CN1196091A - Compositions and method for modulation of gene expression in plants - Google Patents

Abstract

An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of an gene in a plant. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a gene in said plant.

Description

Be used for composition and method in the expression of plant regulatory gene

The application is the part continuation application of following application: 1) denomination of invention of Edington is that ＂ is used to produce the non-provisional application U.S.S.N.08/300 of starch granules in conjunction with the method ＂ of the transgenic plant of glucose sugar transferase active defective, 726 (applying date is on September 2nd, 1994); With 2) denomination of invention of Zwick etc. is the provisional application U.S.S.N 60/001,135 (applying date is July 13 nineteen ninety-five) of " molectron of modified fatty acid saturation ratio and method in plant ".These two parts of applications with its integral body (comprising accompanying drawing) in the lump by this paper reference.

Background of invention

The present invention relates to be used for composition and the method expressed in the plant regulatory gene, more specifically say so and utilize enzymatic nucleic acid molecule regulatory gene is expressed in plant composition and method.

It below is the concise and to the point description that regulatory gene is expressed in plant.These discussion do not mean that it is completely, and the present invention who helps understand subsequently only is provided.This summary is not to recognize that the prior art that following described arbitrary work is invention required for protection.

The various strategies that regulatory gene is expressed in plant are arranged.Sense-rna (Bourque, 1995 plant sciences 105 are summarized among the 125-149) and common suppress (Jorgensen, 1995 science 268 are summarized among the 686-691) have been used for the regulatory gene expression traditionally.The insertion mutagenesis of gene also has been used for silencer and has expressed.Yet this method can not be used for deactivation interest genes specifically.The applicant believes that the ribozyme technology can provide a kind of attractive novel method that changes genetic expression in plant.

More than ten years at first in bacterium, found in the past naturally occurring sense-rna (Simons and Kleckner, 1983 cells 34,683-691).It is considered to a kind of bacterium can regulate the mode of their genetic expression (Green etc., 1986 bioid academic years were commented .55:567-597; Simons1988 gene 72:35-44).The inhibiting demonstration first of the antisense mediation of genetic expression is reported in (Izant and Weintraub 1984 cell 36:1007-1015) in the mammalian cell.Many sense-rna examples that regulatory gene is expressed in plant that utilize are arranged in the literature.Below be some examples:

United States Patent (USP)s such as Shewmaker 5,107,065 and 5,153,566 disclose the sense-rna method that regulatory gene is expressed in plant that adopts.

Someone points out that the inverted defined gene of expressing can serve as the dominance suppressor gene in plant.Produced transgenosis potato plant, these expression of plants are to potato or the cassava particle RNA in conjunction with amylase synthase (GBSS) antisense.In both cases, all constructed have a reduction or do not have active or proteinic transgenic plant of GBSS.These transgenic plant produce the potato of the starch that contains the amylose starch level with obvious reduction, and (Visser etc. 1991, Mol.Gen.Genet.225:2889-296; Salehuzzaman etc. 1993, molecular biology of plants 23:947-962).

Kull etc. 1995, J.Genet.﹠amp; Breed.49,69-76 have reported that expressing the transgenosis potato that is mediated by particle in conjunction with starch synthase (GBSS) gene antisense sequence is the biosynthetic restraining effect of amylose starch in the napiform root.Yet the author points out can not find any activity in vivo of ribozyme at GBSS RNA.

Proved that the sense-rna construct at Δ 9 desaturases in canola increases to 40% (Knutzon etc., 1992 Proc.Natl.Acad.Sci.89,2624) with stearic acid (C18:0) level from 2%.In a high stearic acid system, there is not the reduction of total oil-contg or percentage of germination.The summary (Ohlrogge, J.B.1994 plant physiology .104,821 that have recently the practicality of the plant that the oil of modification forms by several explanations; Kinney, A.J.1994 Curr.Opin.Cell Biol.5,144; 1994 Plant Cell Envir.17 such as Gibson, 627).

Document has at first been put down in writing the homologous transgenosis deactivation in plant, and it is to insert transgenosis and find gene and unpredictable consequence (Napoli etc., 1990, Plant Cell 2:279-289) that transgenosis is reduced with sense orientation as a kind of.As if there are at least two kinds of mechanism of deactivation homology genetic sequence.A kind ofly seemingly transcribe deactivation (the repetition DNA district signal endogenous mechanism of gene silencing) through methylation.This generegulation method relates to be introduced the transgenosis multiple copied or transforms plant (1995 EMBO such as Ronchi J.14:5318-5328) with the transgenosis that has with the interest genes homology.Another kind of suppress mechanism altogether and transcribe after, wherein the aggregate level of expressing from gene and transgenosis is considered to produce high-caliber transcript, the threshold value that it causes two kinds of signals induce degradation (van Bokland etc., 1994 phytology magazines, 6:861-877).The accurate molecular basis that suppresses is unknown altogether.

Unfortunately, antisense and suppress altogether both techniques on the heritability of required proterties, have problems (Finnegan and McElroy 1994 biotechnologys, 12:883-888).Current, not on the DNA of plants level the easy method of specifically inactivating interest genes (Pazkowski etc., 1988, EMBO, J.7:4021-4026).Transposon mutant be poor efficiency and be unsettled incident, and chemomorphosis is highly nonspecific.

It is attractive for property that the applicant believes that ribozyme has, and owing to the machine-processed advantage that has above the competition technology of its katalysis.Yet, the validity that proof ribozyme regulatory gene is expressed in botanical system have any problem (Mazzolini etc., 1992 molecular biology of plants .20:715-731; Kull etc., 1995 J.Genet.﹠amp; Breed.49:69-76).Though bibliographical information the has been arranged activity of ribozyme in vegetable cell, they nearly all involve the gene that the downward modulation external source is introduced, for example the reporter gene in instantaneous measurement (Steinecke etc., 1992 EMBO J.11:1525-1530; Perriman etc., 1993 Antisense Res.Dev.3:253-263; Perriman etc., 1995, Proc. Natl. Acad. Sci.USA, 92,6165).

Several publications [for example, Lamb and Hay, 1990, J.Gen.Virol.71,2257-2264 are also arranged; Gerlach etc., International PCT publication No.WO 91/13994; Xu etc., 1992, Chinese science (Ser.B) 35,1434-1443; Edington and Nelson, 1992, generegulation: sense-rna and DNA biology, editor R.P.Erickson and J.G.Izant, pp 209-221, Raven press, NY.; Atkins etc., International PCT publication No.WO 94/00012; Lenee etc., International PCT publication No.WO 94/19476 and WO 95/03404, Atkins etc., 1995, J.Gen.Virol.76,1781-1790; Gruber etc., 1994, cellular biochemistry supplementary issue 18A, 110 (X1-406) and Feyter etc., 1996, Mol.Gen.Genet.250,329-338] propose to utilize hammerhead ribozyme to regulate the expression of virus replication, virogene and/or reporter gene.These publications are not reported the expression that utilizes ribozyme to regulate plant gene.

Mazzolini etc., 1992, molecular biology of plants 20,715-731; Steinecke etc., 1992, EMBO.J.11,1525-1530; Perriman etc., 1995, Proc. Natl. Acad. Sci.USA, 92,6175-6179; Wegener etc., 1994, Mol.Gen.Genet.245,465-470; With Steinecke etc., 1994, gene, 149,47-54 has described the expression that utilizes hammerhead ribozyme to suppress reporter gene in vegetable cell.

Bennett and Cullimore.1992 nucleic acids research 20,831～837 have been set forth the glna of hammerhead ribozyme mediation, glnb, the external cutting of glng and glnd RNA (glutamine synthetase of encoding) in Phaseolus vulgaris.

Hitz etc. (WO 91/18985) have described the method that adopts soybean Δ-9 desaturase modified plant oil to form.This application has been described with soybean Δ-9 desaturase sequence and has been separated Δ-9 delta 8 desaturase genes from other species.

