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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormones [GH] (Somatotropin)
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8234—Seed-specific, e.g. embryo, endosperm
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
  • C12N15/8254—Tryptophan or lysine
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• This invention relates to seed specific expression casettes for use in regulation and expression of proteins in plants.
• seed storage proteins represent the predominate source of amino acids and nutrition for the germinating plant. It is well known that genes which code for storage proteins are strictly regulated. Seed storage proteins are produced in large quantities at a determined stage of development; expression is both tissue specific and developmentally controlled. These proteins are generally localized in specialized organelles called protein bodies or protein storage vacuoles and are essentially protease free. Because the seeds of many plants can readily synthesize large amounts of these storage proteins it is believed that the biological machinery within seed cells can be put to use synthesizing proteins of importance to pharmacological and industrial needs. Thus, characterization of the storage proteins as well as the ability to direct protein synthesis toward the protein bodies is of continuing experimental, commercial, and nutritional interest.
• the storage proteins of the major grain crops have been particularly well studied.
• the major seed storage proteins are the globulins, represented by two types: the nonglyco- sylated 11 S proteins and the glycosylated 7 S fraction.
• the common bean Phaseolin.
• the major storage protein in known as 0-conglycinin and its subunits (a , a' , and ⁇ ) represent up to 80% of total seed protein in that genus.
• the protein zein constitutes about 80% of corn seed protein. While there are many zein-related genes responsible for zein production, one type, the ⁇ - zeins, are represented in very low copy number and yet account for
• the seeds of many dicot and monocot plants are defl cient in certain essential amino acids which require the addition of these amino acids when these seeds are used as the predominate source of protein in the human or animal diet.
• investigators have attempted to couple the high level expression of seed storage proteins with recombinant engineering techniques to produce plants with enhanced nutritional value.
• the protein is deficient from a nutritional standpoint in that the globulins are relatively low in sulfur-rich amino acids, making legume seed a nutritionally incomplete foodstuff.
• Recombinant techniques could be used to increase the amount of sulfur in the seed storage proteins or direct synthesis of heterologous sulfur-rich proteins.
• non-plant genes in plants has also met with success, but on a more limited level. It has been suggested that the difficulty in attaining expression of non-plant proteins is due to a failure of the plant splicing machinery to recognize introns (also called Intervening sequences) present in the non-native transcribed pre-mRNA, K. Hartmuth and A. Barta, Nucleic Acids Res. 14: 7513-28 (1986), since removal of introns is necessary in order for the mRNA to be functional. See, M. Green, Annu. Rev. Genet. 20: 671-78 (1986). For instance, Barta, et al, Plant Molecular Biol.
• introns also called Intervening sequences
• hGH human growth hormone
• European Patent Application 319 353 discloses a method for expressing a desired protein by modification of the hypervariable region of a determined storage protein by an insert encoding the desired peptide. In this way a modified storage protein is expressed which has contained within it amino acids which code for the foreign protein. The foreign protein is then cleaved by enzyme digestion, isolated and purified. However, the insert must be in reading phase with the non- modified part of the storage protein on either side of the insert, thus effectively limiting the size of the foreign protein which may be expressed to the length of the hypervariable region of the storage protein.
• the present invention overcomes the problems outlined above by providing for seed specific expression of a desired protein comprising fusion of a gene with a seed specific gene regulatory sequence.
• the present invention uses a gene amplification technique which is a modification of the polymerase chain reaction (PCR) method described in U.S. Pat. 4,683,195.
• PCR polymerase chain reaction
• European Patent Application 255 378 provides nucleotide sequences for seed specific transcriptional regulation. However, the reference does not teach the translation cassettes of the present invention.
• European Patent Application 295 959 refers to DNA expression cassettes for transcription of the brazil nut sulfur-rich 2S seed storage protein. The reference neither teaches nor discloses the translational expression cassettes of the present invention.
• European Patent Application 319 353 refers to a process for the production of useful biologically active polypeptides through the modification of plant genes encoding S2 albumins and is discussed above.
• phaseolin mRNA can be produced in tobacco by the use of an mutant phaseolin gene lacking the native introns.
• the gene coding for the ⁇ -zein 15Kd protein and regulatory regions are used in this invention.
