Endogenous Plant Genes Research Articles

Problems that can limit the expression of foreign genes in plants: lessons to be learned from B.t. toxin genes.

It has been more than ten years since the first transgenic plants expressing foreign genes were regenerated following Agrobacterium-mediated transformation. During this time, significant progress has been made toward crop improvement by genetically engineered plants with a variety of desirable traits. New developments in the area of plant transformation and the isolation of agronomically important genes have contributed to this advancement. In some cases, endogenous plant genes have been re-engineered to produce advantageous characteristics, whereas in others, genes from non-plant sources have been used. Prominent examples include the antisense and sense configurations of several genes that have been used to manipulate ripening and senescence processes (1). In addition, genes from bacteria, fungi, plants and viruses have been expressed in plants to achieve resistance to various pathogens (2) or herbicides (3), to alter male fertility (4,5) or lipid composition (6), or to produce new compounds in plants such as biodegradable plastics (7,8). As a result, many exciting transgenic plants and their products should soon reach the market place.

RNase A/T1 protection assay.

AbstractRNase A/T1 mapping provides a sensitive and quantitative method of expression analysis of known gene sequences. It differs from primer-derived analyses such as primer extension and reverse transcriptase-PCR by the use of a probe that is colinear with the transcript under study. This protocol has been applied to the analysis of plant RNA transcripts, for example, in the expression of endogenous plant genes (1), introduced genes (2), mutated or tagged genes (3), antisense gene copies (4), and in the analysis of plant nuclear pre-mRNA splicing (5). There are four steps involved in RNase A/T1 analysis. First, the transcription of a labeled RNA probe complementary to the transcript under study. Second, hybridization of the labeled probe to the target RNA transcript. Third, digestion of the noncomplementary regions of the probe with ribonucleases. Finally, analysis of the digested products on denaturing polyacrylamide gels and autoradiography.KeywordsAmmonium PersulfateIsoamyl AlcoholMolecular Weight ProductRNase DigestionEndogenous Plant GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation.

Endogenous plant genes or transgenes can be silenced on introduction of homologous gene sequences. Here we document a reporter gene-silencing event in Nicotiana tabacum that has a distinctive combination of features--i.e., (i) silencing occurs by a posttranscriptional process, (ii) silencing correlates with DNA methylation, and (iii) this de novo methylation is not restricted to cytosines located in the symmetrical motifs CG and CXG.

An efficient and reliable method for determining the number of transgenes inserted into transgenic plants

A simple and reliable method is described for determining the number of transgenes inserted into transgenic plants. Analysis of tomato plants transformed with an antisense polygalacturonase gene is used to demonstrate the method. The use of the method to analyze other plants transformed with endogenous plant gene coding regions or promoters is discussed.

Import of proteins into the chloroplast lumen.

Plastocyanin is a nuclear-encoded protein that is functional in the thylakoid lumen of the chloroplast. It is synthesized in the cytoplasm as a precursor with an N-terminal transit peptide of 66 amino acids. Its transport route involves two steps, import into the chloroplasts and subsequent routing over the thylakoid membrane into the lumen. Concomitant with the transport, the transit peptide is removed in two successive steps. The transit peptide consists of two functionally different domains. In this study we examine to what extent each domain is involved in import and routing and how far these two processes are linked. For this purpose we made deletions in the N-terminal and C-terminal part of the transit peptide and fusion proteins which only contain one of these parts. The results show that the N-terminal part of the transit peptide is responsible for import into the chloroplast. The N-terminal 43 amino acids are sufficient to direct other proteins into the stroma. The C-terminal part of the transit peptide is a prerequisite for routing inside the chloroplast but not for import. When deletions are made in this part, the transport of plastocyanin stops after import and the intermediate accumulates in the stroma or on the outside of the thylakoids. Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally, expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only. However, in the transgenic plants this foreign plastocyanin protein is found in all tissues examined. The protein is transported into the local plastids of these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue. The precursor of ferredoxin was expressed in Escherichia coli. Surprisingly the precursor interacts with the cytoplasmic membrane and is translocated across this membrane. The unprocessed precursor accumulates in the periplasm.

In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found to be present in all tissues examined. The protein is transported into the local plastids in these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.