ABSTRACT
Due to the laborious and scale-up limitation we have developed a simple method named "seed absorption" to express foreign proteins in plants by means of transient expression. It has been shown that the reporter gene GFP was expressed successfully in tomato (Solanum lycopersicum L.) plants by seed absorbing Agrobacterium suspension containing TMV-based p35S-30B-GFP vector. Various factors influencing the gene expression were optimized including Agrobacterium cell density and other inoculation conditions. This method has the special advantages as simple work process, ease to scale-up, and further expanding the host range of plant bioreactor than previous methods. We assume that the seed absorption method will facilitate the industrial scale production of the recombinant pharmaceutical proteins in plants.
Subject(s)
Agrobacterium/genetics , Gene Transfer Techniques , Seeds/genetics , Solanum lycopersicum/genetics , Tobacco Mosaic Virus/genetics , Transformation, Genetic , Agrobacterium/physiology , Gene Transfer Techniques/instrumentation , Genetic Vectors/genetics , Genetic Vectors/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Solanum lycopersicum/microbiology , Solanum lycopersicum/virology , Seeds/microbiology , Seeds/virology , Tobacco Mosaic Virus/physiologyABSTRACT
A system of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and Nicotiana benthamiana as experimental materiaes. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR; protein levels of rbcS by the antibodies of rbcS and rbcL and photosynthetic pigments wntents in rbcS silenced plants by HPLC method. The results showed that the seedlings at 21-24-day-old and Agrobacterium concentration at OD600 = 1-1.5 gave the best results for gene silencing. The expression level of rbcL was very likely regulated by rbcS, and rbcS gene did not relate to the collection of photosynthetic energy. Probability analysis showed that the tobacco rattle virus vector system is a useful and effective technique to study rbcS gene function via post-transcriptional gene silencing.
Subject(s)
Agrobacterium tumefaciens/genetics , Nicotiana/genetics , Plant Proteins/genetics , RNA Interference , Ribulose-Bisphosphate Carboxylase/genetics , Blotting, Western , Chlorophyll/analysis , Chromatography, High Pressure Liquid , DNA, Complementary/genetics , Genetic Vectors , Lutein/analysis , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/metabolism , Plant Viruses/genetics , Plasmids , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , Ribulose-Bisphosphate Carboxylase/metabolism , Seedlings/genetics , Seedlings/metabolism , Nicotiana/metabolismABSTRACT
Spinach Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 microg antigens were used and 0.3 ml anti-sera with titer of 1:5000 were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthamiana was performed. The expression level of rbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.
Subject(s)
Antibodies/immunology , Protein Subunits/immunology , Ribulose-Bisphosphate Carboxylase/genetics , Ribulose-Bisphosphate Carboxylase/immunology , Animals , Antigen-Antibody Reactions , Antigens/isolation & purification , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Female , Gene Expression Regulation, Enzymologic , Gene Silencing , Mice , Mice, Inbred BALB C , Plant Leaves/genetics , Rhizobium/genetics , Ribulose-Bisphosphate Carboxylase/metabolism , Spinacia oleracea/chemistry , Nicotiana/genetics , Nicotiana/immunologyABSTRACT
Degenerate PCR is introduced in this paper, including what is degenerate PCR, how to design degenerate primers, how to optimize degenerate PCR parameters, how to applying degenerate PCR to obtain full-length gene and which fields can apply degenerate PCR. The limits and recent advances of degenerate PCR are also discussed. Based on this introduction,strategies of gene cloning and applications of degenerate PCR in gene cloning are summarized in brief. Degenerate PCR is a very useful tool for searching and discovering new genes and new members of a protein family.
ABSTRACT
The soil salination is a significant abiotic stress for agricultural production and ecological environment. The research on salt tolerance represents an important part for basic plant biology. Genetic analysis of salt tolerance genes in Arabidopsis has become a central issue in this research areas. In recent years, efforts from some laboratories in the world have led to some significant progresses in this field. In this paper, we will review the progress in salt tolerance genes SOS (salt overly sensitive): SOS1,SOS2 and SOS3.
ABSTRACT
Cellulose is a major component in plant cell wall. About 180 billion tons of cellulose are produced per year in nature. The commercial importance of cellulose makes the genes coding it one of attractive targets for plant genetic engineering. A number of cellulose synthase genes have been first cloned from plant species by Delmer's group in 1996. Recently, research achievement has been obtained in accumulating to understanding the cellulose synthase function,location,and the gene function. The paper summarized the research progress of cellulose synthase genes in higher plants.
