[Improving the production of plant-based recombinant protein: a review].

Recombinant proteins provide new means for disease treatment, while creating considerable economic benefits. Using commercial crops (mainly tobacco), cereal crops, legumes, and vegetable crops to produce recombinant proteins with medicinal value is a hot-spot for research in "molecular farming". Although many recombinant proteins have been expressed in plants, only a small number have been successfully put into use. To overcome the problems that greatly hamper the development of recombinant protein production in plants, researchers have improved expression systems to increase the yield of recombinant proteins. Starting from analyzing the problems of low yield and/or low biological activity of recombinant proteins produced by plants, the optimization strategies to solve these problems were reviewed, and future research directions for improving the yield of recombinant proteins produced by plants were proposed.

A high-quality genome assembly of Jasminum sambac provides insight into floral trait formation and Oleaceae genome evolution.

As one of the most economically significant Oleaceae family members, Jasminum sambac is renowned for its distinct sweet, heady fragrance. Using Illumina reads, Nanopore long reads, and HiC-sequencing, we efficiently assembled and annotated the J. sambac genome. The high-quality genome assembly consisted of a total of 507 Mb sequence (contig N50 = 17.6 Mb) with 13 pseudomolecules. A total of 21,143 protein-coding genes and 303 Mb repeat sequences were predicted. An ancient whole-genome triplication event at the base of Oleaceae (~66 million years ago [Ma], Late Cretaceous) was identified and this may have contributed to the diversification of the Oleaceae ancestor and its divergence from the Lamiales. Stress-related (e.g., WRKY) and flowering-related (e.g., MADS-box) genes were located in the triplicated regions, suggesting that the polyploidy event might have contributed adaptive potential. Genes related to terpenoid biosynthesis, for example, FTA and TPS, were observed to be duplicated to a great extent in the J. sambac genome, perhaps explaining the strong fragrance of the flowers. Copy number changes in distinct phylogenetic clades of the MADS-box family were observed in J. sambac genome, for example, AGL6- and Mα- were lost and SOC- expanded, features that might underlie the long flowering period of J. sambac. The structural genes implicated in anthocyanin biosynthesis were depleted and this may explain the absence of vivid colours in jasmine. Collectively, assembling the J. sambac genome provides new insights into the genome evolution of the Oleaceae family and provides mechanistic insights into floral properties.

Retraction to using a deep recurrent neural network with EEG signal to detect Parkinson's disease.

[This retracts the article DOI: 10.21037/atm-20-5100.].

RNA sequencing reveals transcriptomic changes in tobacco (Nicotiana tabacum) following NtCPS2 knockdown.

BACKGROUND: Amber-like compounds form in tobacco (Nicotiana tabacum) during leaf curing and impact aromatic quality. In particular, cis-abienol, a polycyclic labdane-related diterpenoid, is of research interest as a precursor of these compounds. Glandular trichome cells specifically express copalyl diphosphate synthase (NtCPS2) at high levels in tobacco, which, together with NtABS, are major regulators of cis-abienol biosynthesis in tobacco. RESULTS: To identify the genes involved in the biosynthesis of cis-abienol in tobacco, we constructed transgenic tobacco lines based on an NtCPS2 gene-knockdown model using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis-abienol and labdene diol contents decreased, whereas the gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9514 differentially expressed genes (DEGs; 4279 upregulated, 5235 downregulated) when the leaves of wild-type and NtCPS2-knockdown tobacco plants were screened. Among these DEGs, the genes encoding cis-abienol synthase, ent-kaurene oxidase, auxin/ABA-related proteins, and transcription factors were found to be involved in various biological and physiochemical processes, including diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interactions. CONCLUSIONS: The present study provides insight into the unique transcriptome profile of NtCPS2 knockdown tobacco, allowing for a better understanding of the biosynthesis of cis-abienol in tobacco.

Overexpression of NtDOG1L-T Improves Heat Stress Tolerance by Modulation of Antioxidant Capability and Defense-, Heat-, and ABA-Related Gene Expression in Tobacco.