The above reference of quoting obviously is different from the present invention, because they are open and/or do not relate to use ribozyme in corn.In addition, the applicant believes that these documents are open with the gene in the ribozyme downward modulation vegetable cell, and/or can not reduce gene in the vegetable cell with ribozyme, has let alone plant itself.

Brief summary of the invention

The invention is characterized in and utilize the regulatory gene expression specifically in plant of enzymatic nucleic acid molecule.Preferably, said gene is a native gene.Enzymatic nucleic acid molecule with RNA nicking activity can be organized intron, forms such as RNA enzyme PRNA, Neurospora VS RNA, but be not limited to these forms with tup, hair clip, hepatitis Δ virus, I group intron, II.Enzymatic nucleic acid molecule with RNA nicking activity can be used as monomer or many bodies (preferably many bodies) are encoded.The nucleic acid that coding has the enzymatic nucleic acid molecule of RNA nicking activity can be operatively attached on the open reading frame.Can modify genetic expression in any plant by the nucleic acid transformed plant of enzymatic nucleic acid molecule that has the RNA nicking activity with coding.The technology that many conversion plants are also arranged: these technology include but not limited to be transformed by particulate (coatedmicroprojectiles) bombardment, whisker (whiskers) or electroporation with Agrobacterium, DNA bag.Can modify any target gene with the nucleic acid that coding has an enzymatic nucleic acid molecule of RNA nicking activity.Here two kinds of illustrative targets are that Δ 9 desaturases and particle are in conjunction with starch synthase (GBSS).

Regulate and/or inhibition of gene expression in plant (for example monocotyledons (as corn)) up to the reagent (as enzymatic nucleic acid (ribozyme)) of just having illustrated of finding of the present invention based on nucleic acid.Ribozyme can be used for regulating specific proterties of vegetable cell, for example, and by regulating the enzymic activity that relates in the bio-chemical pathway.In some cases, it may be desirable reducing the expression level of specific gene rather than eliminate its expression fully, and ribozyme can be used for reaching this purpose.The technology that the present invention has developed based on enzymatic nucleic acid makes and can guiding regulatory gene express the vegetable cell, plant tissue or the plant that have the phenotype of change with generation.

Ribozyme (being enzymatic nucleic acid) is to have the nucleic acid molecule that can repeat to cut the enzymatic activity of other different RNA molecule in nucleotide base sequence specificity mode.Such enzymatic RNA molecule can be directed to almost any rna transcription thing, and in vivo with external obtained effective cutting (Zaug etc., 1986, the nature 324,429; Kim etc., 1987, Proc. Natl. Acad. Sci.USA 84,8788; Dreyfus, 1988, Einstein Quarrerly J.Bio.Med..6,92; Hascloff and Gerlach, 1988, nature 334 585; Cech, 1988, JAMA 260,3030; Murphy and Cech, 1989, Proc. Natl. Acad. Sci.USA, 86,9218; Jefferies etc., 1989, nucleic acids research 17,1371).

Because their sequence-specific, and the effective tool that trans cutting ribozyme can be expressed as regulatory gene in various organisms (comprising plant, the animal and the mankind) (Bennett etc., the same; Edington etc., the same; Usman ﹠amp; McSwiggen, 1995 Ann.Rep.Med.Chem.30,285-294; Christoffersen and Marr; 1995 J.Med.Chem.38,2023-2037).Ribozyme can be designed to the specific RNA target of cutting in the cell RNA background.A kind of like this cutting incident makes mRNA not have function, and has suppressed protein from rna expression.In such a way, proteinic synthetic can optionally being suppressed that interrelates with special phenotype and/or morbid state.

Further feature of the present invention and advantage are from its embodiment preferred described below and claim as can be seen.

Accompanying drawing is briefly described

Fig. 1 is the diagram in hammerhead ribozyme well known in the prior art district.Dried II can be 〉=2 base pairs are long.Each N is any Nucleotide, and each represents a base pair.

Fig. 2 a is the diagram in hammerhead ribozyme well known in the prior art district; Fig. 2 b be as Uhlenbeck (1987, nature, 327,596-600) be divided into the diagram of hammerhead ribozyme of substrate and enzyme part; Fig. 2 c be show by Haseloff and Gerlach (1988, nature, 334,585-591) be divided into the similar figure of two-part tup; Fig. 2 d be show by Jeffries and Symons (1989, nucleic acids research, 17,1371-1371) be divided into the similar figure of two-part tup.

Fig. 3 is the diagram of the general structure of hair clip ribozyme.Spiral 2 (H2) has at least 4 base pairs (being that n is 1,2,3 or 4), and spiral 5 can have 2 or a plurality of base (preferably 3-20 base, promptly m is 1-20 or more) not essentially. Spiral 2 and 5 can be covalently bound through one or more bases (being that r is 〉=1 base).Spiral 1,4 or 5 also can be extended by 2 or a plurality of base pair (as 4-20 base pair), with the stable nucleus enzymatic structure, and protein binding site preferably.In each example, each N and N ' are respectively any normal or bases of modifying, and on behalf of potential base pairing, each dash interact.These Nucleotide can be modified on glycosyl, base and phosphate.Base pairing is optional completely in spiral, but is preferred.Spiral 1 and 4 can be any size (is that o and p are respectively 0 to any number separately, for example 20), as long as some base pairing is held.Essential base shows as particular bases in the structure, but one skilled in the art will realize that one or more bases can replace through chemically modified (dealkalize base, base, glycosyl and/or phosphate are modified) or with the another kind of base that does not have obviously influence.Spiral 4 can form from two independent molecules, does not promptly have shack.Shack can be to be with or without the ribonucleotide that base, glycosyl or phosphate are modified when existing." q " is 〉=2 bases.Shack also can be substituted by the non-nucleotide linkers.H refers to base A, U or C.Y refers to pyrimidine bases."-" refers to covalent linkage.

Fig. 4 is the diagram of the general structure in hepatitis Δ virus ribozymal well known in the prior art district.

Fig. 5 is from the diagram of cutting the general structure in VS RNA ribozyme district.

Fig. 6 is the synoptic diagram that RNA enzyme H accessibility is measured.Specifically, the left side of Fig. 6 is the figure that is attached to the complementary DNA oligonucleotide on the accessible site of target RNA.The complementary DNA oligonucleotide is represented by the wide line that is marked with A, B and C.Target RNA is represented by thin line of torsion.The right side of Fig. 6 is the gel separation synoptic diagram that does not cut target RNA from the target RNA of cutting.The detection of target RNA is undertaken by the radioautograph of the T7 transcript of body tag.Each road common band is represented uncut target RNA; The distinctive band in each road is represented cleaved products.

Fig. 7 is the diagram of the RNA enzyme H accessibility of GBSS RNA.

Fig. 8 is the diagram of the GBSS RNA cutting undertaken by ribozyme under different temperature.

Fig. 9 is the diagram of being cut by the GBSS RNA that the multinuclear enzyme carries out.

Figure 10 has listed from the nucleotide sequence of the isolating Δ 9 desaturase cDNA of corn.

Figure 11 and 12 is fatty acid biological synthetic diagrams in plant.Figure 11 is from Gibson etc. 1994, Plant Cell Envir.17,627 reorganizations.

Figure 13 and 14 is the diagram of the RNA enzyme H accessibility of Δ-9 desaturation ribozyme.

Figure 15 shows the cutting of the external Δ-9 desaturation ribozyme that carries out through ribozyme.10/10 represents the length of the brachium conjunctivum of tup (HH) ribozyme.10/10 refers to spiral 1 and spiral 3 each and target RNA formation 10 base pairs (Fig. 1).On behalf of the spiral 2/ spiral I between hair clip ribozyme and its target, 4/6 and 6/6 interact.4/6 refers to that hair clip (HP) ribozyme and target form the spiral 2 of 4 base pairings and spiral 1 mixture (referring to Fig. 3) of 6 base pairings.6/6 refers to that hair clip ribozyme and target form the spiral 2 of 6 base pairings and spiral 1 mixture of 6 base pairings.Cleavage reaction carried out under 26 ℃ 120 minutes.

