JPH01501038A - Intracellular directed transport of expression products - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] This application is part of Application No. 912.408 filed on September 26, 1986. This is a continuation application.

Relating to techniques for directing the transport of proteinaceous gene products.

background Plant cells contain individual accessory cell chambers bounded by membranes. to photosynthetic plants The most prominent organelle was the chloroplast. present in leaf cells The chloroplast is one growth stage of this organelle. Proplasty De, ethioblast, amyloblast and chromoblast are different stages . Embodiments of the invention relate to organelles that may be referred to as "chloroplasts." Generally applicable.

Most of the chloroplast proteins are synthesized in the cytoplasm and transferred to nuclear genes. and then taken up into chloroplasts. The import is It is related to the removal of the mino-terminal part, ie the transfer peptide. The transfer peptide is Recognized by specific proteases associated with chloroplasts, usually at least is also attached to the mature peptide by an amino acid sequence that requires two amino acids.

Therefore, the mature peptide profile is translocated into the chloroplast and and processed as a result of recognition by one or more proteins.

There are many techniques for manipulating and transforming plant cells to confer specific functions on them. For this purpose, genes introduced into plant cells are translocated into chloroplasts. , and it is desired to yield a product that functions in the chloroplast. Probably. Therefore, constructing chimeric constructs that provide efficient expression in plant hosts, Production of novel products that are translocated to and functional in the chloroplast of the plant host or is desired to result in enhanced production of a particular product.

to provide chimeric genes that are shown to be able to be transposed into plant hosts. , the small subunit of Japanese pea paste broth linked to the NPT-II gene. The use of transfer peptides of 1,5-bisphosphate carboxylase is described. Lub ben and [eegstra, Proc, Natl, Acad, Sci , USA (1986) 83: 5502-5506, chloroplast The soybean SSU transfer peptide Fentou SU aged protein for rearrangement to We report on the use of 13 amino acids from wood and heat shock proteins. Also, that See also Volume 5, Nα1, pages 9-13. mutated herein by reference incorporated into the book. See also εPA No. 218.571.

Summary of the invention DN, which results in the translocation of proteins expressed in the cytoplasm into the chloroplast. A constructs and their resulting proteinaceous products expressed from such constructs. is provided.

The construct is used to transport expressed cytoplasmic proteins to the chloroplast. Transfer peptides recognized by host plant cells, at least two positively charged a short polar or hydrophilic region with amino acids fused to the short polar region; Contains an array that codes the part to be processed. Polar regions are naturally occurring or May be synthetic and usually requires effective processing of the transfer peptide provides effective transport from the cytoplasm to the chloroplasts with can act for.

Description of specific aspects The methods and compositions of the present invention involve introducing heterologous or homologous genes into plant cells; the gene is expressed and the resulting polypeptide product is extracted from the cytoplasm. Providing that it is transported to the chloroplast. The genetic construct The sense strand or positive transcription region and the transcription termination region recognized by the plant host cell. Contains the polarity in the direction of transcription. The translated region is transferred from the cytoplasm to Chloroplus. A transfer peptide that is recognized by a host to transport a polypeptide into a host, usually , after transport into the chloroplast, the translocated peptide is cleaved to cleave it. The processing signal may be the same or different from that originally associated with the transferred peptide. at least two positively charged amino acids comprises a sequence encoding a polar amino acid with a polar amino acid and a gene of interest, where the translocation The transferred peptides, processing sequences, polar region sequences, and target genes are all The reading frames are aligned correctly.

Many biological processes are involved in chloroplasts. Therefore, in chloroplast There is substantial interest in having a wide variety of proteins introduced into Here, the protein can lead to denaturation of biological processes, causing chloroplasts to confer new capabilities on biological processes or protect biological processes from interference.

Since chloroplasts have sites for the production of fatty acids, the chloroplast By introducing various proteins into the protein, fatty acid production is increased and fatty acid production is increased. Modifying the size distribution or modifying the unsaturated nature of the fatty acids both in number and location can be done. Various enzymes that may be involved in such functions include acyl carriers. -protein (ACP), acetyl-CoA ACP transacylase, Oesterase, malonyl-CoA ACP) lance acylase, β-ketoa Contains sil-ACP, synthetase, etc.

Another biological process associated with chloroplasts is starch synthesis. child The process involves the use of various enzymes, namely starch phosphorylase, N0PG) cosylase, D-enzyme glucose bipophosphorylase, etc.

Other proteins produced in the cytoplasm and translocated to the chloroplast are Small subunit of rose-1,5-bisphosphate carboxylase, chlorophyll A and B binding proteins, ferredoxin, shikimate pathway enzymes and amino acid production Contains synthetic enzymes.

The enzyme 5-enolbilvir-3-phosphoshikimate synthetase is involved in the pathway. Of particular interest are biological processes for the synthesis of aromatic amino acids. This fermentation The compound is of particular interest because it is the target for the commonly used herbicide glyphosate. is of interest.

Expression products are paired by peptide sequences that are recognized as processing signals. A lead that is recognized by the host for translocation from the cytoplasm is bound to the elephant's gene. A precursor or proform of the desired peptide having the following sequence. produced in the cytoplasm The resulting peptide proform is translocated to the chloroplast, where it is , processed by peptide degradation.