Drought and heat stresses are two major environmental stress factors that severely threaten crop growth and productivity. Plant delay of germination 1-like (DOG1L) family genes play important roles in various developmental processes and stress responses. In our previous study, a tobacco DOG1L gene (NtDOG1L-T) was found to regulate seedling growth and drought response. Unfortunately, the role of DOG1L genes in heat stress response is yet to be studied. Here, we present data supporting the role of DOG1L genes in heat stress and possible underlying molecular mechanisms. Transcript levels of NtDOG1L-T were rapidly induced by heat or abscisic acid (ABA) treatment. Furthermore, NtDOG1L-T promoter activity was markedly activated by ABA or heat stress, as revealed by histochemical staining in transgenic tobacco seedlings. Overexpression of NtDOG1L-T in transgenic lines improved heat stress tolerance. The NtDOG1L-T-transgenic plants exhibited lower levels of reactive oxygen species (ROS) and lipid peroxidation but higher antioxidant enzyme activities in response to heat stress. Furthermore, transcript abundance of some defense-, heat-, and ABA-responsive marker genes was significantly upregulated, as shown by reverse transcription quantitative PCR (qPCR) in these transgenic plants. In conclusion, NtDOG1L-T positively regulates heat stress tolerance possibly by modulation of antioxidant capability and defense-, heat-, and ABA-related gene expression in tobacco. This study may provide valuable resource for the potential exploitation of DOG1Ls in genetic improvement of heat stress tolerance in crops.

Using a deep recurrent neural network with EEG signal to detect Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) gradually degrades the functionality of the brain. Because of its relevance to the abnormality of the brain, electroencephalogram (EEG) signal is used for the early detection of this disease. This paper introduces a novel computer-aided diagnosis method to detect PD, which is an efficient deep learning method based on a pooling-based deep recurrent neural network (PDRNN). Therefore, the purpose of this study is to detect Parkinson's disease based on deep recurrent neural network of EEG signal. METHODS: The EEG signals of 20 patients with Parkinson's disease and 20 healthy people in Henan Provincial People's Hospital (People's Hospital of Zhengzhou University) were examined, and a PDRNN learning method was applied on the dataset for managing the demand of the traditional feature presentation step. RESULTS: The suggested DPRNN network gives the precision, sensitivity and specificity of 88.31%, 84.84% and 91.81%, respectively. Nevertheless, 11.28% of the healthy cases are wrongly categorized in Parkinson class. Also, 11.49% percent of Parkinson cases are classified wrongly in the healthy class. CONCLUSIONS: The experimental model has high efficiency and can be used as a reliable tool for clinical PD detection. In future research, more cases should be used to test and develop the proposed model.

Activation of potassium released from soil by root-secreted organic acids in different varieties of tobacco (Nicotiana tabacum).

Organic acids secreted from the roots of plants play important roles in nutrient acquisition and metal detoxification; however, the precise underlying mechanisms of these processes remain poorly understood. In the present study we examined the content of organic acids exuded from roots and the effects of these organic acids on the activation of slowly available potassium (K) at different K levels, including normal K supply and K-deficient conditions. In addition, the study system also comprised a high-K tobacco variety (ND202) and two common ones (K326 and NC89). Our results showed that high-K varieties exhibited significantly higher contents of organic acids in its root exudates and available K in both rhizosphere and non-rhizosphere soils than the other varieties. This research also suggested that a cyclic process in which soil was acidified after being complexed by organic acids was involved in the release of slowly available K, and that this process primarily depended on the soil pH at high organic acids concentrations, but the complexation of organic ligands became dominant at low concentrations. In conclusion, tobacco roots secrete organic acids to increase available K content and improve the utilisation rate of soil K. High-K varieties probably enhance slowly available K activation by secreting relatively high amounts of organic acids, thus leading to more available K in soil for absorption by plants.

Genome-wide identification and functional prediction of tobacco lncRNAs responsive to root-knot nematode stress.

Root-knot nematodes (RKNs, Meloidogyne spp.) are destructive plant parasites with a wide host range. They severely reduce crop quality and yield worldwide. Tobacco is a versatile model plant organism for studying RKNs-host interactions and a key plant material for molecular research. Long noncoding RNAs (lncRNAs) play critical roles in post transcriptional and transcriptional regulation in a wide range of biological pathways, especially plant development and stress response. In the present study, we obtained 5,206 high-confidence lncRNAs based on RNA sequencing data. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the target genes of these lncRNAs are mainly involved in plant biotic and abiotic stresses, plant hormone signal transduction, induced systemic resistance, plant-type hypersensitive response, plant-type cell wall organization or biogenesis. The 565 differentially expressed lncRNAs found to be involved in nematode stress response were validated by quantitative PCR using 15 randomly-selected lncRNA genes. Our study provides insights into the molecular mechanisms of RKNs-plant interactions that might help preventing nematode damages to crops.

Comparative Transcriptome Profiling Reveals Defense-Related Genes against Meloidogyne incognita Invasion in Tobacco.