Figure 16 shows that arm lengths changes the influence to HH and HP ribozyme external activity.7/7,10/10 and 12/12 in fact as above description to the HH ribozyme.6/6,6/8,6/12 represents spiral 1 length and constant (6bp) spiral 2 of the variation of hair clip ribozyme.Cleavage reaction carries out as above description in fact.

Figure 17,18,19 and 23 is the diagrams that make up the unrestricted strategy of the transcript that comprises multinuclear enzyme primitive (identical or inequality, the various sites in the guiding Δ-9 desaturation ribozyme).

Figure 20 and 21 shows the external cutting of the Δ 9 desaturation ribozymes that undertaken by the ribozyme that adopts the phage t7 RNA polymerase to transcribe from dna profiling.

Figure 22 is the diagram that makes up the unrestricted strategy of the transcript that comprises multinuclear enzyme primitive (identical or inequality, the various sites in the guiding GBSS RNA).

Figure 24 shows the cutting of the Δ 9 desaturation ribozymes that undertaken by ribozyme.The representative of body more than 453 is at many bodies ribozyme construct of hammerhead ribozyme site 453,464,475 and 484.The representative of body more than 252 is at many bodies ribozyme construct of hammerhead ribozyme site 252,271,313 and 326.The representative of body more than 238 is at many bodies ribozyme construct in 252,259 and 271 and hair clip ribozyme sites 238, three hammerhead ribozyme sites (HP).The representative of body more than 259 is at many bodies ribozyme construct in two hammerhead ribozyme sites 271 and 313 and hair clip ribozyme sites 259 (HP).

The level of Figure 25 explanation GBSS mRNA in ribozyme negative control (C, F, I, J, N, P, Q) and active ribozyme RPA63 transformant (AA, DD, EE, FF, GG, HH, JJ, KK).

The Δ 9 desaturase mRNA levels of Figure 26 explanation in (irrelevant ribozyme) control plant (RPA63-33, RPA63-51, RPA63-65) of unconverted plant (NT), 85-06 high stearic acid plant (1,3,5,8,12,14) and conversion.

Figure 27 illustrates the level of Δ 9 desaturase mRNA in unconverted plant (NTO), 85-15 high stearic acid plant (01,06,07,10,11,12) and the normal stearic acid plant of 85-15 (02,05,09,14).

Figure 28 illustrates the level of Δ 9 desaturase mRNA in unconverted plant (NTY), the 113-06 non-activity ribozyme plant (02,04,07,10,11).

The level of Figure 29 a and 29b explanation Δ 9 desaturase proteins in maize leaf (R0).(a) unconverted HiII is that the RPA113-17 of plant a-e and ribozyme inactivation is plant 1-6.(b) RPA85-15 of ribozyme activity is plant 1-15.

The stearic acid of Figure 30 explanation in the RPA85-06 plant leaf.

The stearic acid of Figure 31 explanation in the RPA85-15 plant leaf, the result of three kinds of tests.

The stearic acid of Figure 32 explanation in the RPA113-06 plant leaf.

The stearic acid of Figure 33 explanation in the RPA113-17 plant leaf.

Stearic acid in the blade of Figure 34 explanation in control plant.

Figure 35 illustrates the blade stearic acid in the R1 plant (deriving from the high stearic acid plant hybridization) (RPA85-15.07 selfing).

The level of Figure 36 explanation Δ 9 desaturases in maize leaf of future generation (R1). ^*Expression demonstrates those plants of high stearic acid content.

Figure 37 illustrates with the stearic acid in the individual body embryo of the culture (308/430-012) of antisense Δ 9 desaturases conversion.

Figure 38 illustrates with the stearic acid in the individual body embryo of the culture (308/430-015) of antisense Δ 9 desaturases conversion.

Figure 39 illustrates the stearic acid from the individual blade of culture (308/430-012) the regenerated plant that transforms with antisense Δ 9 desaturases.

Figure 40 explanation is a amylose content in the single grain of 308/425-12.2.1 at unconverted control series (Q806) and antisense.

The GBSS activity of Figure 41 explanation in the single grain of negative system of Southern (RPA63-0306) and the positive system of Southern (RPA63-0218).

Figure 42 explanation can be used for expressing the conversion carrier of enzymatic nucleic acid of the present invention.

Detailed Description Of The Invention

The present invention relates to be used for composition and the method expressed in the plant regulatory gene, more specifically say so and utilize enzymatic nucleic acid molecule regulatory gene is expressed in plant composition and method.

Following phrase and term definition are as follows:

＂ suppresses ＂ or ＂ to be regulated ＂ to mean enzyme (as GBSS and Δ 9 desaturases) active or be reduced to by the mRNA level of these genes encodings and be lower than observed level when not having enzymatic nucleic acid, and preferably be lower than observed level when having non-activity RNA molecule (can be attached on the same loci on the mRNA, but can not cut this RNA).

" enzymatic nucleic acid molecule " means the complementarity that has appointment gene target in the substrate land, and also has the nucleic acid molecule that specificity is cut the enzymic activity of this target.That is, this enzymatic nucleic acid molecule can intermolecular cutting RNA (or DNA) and is made target RNA molecular inactivation thus.This complementarity can make the enzymatic nucleic acid molecule fully hybridize on the target RNA, and cutting is taken place.Hundred-percent complementarity is preferred, but the complementarity that is low to moderate 50-75% in the present invention also is useful.Nucleic acid can be modified on base, glycosyl and/or phosphate.Term enzymatic nucleic acid and the replaceable use of following phrase, for example RNA, self-cleaving RNA, cis cutting RNA, self-dissolving RNA, endoribonuclease, small-sized enzyme, guide's enzyme (leadzyme) are cut, engaged certainly to ribozyme, catalysis RNA, enzymatic RNA, catalytic dna, nucleosidase (nucleozyme), DNA enzyme (DNAzyme), RNA enzyme, multinuclear enzyme, molecule.The nucleic acid molecule with enzymatic activity all described in all these terms.This term comprises enzymatic RNA molecule, comprises one or more ribonucleotides, and can comprise the Nucleotide or the dealkalize base section of other type of great majority, as described below.

The complementary ＂ of ＂ means nucleic acid and can interact through the base pairing of traditional Watson-Crick or other unconventional type (for example Hoogsteen type) and other RNA sequence formation hydrogen bond.

＂ carrier ＂ means any technology based on nucleic acid and/or virus that is used to transmit and/or express required nucleic acid.

＂ gene ＂ means the nucleic acid of coding RNA.

＂ plant gene ＂ means the coded gene of plant.

The endogenous ＂ gene of ＂ means the gene of finding at the genomic natural place of vegetable cell usually.

＂ external source ＂ or ＂ allos ＂ gene mean can not be found in the common host plant cell, but the gene of introducing by the gene transfer technique of standard.

" nucleic acid " means a kind of like this molecule, and it can be strand or two strands, form by the Nucleotide that contains glycosyl, phosphate and purine or pyrimidine bases (can be identical or different), and can be modify or unmodified.

＂ genome ＂ means the genetic material that is included in each organism and/or the virocyte.

＂ mRNA ＂ means can be by the proteinic RNA of cell translation becoming.

＂ cDNA ＂ means the DNA that is complementary to and derives from mRNA.

＂ dsDNA ＂ means double-stranded cDNA.

＂ has adopted ＂ RNA to mean the rna transcription thing that comprises the mRNA sequence.

＂ sense-rna ＂ means and comprises the sequence that is complementary to all or part of target RNA and/or mRNA, and blocks the rna transcription thing of expression of target gene by processing, transportation and/or the translation of disturbing primary transcript and/or mRNA.Complementarity may reside in any part of target RNA, that is, and and on 5 ' non-coding sequence, 3 ' non-coding sequence, intron or encoding sequence.Sense-rna normally has the mirror image of adopted RNA.