The DNA sequence of the processing signal is an oligonucleotide recognized by pepdase. encodes a oligopeptide. Transport of chloroplasts through membranes and chloroplasts Upon entry into the storage organometa, the expression product is cleaved by peptidases. and performs its intended function as a mature protein.

All genes of interest transfer peptides through binding sequences encoding polar regions. and a processing signal. Above protein Any gene encoding can be used. As pointed out, these genetic The child modifies the composition of the natural composition and provides protection from stress, e.g. herbicides. may be included to provide, enhance a particular function, or the like.

- involved in the expression of an enzyme that catalyzes the conversion to rubyrubyl-3-phosphoshikimate.

The enzyme is 5-enolbilvyl-3-phosphoshikimate synthetase (EC : 2.5.1.19>; hereinafter referred to as ES-3-P synthetase do. Natural gene products increase shikimic acid production in metabolic pathway products It is especially useful. glyphosate) (N-phosphonomethylglycine), i.e. The genes that express enzymes that are important and are resistant to commonly used herbicides are structural genes. Confers glyphosate resistance to normally glyphosate-sensitive cells in which the gene is expressed Especially useful. U.S. Patent No. 4.535.060 addresses such sudden changes. Describe the catabolized structural genes.

Transfer peptides and processing signals are expressed in the cytoplasm and chloro It can be derived from any plant protein that is translocated into the plast. certain polypes The mature protein was determined by comparing the mRNA for the peptide and the mature product. methionine is absent in the protein and is usually caused by the methsenger starting at the start codon. The amino acid sequence encoding the protein will normally be a transposed sequence. often with others It may be desirable to use one transposition sequence that is different. Typical transition sequence is the small subunit of ribulose-1,5-bisphosphate carboxylase (SS U) from the transfer peptide and acyl carrier protein (ACP). Ru. Fragments from natural translocation sequences that retain their transport activity are also used. can be done. The transfer peptide chloroplasts the peptide that is bound to the transfer peptide. sequence that can be transposed to the last, and the complete wild-type transposed peptide , a functional fragment thereof or a functional mutant thereof. completely innate This sequence is also included in nature. For the most part, transferred peptides from one plant are commonly It is also 'RWi' by other plants. Therefore, the transfer peptide is a chimeric gene. The offspring may be homologous or xenologous to the primary host into which they are introduced. metastasis pep Chido is soybean, corn, petunia, tobacco, rapeseed, tomato, and comb. It arises from gi, lentil, etc. The transfer peptide usually has at least about 20 amino acids and no more than about 100 amino acids.

The translated region consists of three components: (1) a transfer peptide and a normal processing sequence; translocation of the expression product from the cytoplasm to the plast and removal of the transferred peptide (2) translocation and stability of the gene of interest; (3) provide a change in chloroplast function; Chimeric genetics with the gene of interest supplying a functional protein product that would would provide a child.

The pole that would separate the gene of interest from the transferred peptide and processing signal. The sexual region usually has at least six, usually eight, codons and about It will contain no more than 20 codons. The polar region is unique to the transferred peptide. The naturally occurring region of the mature peptide that is naturally attached to the transferred peptide. and the different proteins that confer the N-terminus of such mature proteins. A region derived from a protein or encoding a non-naturally occurring polypeptide sequence. It can be a synthetic sequence that is coded.

In addition to the number of codons, the sequence is hydrophilic and optionally at least about by having 40%, preferably at least about 50% polar amino acids; It will be further characterized. A polar amino acid has a petro atom in its chain. It consists of charged and neutral amino acids. These amino acids are charged amino acids such as lysine (K), arginine (R), aspartic acid (D ), glutamic acid (E) and histidine (H) and neutral polar amino acids, e.g. Baserine (S), Threonine (T), Asparagine (N), Glutamine (Q) including. Usually, the sequence consists of two positively charged amino acids, preferably preferably at least 3 amino acids and up to 5 positively charged and usually no more than 25%, preferably 20% No more sequences than will be positively charged amino acids. charged amino acids are present in the range of 6 to 15 amino acids and also contain processing signals. It can also be present as the first amino acid after. Also, regarding acp , whose polar region can be the N-terminal 12 to 30 amino acids of the mature aCp. . To define a hydrophilic region, the hydrophilic or hydrophobic nature of the tambatar region is yte and Doolittle, J. Mo], Bio, (1982) 157 : 105-132. This reference is , pointing out the exposed nature of the area.

As a specific exception, the N-terminal region of plant acyl carrier proteins is used. and up to 35 codons, preferably no more than about 30 codons. and can range from about 12 to 30 codons. that polar territory A region is usually predetermined before the hydrophobic region, which is the last positively charged amino acid region. The amino acid immediately following the acid contains at least about 50% hydrophobic amino acids, preferably less It will have at least about 60% hydrophobic amino acids. Certain hydrophobic amino acids are glycine (G), alanine (A), proline (P) and more particularly leucine ( L), isoleucine (I), valine (V) and aromatic amino acids such as phenyl Contains lualanine (F), tyrosine (Yo) and tryptophan (W). as desired 5 amino acids following the last positively charged amino acid of the polar region, Preferably the 10 amino acids will not be positively charged amino acids . This hydrophobic region contains the appropriate amino acids that are naturally present as part of the gene of interest. , a polar region having a wild-type sequence, a synthetic sequence, or a combination thereof related to the transferred peptide. This can be achieved by using the range.