Root-knot nematodes Meloidogyne incognita are one of the most destructive pathogens, causing severe losses to tobacco productivity and quality. However, the underlying resistance mechanism of tobacco to M. incognita is not clear. In this study, two tobacco genotypes, K326 and Changbohuang, which are resistant and susceptible to M. incognita, respectively, were used for RNA-sequencing analysis. An average of 35 million clean reads were obtained. Compared with their expression levels in non-infected plants of the same genotype, 4354 and 545 differentially expressed genes (DEGs) were detected in the resistant and susceptible genotype, respectively, after M. incognita invasion. Overall, 291 DEGs, involved in diverse biological processes, were common between the two genotypes. Genes encoding toxic compound synthesis, cell wall modification, reactive oxygen species and the oxidative burst, salicylic acid signal transduction, and production of some other metabolites were putatively associated with tobacco resistance to M. incognita. In particular, the complex resistance response needed to overcome M. incognita invasion may be regulated by several transcription factors, such as the ethylene response factor, MYB, basic helix⁻loop⁻helix transcription factor, and indole acetic acid⁻leucine-resistant transcription factor. These results may aid in the identification of potential genes of resistance to M. incognita for tobacco cultivar improvement.

Transcriptome analysis of resistant and susceptible tobacco (Nicotiana tabacum) in response to root-knot nematode Meloidogyne incognita infection.

The root-knot nematode (RKN) Meloidogyne incognita reproduces on the roots of tobacco (Nicotiana tabacum), damaging crops, reducing crop yield, and causing economic losses annually. The development of resistant genotypes is an alternative strategy to effectively control these losses. However, the molecular mechanism responsible for host pathogenesis and defense responses in tobacco specifically against RKNs remain poorly understood. Here, root transcriptome analysis of resistant (Yuyan12) and susceptible (Changbohuang) tobacco varieties infected with RKNs was performed. Moreover, 2623 and 545 differentially expressed genes (DEGs) in RKN-infected roots were observed in Yuyan12 and Changbohuang, respectively, compared to those in non-infected roots, including 289 DEGs commonly expressed in the two genotypes. Among these DEGs, genes encoding cell wall modifying proteins, auxin-related proteins, the ROS scavenging system, and transcription factors involved in various biological and physiochemical processes were significantly expressed in both the resistant and susceptible genotypes. This work is thus the first report on the relationships in the RKN-tobacco interaction using transcriptome analysis, and the results provide important information on the mechanism of RKN resistance in tobacco.

[Difference of potassium absorption characters and mechanism of tobacco in genotypes].

Tabacco (Nicotiana tabacum) has a relatively high requirement for potassium (K). The difference in root characteristics and ability to release K from minerals of flue-cured tobacco in four genotypes ND202, NC628, G28 and NC628 x ND202 were analyzed. The results showed that the more available K released from minerals by roots, the more K would be accumulated by plants. ND202, which was K-enriched genotype, had well developed root system and was easily influenced by K content in the environment. Compared with ND202, NC628 significantly enhanced the effectiveness of K in substrate by releasing K from minerals, though the ability to absorb K was weak. Moreover, the hybrid of NC628 x ND202 had a higher ability to release K and to tolerate low-level K than maternal parent. It was higher in dry matter accumulation and K enrichment than male parent. So hybridization had the utility value in high-K variety cultivation. These results provided evidence that parents should have the ability to enhance available K from soil besides the high combining ability for breeding the high-K variety.

Overexpression of a maize E3 ubiquitin ligase gene enhances drought tolerance through regulating stomatal aperture and antioxidant system in transgenic tobacco.

Drought is one of the most important limiting factors in crop production. In our previous study, a putative Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (AtSDIR1) homolog encoding a RING-finger protein from Zea mays (ZmRFP1) was cloned and its expression pattern and Ub E3 ligase activity were characterized. However, it is uncertain that ZmRFP1 acts as a positive regulator during drought stress. In this study, we further characterized ZmRFP1 in transgenic tobacco to investigate drought tolerance and possible function mechanisms. Overexpression of ZmRFP1 enhanced drought tolerance in tobacco. The transgenic tobacco lines had more closed stomatal pores, higher proline accumulation, but lower levels of malondialdehyde (MDA) when compared with the wild type (WT) under drought stress. Further investigation showed that ZmRFP1 transgenic plants displayed higher SOD and CAT activities, increased NtSOD and NtCAT transcript levels, and decreased reactive oxygen species (ROS) accumulation under drought stress. Taken together, our results demonstrate that ZmRFP1 confers drought stress tolerance in transgenic tobacco not only by increasing the ability to retain water, but also by reducing ROS accumulation and membrane damage through enhancing the antioxidant system. ZmRFP1 might serve as a candidate gene in genetic improvement for drought tolerance engineering in cereal crop plants.