As used herein ＂ expresses ＂ and means transcribing of vegetable cell interior enzymatic nucleic acid molecule, mRNA and/or sense-rna and stable accumulation.Expression of gene comprises gene transcription and mRNA translation is become precursor or mature protein.

＂ suppresses ＂ altogether and means expression of exogenous gene, and this foreign gene and certain gene have substantial homology, and causes that in vegetable cell the activity of external source and/or endogenous protein product reduces.

The horizontal ＂ that ＂ changes means the generation level of gene product in the transgenosis organism different with normally or in the non-transgenic organism.

＂ promotor ＂ means the nucleotide sequence elements in this expression of gene of gene inner control.Promoter sequence provides RNA polymerase and effectively transcribes the recognition reaction of other required transcription factor.The promotor in various sources can be used for vegetable cell effectively, with ribozyme expression.For example, the promotor of bacterial origin is as octopine synthase promoter, nopaline synthase promoter, manopine synthase promoter; The promotor of viral source is as Cauliflower mosaic virus (35S); Plant promoter, as ribulose-1,5-bisphosphate, the little subunit of 6-bisphosphate (RUBP) carboxylase (ssu), β-conglycinin promotor, phaseollin promotor, ADH promotor, heat-shocked promotor and tissue-specific promoter.Promotor also can comprise some can improve the enhancer sequence element of transcribing efficient.

＂ enhanser ＂ means the nucleotide sequence elements (Adh) that can stimulate promoter activity.

＂ constitutive promoter ＂ means the promoter element (Actin muscle, ubiquitin, CaMV 35S) that instructs successive genetic expression in the neutralization of all cells type at any time.

＂ tissue specificity ＂ promotor means the promoter element (zein, oleosin, napin, ACP) of being responsible for genetic expression in specific cells or types of organization such as blade or seed.

＂ development-specific ＂ promotor means in the specific development of plants stage (taking place as early stage or late embryo) and is responsible for the promoter element of genetic expression.

＂ inducible promoter ＂ means and is responsible for response signal specific (as: physical stimulation (heat shock gene); Light (RUBP carboxylase); Hormone (Em); Metabolite; The promoter element of genetic expression and adverse circumstance).

＂ plant ＂ means eucaryon and protokaryon photosynthetic organism.

＂ angiosperm ＂ means its seed and is encapsulated in plant (for example coffee, tobacco, soybean, pea) in the ovary.

＂ gymnosperm ＂ means its seed and exposes rather than be encapsulated in plant (for example, pine tree, dragon spruce) in the ovary.

It is the plant of feature that ＂ monocotyledons ＂ means only there to be a slice cotyledon (the original blade of embryo).For example, corn, wheat, paddy rice and other.

＂ dicotyledons ＂ means the plant that produces the seed with two cotyledons (the original blade of embryo).For example, coffee, canola, pea and other.

＂ transgenic plant ＂ means the plant of expression alien gene.

＂ open reading frame ＂ means does not have intron, encoding amino acid sequence, have the definite translation initiation and the nucleotide sequence of terminator.

The invention provides the method that is used to produce the short cutting agent of class of enzymes, described cutting agent has the specificity of height to the RNA of required target.Described enzymatic nucleic acid molecule can be directed to the sequence area of the high degree of specificity of target, so that can obtain specific gene inhibition.Enzymatic nucleic acid can be directed to the high conservative region of gene family in addition, to suppress the genetic expression of relevant enzyme family.Said ribozyme can be expressed in by carrier (expressing nucleic acid of the present invention) plant transformed.

The enzymatic character of ribozyme is more favourable than other technology, because it is lower to influence the essential ribozyme concentration of treatment.This advantage has reflected that ribozyme plays the ability of enzymatic action.Like this, single ribozyme molecule can cut the molecule of many target RNA.In addition, ribozyme is the inhibitor of high degree of specificity, and it suppresses specificity and not only depends on the base pairing mechanism that is attached on the target RNA, and depends on target RNA cutting mechanism.The single mispairing of contiguous cleavage site or base replace the catalytic activity that can eliminate ribozyme fully.

6 basic variants of naturally occurring enzymatic RNA are known at present.The hydrolysis (and can cut other RNA molecule thus) of each variant trans phosphodiester bond of energy catalysis RNA under physiological condition.Table I is summed up some features of these ribozymes.In general, enzymatic nucleic acid works by at first being attached on the target RNA.Such combination takes place in the target bound fraction by enzymatic nucleic acid, this part very near this molecule play cutting target RNA effect the enzymatic part.Like this, enzymatic nucleic acid then in conjunction with it, in case be attached to correct site, is just brought into play enzymatic action cutting target RNA at first by complementary base pairing identification target RNA.The tactic cutting of a kind of like this target RNA will destroy it and instruct encoded protein matter synthetic ability.Behind combination of enzymatic nucleic acid and its RNA target of cutting, it discharges from this RNA, seeks another target, and can repeat combination and the new target of cutting.

In a preferred embodiment of the invention, the enzymatic nucleic acid molecule forms with tup or hair clip primitive, but also can form with hepatitis Δ virus, I group intron, II group intron, RNA enzyme PRNA (relevant with the RNA guide sequence) or Neurospora VS RNA.The example of such tup primitive is by Dreyfus, and is the same, Rossi etc., and 1992, AIDS research and human retrovirus 8,183 describe; The example of hair clip primitive is by Hampel etc., and EP 0360257, Hampel and Tritz, and 1989 biological chemistries 28,4929, Feldstein etc. 1989, gene 82,53, Haseloff and Gerlach, 1989, gene, 82,43 and Hampel etc., 1990 nucleic acids research, 18,299 describe; The example of hepatitis Δ virus primitive is by Perrotta and Been, and 1992, biological chemistry, 31,16 describe; The example of RNA enzyme P primitive is by Guerrier-Takada etc., 1983 cells 35,849; Forster and Altman, 1990, science 249,783; Li and Altman, 1996, nucleic acids research 24,835 is described; The example of Neurospora VS RNA ribozyme primitive is by Collins (Saville and Collins, 1990 cells, 61,685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci.USAs 88,8826-8830; Collins and Olive, 1993 biological chemistries 32,2795-2799; Guo and Collins, 1995, EMBO.J.14,363) describe; The example of II group intron is by Griffin etc. 1995, Chem.Biol.2,761; Michels and Pyle, 1995, biological chemistry 34,2965 is described; The example of I group intron is by Cech etc., United States Patent (USP) 4,987, and 071 describes.These specificity primitives are not restrictive in the present invention, one skilled in the art will realize that, it is important in enzymatic nucleic acid molecule of the present invention that just it will have the specific substrate binding site that is complementary to one or more target gene RNA district, and in substrate binding site or its have on every side and give molecule RNA the nucleotide sequence of nicking activity.

Enzymatic nucleic acid molecule of the present invention from eukaryotic promoter at cell inner expression [Gerlach etc. for example, International PCT publication WO 91/13994; Edington and Nelson, 1992, generegulation effect: sense-rna and DNA biology, editor R.P.Erickson and J.G.Izant, pp 209-221, Raven press, NY.; Atkins etc., International PCT publication WO94/00012; Lenee etc., International PCT publication WO 94/19476 and WO 9503404, Atkins etc., 1995, J.Gen.Virol.76,1781-1790; McElroy and Brettell, 1994, TIBTECH 12,62; Gruber etc., 1994, cellular biochemistry supplementary issue 18A, 110 (X1-406) and Feyter etc., 1996, Mol.Gen.Genet.250,329-338; All these document this paper are reference in the lump].By the instruction of this paper, one skilled in the art will realize that any ribozyme can both be from suitable promoter expression in the eukaryote cell.Ribozyme is expressed under the control of constitutive promoter, tissue-specific promoter or inducible promoter.