It is desirable to use fewer amino acids to obtain the desired transposition efficiency. be done. The final product is a fusion protein comprising a polar region and the gene of interest. Therefore, its polar region depends on its stability, activity, location in the chloroplast, and can influence the genes of interest with respect to other properties. Therefore, most In addition, its polar region has an opposite role for the gene of interest in the chloroplast. It will be chosen to minimize the effect.

Depending on the nature of the polar region, various methods are used to synthesize chimeric genes. can be done. The naturally occurring N-terminus of the mature protein normally associated with the transfer peptide If end codons are used, the gene of interest aligns the appropriate reading frame and includes polar regions. will be combined with If a convenient restriction site exists at the end of that polar region, The gene containing the transferred peptide is cleaved at that site and bound to the gene of interest. can be manipulated to On the other hand, if no convenient restriction site exists, other restriction sites is used, and an adapter can be used to regenerate the deleted codon. Ru. In many cases, the cutting is carried out by the fallen Yu 31, and the target is turned into a plant terminal. A plant end can be provided that can be attached to a gene.

The particular manner in which the sequences are joined and linked is not critical to the invention. The polar region changes If not naturally linked to the transferred peptide, it can be synthesized or obtained from a convenient source and adapted to link the transfer peptide to the gene of interest. can be used as a filter or bridge. Transfer peptides and processing signals In the absence of convenient restriction sites in the target gene and/or gene of interest, The sexual region acts as an adapter to regenerate deleted codons by restricting can act.

The DNA sequence encoding the transfer peptide is a complete DNA sequence, including processing signals. a sequence encoding a transfer peptide or about 1 to 10 codons or from the 3'-terminus It may be a sequence encoding a truncated transfer peptide that deletes a portion of the codons of Ru. Furthermore, numerous changes in the transferred peptide and processing signals caused by mutations, deletions or insertions, where such changes can provide convenience, i.e. provide convenient restriction sites or bad restriction sites can be removed. Mutations can be conservative or non-conservative. As a result, the transferred peptide may be the same or different from the wild-type transferred peptide. can.

Individual sequences are obtained from naturally occurring sources and modified from naturally occurring sources. sequence, and can be a combination of naturally occurring and synthetic sequences. and the like. Various techniques have been used to modify these sequences. For example, in vitro mutagenesis, primer repair, re-cleavage, ligation, termination, etc. can be used. Those various techniques are performed according to conventional methods. obtain.

The structural gene of interest may be derived from cDNA, chromosomal DNA, or wholly or partially. It can be done. All or part of the structural gene may be a natural protein encoding the wild-type peptide. naturally occurring sequences or wild-type peptides or as a result of point mutations, insertions or deletions. It may be a synthetic sequence that encodes a mutant, completely or partially. Some In some situations, all or some of the codons may be modified, e.g. It is desirable to use DON to enhance expression. non-plant sources, e.g. microorganisms, For example, bacteria and fungi, non-transparent animals such as insects, and vectors such as mammals. Heterologous genes from mammals and fish are of interest.

The various segments of the chimeric gene can be linked in a variety of ways, usually in one or more More fragments are inserted into the vector, cloned, and The cloned plasmid is restricted, then appropriately manipulated, and the flanking plasmid ligating the fragment to the cloned fragment. this These steps can be repeated until the complete chimeric gene is completed.

A particularly preferred chimeric gene of the present invention is ribulose-1°5-bisphosphate carboxylic acid. Transfer peptide from xylase small subunit (SSU) and glyphosate resistance It is formed by adding a weight. Many publications have shown that the SSU transfer peptide is , without considering how it is bound to the structural gene, Although we have pointed out that this may be due to the SSU transfer peptide gene, turns out to be untrue when the offspring is linked to the aroA gene and other genes. Ta.

10 to 20 copies of the 5SU 5'-end of the sequence encoding the mature SSU protein. Don and no more than 20 codons, preferably 14 to 18 codons, and more Of particular interest is the use of preferably 16 codons.

For example, Van den Broeck et al., Nature (1985) 313: 358-363 describe chimeric genes, where small subunits are The NPT-IIn gene is a bacterial neomycin phosphotransferase. ■ is connected to the code). encoded by both plasmids described in that document. The resulting fusion protein consists of 57 amino acid transitions of the mature small subunit polypeptide. the transpeptide and the first methionine, the seven amino acid linker fragment and the first Consists of NPT-II lacking methionine. Apparently this unique fusion product is N The PT-IIn gene could be transferred to chloroplasts. similar EMBO Journal (1985) 4: 25-32, but the structure used was Van d The same NPT-n gene described by ep Broeck has six artificial 57 amino acids from the mature small subunit gene linked through codons It contained a transposed sequence and an additional 22 amino acids.

peptide and the N-terminal 13 amino acids of the SSU mature peptide of Ento We used soybean heat-shock protein (Dalton for 17.5) that was cleaved to . Structure with Ento SSU mature peptide instead of heat shock protein In comparison, translocation of heat-shock proteins was substantially less effective.

The method of Van den Broeck et al. is attempted with the aroA gene. In this case, the gene product was not transported into the plant chloroplast. Therefore , small subunit transfer peptides generally transport peptides into chloroplasts. Van den Broeck et al. The general point that er etc. seems to be wrong. Transfer peptide sequence, pro Processing sequence and post-processing amino terminus of the mature small subunit peptide Reduction of small subunit genes into segments, including partial or other polar sequences oA There appears to be a prerequisite for gene fusion. A functional mutant may be used, where one or more, preferably 1 to 3, amino acids are , preferably conservatively substituted by other amino acids, so that the transfer peptide gene is ar It is linked to the oA gene.

