolyte accumulation, and the higher activity of antioxidant enzymes to scavenge reactive oxygen species, ultimately suppressed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2-), and malondialdehyde (MDA), conferring higher drought tolerance in gmhdz4 compared with both GmHdz4-oe and NT. Together, our results provide new insights for future research on the mechanisms by which GmHdz4 gene-editing via CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.

Impact and mechanism of sulphur-deficiency on modern wheat farming nitrogen-related sustainability and gliadin content.

Two challenges that the global wheat industry is facing are a lowering nitrogen-use efficiency (NUE) and an increase in the reporting of wheat-protein related health issues. Sulphur deficiencies in soil has also been reported as a global issue. The current study used large-scale field and glasshouse experiments to investigate the sulphur fertilization impacts on sulphur deficient soil. Here we show that sulphur addition increased NUE by more than 20% through regulating glutamine synthetase. Alleviating the soil sulphur deficiency highly significantly reduced the amount of gliadin proteins indicating that soil sulphur levels may be related to the biosynthesis of proteins involved in wheat-induced human pathologies. The sulphur-dependent wheat gluten biosynthesis network was studied using transcriptome analysis and amino acid metabolomic pathway studies. The study concluded that sulphur deficiency in modern farming systems is not only having a profound negative impact on productivity but is also impacting on population health.

[Studies on the seed embryo germination and propagation of Dendrobium candidum in vitro].

OBJECTIVE: To determine optimum culture conditions for the seed embryo culture and rapid propagation of Dendrobium candidum. METHOD: Seed embryos of D. candidum were incubated in the medium containing a combination of 6-benzylaminopurine (BA) and 1-naphthaleneacetic acid (NAA), potato extract, banana extract and activated carbon in order to induce seed embryo germination, protocorm differentiation, plantlet propagation and plantlet rooting. RESULT AND CONCLUSION: The maximum embryo germination percentage was obtained in the 1/2 MS media supplemented with 20% potato extract. The 1/2 MS medium supplemented with 1.0 mg x L(-1) BA and 0.1 mg x L(-1) NAA was very beneficial to the protocorm differentiation and propagation of D. candidum. The highest protocorm propagation index was obtained from the medium containing the activated carbon. The highest root numbers and length were observed in plants growing in 1/2 MS medium containing 0.5 mg x L(-1) NAA.

Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

[Effects of modafinil on visual and auditory reaction abilities and subjective fatigue level during 48 h sleep deprivation].

OBJECTIVE: To investigate the effects of modafinil, a new central stimulant, on visual and auditory motion reaction ability and subjective fatigue level during 48 h sleep deprivation (SD). METHOD: Six male healthy young volunteers were exposed to two 48 h periods of continuous wakefulness during the crossover experiment. In one period, three 200 mg doses of modafinil were given and in the other, separated by two weeks, matching placebos were administered. The SD time started from 7:00 of the first day to 7:00 of the third day. Drugs were given at 0:00, 16:00 of the second day and 0:00 of the third day. Variables, including visual and auditory motion reaction time, attention distribution, critical flicker fusion frequency (CFF), Stanford sleepiness scale (SSS) and rating of perceived exertion (RPE), were tested at 21:00 of the first day and 1, 3, 5, 7 h after each drug administration. RESULT: After 2-3 does of modafinil, the CFF level was obviously enhanced while the scores of Stanford sleepiness scale and RPE were markedly reduced. The visual and auditory motion reaction ability, as well as the attention distribution, showed no significant change. CONCLUSION: Modafinil can effectively reduced the subjective fatigue and sleepiness levels during SD.

Preliminary studies on the structure-activity and time-effect relations of the central stimulating effects of modafinil in mice.

OBJECTIVE: To observe the structure-activity and time-effect relations of the central stimulating effects of modafinil for further pharmaceutical design and development and rational use of this kind of stimulant. METHOD: Male mice served as subjects. The experiment was divided into two parts: 1) comparison between the dose (60, 120, 240 mg/kg) and temporal features of the effects of modafinil and its two derivatives on locomotor activity of mice; 2) Observation of the stimulating effects of modafinil (120 mg/kg) given during the day or night. RESULT: Modafinil could significantly increase the locomotor activity of mice in a dose-dependent way, the effect was best observed at the concentration of 120 mg/kg. The stimulating action of modafinil was increased by chlorination, but decreased by methylation. The central stimulating effect of modafinil showed no significant circadian difference. CONCLUSION: The central stimulating effect of modafinil shows dose and time-related features. The effect tends to be enhanced by Chlorination but reduced by methylation.

Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability.

The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and ß-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants.

[Seed germination characteristics of parasitic plant and its host recognition mechanisms].

Parasitic plants are widely distributed in various ecological environments, with different growth habits and host recognition mechanisms. This paper discussed the distinctive seed germination characteristics of root parasitic plants such as Orobanche and Striga, summarized the signals for parasitic seed germination discovered up to now, and reviewed the effects of various germination signals, plant hormones and several fungal metabolites on the host recognition of parasitic plants, as well as the respiration characteristics during the conditioning, and the activating mechanism of the signals for parasitic seed germination. The induction of various differentiated calli in different Orobanche species, and the establishment of novel in vitro aseptic infection system and its application in the host recognition of parasitic plants were also discussed, with the present problems in researching the recognition mechanisms between parasitic plants and hosts put forward, and the further work prospected.

[Gene flow and its ecological risks of transgenic oilseed rape ( Brassica napus)].

Transgenic oilseed rape Brassica napus, one of the first genetically modified crops, has now been released to commercial use in Canada and Australia. As a cross-pollinating crop, its natural crossing rate is 30%, and it is liable to cross with other Brassica species. The ecological risk of transgenic oilseed rape has been concerned by the scientists all over the world. There are two ways for the pollens flow of transgenic oilseed rape, one takes place between transgenic oilseed rape and other related wild species, and the other occurs between transgenic and nontransgenic oilseed rape. The gene may flow to other related wild species, but it is unlikely to get hybrids in field. Because the gene can really flow to the conventional oilseed rape, it is necessary to have a sufficient isolation distance in cultivating transgenic oilseed rape.