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*

Nicotine is the main component for smoking addiction. It is widely believed that nicotine dependence is heritable. Many studies are committed to study the effects of specific gene polymorphisms connect with nicotine dependence. Release of dopamine has been considered the most important channel for nicotine dependence. This paper provides a review for recent advance in studies on dopamine system related genetic polymorphisms associated with nicotine dependence.
Animals ; Dopamine ; metabolism ; Humans ; Nicotine ; metabolism ; Polymorphism, Genetic ; Tobacco Use Disorder ; genetics ; metabolism

Studies have shown that transgenic plants expressing antiapoptotic genes from baculovirus and animals increase resistance to biotic and abiotic stress. However, the mechanism under these resistances is conjectural, or in some cases even controversy. In the present study, the p35 gene from baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was expressed in tobacco, and for the first time P35 protein was detected in transgenic plants by Western blotting. Inoculation of T1 transgenic tobacco leaves with tobacco mosaic virus (TMV) showed enhanced resistance, and DNA laddering was observed after TMV infection in control but not in transgenic plants. DAB staining showed that TMV infection did not affect peroxide induction of transgenic plants, Western blotting analysis of PR1 protein also showed no difference of control and transgenic plants. Inoculation of fungus (Sclerotinia sclerotiorum) using a detached leaf assay showed enhanced resistance of transgenic leave tissue. RT-PCR analysis demonstrated that p35 gene expression induced earlier expression of PR1 gene after S. sclerotiorum infection. Taken together, our results suggest that the mechanism under enhanced disease resistance by P35 protein is possibly related to the activation of PR-related proteins in addition to the inhibition of programmed cell death, depending on the pathogens challenged.
Gene Expression Regulation, Plant ; Immunity, Innate ; Plant Diseases ; genetics ; Plants, Genetically Modified ; genetics ; immunology ; virology ; Tobacco ; genetics ; immunology ; virology ; Tobacco Mosaic Virus ; Transformation, Genetic ; Viral Proteins ; genetics ; metabolism

Palmitoleic acid (16:1delta9), an unusual monounsaturated fatty acid, is highly valued for human nutrition, medication and industry. Plant oils containing large amounts of palmitoleic acid are the ideal resource for biodiesel production. To increase accumulation of palmitoleic acid in plant tissues, we used a yeast (Saccharomyees cerevisiae) acyl-CoA-delta9 desaturase (Scdelta9D) for cytosol- and plastid-targeting expression in tobacco (Nicotiana tabacum L.). By doing this, we also studied the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1delta9) and cis-vaccenic (18:1delta11) were significantly enhanced in the Scdelta9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1delta9 and 18:1delta11 in the Scdelta9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1delta9 levels. Our data indicate that yeast cytosolic acyl-CoA-delta9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1delta9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. The present study developed a new strategy for high accumulation of omega-7 fatty acids (16:1delta9 andl8:1delta11) in plant tissues by protein engineering of acyl-CoA-delta9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.
Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Monounsaturated ; metabolism ; Plants, Genetically Modified ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Tobacco ; genetics ; metabolism

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

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*

Acid fibroblast growth factor (aFGF) has great potential in clinical application, but it is very expensive. In order to reduce the cost of production and to make full use of the merits integrated with plant bioreator, we have explored the aFGF in transgenic Tobacco expression. AFGF gene was inserted into plant expression vector pBI121; the acquired plants contained aFGF gene expression vector pBI121-TOAB-aF. Using Agrobacterium-mediated gene transformation of Tobacco and using transgenic Tobacco containing kanamycin and cephalosporin culture medium, we obtained kanamycin resistant transgenic Tobacco plants. PCR detection, RT-PCR detection and Western blot detection confirmed that foreign genes were successfully expressed in Tobacco. These data could serve as a theoretical foundation on which to use the plant bioreactor for production of aFGF.
Agrobacterium ; genetics ; Fibroblast Growth Factor 1 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Tobacco ; genetics ; metabolism

A novel practical binary vector to get marker-free transgenic plant was constructed. The estrogen-inducible Cre/loxP DNA recombination system was adopted in this system. All non-target genes located between two identical orientation loxP sites could be excised from the transgenic genome by the Cre expression. In order to analyze this system, the target gene, GUS expression box (CaMV35s: :GUS), was inserted in the MCS outside the region franked by two loxP sites. Then it was introduced into the tobaccos. Results showed that the high-efficiency DNA recombination had take place and the target gene was working order after DNA excitation.
Base Sequence ; Genetic Vectors ; genetics ; Integrases ; metabolism ; Molecular Sequence Data ; Plants, Genetically Modified ; genetics ; Recombination, Genetic ; Tobacco ; genetics

OBJECTIVETo test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana.

METHODSSeven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.

RESULTSThe 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression.

CONCLUSIONTransient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Plant Viruses ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Potexvirus ; genetics ; RNA Interference ; Tobacco ; genetics ; metabolism

Amorpha-4,11-diene synthase (ADS) can convert farnesyl pyrophosphate (FPP) to amorpha-4, 11-diene, a precursor of artemisinin. ADS plays an important role in the biosynthesis of artemisinin. This review summarizes the molecular biology and metabolic engineering study of ADS in recent years. The genomic DNA and its cDNA sequences of amorpha-4, 11-diene synthase were cloned from Artemisia annua L. The cDNA encoding amorpha-4, 11-diene synthase contains a 1 641 bp open reading frame coding for 546 amino acids. ADS shows a broad pH optimum and an absolute requirement for divalent metal ions as cofactors. The specificity of ADS to the substrates and products is not high and the formation of amorpha-4, 11-diene by ADS from FPP is achieved by an initial 1, 6-closure with subsequent 1, 10-closure. The ADS cDNA cloned from Artemisia annua L, or totally synthesized by PCR, was introduced into different hosts including E. coli, S. cerevisiae, Nicotiana tabacum L. Arabidopsis thaliana and A. nidulans resulting in varied engineering microorganisms and cells producing amorpha-4, 11-diene. The way to improve the production of amorpha-4, 11-diene was investigated by two strategies such as improving the supply of substrate and directing FPP flux to amorpha-4, 11-diene production from competing pathways.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Amino Acid Sequence ; Antimalarials ; metabolism ; Arabidopsis ; enzymology ; genetics ; Artemisia annua ; enzymology ; genetics ; Artemisinins ; metabolism ; Aspergillus ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; Saccharomyces cerevisiae ; genetics ; metabolism ; Tobacco ; enzymology ; genetics