For expression, it will be necessary to have transcriptional and translational control signals. convenience For example, a chimeric gene with its own start and stop codons can be prepared. , so that expression will be initiated and terminated at the appropriate codon. Sara the transcription initiation region and terminus, including the RNA polymerase binding site and the transcription initiation site; minator regions will be added to the 5' and 3' of the chimeric gene, respectively. .

Conveniently, the transcription initiation region is the native promoter region associated with the transfer peptide. Something can happen. However, in many cases the native transfer peptide transcriptional activator The originating region is characterized in that it does not provide the desired degree of transcription or that constitutive transcription is desired ( Here transcription of the leader peptide is inducible) and therefore can be tolerated or vice versa. The same is true. Therefore, its native transcription initiation region may be different regions or region upstream from the remarase binding site, and transcription initiation is triggered can be substituted to confer sexual transcription.

Transcription initiation regions that may be used include small subunit (SSU) carboxylase, Contains plant genes such as acyl carrier protein (ACP) and elongation factor 1. are derived from various plant gene promoter regions including, or are functional in plants. Bacterial plasmid gene promoters, such as Agrobacterium bacterium) Ti- or Ri-plasmid opine synthase protein It can also be a motor or viral promoter. These promoters pin synthesis, e.g. octovin synthase, tubalin synthase, mannovin synthase Contains promoters related to agrobin synthase, aglobin synthase, etc. virus The promoter is cauliflower mosaita virus 35S promoter and region ■ ζ・mu the promoter.

In the same manner as chimeric gene construction, the chimeric gene provides the desired transcriptional regulation. Expression cassettes can be developed that flank transcription initiation and termination regions. many In some cases, and frequently, expression constructs are developed in which transcription initiation and termination regulatory regions are regions are provided, separated by a polylinker with many restriction sites, where the Chimeric genes can be created by inserting a small linker fragment or replacing it with a chimeric gene. The offspring can be linked such that they are under the regulatory control of the expression cassette. The key to its expression The kit can be replicated and selected in one or more hosts, including especially prokaryotic hosts. It is usually carried on a vector that can be used.

Depending on the manner in which the expression cassette is introduced into the plant cell, the expression cassette may be Other DNA regulatory regions may be initially linked. Typically, the expression cassette is isolated, Cloning of the expression cassette for sequencing, analysis and the like To make it possible, prokaryotes, especially E, co! Functional in J (E, coli) It will be linked to the replication system. Regarding the replication system, selection can be made in the host. one or more markers for biocide resistance, e.g. quality; heavy metal tolerance; toxicity tolerance; complementarity; provision of prototrophy to auxotrophic hosts; immunity will typically include markers such as gender;

When DNA is microinjected into host cells, the injected DNA Markers that allow selection of these cells that become integrated and functional are becoming commonplace. It would be highly desirable. Therefore, markers that can be detected in the plant host are selected. There will be.

On the other hand, armed (causing bile formation) or unarmed (causing bile formation) tumor-inducing plasmids, such as Ti- or Ri-plasmids can be used. ) loekema, Nature (1983) 303: 179-180; EPA No. 116718 and Wo 861 See 03776. There are various construction methods involving T-DNA, and we will discuss them here. The elephant expression cassette has a single T-DNA border, i.e. the right border. , or can be bordered on both the right and left sides by a T-DNA border.

The border is at least about 50 bp, usually at least about 100 bp T- It will contain DNA. Optionally, one or more expression systems thereof. can contain more T-DNA structural genes than in the plant host, resulting in can be used as a marker for the presence of T-DNA integration. It will bring about the manifestation of Obin. Specification of the expression cassette being introduced into the host nucleus An embodiment of the invention is that the expression cassette is present and that the expression cassette is present and It is not critical to the invention as long as it can function.

Expression vectors contained in prokaryotic vectors having one or both T-DNA borders. Ti- or Ri-bra, respectively, such as Zambryski, et al. , Genetic Engineering, Pr1n-cjples an d Methods, Volume 6 (Setlow and Ho 11aender edition) 2 53-278 (Plenum, N, Y, 1984); Binary Vector Application No. 834,161; and A. Hoekema, Th. e Binary Plant Vector Systems (1985), (Offsetdrukkerij Kanters, B, V, Albles- sardum). The expression cassette is T - integrated into the tumor-inducing plasmid in the DNA, and then the cassette is The bacteria can be used to infect plant cells or tissues.

Determine whether the desired product is present in a plant cell that has been integrated into the genome. Various techniques exist and will be described. For example, the Northern method can be used to detect mRNA encoding a given peptide. moreover, The presence of expression can be detected in a variety of ways. The expression product is a detectable expression type, e.g., if it provides a novel phenotype or an enhancement of an endogenous phenotype, the desired production Expression of a substance can be determined by detecting the phenotype. A detectable phenotype If not available, antibodies specific for the mature product can be used. Chlorop The rust is isolated according to conventional methods, destroyed and processed into Western or other Techniques can be used to identify the presence of the desired product.

After transformation, cellular tissues (e.g. protoplasts, explants or cotyledons) are transferred to regeneration medium for the formation of cells.