In order to obtain the adjusting of ribozyme mediation, with the ribozyme rna introduced plant.Though the example of the plasmid that makes up the transformation experiment that is used for this paper explanation below is provided, has designed many dissimilar plasmids that can be used for Plant Transformation fully in technician's ken.Referring to Bevan, 1984, nucleic acids research 12,8711-8721 (this paper is reference in the lump).The mode that many conversion plants are also arranged.Embryo's generation corn culture is handled with helium in following example.Except utilizing outside the particle gun (United States Patent (USP) 4,945,050 of Cornell and the United States Patent (USP) 5,141,131 of DowElanco), plant can be used following technical transform: the edaphic bacillus technology, referring to the United States Patent (USP) 5,177,010 of Toledo university, Texas A﹠amp; The United States Patent (USP) 5,104,310 of M, european patent application 0131624B1, the european patent application 120516 of Schilperoot, 159418B1 and 176,112, the United States Patent (USP) 5 of Schilperoot, 149,645,5,469,976,5,464,763,4,940,838 and 4,693,976, the european patent application 116718 of MaxPlanck, 290799,320500, the european patent application 604662 and 627752 of Japan Tobacco, the european patent application 0267159 of Ciba Geigy and 0292435 and United States Patent (USP) 5,231,019, the United States Patent (USP) 5,463,174 and 4 of Calgene, 762,785, United States Patent (USP) 5,004,863 and 5 with Agracetus, 159,135; The whisker technology is referring to the United States Patent (USP) 5,302,523 and 5,464,765 of Zeneca; Electroporation technology, referring to WO 87/06614, the Dekalb of Boyce Thompson Institute 5,472,869 and 5,384,253, WO9209696 and the WO9311335 of PGS; All these document this paper references in the lump.Except that the many technology in order to the conversion plant, the type of the tissue that contacts with allogenic material (plasmid that comprises RNA or DNA typically) also can change.Such tissue includes but not limited to that the embryo organizes, callus I type and II type and to transforming and the acceptable any tissue of regeneration of transgenic plant thereafter.Another variable factor is to select selected marker.Preferred special marking is that the professional can judge, but any following selected marker can together be used with unlisted any other gene that selected marker works that can be used as of this paper.Such selected marker includes but not limited to chlorosulfuron, Totomycin, PAT and/or bar, bromoxynil, kantlex etc.The bar gene can separate from streptomyces, particularly separates from streptomyces hygroscopicus or green color-producing streptomycete kind.Bar genes encoding phosphinothricin Transacetylase (PAT), the activeconstituents among this enzyme-deactivating weedicide bialaphos phosphinothricin (PPT).Like this, the combination of many technology can be used to adopt the adjusting of ribozyme mediation.

Ribozyme can be used as monomer and is individually expressed, that is, expressed as a kind of transcript at a kind of ribozyme in a site.In addition, expressed as the part of single rna transcription thing at two or more ribozymes of an above target site.More than one the single rna transcription thing of ribozyme that comprises at an above cleavage site is easy to generate to obtain effective adjusting of genetic expression.It is identical or different making up intravital ribozyme at these many bodies.For example, many bodies construct can comprise many hammerhead ribozymes or hair clip ribozyme or other ribozyme primitive.In addition, many bodies construct can be designed to comprise many different ribozyme primitives, for example tup and hair clip ribozyme.More particularly, design so many bodies ribozyme construct, wherein a series of ribozyme primitives are cascaded in single rna transcription thing.Ribozyme interconnects by the Nucleotide joint sequence, and wherein said joint sequence can also can not be complementary to target RNA.Many bodies ribozyme construct (multinuclear enzyme) improves the validity of the genetic expression adjusting of ribozyme mediation probably.

The activity of ribozyme also can they be released from primary transcript be enhanced owing to second ribozyme (Draper etc., PCT WO 93/23569 and Sullivan etc., PCT WO 94/02595, both with its integral body by this paper reference in the lump; Ohkawa, J. etc., 1992, Nucleic Acids Symp.Ser., 27,15-6; Taira.K. etc., 1991, nucleic acids research, 19,5125-30; Ventura, M. etc., 1993, nucleic acids research, 21,3249-55; Chowrira etc., 1994 journal of biological chemistry .269,25856).

The adjusting of the genetic expression of ribozyme mediation can be implemented in various plants (comprising angiosperm, gymnosperm, monocotyledons and dicotyledons).Plant includes but not limited to interest: cereal, as paddy rice, wheat, barley, corn; The produce oil farm crop are as soybean, canola, Sunflower Receptacle, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor-oil plant, peanut; The Botanical gardens farm crop are as coffee ﹠ tea; Fruit is as pineapple, pumpkin, mango, banana, grape, orange, apple; Vegetables are as Cauliflower, wild cabbage, melon, green peppers, tomato, Radix Dauci Sativae, lettuce, celery, potato, cabbage; Leguminous plants is as soybean, Kidney bean, pea; Fresh flower is as carnation, chrysanthemum, daisy, turmeric, silk China pink, alstromeria, mary bush, Petunia, rose; Tree is as olive, softwood trees, willow, pine tree; Nut is as English walnut, pistachio fruit etc.Below be the more general purpose unrestricted examples that are described in ribozyme in the adjusting of genetic expression.

The energy-conservation enough caffeine concentration that in coffee berry, obviously changes of the downward modulation of related genetic expression in caffeine is synthetic of ribozyme mediation.Gene (as heteroxanthine nucleosides in the coffee plants and/or 3-methyltransgerase) is expressed and can easily be regulated by enough ribozymes, to reduce caffeine synthetic (Adams and Zarowitz, United States Patent (USP) 5,334,529; This paper is reference in the lump).

Expression has generation in the blade of the nicotine concentration of change at the rotaring gene tobacco plant of the ribozyme of the gene (as N-methyl putrescine oxidase or putrescine N-methyltransferase gene (Shewmaker etc., the same)) that relates in Nicotine produces.

Expression will postpone the maturation of fruit (as tomato and apple) at the transgenic plant of the ribozyme of the gene that relates in the fruit maturation, described gene such as ethene form enzyme, pectin methyl transferring enzyme, Rohapect MPE, polygalacturonase, 1-1-aminocyclopropane-1-carboxylic acid (ACC) synthase, acc oxidase gene (Smith etc., 1988, nature, 334,724; Gray etc., 1992, Pl.Mol.Biol., 19,69; Tieman etc., 1992, vegetable cell, 4,667; Picton etc., 1993, plant magazine 3,469; Shewmaker etc. the same; James etc., 1996, biotechnology, 14,56).

Expression has generation at the transgenic plant of the ribozyme of the gene that relates in the fresh flower pigmentation fresh flower (as rose, petunia) of the color of change, said gene such as chalcone synthase (CHS), phenyl styryl ketone flavanone isomerase (CHI), phenylalanine ammonia-lyase or dehydrogenation flavonol (DF) hydroxylase, DF reductase gene (Krol van der etc., 1988, nature, 333,866; Krol van der etc., 1990, Pl.Mol.Biol.14,457; Shewmaker etc., the same; Jorgensen, 1996, science, 268,686).

Lignin is for the essential organic compound of physical strength that keeps cell walls in plant.Though be essential, lignin has some shortcomings.They make the silage indigestibility, and for being unwelcome from wood pulp and other material production paper.The transgenic plant of the ribozyme of the gene that expression relates in producing at lignin will have the lignin level of change, said gene such as O-methyltransgerase, cinnyl coenzyme A: NADPH reductase enzyme or cinnyl ethanol dehydrogenase (Doorsselaere etc., 1995, plant magazine, 8,855; Atanassova etc., 1995, plant magazine, 8,465; Shewmaker etc., the same; Dwivedi etc., 1994, Pl.Mol.Biol.26,61).

Other useful targets of useful ribozyme: Draper etc. are disclosed in following document, International PCT publication WO 93/23569, Sullivan etc., International PCT publication WO94/02595, and Stinchcomb etc., International PCT publication WO 95/31541, these documents with its integral body in the lump by this paper reference.

