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In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. 11.77 New.An efficient and high yielding expression system is required to produce recombinant proteins. 4.9 out of 5 stars (33) Total Ratings 33, 100 agree - Would recommend. Las series con mas exito son las conocida Delica, Twist, Drop y las Bugle Beads y todas tienen una infinidad de colores, se cuentan mas de 2000 colores distintos.Xuron 170-ii Micro-shear Flush Cutter Jewelry Beads Beading Wire Work Tools. Pero el exito llego con su hijo Hiroshi, que llego a meter a punto la fabricacion en serie mas grande de Japon, que desde entonces no ha parado de crecer , en surtido, colores y calidad.
The replication system of plant viruses results in high-level expression of foreign proteins within a few days 1. Alternatively, transient expression systems with virus-based vectors have the advantage of rapid and high-level expression of the recombinant proteins. However, substantial time is required to generate transgenic plants, and the yield of the expressed protein is relatively low. A revolutionary tool for biophysics.Transgenic plants are generally used to obtain recombinant proteins or identify the localization of proteins.
Thus, the magnICON system is very useful system in tobacco, including Nicotiana benthamiana. ZMapp for Ebolavirus infections, which was used during a recent outbreak, was also produced by the magnICON system 4. A tobamovirus (TMV)-based deconstructed viral system (magnICON) has been extensively engineered to achieve high levels of recombinant protein accumulation in tobacco leaves 3.
A recent study demonstrated that introduction of PsaK 5′-UTR, extensin (Ext) terminator, and the tobacco Rb7 matrix attachment region into the geminivirus replicon vector greatly improved the expression level of protein with the Agrobacterium strain EHA105 9. Some geminivirus species can replicate in non-host plant cells and BeYDV has a broad host range in dicotyledonous plants 8. Benthamiana leaves 7 and in lettuce leaves 5. This mechanism has been used for boosting protein expression in transgenic plants 6 and for transient expression of foreign proteins with efficiency in N. Because of TMV-based viral vector, host factors from a plant species must recognize and interact with TMV elements or factors.Bean yellow dwarf virus (BeYDV), a Mastrevirus of the Geminiviridae family, contains a single-stranded circular DNA genome and uses a rolling circle mechanism to replicate its genome, resulting in a very high yield of copies.
The less efficient polyadenylation of mRNA leads to reduction of translatable mRNAs and, consequently, decrease in protein production 16, 17. Read-through transcripts may cause inhibition of 3′-end cleavage/polyadenylation processing. The introduction of a second terminator possibly detects read-through transcripts and traps it by the hairpin structure 15. Furthermore, the expression level of the gene was increased with double transcription terminators, the CaMV 35S terminator and NOS terminator 13, 14 however, these results were obtained from stable transformants.
Each vector was transformed into Agrobacterium tumefaciens GV3101 and green fluorescent protein (GFP) was transiently expressed in N. These results indicated that this system is a useful tool, with which to express specific proteins in plant cells.A comparison of the expression level of GFP between pBYR2HS and pBYR2fp geminiviral replicon vectorIn the pBYR2HS vector, the alcohol dehydrogenase (AtADH) 5′-UTR region was replaced with tobacco mosaic virus (TMV) Ω and the HSP terminator was inserted into pBYR2fp, resulting in a double terminator construct (Fig. Benthamiana, but also tomatoes, eggplants, lettuce, hot peppers, melons, and orchids. Furthermore, this system could be applicable to not only N. In particular, when the HSP and Ext terminators were used as a double terminator, the expression level was the highest and reached approximately 3.7 mg/g fresh weight (FW) in N.
Transfection with pBYR2HS-EGFP improved expression of GFP in these plants except for the rose, compared with pBYR2fp-EGFP. 2G), orchids Phalaenopsis aphrodite (Fig. 2F), melons Cucumis melo (Fig. 2E), hot peppers Capsicum frutescens (Fig. 2C), tomato Solanum lycopersicum fruits (Fig. 2B), eggplants Solanum melongena (Fig.