• the sequence and isolation of this sulfur-rich zein protein is disclosed in Pedersen, et al, "Sequence Analysis and Characterization of a Maize Gene Encoding a High-Sulfur Zein Protein of M r 15,000", J. Biol. Chem. 261:6279-6284 (1986).
• the present invention uses specific linkage sequences not taught by the reference.
• the present invention provides a seed specific expression cassette which has a promoter derived from either phaseolin, ⁇ '- subunit of ⁇ -conglycinin, or ⁇ -zein 15Kd; a translation initiation signal from either phaseolin, the ⁇ '-subunit of ⁇ -conglycinin, ⁇ -zein 15Kd, or an animal gene; a gene derived from brazil nut sulfur-rich seed storage protein or an animal gene; a translation termination region derived from either the animal gene, phaseolin, or ⁇ -zein 15Kd; and at least one polyadenylation region from phaseolin, the animal gene, or ⁇ -zein 15Kd; wherein the regulatory sequences are operably linked to one another in such a manner that the gene is expressed in seed or seed storage protein bodies.
• the invention further provides enzyme restriction sites incorporated into the sequences which link the regulatory elements and genes.
• the invention also provides that certain of the regulatory elements be derived from the same genetic source.
• the present Invention provides an expression cassette in which the promoter is from phaseolin or ⁇ '-subunit of ⁇ - conglycinin, the animal gene and remaining elements are from bovine somato-tropin, and the restriction sites contained within the linkers are Ncol and Hindlll.
• the present invention provides an expression cassette in which the promoter is from phaseolin or ⁇ '-subunit of ⁇ - conglycinin, one poly-A signal is from phaseolin, the remaining regions, including a second poly-A site, are from bovine somatotropin and the restriction sites are Ncol and Hindlll.
• the present invention provides an expression cassette for the expression and regulation of bovine somatotropin in seed storage bodies.
• the gene is from brazil nut 2S storage protein or subunits thereof, the promoter and translation initiation signal are derived from the same genetic source, and the translation termination signal and poly-A region are. from phaseolin or ⁇ -zein 15Kd.
• the invention also provides for cells, plants, and seeds containing the expression cassettes disclosed here.
• the present invention provides a plant In which a gene coding for a specified protein is Incorporated into the plant's genome in a manner such that regulation and expression of the specified protein Is under the control of a seed specific regulatory sequence.
• the protein which is of Interest and for which expression is desired may be a plant protein homologous or heterologous to the host plant, or on the other hand, may be normally produced in a virus, microorganism, or other animal tissue.
• Corn and soybean represent ideal hosts and seed storage protein regulatory regions isolated from Phaseolus vulgaris (phaseolin gene), Glycine max (promoter from the ⁇ '-subunit of ⁇ -conglycinin gene) and Zea mays (zein 15 Kd peptide gene) are preferred as sources of regulatory regions. Proteins which would have value when expressed in plant seeds include those designed for nutritional improvement (e.g., high sulfur soybean and common bean, high lysine corn, etc.), pharmacological importance (enzymes and hormones), and industrial value (enzymes).
• Bst bovine somatatropin
• cloning vectors which have an appropriate restriction site, a replication system, and a marker.
• a cloning vector we have selected pUC18, Norrander et al, Gene (1983)26:101- 106, containing an ampicillin resistance marker.
• Other suitable vectors are well known and may be easily adapted for use with the specific restriction sites of the present expression cassettes.
• CPCR custom polymerase chain reaction
• cassettes for transcriptional and translational fusion Two dicot specific seed promoter cassettes; and one monocot specific seed promoter cassette.
• these cassettes may be utilized in a variety of hosts.
• incorporation of these cassettes into a host plant genome may be accomplished by a variety of conventional methods. Such methods include transformation with Ti-DNA (A. tumefaciens, A. rhizopenes, etc.), protoplast fusion, injection, electroporation, and microprojectile bombardment.
• These plant seed expression cassettes are used to express a high sulfur seed storage protein gene in the seeds of both dicot and monocot plants and to express a non-plant gene, bovine somatotropin (Bst), in the seeds of dicot plants.
• Bst bovine somatotropin
• Transgenic plants containing the heterologous gene of Interest are obtained through the Rl generation. Seeds from these plants, R2 seeds, are stained with protein antibody for TEM visualization of the heterologous protein localized within the protein storage bodies.