Particle in plant is in conjunction with the adjusting of starch synthase genetic expression:

In plant, the starch biosynthesizing takes place in chloroplast(id) (storage of short-term starch) and amyloplast (long-term starch storage).Starch granules is usually by alpha-D-glucose unit's (amylose starch) of straight chain alpha (1-4) connection and being made up of the crosslinked amylose starch (amylopectin) of α (1-6) key of side chain form.The enzyme that relates to during starch in plant is synthetic is a starch synthase, and this enzyme utilizes ADP-glucose to produce α (the 1-4)-glucose of linear chain.Find the starch synthase of two kinds of principal modes in plant: particle is in conjunction with starch synthase (GBSS) and a kind ofly be arranged in chloroplast stroma and a kind of soluble form amyloplast (soluble starch synthase (sss)).This kind of enzyme of two kinds of forms utilizes ADP-D-glucose, and particle combining form also utilizes UDP-D-glucose, and the former is preferential.The GBSS that is called waxy proteins matter has the molecular weight 55 to about 70 kDa in the various plants of determining features.The sudden change that influences gbss gene in the several plant kind once caused the amylose starch loss, and the maintenance of the total amount of starch is constant relatively.Except that the active loss of GBSS, that these mutanies also contain the level that changes, reduce or do not have GBSS protein (Mac Donald and Preiss, plant physiology, 78:849-852 (1985), Sano, Theor.Appl.Genet.68:467-473 (1984), Theor.Appl.Genet.75:217-221 such as Hovenkamp-Hermelink 91987), Shure etc., cell 35,225-233 (1983), Echt and Schwartz, genetics, 99:275-284 (1981)).The existence of side chain enzyme and solubility ADP-glucoamylase glycosyltransferase shows the approach independent of each other that has formation branched chain polymer amylopectin and straight-chain polymer amylose starch in GBSS mutant plant and wild-type plant.

The particle that relates in the biosynthesizing of Wx (wax) locus coding starch is in conjunction with Transglucosylase.Being expressed in of this kind of enzyme is limited in the corn in endosperm, pollen and the blastular.Because the outward appearance of sudden change grain (amylose starch is formed the phenotype due to reducing in the grain), the sudden change in this locus is called as wax.In corn, this kind of enzyme is transported among the amyloplast of endosperm of growth, the generation of its catalysis amylose starch there.Corn grain about 70% is a starch, and wherein 27% is linear straight chain starch, the 73%th, and amylopectin.Wax is in conjunction with a kind of recessive mutation in starch synthase (GBSS) gene at the coding particle.The plant of isozygotying of this recessive mutation produces the starch that contains 100% amylopectin form.

Ribozyme with locus specificity (as described below) of catalytic activity and increase is, a perhaps more specific inhibition molecule more more effective than antisense oligonucleotide.In addition, these ribozymes can suppress the GBSS activity, and the catalytic activity of ribozyme is essential to restraining effect.To those skilled in the art, can obviously find out, can design other ribozymes that in the plant variety beyond the corn, cut the required said target mrna of GBSS activity from embodiment.

Like this, in a preferred embodiment, feature of the present invention is to suppress amylose starch to produce related ribozyme in (for example by reducing the GBSS activity).The RNA molecule of these endogenous expression comprises the substrate land, and it is in conjunction with Ji the district of said target mrna.This RNA molecule also contains the district of catalysis RNA cutting.Said RNA molecule is the ribozyme of tup or hair clip primitive preferably.In case said target mrna is just cut in combination, ribozyme, stop translation and protein accumulation.Lacking under the situation of expression of target gene, amylose starch produces and is lowered or is suppressed.The back provides specific embodiment.

Embodiment preferred comprises the ribozyme with the brachium conjunctivum that is complementary to the binding sequence among Table III A, VA and the VB.The example of such ribozyme shows in Table III B-V.Though those of skill in the art will recognize that this example is at the mRNA of a kind of plant (for example corn) design, can prepare the similar ribozyme of the mRNA that is complementary to other plant variety.Complementation means brachium conjunctivum ribozyme can be interacted with sequence-specific mode and target RNA like this, thereby causes the cutting of plant mRNA target.The example of such ribozyme is made up of sequence shown among Table III-V basically.

Embodiment preferred is ribozyme and they is used for suppressing starch granules in conjunction with ADP (uridine diphosphate (UDP))-glucose: α-1 plant, the active method of 4-D-Dextran 4-alpha-glucosyl transferring enzyme (being that particle is in conjunction with starch synthase (GBSS)).This is by finishing with the ribozyme inhibition of gene expression, and this inhibition causes reducing or removing the GBSS activity in plant.

In another aspect of the present invention, the transcription unit of ribozyme from be inserted into Plant Genome that cutting target molecule and inhibition amylose starch produce expresses.Preferably, can advance Plant Genome and select the recombinant vectors of the conversion department of botany of ribozyme expression in vegetable cell, to express by stable integration by composing type or inducible promoter.In case expressed, ribozyme just cuts their said target mrna, and makes their host cell reduce the generation amylose starch.Ribozyme expressed in vegetable cell is under constitutive promoter, tissue-specific promoter or inducible promoter control.

The modification of W-Gum is the important application of ribozyme technology, and it can reduce expression of specific gene.High-caliber amylopectin is desirable to corn wet milling technology, thereby but also has the high amylopectin starch corn to cause the digestibility that increases to increase some evidence of the energy availability of food.About 10% wet-milling starch has the wax phenotype, but because its recessive character, traditional wax kind is difficult to be handled by the grower.Be used for cutting GBSS mRNA and reduce thus plant particularly the active ribozyme of corn embryosperm GBSS will show as the dominance characteristic, and produce and to have the maize plant that is easy to the wax phenotype handled into the grower.

The modification of the saturated distribution of lipid acid in the plant:

The biosynthesizing of lipid acid originates in chloroplast(id) in the plant tissue.Lipid acid is synthesized by the thioesters of Fatty acid synthetase mixture (FAS) as acyl carrier protein (ACP).Chain length is that the lipid acid of 16 carbon atoms is followed one of following three kinds of approach: 1) they are right after after synthetic and are released, and are transformed into glyceraldehyde-3 phosphate (G3P) by the chloroplast(id) acyltransferase, for further modifying in chloroplast(id); 2) after plastid output, they are released and are transformed into coenzyme A (CoA) by thioesterase; Or 3) they further extend to the C18 chain length.The C18 chain by stearyl-ACP desaturase at the rapid desaturation in C9 position, then oleic acid (18: 1) base is transformed into G3P immediately in chloroplast(id), or is transformed into oleyl coenzyme A (Somerville and Browse, 1991 from chloroplast(id) output and by thioesterase, science, 252:80-87).The great majority of C16 lipid acid are followed the 3rd approach.

In corn seed oil, the triglyceride level that has superiority produces in endoplasmic reticulum.Most of oleic acid (18: 1) and some palmitinic acids (16: 0) are transformed into G3P from phosphatidic acid, are transformed into DG ester and phosphatidylcholine then.Occur in the endoplasmic reticulum by the further desaturation of film in conjunction with the acyl chain on the phosphatidylcholine of desaturase generation.Then in the generation of triglyceride level; two and triunsaturated chain be released in the acyl-CoA set; and transfer to C3 position (Frentzen on the glycerol backbone in the DG; lipid metabolism in 1993 plants; p.195-230, (editor Moore, T.S.) CRC press; Boca Raton, FA.).The synoptic diagram of the biosynthetic pathway of vegetable fatty acid shows in Figure 11 and 12.Three kinds of lipid acid that have superiority in corn seed oil are linolic acid (18: 2, about 59%), oleic acid (18: 1, about 26%) and palmitinic acid (16: 0, about 11%).These are mean value, can some difference according to genotypic difference.Yet, the biased sample of the oil of the US Corn Belt production that past 10 years was analyzed has this composition (Glover and Mertz always, the nutritional quality of 1987 grain: heredity and agronomy improvement, p183-336, (editor Olson, R.A. and Frey K.J.), U.S. agronomy meeting company, Madison, WI.; Fitch-Haumann, 1985 U.S. POL chemistries meeting magazine 62:1524-1531; Strecker etc., 1990, edible fat and oil processing: ultimate principle and modern practice (editor Erickson, D.R.), U.S. POL chemistry man association, Champaign, II).This advantage of C18 chain length can reflect the abundance and the activity of several key enzymes, as being responsible for Fatty acid synthetase that the C18 carbochain produces, being responsible for the stearyl-ACP desaturase (Δ 9 desaturases) that produces at 18: 1 and responsiblely being transformed into 18: 2 microsome Δ-12 desaturase at 18: 1.