The regeneration medium usually contains a fungicide, such as carbenicillin (500 μ/l). can contain selection reagents to select transformed cells. . For example, regarding the kanamycin resistance gene (APH3' II), syn, at least about 30 mg/l and usually no more than about 500 mg/l; Preferably it will be added to the selection medium at about 50-100 mg/A. its rebirth The medium is supplemented with a suitable salt source, such as Murash ige-3koog salt medium, carbon sources, such as sucrose and other suitable additives, such as hormones, such as enzymes. atin, etc. (at about 0.75-2.25 mg/l), myo-inositol (at about 50 mg/l), etc. ~200 µ/l), etc. Also, vitamins such as N1tsch vitamin , about 0.5 to 100OX stock, as conventionally present in regeneration media. It can be added at 1.5 mf/β (usually 1.0 mj2.#). N1tsch Bi Tamin's 1000X stock has the following final volume of 100m7! Contains in: Thiamine HCl 5 Q mg, glycine 200 mg, nicotinic acid 50 cm, pyrido Xin 50■, folic acid 50mg, biotin 5mj2 and water (final volume 100rn1 amount remaining). The carbon source will be present at 10-30 g/β. Conveniently, re The live medium contains about 0.5-1.0% agar and the regeneration medium contains about 6±0. 5 p) Buffered with I.

In 2-3 weeks, shoots usually grow. After the shoots reach about 1-2 marks, they are cut at the base and transferred to a rooting medium, and this medium is used as the seedling grows. The medium is the same as the medium used in this study, and supplemented with carbenicillin and kanamycin sulfate. added.

Once modified plants are obtained, they can be grown to sufficient maturity to produce the desired to determine whether the product is still produced from all or part of the plant cells. be able to provide sufficient material for Expression of the desired product is shown in the plant. After that, the plants are grown to obtain seeds to obtain the desired expression of the expression cassette. used for crossbreeding with other plants to produce hybrids with current ability. A variety of embryos can be generated for further reproduction.

Chloroplasts are very important cell organelles. Therefore, chloroplast Transport of proteins to the last layer is important for improving herbicide resistance, improving amino acid metabolism, and improving photosynthesis. Good, can enable improvement of fatty acid metabolism and improvement of other important metabolic functions .

Ribulose-1,5-bisphosphate carboxylase small subunit leader chain (more preferably small subunits from soybean) Of particular interest is the construction method of Berry-Lowe et al., JIM. Ol, Appl, Genet, (1982) Trochanter, especially as above, wild-type For enzymes, ar expressing enzymes that are resistant to inhibition by enzyme inhibitors. It can be linked to the oA gene.

Along with chimeric plasmids, transcriptional regulatory systems such as those associated with T-DNA, etc. For example octobin, tubarin, mannovin or aglobin synthase, or small A block unit transcriptional regulation system can be used. Typically, the configuration is a reader page. peptide or the terminator region of the transcription initiation region, and which , can be introduced downstream from the structural gene by conventional means. Usually, its termination The tar region will be about 50 bp to about 1000 bp downstream from the stop codon. cormorant.

The methods of the invention result in the production of novel proteins. N-terminal 1 of the mature product ~20 amino acids with a polar region or an N-terminus extended by a polar region. May be replaced. These proteins are naturally occurring proteins encoded by hydrophilic regions. They are distinguished by having an absent N-terminus. Therefore, typically ar The oA gene contains 8-20 heterologous amino acids at its N-terminus. SSU For Buunit, the protein contains 14 to 1 g of mature SSU protein. , preferably will contain 16 amino acids. Similarly, other Chloroplus Endogenous proteins have their own structure as a result of the replacement of their native N-terminus with a polar region. polar region by lacking the N-terminus or extending the wild-type N-terminus. It can be characterized by being fused into a region. The following examples are illustrative and limiting. It's not something you do.

Three types of RuBP 5SII clones were used. pSR32,1 is the genome tobacco clone [Berry-Lowe et al. (1982) supra]. TSSU 3-8 and 5SU3-2 are genomic tobacco clones [0° Nea I etc. .

Nucl, Ac1d, Res, (1987)). pss15 is Ento's The cDNA clone [Coruzzi et al., J. Biol, Chem. , (1983) 258:1399]. Transfer peptides of soybean and tobacco SSU The regions are 75% homologous. The former is used for in vitro uptake studies. , the latter was used for plant expression in 7-H Molecular Clo ning: A Laboratory Manual, Co1d Spr ingHarbor Laboratory, Co1d Spring Ha rbor, NY]. Manufacturer's specifications, if applicable. Accordingly, pHc12 (Cm”) and pUc13 (Cm”) (Ken Buckley (Ph.D., thesis; U.C., Sanjaygo), EcoRI and HindII and pUc18 and pUc19 respectively. inserting a polylinker into linearized pUc12 and pUc13, respectively; pCGN565 and pCGN566 were obtained, respectively. Each plasmid is Carrying a resistant loramphenicic marker. 'RIIBPCssu5'- The EcoRI fragment containing the untranslated region and the 5′-coding region, respectively. into the EcoR1 site of pCGN565 and pCGN566, and RuBPcssu gene carried on GN330 and hippCGN331 to obtain the amino terminal part of.

did. - The resulting fragment contains 8 of the 5'-untranslated region of the phage M1 3-65848 was obtained.