• Bst means bovine somatotropin protein.
• BST means the gene encoding Bst.
• Bz means a sulfur-rich protein isolated from Brazil nut seed.
• Bz-1", Bz-2", and “Bz-3” are subunits of Bz. These proteins are described in further detail below.
• BZ, “BZ-1”, “BZ-2”, and “BZ- 3” are the genes encoding the respective proteins.
• Seed specific expression cassette means a DNA construct containing gene regulatory elements and a gene encoding a protein of interest, in a suitable vector for incorporation into a plant genome, such that the expression of the gene is under the control of the gene regulatory regions, the regions being of the specificity, orientation, and reading frame such that expression of the gene is in the plant seed.
• “Expression cassette” means a DNA construct lacking at least a gene encoding a protein of interest.
• “Oligomer primer” means a nucleotide sequence needed for PCR amplification of a gene or gene regulatory element. The primers used in this invention have been synthesized to incorporate specific restriction enzyme sites into the primer.
• Charts 1 through 22 illustrate the constructions of the present invention. The following conventions are used to illustrate plasmids and DNA fragments:
• the single line figures represent both circular and linear double-stranded DNA.
• phaseolin (common bean) is known as phaseolin.
• the complete sequence, including 5' and 3' regulatory elements, and cDNA counterpart have been published by Slightom et al. (1983).
• phaseolin regulatory regions we use the clone pPhasmin which has the intronless cDNA counterpart in place of the genomic coding and intron regions, as described by Cramer et al., Proc. Natl. Acad. USA, 82:334-338 (1985).
• Oligomer primers for amplification of phaseolin promoter (pvP) are constructed to contain Hindlll (in the 5' primer) and Ncol (in the 3' primer) sites.
• the Ncol site is designed to include a translation initiation site which conforms to the rules described by Kozak (1986) by containing the sequence AAXXATGG where X represents G, A, T or C. Sequences of the oligomers used for the amplification and engineering of the phaseolin promoter are shown in Chart 1(a). Using CPCR technology the restriction sites are fused to the 500 b.p. phaseolin promoter region resulting in the fragment designated pvP in Chart 1(b).
• oligomer primers are synthesized to flank the phaseolin polyadenylation (poly-A) signal.
• poly-A phaseolin polyadenylation
• the 5' primer incorporates a Ncol site and the 3' primer a Hindlll site.
• the nucleotide sequences of these two primers is shown in Chart 1(c).
• Amplification results in a 1100 b.p. fragment designated pvS in Chart 1(d).
• the pvP and pvS fragments were subjected to digestion by Ncol followed by ligation of the fragments (Chart 1(e)).
• the ligated pvP + pvS fragment (1600 b.p.) is subjected to Hindlll digestion, purified on polyacrylamide gel, and cloned into Hindlll digested pUC18.
• the clone containing the correct pvP + pvS sequences (confirmed by restriction enzyme mapping and nucleotide sequencing) is referred to as pUC18pvPpvS in Chart 1(f).
• This expression cassette is used with coding regions which either contain their own translation initiation signal or do not need a signal for proper targeting of the protein product. in addition, any coding regions inserted into this cassette must include a translation termination signal.
• oligonucleotide primers are designed for the amplification of promoter and terminator fragments from pPhasmin which contain the phaseolin signal peptide (SP) and translational termination (TT) signals, respectively.
• SP phaseolin signal peptide
• TT translational termination
• phaseolin 3' regulatory regions are obtained using the primers shown in Chart 2(c). Again, these primers contain an inframe Nco I site in the 5' oligomer and a Hindlll site in the 3' oligomer. Amplification yields a 1200 b.p. fragment referred to as pvTTpvS in chart 2(d).
• Both fragments, pvPpvSP and pvTTpvS, are subjected to digestion by Ncol and the exposed Ncol sites are ligated together to yield the fragment referred to as pvPpvSPpvTTpvS in chart 2(e).
• This fragment is then digested with Hindlll to expose CPCR engineered 5' and 3' sites followed by cloning into Hindlll digested pUC18.
• the phaseolin expression cassette referred to as pUC18pvPpvSPpvTTpvS in chart 2(f) is obtained and checked by restriction enzyme mapping and nucleotide sequencing to determine that it is correct.