Δ 9 desaturases (being also referred to as stearyl-ACP desaturase) of plant are a kind of solubility chloroplast enzymes; it be connected with acyl carrier protein (ACP) C18 (also adopting C16 once in a while)-acyl chain is substrate (McKeon; T.A. and Stumpf; P.K., 1982 journal of biological chemistry 257:12141-12147).This is opposite with Mammals, eucaryon such as low and cyanobacteria Δ 9 desaturases.Rat and yeast Δ 9 desaturases be film in conjunction with microsomal enzyme, it is a substrate with the acyl-CoA chain, and cyanobacteria Δ 9 desaturases are substrate with the acyl chain on the DG.Separate several Δ 9 desaturase cDNA clones that derive from dicotyledons so far and determined feature (Shanklin and Somerville, 1991 Proc. Natl. Acad. Sci.USA 88:2510-2514; Knutzon etc., 1991 plant physiology 96:344-345; Sato etc., 1992 plant physiology 99:362-363; Shanklin etc., 1991 plant physiology 97:467-468; Slocombe etc., 1992 molecular biology of plants 20:151-155; Taylor etc., 1992 plant physiology 100:533-534; Thompson etc., 1991 Proc. Natl. Acad. Sci.USA 88:2578-2582).The enzyme that relatively holds itself out to be a kind of high conservative of the Δ 9 desaturase sequences of different plants.Has high-caliber identity property at amino acid levels (about 90%) with at nucleotide level (about 80%) on both.Yet,, between plant and other Δs 9 desaturases, do not have sequence similarity (Shanklin and Somerville, the same) as what can expect from its different physics and zymetology feature.

The purifying of Semen Ricini desaturase (with other enzymes) and characterized show that Δ 9 desaturases are that activated, sub-unit molecule amount is about 41 kDa as homodimer.To its external activity, this enzyme require molecular oxygen, NADPH, NADPH ferredoxin oxide-reductase and ferredoxin.Fox etc., 1993 (Proc. Natl. Acad. Sci.USA, 90:2486-2490) each homodimer of Semen Ricini enzyme that is presented at expression in escherichia coli comprises four ferrous atoms of catalytic activity.The enzyme of oxidation comprises two two identical iron bunch, and they are reduced to two ferrous states in the presence of hyposulfite.In the presence of stearoyl-CoA and oxygen, this bunch got back to two iron states again.This illustrates that this desaturase belongs to the oxygen activator matter group that contains two iron-oxygen bunch.Other members of this group are ribonucleotide reductase and methane monooxygenase hydroxylase.In these catalysis discrepant proteinic expection primary structure relatively show they all contain by about 80-100 amino acid separate conservative aminoacid sequence (Asp/Glu)-Glu-Xaa-Arg-His is right.

The traditional plant type of rearing shows the stearic acid level that can obtain plant is not had the raising of toxic action.(Ladd and Knowles, 1970 farm crop science, (Hammond and Fehr, the 1984 magazine 61:1713-1716 of the U.S. POL chemistry association 10:525-527) and in soybean in safflower; Graef etc., 1985 farm crop science 25:1076-1079) the stearic acid level increases significantly.The mutability that this explanation is formed at the lipid acid of seed oil.

Obtained the active increase of Δ 9 desaturases by Δ 9 delta 8 desaturase genes transformation of tobacco with yeast (Polashock etc., 1992 plant physiology 100,894) or rat (Grayburn etc., 1992 biotechnologys, 10,675).Two groups of transgenic plant all have tangible change on lipid acid is formed, yet identical with control plant on the phenotype.

Corn seldom is used to produce the margarine production, because in that it is not used as oil crops traditionally, and relatively has lower seed oil content with soybean and canola.Yet Semen Maydis oil has the palmitinic acid (16: 0) (being desirable) of low-level linolenic acid (18: 3) and higher level in oleomargarine.The applicant believes by downward modulation Δ 9 desaturases active reduction oleic acid and linolic acid level will make corn replace soybean and canola on saturated oil market effective ground.

Oleomargarine and confectionery fat are produced by the chemical hydrogenation of plant (as soybean) oil.This process has increased produces the cost of oleomargarine, and also causes the cis and the trans-isomer(ide) of lipid acid.Trans-isomer(ide) is not naturally occurring in the oil that plant produces, and it increases the worry in the potential Health hazard.The applicant believes that a kind of elimination is that plant is carried out genetic engineering procedure to the method for the needs of chemical hydrogenation, with the downward modulation desaturase.Δ 9 desaturases are incorporated into first pair of key in 18 carbon fatty acids, and are the key steps that realizes the fatty acid desaturation degree.

Like this, in a preferred embodiment, the present invention relates in plant, change the composition (and using method) that lipid acid is formed.This be by with ribozyme, antisense nucleic acid, suppress altogether or triple helical DNA inhibition of gene expression causes reducing in plant or the activity of eliminating some enzyme (for example Δ 9 desaturases) is finished.This activity reduces in monocotyledons (as corn, wheat, paddy rice, palm, coconut etc.).Δ 9 desaturase activity also can reduce in dicotyledons (as Sunflower Receptacle, safflower, cotton, peanut, olive, sesame, sepal distance flower spp, flax, Jojoba genera, grape etc.).

Like this, on the one hand, feature of the present invention is to suppress related ribozyme in the fatty acid desaturation (for example by reducing Δ 9 desaturase activity).The RNA molecule of these endogenous expression comprises the substrate land, and this land is in conjunction with Ji the district of said target mrna.This RNA molecule also contains the district of catalysis RNA cutting.Said RNA molecule is the ribozyme of tup or hair clip primitive preferably.In case said target mrna is just cut in combination, ribozyme, stop translation and protein accumulation.Lacking under the situation of expression of target gene, the stearic acid level increases, and the generation of unsaturated fatty acids is lowered or is suppressed.Specific example provides in following listed table.

In preferred embodiments, ribozyme has the brachium conjunctivum of the sequence among Table VI of being complementary to and the VIII.Though those of skill in the art will recognize that this example is at the mRNA of a kind of plant (for example corn) design, can prepare the similar ribozyme of the mRNA that is complementary to other plant.Like this, complementation means brachium conjunctivum can interact ribozyme with sequence-specific mode and target RNA, thereby causes the cutting of plant mRNA target.The sequence that the example of such ribozyme limits in Table VII and the Table V typically.Active ribozyme typically comprises those enzymatic centers of being equal in the example and can be in conjunction with the brachium conjunctivum of plant mRNA (so that cutting takes place) on target site.Do not disturb other sequence of such combination and/or cutting to exist.

Useful especially ribozyme sequence shows in Table VII and VIII in this research.

Those of skill in the art will recognize that ribozyme sequence listed in described table only is the representative of many these class sequences, wherein the enzymatic of ribozyme part (all parts except that brachium conjunctivum) is changed (to influence activity).The stem ring II sequence (5 '-GGCGAAAGCC-3 ') of for example listing in the hammerhead ribozyme in the Table IV can be changed (replace, disappearance and/or insert), to contain any sequence, preferably as long as the stem structure of two minimum base pairings can form.Similarly, the stem ring IV sequence (5 '-CACGUUGUG-3 ') of listing in the hair clip ribozyme among Table V and the VIII can be changed (replace, disappearance and/or insert), to contain any sequence, preferably as long as the stem structure of two minimum base pairings can form.These ribozymes are equivalent to specifically described ribozyme in described table.

