Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.
Drought Resistance ; Tobacco/genetics* ; Soybeans/genetics* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Stress, Physiological/genetics* ; Droughts ; Gene Expression Regulation, Plant

BACKGROUND: Head and neck cancers (HNCs) are a heterogeneous group of tumors that progress owing to varied enviromental and genetic risk factors. Viral infections are threatening and adept at altering the expression of cellular transcription factors such as nuclear factor kappa B (NF-κB) and deregulation of other cellular proteins like NF kappa B inhibitor alpha (IκBα). The present study was conducted to detect high-risk genotypes of human papillomavirus (HPV) and protein expression of NF-κB signaling pathway in HNC patients with HPV infection. METHODS: For HPV detection, genomic DNA from 152 HNC tumors was extracted formalin-fixed paraffin-embedded tissue DNA kit. For genotyping, polymerase chain reaction (PCR) using a general primer, HPV type-specific primers and agarose gel electrophoresis were performed. Immunohistochemistry (IHC) was also performed on 4-μm thick tissue sections using HPV E6 monoclonal antibody. Protein expression analysis of NF-κB signaling pathway including p50, p65, and IκBα was performed using IHC. RESULTS: PCR analysis showed that 24.3% (37/152) of HNC cases were HPV positive. Among HPV positive, 86.5% (32/37) were tobacco users, while among HPV negative, 66.9% (77/115) were tobacco users. A significant association of HPV positivity and tobacco user was observed by univariate analysis [ P   <  0.01; odds ratio (OR): 0.310, 95% confidence interval (CI): 0.110 to 0.870]. More HPV positive patients were with poor oral hygiene (78.3%) when compared with patients with good oral hygiene (21.6%) [ P  < 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis showed that age, tobacco use and oral hygiene are significant predictors ( P  < 0.02). PCR and IHC staining results confirmed that HPV16 was predominant among HNC cases (64.8%) when compared with HPV18 (35.2%). Expression of NF-κB proteins (p50, p65, and IκBα inhibitor) were also observed in HPV and non-HPV infected HNC tissues. IHC expression of p50, and p65 showed nuclear staining, while IκBα inhibitor showed cytoplasmic staining. Protein expression in HPV cases was higher as compared to HPV naive cases ( P  < 0.05). CONCLUSIONS From the study, it can be established that the use of tobacco, oral hygiene, and HPV infection may be synergistically involved in modulating the expression of NF-κB signaling pathway for the development and progression of HNC in the Pakistani population.
Alphapapillomavirus ; Antibodies, Monoclonal ; DNA ; DNA, Viral/genetics* ; Formaldehyde ; Head and Neck Neoplasms ; Humans ; NF-KappaB Inhibitor alpha/genetics* ; NF-kappa B/metabolism* ; Oral Hygiene ; Pakistan ; Papillomaviridae/metabolism* ; Papillomavirus Infections/metabolism* ; Signal Transduction ; Tobacco ; Tobacco Use ; Transcription Factors/metabolism*

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.
Crops, Agricultural/genetics* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Recombinant Proteins ; Tobacco/genetics*

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.
Arachis/genetics* ; Fabaceae/genetics* ; GTP-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Salt Stress ; Stress, Physiological/genetics* ; Tobacco/genetics*

Perilla (Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2. The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E. coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E. coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco (Nicotiana tabacum, variety: Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E. coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.
Acyltransferases ; Cloning, Molecular ; Escherichia coli/metabolism* ; Fatty Acids ; Perilla frutescens/metabolism* ; Plant Oils ; Plant Proteins/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Seeds/chemistry* ; Tobacco/genetics*

Hd-Zip, a unique transcription factor in plant kingdom, influences the growth, development, and secondary metabolism of plants. Hd-zip Ⅳ is thought to play an important role in trichome development of Schizonepeta tenuifolia. This study aims to explore the functions of StHD1 and StHD8 in Hd-zip Ⅳ subfamily in peltate glandular trichome development. To be specific, the expression patterns of the two genes and interaction between the proteins encoded by them were analyzed based on transcriptome sequencing and two-hybrid screening. The subcellular localization was performed and functions of the genes were verified in tobacco and S. tenuifolia. The results showed that StHD1 and StHD8 had high similarity to HD-Zip Ⅳ proteins of other plants and they all had the characteristic conserved domains of HD-Zip Ⅳ subfamily. They were located in the nucleus. The two genes mainly expressed in young tissues and spikes, and StHD1 and StHD8 proteins interacted with each other. The density and length of glandular trichomes increased significantly in tobacco plants with the overexpression of StHD1 and StHD8. Inhibiting the expression of StHD1 and StHD8 by VIGS(virus-induced gene silencing) in S. tenuifolia resulted in the reduction in the density of peltate glandular trichomes, the expression of key genes related to mono-terpene synthesis, and the relative content of limonene and pulegone, the main components of monoterpene. These results suggested that StHD1 and StHD8 of S. tenuifolia formed a complex to regulate glandular trichomes and affect the biosynthesis of monoterpenes.
Trichomes/metabolism* ; Lamiaceae/genetics* ; Tobacco/genetics* ; Monoterpenes/metabolism* ; Cloning, Molecular ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.
Biomass ; Gene Expression Regulation, Plant ; Plant Breeding ; Plant Leaves/genetics* ; Plants, Genetically Modified/metabolism* ; Starch ; Tobacco/metabolism*