Expression levels of GFP from plants agroinfiltrated with pBYR2HS-EGFP were higher than that from plants agroinfiltrated with pBYR2fp-EGFP (Fig. The GFP was also detected by immunoblot analysis with anti-GFP antibodies. The total soluble proteins were detected with Coomassie Brilliant Blue (CBB) staining. Benthamiana and 1 mg FW of lettuce, eggplant, tomato, hot pepper, and rose. 2I).Then, total soluble proteins were prepared from 0.2 mg fresh weight (FW) of N. No fluorescence was detected in the rose (Fig.
Because a clear GFP band with CBB staining was not observed in tomatoes, hot peppers, and roses, western blot analyses were performed. Similarly, the expression level of GFP from lettuce or eggplant agroinfiltrated with pBYR2HS-EGFP (0.37 mg/g FW or 0.46 mg/g FW, respectively) was higher than that of lettuce or eggplant agroinfiltrated with pBYR2fp-EGFP (0.20 mg/g FW or 0.42 mg/g FW, respectively, Fig. Benthamiana agroinfiltrated with pBYR2fp-EGFP. Conversely, approximately 1.5 mg of GFP was expressed in 1 g FW in N. Benthamiana agroinfiltrated with pBYR2HS-EGFP (Fig.
Coomassie Brilliant Blue (CBB) staining and immunoblot analysis with anti-GFP antibodies were performed by using agroinfiltrated leaves of N. Total soluble proteins were extracted from agroinfiltrated plant leaves with pBYR2HS-EGFP or pBYR2fp-EGFP. These results indicated that introduction of the HSP terminator improved the expression level of GFP in the agroinfiltrated plants and this system can be used for several species.Effect of HSP terminator on transient GFP expression at 3 days post-agroinfiltration. GFP expression in roses agroinfiltrated with pBYR2HS-EGFP was not detected even when the western blot analysis was performed.
Data represent the means ± SD ( n = 3 to 4). The band clearly seen at 55 kDa in a CBB staining gel is corresponding to large subunit of Rubisco. Arrowheads indicate bands corresponding to GFP protein. The amount of protein was measured according to band intensity from CBB staining gel using ImageJ software. NT indicates non-transfected plants. The numbers at the top of the gels indicate different samples taken from different leaves from different plants.
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These plasmids were used for agroinfiltration into N. First, pBYR2H-EGFP containing only the HSP terminator, pBYR2fp-EGFP containing only Ext 3′, and pBYR2HS-EGFP containing both the HSP terminator and Ext 3′ were compared. Full-length gels and full-length blots are presented in Supplementary Figures S1 and S2.Full size image A double terminator with geminiviral replication enhances transient protein expressionTo confirm whether the HSP terminator improved expression of the protein or double terminator enhanced expression, several kinds of plasmids were prepared (Fig.
However, if the plasmid contained both the HSP terminator and Ext 3′, the expression level was significantly higher than with plasmids containing the single terminator. 4G), suggesting that the HSP terminator enhanced expression of GFP. 4G) was higher than with pBYR2fp-EGFP (1.7 mg/g FW, Fig. Benthamiana with pBYR2H-EGFP (2.2 mg/g FW, Fig. The expression level of GFP from N.
Interestingly, the expression level of GFP with pBYR2HTS-EGFP was statistically lower than that with pBYR2HS-EGFP (Fig. In conclusion, the expression level of GFP with pBYR2HS-EGFP (3.9 mg/g FW) was the higher than that obtained with other plasmids, such as pBYR2HH-EGFP (3.4 mg/g FW), pBYR2EE-EGFP (3.7 mg/g FW), pBYR2TN-EGFP (3.2 mg/g FW), pBYR2HT-EGFP (2.9 mg/g FW), and pBYR2HTS-EGFP (2.9 mg/g FW) (Fig.
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