• This seed specific expression cassette is used with coding regions which need a seed specific signal peptide and an inframe translation termination region.
• ⁇ -conglycinin represents the major seed storage protein. The level of expression is very high; each of the two to four subunits account for between 12 to 20% of the total protein in the seed.
• the nucleotide sequence of the promoter region of Glycine max ⁇ '-subunit of ⁇ -conglycinin has been published by Doyle et al., J. Biol. Chem. 261:9228 (1986).
• the promoter (gmP) is amplified from 250 ng total Glycine max DNA and, by using CPCR, a Hindlll site is added to the 5' end as well as an Ncol site at the translation initiator codon (ATG) .
• oligomers are designed for amplification of the promoter (oligomer sequences are shown in Chart 3(a)).
• the sequence of the amplified 600 b.p. gene, designated gmP in Chart 3(b) is verified by restriction enzyme mapping and nucleotide sequencing for accuracy.
• the fragment gmP was subjected to digestion by Ncol and ligated to the Ncol digested fragment pvS (described In Example 1) to obtain the fragment referred to as gmPpvS in Chart 3(c).
• the resulting fragment, gmPpvS is digested with Hindlll, ligated into Hindlll digested pUC18, and the clone pUC18gmPpvS is obtained.
• This expression cassette is used to direct seed specific expression of coding regions which require transcriptional fusions, similar to that shown in Example 1. However, when incorporated into the Glycine genome the level of expression may be somewhat higher than the Phaseolus expression cassette of Example 1, due to the more native environment for the Glycine cassette.
• the same 5' primer as described in Example 3 is used.
• the 3' oligomer primer is designed to hybridize just beyond the signal peptide cleavage point described by Doyle et al, (1985), which occurs between amino acid residues 25 and 26. Therefore, we have selected to add the Ncol site after amino acid residue 29.
• the primers used for this amplification are shown in Chart 4(a).
• gmPgmSP in Chart 4(b) The result of this amplification, referred to as gmPgmSP in Chart 4(b), is a fragment of 690 b.p. Authenticity of this fragment is checked by nucleotide sequencing. It is then subjected to digestion by Ncol and ligated to the Ncol cut fragment pvTTpvS described in Example 2. The resulting fragment, referred to as gmPgmSPpvTTpvS in Chart 4(c), was subjected to digestion by Hindlll and cloned into Hindlll digested pUC18. The clone (designated pUC18gmPgmSPpvTTpvS in Chart 4(d)) is isolated and its structure checked by restriction enzyme mapping and nucleotide sequencing. This expression cassette is used for the expression of modified seed storage protein genes or foreign genes in the seeds of dicotyledonous plants.
• the zein proteins constitute about 80% of the total seed protein of Zea mays (corn); however, there are many zein-related genes responsible for the production of these zein proteins. Recently, one particular member of the zein gene families was found to be represen- ted in low copy number, but responsible for the production of about 10% of the total zein proteins. These genes are referred as ⁇ -zeins (or 15 Kd) genes and the sequence of one member has been described by Pedersen et al. (1986). However, the nucleotide sequence of the ⁇ - zein gene reported by Pedersen only extends about 233 b.p. 5' of the translation initiation, thus the complete ⁇ -zein gene promoter may not be present.
• Clone pZein15K (Chart 6(a)) provides the starting material for the CPCR amplification of its promoter and polyadenylation regions.
• an indigenous Ncol site in the promoter region was removed by flushing with mung bean nuclease following the procedure of Maniatis, et al, (1982), which caused a loss of four base pairs (CATG). These four base pairs are located between the CCAAAT and TATAA elements and thus should not affect the function of the promoter.
• This modified ⁇ -zein gene promoter region was amplified using CPCR to add a Sail site and a Ncol site to the 5' and 3' primers, respectively.
• the sequences of the oligomers used to accomplish this are shown in Chart 6(b).
• the amplified product was a 350 b.p. fragment which is referred to as zmP in Chart 6(c).
• Additional CPCR oligomer primers were used to amplify the polyadenylation region of pZein 15K so as to add a (5') Ncol site and a (3') Sail site.
• Oligomers sequences are shown in Chart 6(d).
• the amplified product was a fragment of approximately 700 b.p. which Is referred to as zmS in Chart 6(e).