[A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

OBJECTIVE: To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. METHODS: To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. RESULTS: Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. CONCLUSION: We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

[Nitrogen uptake and allocation characteristics of flue-cured tobacco in Nanxiong tobacco-planting area of Guangdong Province].

By the method of field in situ culture and 15N isotopic tracer technique, and taking flue-cured tobacco (Nicotiana tobacum) cultivar K326 as test material, a field experiment was conducted in the Nanxiong tobacco-planting area of Guangdong Province to study the characteristics of soil nitrogen (N) mineralization, the patterns of N accumulation and allocation in tobacco plants, and the allocation of plant-absorbed fertilizer N applied in current growth season. In the study area, the amount of soil mineralized N increased with tobacco growth, peaked at 75 days after transplanting, and decreased thereafter. The soil mineralized N at each tobacco growth stage was significantly higher in the control than in the N fertilization treatment. The N accumulation in tobacco plant organs was in the order of leaf > stalk > root. Tobacco plants mainly absorbed fertilizer N at rosette stage and topping stage, and mainly absorbed soil N at mature stage. The absorbed N in tobacco whole growth period was mainly derived from soil N, and the absorbed soil N and its proportion to the total absorbed N increased evidently with extending growth stage and ascending leaf position. The fertilizer N use efficiency per plant and the residual rate and loss rate of applied fertilizer N were 30. 8%, 32. 3% , and 36. 9% , respectively. In the study area, soil N mineralization rate was relatively high, and soil N had greater effects on the quality of upper tobacco leaves. Under the application rate of 150 kg N x hm(-2), the residual amount and loss amount of applied fertilizer N were relatively high.

[Effects of nitrogen fertilization on leaf senescence, photosynthetic characteristics, yield, and quality of different flue-cured tobacco varieties].

Taking three flue-cured tobacco (Nicotiana tabacum L.) varieties Y5, Y7, and NC89 as test objects, this paper studied the effects of nitrogen fertilization on their leaf senescence, photosynthetic characteristics, yield, and quality. Increasing nitrogen supply increased the leaf superoxide dismutase (SOD) activity, chlorophyll content, and photosynthetic performance, and decreased the leaf malondialdehyde (MDA) content significantly. Accordingly, the leaf senescence was delayed. Compared with Y5 and NC89, variety Y7 had higher leaf MDA content and lower leaf SOD activity, chlorophyll content, and photosynthetic performance, and thus, its leaves senesced faster. High quality tobacco leaves with higher average price, gross value, superior leaves percentage, and reasonable chemical constituents were harvested under the application of 45 kg N x hm(-2), compared with applying 60 and 75 kg N x hm(-2). It was suggested that different flue-cured tobacco varieties had obvious differences in their leaf senescence physiological characteristics, and nitrogen fertilization played important roles in regulating their leaf senescence, yield, and quality. Appropriate nitrogen fertilization could improve the quality of tobacco leaves, and achieve higher economic benefits.

[Effects of different planting patterns on the senescence characteristics of flue-cured tobacco roots and leaves].

With flue-cured tobacco (Nicotiana tabacum) cultivar K326 as test material, a field experiment was conducted at Nanxiong area of Guangdong Province in 2008 to study the effects of four planting patterns, i.e., single-row ridge + film mulching at early stage, single-row ridge + film mulching at early and late stages, double-row concave ridge + film mulching at early stage, and double-row concave ridge + film mulching at early and late stages, on the senescence characteristics of its roots and leaves, and the economic traits of its leaves. Comparing with other three planting patterns, double-row concave ridge + film Aulching at early and late stages promoted the root growth of test cultivar during its whole growth period, with the root vigor increased significantly. Meanwhile, the leaf chlorophyll content and protective enzymes (SOD and POD) activities were higher, and the MDA content was lower. The leaf yield, output value, average price, and the proportion of superior leaves were also higher. Double-row concave ridge + film mulching at early and late stages alleviated the senescence characteristics of roots and leaves, and improved the economic traits of flue-cured tobacco leaves, being the efficient planting pattern to product high-quality flue-cured tobacco leaves in Nanxiong tobacco-growing area of Guangdong Province.