AccI and and cloned into pUcg cut with PstI, and the aroA gene 14 amino acids at the N-terminus are substituted. pPMG34 has not been previously described. [5tarker et al. 1, J. Biol, Chem, (1985)] 260:4724-4728], BamHT-5 containing the aroA gene The alI fragment was cloned into pHc9, resulting in pPMG34.1.

Starting from the BamHI site, the 5'-untranslated region of the aroA gene was 74 upon treatment with DNA polymerase in the presence of cleoside tribosphate. It was further degraded and then digested with mung bean nuclease. So After treatment with each 74 DNA polymerase and mung bean nuclease , the fragment was inserted into the pUC vector as a 5all insert plant. The transformants were cloned and the transformants were screened for the expected product.

These 2 are cleaved by T4 polymerase and mung bean nuclei in the presence of dGTP. Treated with crease and T4 DNA ligase. That DNA, E, Wang I JLC3, i.e. aroA mutant [Coma 1 fee, Natur (1983) 31 Uni 741-744] and supplemented with several The plasmid was characterized. Plasmid pPMG34.3 was digested with crease. , remove the overhang, and insert into the site a chloramphenicol-resistant pPMG64 containing the genetic gene is obtained.

The site is close to the 3'-end of exon 1 of the RUBPC, Su gene.

-Provides peptides and processing signals. The array is: Ru: ,,,ACAAT I GCATGCI CI GGATCCI CG IT GACTTTCI ATGGAA.

5phl BamHI 5'-UT aroA aroA: 7-do area area R [IBPCS, code 1 linker region 1 region The resulting plasmid pPMG70 was digested with )IindIII and the small The untranslated (UT) region upstream of the unit was removed to obtain plasmid pPMG72. . Plasmid pPMG72 was digested with HindIII and EcoRI, and the The 5SU-aroA promoter/gene fragment was transformed into HindIIr and [Melton et al., NI Jcleic Ac1d Re5earch (1986) 12 Near 035] The plasmid pcGN1068 was obtained. The vector pSP6 4 allows in vitro transcription of cloned DNA.

Configuration of 5SU-aroA fusion 3 This fusion combines 24 amino acids of the mature SSU peptide into a small subunit transfer peptide. tido and aro/ were present and connected between the sequences. This is μ+o) and caused deletion of the BamHI site located between the Sme1 site and the intermembrane. the The resulting plasmid pcGNIO75 was digested with EcaRI and )lindlI [ The ss-ro aroA chimeric gene (fusion 1) was isolated. This gene, EcoR1 (Vector Cloning Systems, San Di pcGN1076 was obtained. Plasmid pcGN1. 076 was cleaved with sphI and BamHI, and pea SSU cDNA clones were isolated from the cDNA clones and the resulting fragments were placed on agarose. Separation was performed by gel electrophoresis and electroelution from the gel.

The chimeric gene in pcGNI077 contains the transfer peptide of soybean SSU, the mature It consists of a part of the SSU of Japanese bean (24 amino acids) and the aroA gene. So This was called Fusion 3. pcGN1077 by EcoR1 and old ndIII pSP64 [0, A. Melton et al., Nucleic Ac1dResearch (1 984) 12:7035], the plasmid pcGN1086 was constructed. Cloning into the texture of pcGN1086 pcG81008 was obtained.

aroA of pPMG34.3 to construct the 5SU-aroA fusion The gene was excised as a BamHl-5alI fragment and gel purified. Manufactured. Plasmid pPMG72 was digested with BamHI-3alI.

5SU-a lacking the flag Met codon containing vector and SSU coding region contained the roA fusion.

In vitro synthesis of wild type aroA, fusion 1 and fusion 3. Then it etc. according to the manufacturer's specifications (Promego-B 1otec.

5P6-RNA polymer according to Medison, Wisconsin) Transcription was performed by adding enzyme and nucleotide precursors. Next, the RNA was extracted from wheat. Added to in vitro translation extracts from embryos BRL, Bethesda, M. D), and was translated in the presence of 535-methionine. its obtained pep Tides were analyzed by 5DS-polyacrylamide gel electrophoresis. pcGNl Translation of mRNA from ooB is comparable in mobility to the wild-type aroA product. This resulted in the synthesis of a 43 kd peptide. pcGN1068 and pCGN10 Translation of mRNA from 86 results in peptides of 50 and 53 kd, respectively. Ta. Furthermore, the 43 kd peptide was probably translated at the original aroA start codon. Translation of 0I RNA from pcGN1068 due to spurious liposome initiation Generated from the translation. Isolated spinach as described by each Sea urchins were incubated with Shiroblasts. Next, try the chloroplast. treated with lipsin to digest all peptides except the chloroplasts. I met. The stroma and membrane fractions were separated as described in the reference above and their and analyzed by 5DS-gel electrophoresis.

Constituent nuclease of 5SU-aroA fusion production vector pcGN1096 To generate many fusions by cleavage with 31, we called it pcGN1096. A specialized vector was constructed. The following is the configuration scheme: pcG The aroA component of N1077 was removed by digestion with sphI and 5alI. . I was in that place. The resulting plasmid pcGN1094 was Genet, which has the transfer peptide of the clone and the mature part of the pea clone ( 1979) 177:65) Hind m-BamHI region was transformed into pDs7 pBR322 [Bolivar et al., Gene (1977)] 2:95] into the same site. Kanamycin resistance gene pc ligated into the flank site to generate GN1093. Plasmid PPM634. 3 was digested with 5all and cut out from the fill-in as above. The mycin gene was ligated to obtain pcGN1095. kanamycin and aro A gene, Sst I and EcoRI 硼---■暉----- pcGN1095 due to digestion by 騨-Nawate-Ikunate■drunk-kaimei■■■Akebono-■-■- It was cut out as a fragment and inserted into the SstI and EcoRI sites of pcGN1094. pcGN1096 was obtained. In summary, pcGN1096 is 5' 3' contains the following pertinent features: various amounts of the SSU code region have been deleted.