• the two amplified fragments, zmP and zmS are subjected to digestion with Ncol followed by ligation and isolation of the fragment referred to as zmPzmS in Chart 6(f).
• zmPzmS Is digested with Sail and cloned into Sail digested pUC18.
• Clone pUC18zmPzmS (Chart 6(g)) is isolated and its structure verified using restriction enzyme mapping and nucleotide sequencing.
• the expression cassette pUC18zmPzmS is used for seed specific expression of modified seed storage protein genes or other genes which do not require a zein signal peptide and/or a zein translation terminator.
• CPCR amplification of the ⁇ -zein transla tion terminator and polyadenylation signal regions of pZein 15K is done using a linker engineered to contain a 5' inframe Ncol site and a 3 ' Sail site; sequences are shown in Chart 7(c).
• the fragments zmPzmSP and zmTTzmS are subjected to digestion by Ncol and ligated together to obtain the fragment referred to zmPzmSPzmTTzmS in Chart 7(e).
• This fragment is then subjected to digestion by Sail and cloned into Sail digested pUC18.
• the resulting clone, pUC18zmPzmSPzmTTzmS (Chart 7(f)) is confirmed by restriction enzyme site mapping and nucleotide sequencing.
• This seed specific expression cassette pUCzmPzmSpzmTTzmS is used to regulate the expression of modified seed storage protein genes or other genes which may require a monocot or ⁇ -zein signal peptide and inframe translation terminator signal.
• BZ-1 was synthesized as a single 144 base oligomer, BZ-2 as a 210 base oligomer and BZ-3 as a
• brazilnut gene fragments were complemented, purified, and had Ncol sites added on both 5' and 3' ends using CPCR amplification. As is true of the BZ gene in Example 7, each inframe Ncol site yields the codon TGG which encodes tryptophan, making resulting plant seed a more nutritionally complete foodstuff.
• the primers used for amplification of BZ-1 are shown in Chart 11(a), those for BZ-2 in Chart 11(b), and those for BZ-3 in Chart 11(c). Because of the addition of the extra nucleotides the fragments isolated after amplification are about 15 b.p. larger than the original synthesized fragments BZ-1, BZ-2, and BZ-3. These fragments are subjected to digestion with Ncol and cloned into the Ncol cut expression cassettes described in Examples 2, 4 and 6.
• the following clones are isolated, pUC18pvPpvSPSZ-1, -2 , and -3pvTTpvS (Charts 12(a) -(c)), pUC18gmPgmSPBZ-2, -2, and -3pvTTpvS (Charts 13(a)-(c)), pUC18zmPzmSPBZ-1, -2 , and -3pvTTpvS (Charts 14(a) -(c)).
• the structure of these clones is checked by restriction enzyme mapping and nucleotide sequencing.
• the expression cassettes contained within these plasmids may removed and transferred into vectors used for Agrobacterium systems or may used with microprojectile bombardment of plant tissues.
• Bst bovine somatotropin gene
• Bgh bovine growth hormone
• This clone is referred to as pSVGH-1 (delta IVS) and Is used to provide the Bst coding region for CPCR engineering.
• pSVGH-1 delta IVS
• Is used to provide the Bst coding region for CPCR engineering.
• CPCR allows the coding region to be engineered in such a manner to be accepted by any of the expression cassettes described in Examples 1 to 6. These arrangements can include the use of different gene regulatory regions which can be provided by the expression cassette or even from the Bst gene.
• Bst protein is encoded in a gene which contains a 26 amino acid signal peptide followed by a coding region containing 191 amino acids.
• the clone pSVGH-1 also contains the indigenous Bst gene polyadenylation signal.
• For the Bst protein to be active its signal peptide must be removed, thus by using CPCR in combination with the cassettes described above we have the option of expressing It with or without its signal peptide, with or without a seed protein signal peptide, and with or without its polyadenylation signal.
• This example illustrates several of these expression cassettes coding for Bst.
• the first series of constructions uses the seed storage protein gene expression promoters, described in Examples 1, 3 and 5, to construct transcriptional fusions genes which use the Bst signal peptide and polyadenylation signal.
• pSVGH delta-1
• CPCR is used to add an Ncol site to its 5' end (retaining the translation initiation signal) and adding a Hindlll site 3' of the Bst polyadenylation signal.