OBJECTIVETo explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants.

METHODSThe HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR.

RESULTSThe HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells.

CONCLUSIONHFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.

Endoplasmic Reticulum ; Genetic Engineering ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; Imidazoles ; chemistry ; Plant Leaves ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Tobacco ; genetics ; metabolism

To explore the influence of low light on the synthesis of carotenoids, chlorophyll and the adaptability of transgenic plants with tomato Solanum lycopersicon L. GGPS2 gene, we constructed a vector containing a GGPS2 gene with green fluorescent protein (GFP) as report gene under the control of a cauliflower mosaic virus 35S promoter and introduced it into tobacco Nicotiana tabacum L. cv. Wisconsin 38 by Agrobacterium tumefaciens-mediated transformation. PCR analysis of the DNA from kanamycin resistant tobacco indicated that the transgenic tobacco containing the nptII gene, SlaGGPS2 gene and without contamination of Agrobacterium. We also detected the root tip of kanamycin resistant tobacco showing characteristic fluorescence. The contents of carotenoid, chlorophyll and photosynthesis of transgenic tobacco increased in comparison with wild tobacco after low light treatment. In addition, leaf mass per unit area, total dry weight, ratio of root to shoot in transgenic tobacco were all higher than that of the wild tobacco, which proved that the transgenic tobacco could increase the accumulation of biomass and promote it transport to root. The transgenic tobacco with SlaGGPS2 gene can increase the contents of carotenoid, chlorophyll, enhance the photosynthetic rate, promote the biomass accumulation and its distribution to root. Hence, the transgenic tobacco with SlaGGPS2 gene had increased low light tolerance and the SlaGGPS2 gene maybe can be used in other crops.
Agrobacterium tumefaciens ; Carotenoids ; analysis ; Chlorophyll ; analysis ; Gene Expression Regulation, Plant ; Genetic Vectors ; Light ; Lycopersicon esculentum ; genetics ; Photosynthesis ; Plants, Genetically Modified ; metabolism ; radiation effects ; Tobacco ; metabolism ; radiation effects

This study is to investigate the protective effect of mangiferin on NF-kappaB (P65) and IkappaBalpha expression in peripheral blood mononuclear cell (PBMC) in rats with cigarette smoke induced chronic bronchitis. The rat model with chronic bronchitis was established by cigarette smoke. Real-time fluorescence RT-PCR was executed for evaluating the NF-kappaB (P65) and IKkappaBalpha gene expression in mononuclear cell, and flow cytometry for their protein expression. The serum hs-CRP (high-sensitivity C-reactive proteins) and TNF-alpha (tumor necrosis factor-alpha) were detected by enzyme-linked immunosorbent assay. The histopathological score was obtained from lung tissue HE staining slides of lung tissue. The results showed that mangiferin could markedly suppress the NF-kappaB (P65) mRNA and protein expression in mononuclear cell, while promote the IkappaBalpha mRNA and protein expression. Furthermore, mangiferin could lower serum hs-CRP and TNF-alpha level, and reduce the chronic inflammatory damage of bronchiole. These results suggested that mangiferin could notably ameliorate chronic bronchiole inflammation induced by cigarette smoke, and this protective effect might be linked to the regulation of NF-kappaB (P65) and IkappaBalpha expression in mononuclear cell.
Animals ; Bronchi ; pathology ; Bronchitis, Chronic ; blood ; etiology ; metabolism ; pathology ; C-Reactive Protein ; metabolism ; I-kappa B Kinase ; genetics ; metabolism ; Leukocytes, Mononuclear ; metabolism ; pathology ; Male ; Mangifera ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tobacco Smoke Pollution ; Transcription Factor RelA ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; Xanthones ; isolation & purification ; pharmacology