It also shows the cross-species translocation of the kanamycin resistance gene starting from the 5' end of the gene. was connected to a point within the SSU code region. One of the three types of fusion is S A translationally active fusion should be generated between the SU coding region and a r o'A. be. These events were observed in E. coli LC3, an aroA mutant [(o Transform the resulting plasmid in Mai et al. (1983) supra]. and on its plasmid and minimal medium by ampicillin resistance. by Sanger dideoxy sequence analysis for translationally correct fusions by propagation of Decided.

It was configured in order to pcGNlsOL cleaves spinach ACP-1 was cloned into pUc18, resulting in pcGN1919.

The - code region was cloned to obtain pcG81608. This plasmid's associated The region is listed as follows from 5' to 3' and the excised plasmid is Sm aI and retransported to delete various amounts of the acp coding region. So It also amplifies the kanamycin resistance gene starting from the 5' end of the gene. It was linked to points within the cp coding region. One of the three fusions is acp A translationally active fusion should be generated between the coding region and aroA. this The events of E. rlJLC3, the aroA mutant, by transforming the plasmid and its resistance to ampicillin. Select for translationally correct fusions by plasmid and growth on minimal medium. selected by Isolate the complementary plasmid and undergo restriction digestion more characterized. The sequence of the fusion was imprinted with primers homologous to the amino-terminal region of aroA. It was determined by Sanger dideoxy sequence analysis using

Testing of fusion structures Fusions selected for characterization are cloned into expression vectors as follows. It has become an online version. The fusion derived from pcGN1608 was transformed into a SalI-BcoRI fragment. As a fragment, it was cloned into pcGN1906 cut by the same enzyme. It has become an online version. pcGN1906 was cloned through a linker into the pUC host. CoMV35S promoter (nucleotides 7146-7546) and O This is an expression vector having a C33' region 2:335:l. 5ail and The EcoR,I site is a 5' Located between the promoter and 3' termination region. The obtained plasmid was C fusion under the control of the aL4ν35S promoter and tml polya The denylation signal was cloned as a subura fragment. The latter is CaM By V35S promoter and Syl II, Sal I and EcoR1 sites It carries a separate OC33' polyadenylation region. The resulting plasmid were characterized by restriction endonuclease digestion (porate). 48 After incubation for an hour, the electroborated protoplasts Western buff fusions 1 and 3 contain the first 1 and 24 apical cells of mature 5SL7. Incorporate each amino acid. Fusion 4 is from linker code pcGN1008 The translation of the mRNA of 43 is comparable in mobility to the wild-type aroA product. This resulted in the synthesis of a peptide of KDa. pcGN1068 and pcGNI086 Translation of mRNA from . The radiolabeled precursor was then added to isolated spinach chloroplus Incubated with After that incubation, the chloroplasts Wash, treat with trypsin if necessary, and separate membrane and stromal fractions. , and analyzed by SDS RAGE. Fusion 1 protein is negligible in the chloroplast if either the membrane or stromal fraction is found in the amount has not been effectively transposed. Fusion 3 is transposed: two polypeptides are transposed Molecular landscape 47 and 46 for the products found in the troma fraction and processed It has a prephase size of KDa. The change in mobility of the translocated protein is After thin treatment, it is obvious. This is because transport is not complete and the protein Point out that some still have access to proteases. support this If so, a small amount of the 47 KDa species of EPSP synthase is present in the membrane fraction.

Partial digestion based on trypsinization of chloroplasts also This may be explained by the limited access to the stroma. On the other hand, the 47KDa species is the result of a complete transport process and is therefore partially exposed to protein hydrolysis. It will be done.

Chloroplasty of chimeric fusion proteins by transient expression in leaf protoplasts Transport to strike Combines 12,16,19,34,64 and 92 amino acids of the mature small subunit. The engulfing fusion was isolated and characterized. This is used to facilitate transportation analysis. The fusion was cloned into an expression vector and injected into leaf protoplasts. lectroporated. After 48 hours, the protoplasts were treated with anti-aroA antiserum. was analyzed using Western plot. Chloroplasts are not isolated However, the Western plot shows the size between the precursor and mature protein. Since the difference in It was clearly proven that it was done. In experiments, precursors are never detected. It was.

Absence of detectable precursor in the case of effective transport or in the case of no transport Currently, we point out that this species has a very short half-life under transient assay conditions. Ru. It may be transported with good results or rapidly turned over. Ru. The amount of mature small subunit present in the fusion is sufficient for its transport efficiency. effective: the most effective construct is the mature small subunit 16 It was a structure that incorporated several amino acids. Given an arbitrary unit whose efficiency is 1 , then the following are the standardized efficiencies: 12aa=0.3, 19aa= 0.09,24aa=0.07,1aa=0.0'05, 64 and 92aa <0.003゜Furthermore, observed by fusion bodies 3 (19) and 4 (34) No processing heterogeneity was seen with the 12 or 16 aa fusions. .