• the oligomer primers of this construction are shown in Chart 15(a) and are used to amplify a 840 b.p. fragment referred to as bvSPBSTbvTTbvS. This fragment is subjected to digestion by Ncol and cloned into the seed promoter fragments described in Examples 1 and 3, see Charts 15(b) and 16(a).
• ligated seed promoter-Bst fragments are then digested with Hindlll followed by ligation to Hindlll digested pUC18 and the following clones isolated, pUC18pvPbvSPBSTbvTTbvS and pUC18gmPbvSPBSTbvTTbvS, as shown in Charts 15(c) and 16(b), respectively.
• the correct sequence is verified using restriction enzyme site mapping and nucleotide sequencing.
• the inserts from these clones can be removed and transferred into either an Agrobacterium vector system or into a vector to be used for mlcroprojectile bombardment of plant tissues.
• the Bst gene can be cloned directly into the Ncol site located within the seed expression cassettes shown in Examples 1 and 3.
• the 3' oligomer primer needs to be changed to include Ncol. This is done with the use of the oligomer primer shown in Chart 17(a) and CPCR engineering.
• the fragment containing the Bst gene obtained from this amplification is also about 640 b.p. in length, and is referred to as bvSPBSTbvTTbvS in Chart 17(b). This fragment is digested with Ncol and cloned into the Ncol site of the plant expression cassette shown in Example 1.
• Bst does not include its 19 amino acid signal peptide, which is cleaved soon after synthesis (Woychik et al., 1982). However, in a plant system it is doubtful that this signal peptide would be removed, thus its removal would have to be done as part of a purification procedure.
• the above constructions do not include the normal signal peptides for the plant seed storage protein genes to ensure correct targeting of the Bst peptide.
• the following constructions illustrate a translational fusion, made with the Bst protein gene, which does not include the
• Bst signal peptide but d'oes contain the signal peptides from the plant seed storage protein genes. This is accomplished by using CPCR to amplify the part of the Bst gene which does not Include the Bst signal peptide and cloning it into the expression cassettes described in Examples 2 and 4.
• the polypeptide resulting from expression from these fused gene is expected to initially include the seed storage signal peptides, which should be removed as a result of normal proteolytic cleavage to yield a Bst polypeptide which would be identical to the naturally occurring peptide except for the presence of two to four additional amino acids at its N-terminus. These additional amino acids are needed to ensure that the signal peptide cleavage site is recognized (see Examples 2, 4 and 6).
• the appropriate Bst gene fragment can be obtained using CPCR amplification using the two primers shown In Chart 21(a) which amplifies a 600 b.p. fragment referred to as BSTbvTT in Charts 21(b) and 22(a). This fragment is subjected to digestion by Ncol and then ligated into the seed storage protein gene cassettes described in Examples 2 and 4 to obtain clones, pUC18pvPpvSPBSTbvTTpvS (Chart 21(c)) and pUC18gmPgmSPBSTbvTTpvS (Chart 22(b)).
• the engineered expression cassettes contained within these clones can be removed by digestion with Hindlll followed by transfer to vectors designed to be used with Agrobacterium or microprojectile gene transfer systems.
• a plant was produced which contained an expression cassette coding for the bovine somatotropin gene, BST.
• BST bovine somatotropin gene
• Plasmid pSVbGH-1 contains a single restriction site for BamHI which lies between the SV40 promoter and the Bst gene.
• pPhasBst is made by digestion of pSVbGH-1 with BamHI followed by subsequent incorporation of the purified promoter region at the restriction site.
• the region of the pPhasBst plasmid containing the Bst genes and the phaseolin regulatory region is removed by digestion with Kpnl and inserted at the Kpnl site of Aprobacterium binary vector pGA482G to make plasmid pGA482G/PhasBst.
• pGA482G/PhasBst is used to transform Agrobacterium strain C58Z707.
• the resultant Agrobacterium is used to infect cultured tobacco leaf pieces, from which whole plants were derived.
• Transgenic tobacco plants containing the PhasBst gene are obtained through the R1 generation. Seeds from theses plants, R2 seeds, are stained with Bst antibody for TEM visualization. Results show localization of Bst protein within the protein storage bodies.

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