Constructs that incorporate the 35 amino acids of mature ACP, although with relatively low efficiency, , was conveniently transported during the Rishiroblast. A second fusion incorporating 42 amino acids The coalescence was not transported at all. Transfer peptide and 12 ACP usage patterns is based on a fusion of only two species, one of which is a small subunit. resemble. The peptide region at the amino terminus of the transported mature protein is optimally required for efficiency. This peptide has remarkable characteristics; is located on the surface of the protein in both SSU and aCp. Kyte and Doolittle [J,! , 4o1. Bio1. (1982) 157:105). Furthermore, it is a series of positive carry charged amino acids; in the case of the small subunit they are at position 9.10 and 11 (all Lys), for ACP, positions 8, 9.14, 20 ．． There is a Lys residue at 22 and an Arg residue at position 1. more positively charged Significant evidence of positive amino acid residues is located at 11 and 18. the amino-terminal region of plant EPSP synthase (with chloroplast as a precursor) Based on Della C1 oppa etc. 0, Biot Technology (1987) 5:579-584. It will be done. Lubben and Keegstra.

The above describes the transfer of SSU peptide and soybean to demonstrate in vitro transport. Thirteen amino acids of mature SSU fused to a heat shock protein were used.

However, no information is provided on the effect of the mature part of SSU on transport. won.

Improved efficiency of translocation of proteins, especially those with hydrophobic N-termini , using a polar binding group between the processing signal and the protein of interest. It is clear from the above results that this can be achieved by In this embodiment, the The N-terminus of a protein allows for virtually complete translocation of the protein. Can be selected to retain wild-type characteristics. Furthermore, the linker is a protein Enhanced or modified properties can be imparted. Controlling changes at the N-terminus However, it provides substantial flexibility in the composition of genes and their expression products. It will be done.

All publications cited herein are incorporated by reference within the level of skill of those skilled in the art with respect to the present invention. Each of the 6 publications that are attributable to the It's included.

The foregoing invention has been described in some detail, illustratively and illustratively, for clarity of understanding. However, modifications and changes can be made within the scope of the claims.

international search report 12^"51"-pC〒/1isQfu102401"=j111M-hamu- =-m, PCT/US87/02401-PCT/US87102 401 Attachment To Form PCT/Engineering SA/210. Part 1 construction.

PC? /υ587102401 A set of people to ForTnPCIISA/210. Part V LPσ/υ587102401 xttac) lIIIentto For+++ PCT/ISA/210.　 part Vl, 1

Claims (11)

[Claims] 1. A DNA structure; 1) Functional transcription and translation initiation regions in plants; 2) Transfer peptides and processes a first DNA sequence encoding a signal; 3) Encodes a polar region comprising at least two positively charged amino acids Approximately 6 to 20 codons or maturation. Encodes the N-terminal region of the acp gene a second DNA sequence of 12 to 30 codons; 4) a third DNA sequence encoding a gene of interest from a source other than a plant; and A DNA structure comprising functional transcription and translation termination regions in the direction of transcription in plants. Creation. 2. The first sequence is a small sample of reprose-1,5-bisphosphate carboxylase. 2. The DNA construct according to claim 1, which is a transfer peptide of buunit. 3. the second sequence is a native sequence linked to the first sequence, and about 1 2. A DNA construct according to claim 1, which has 4 to 18 codons. 4. armed or unarmed Ti- or Ri-plasmids at their ends. The DNA structure according to claim 1, which is linked to the DNA structure of claim 1. 5. The first sequence is a small subunit of ribulose-1,5-bisphosphate carboxylase. a transfer peptide of the unit; and said third sequence is replaced by said second sequence. Claim 1, which is an aroA gene having up to 20 first codons The DNA constructs described. 6. (1) Functional transfer peptides and processing signals in plant cells (2) a polar region of about 6 to 20 amino acids; and ( 3) Second peptide region consisting of a peptide having the function of chloroblast protein A protein consisting of a region. 7. 2. The polar region is a natural sequence linked to the first peptide region. Protein according to range item 6. 8. The transfer peptide is a small protein of ribulose-1,5-bisphosphate carboxylase. a transfer peptide of the subunit, and the second peptide region is the polar region. In the aroA gene with up to 20 N-terminal amino acids replaced by A protein according to claim 6. 9. (1) From the transferred peptide and processing signal of the plant acp gene (2) 12 to 30 peptide regions encoding the N-terminus of mature ACP; codon; and (3) a peptide having the function of a chloroblast protein other than acp. A protein comprising a second peptide region consisting of a peptide. 10. For introducing the target gene into chloroplast organelles in plants The law is: 1) Functional transcription and translation initiation regions in plants; 2) Transfer peptides and processes a first DNA sequence encoding a signal; 3) Encodes a polar region comprising at least two positively charged amino acids DNA sequence of about 8 to 20 codons; 4) a third DNA sequence encoding a gene of interest other than a plant gene; and A DNA structure comprising functional transcription and translation termination regions in the direction of transcription in plants. grow plants that have DNA sequences in their genomes that contain the The DNA sequence is translated by the transfer peptide, the processing signal A protein comprising a polar region and a translation product of said gene of interest is produced. and the protein is translocated to the chloroblast, and the translocated A method comprising: the peptide being removed. 11. A plant cell comprising a structure according to claim 1.