In another aspect of this invention, the transcription unit of ribozyme from be inserted into Plant Genome of cutting target molecule and minimizing unsaturated fatty acid content expresses in plant.Preferably, can advance Plant Genome and select the recombinant vectors of the conversion department of botany of ribozyme expression in vegetable cell, to express by stable integration by composing type or inducible promoter.In case expressed, ribozyme just cuts their said target mrna, and makes their host cell reduce the generation unsaturated fatty acids.The ribozyme of expressing in vegetable cell is under constitutive promoter, tissue-specific promoter or inducible promoter control.

The change that lipid acid is formed is the important application based on the technology of nucleic acid that can reduce expression of specific gene.High-caliber saturated fatty acid is desirable in the plant that produces commercially important oil.

One relevant aspect, feature of the present invention is the method for separating the cDNA sequence of coded delta 9 desaturases in the corn.

Hair clip and the hammerhead ribozyme of cutting Δ 9 desaturase mRNA have also been described in preferred embodiments.It will be understood by those skilled in the art that from example described below be easy to design other ribozymes of the required said target mrna of cutting Δ 9 desaturases activity now, these ribozymes within the scope of the invention.

Though the object lesson of corn RNA is provided,, one skilled in the art will realize that these instructions are not limited to corn.In addition, identical target can be used for the other plant kind.The complementary arms of specified plant RNA sequence of being suitable for leading can be used for the ribozyme at the sort of specific RNA.The embodiment of this paper and explanation are not intended to limit the present invention, and those skilled in the art should be realized that and can be easy to produce similar embodiment on multiple different plant, so that use the description of this paper to regulate the expression of various different plant genes, and these contents within the scope of the invention.

The embodiment of this paper has followed standard molecular biological technique.Other information is seen Sambrook, J., Fritsch, E.F., Maniatis, T. (1989), molecular cloning laboratory manual, second edition, cold spring port: press of cold spring harbor laboratory, its content this paper reference in the lump.

EXAMPLE Example 1: the separation of the Δ 9 desaturase cDNA of corn

Design and synthesize the degenerate pcr primer at two conservative peptides relevant with the combination of the two iron-oxygen groups of plant Δ 9 desaturases.By one 276 bp dna fragmentation of maize cDNA pcr amplification, and it is cloned in the carrier.The aminoacid sequence of this fragment supposition and sequence similarity by proteic two the isolating districts of conservative peptide of dicotyledons Δ 9 desaturases.This fragment is used for screening maize cDNA library.16 clones have been separated altogether.Further a clone has been identified in estriction map mensuration and hybridization, and this clone is checked order.CDNA inserts segmental characteristic: 1621nt cDNA, 145 nt5 ' and 294 nt, 3 ' non-translational region (comprising the poly-A tail of 18nt); Encode 394 amino acid open reading frame of 44.7 kD polypeptide; Semen Ricini Δ-9 delta 8 desaturase genes of the mature protein of inferring with coding has 85% amino acid identity property.Figure 10 has listed its full sequence.Embodiment 2: the potential ribozyme cleavage site of identifying Δ 9 desaturases

About 250 HH ribozyme sites and about 43 HP sites in corn Δ 9 desaturase mRNA, have been identified.The HH site is made up of uridine and any Nucleotide (except the guanosine (UH)).Table VI and VIII have listed HH and HP ribozyme cleavage site.Numbering system begins by 1 at 5 ' end with the Δ of sequence shown in Figure 10-9 desaturase cDNA clone.

Can design at an easy rate and synthetic ribozyme (as listed those among Table VII and the VIII), with at 5-100 or more a plurality of base cleavage site (referring to Fig. 1-5) as the substrate brachium conjunctivum.These substrate brachium conjunctivums make ribozyme to interact in the sequence-specific mode with their target in ribozyme.Embodiment 3: the selection of Δ 9 desaturase ribozyme cleavage sites

Use algorithm (as the secondary structure of M.Zuker (Zuker.M., 1989 science, 244,48-52)) by Computer Analysis evaluation Δ 9 desaturase mRNA.Secondary pleated sheet structure that does not form the RNA/RNA stem district that has 8 above Nucleotide and the mRNA district of containing potential hammerhead ribozyme cleavage site have been identified.By RNA enzyme H determination and analysis the oligonucleotide accessibility in these sites (referring to the following examples 4).Embodiment 4: the RNA enzyme H of Δ 9 desaturases measures

49 DNA oligonucleotide (each long 21 Nucleotide) are used for RNA enzyme H to be measured.These oligonucleotide have covered 108 sites in the Δ 9 desaturation ribozymes.Utilize the Δ 9 desaturase cDNA transcripts of total length to carry out RNA enzyme H mensuration (Fig. 6).The reached cleavage site of the method screening RNA of the Draper general description by above.In brief, synthesized the DNA oligonucleotide of representing the ribozyme cleavage site.Use polymerase chain reaction to produce and be used for from the substrate of corn cDNA clone generation T7 rna polymerase transcribe.At external rna transcription thing by these template complex signs.With the annealing of the transcript of oligonucleotide and mark, add RNA enzyme H, and with mixture 37 ℃ of following incubations 10 minutes.Termination reaction and the order-checking polyacrylamide gel on isolation of RNA.Use Molecular Dynamics phosphorescent substance imaging system to measure the percentage composition (Figure 13 and Figure 14) of the substrate of cutting by the radioautograph quantivative approach.Embodiment 5: the tup and the hair clip ribozyme that are used for Δ 9 desaturases

Design tup (HH) and hair clip (HP) ribozyme make it the site that oligonucleotide covered of being cut by fullest in RNA enzyme H measures, and analyze these ribozymes by the computer folding algorithm then, and superseded ribozyme with obvious secondary structure.

Synthesized ribozyme by chemical process.Once the someone had described RNA synthetic general process (Usman etc., 1987, Journal of the American Chemical Society, 109,7845-7854 and Scaringe etc., 1990, nucleic acids research, 18,5433-5341 in the past; Wincott etc. 1995; nucleic acids research; 23; 2677), 394 type synthesizers (Applied Biosystems, Inc.) go up to use improved 2.5 μ mol scale schemes (have 5 minutes the coupling step of the Nucleotide that is used for the silyl protection and 2 minutes be used for 2 '-the coupling step of O-methylated nucleotide) carried out small-scale synthetic.Table II has been summarized the quantity and the duration of contact of the reagent that is used for synthesis cycle.In each coupling circulation, used with respect in conjunction with the phosphamide of polymeric 5 ' hydroxyl 6.5 times excessive (0.1M, 163 μ l=16.3 μ mol) and the S-ethyl tetrazolium of 24 times excessive (0.25M, 238 μ l=59.5 μ mol).Measure by the quantitative analysis method of trityl component colorimetric, the average coupling productive rate on the 394 type synthesizers is 97.5-99%.Other be used for 394 oligonucleotide synthetic agent: detritylation solution is 2% trichoroacetic acid(TCA) that is dissolved in methylene dichloride (ABI); Use is dissolved in the 16%N-Methylimidazole and 10% acetic anhydride/10%2 that are dissolved among the THF (ABI), 6-lutidine end-blocking among the THF (ABI); Oxidizing solution is the 16.9mM I that is dissolved among the THF (Millipore) ₂, 49 mM pyridines, 9% water.Directly use B and the synthetic level of J acetonitrile in the reagent bottle.The solid medicine preparation S-ethyl tetrazolium solution (0.25M acetonitrile solution) that use obtains from the international chemical drug company of the U.S..

Carry out the deprotection of RNA as follows.Transferring to the screw plug vial of 4mL from synthetic post, and in methylamine (MA) solution, suspending 10 minutes under 65 ℃ in conjunction with polymeric oligoribonucleotide (no trityl).After being cooled to-20 ℃, from the polymer carrier, remove supernatant liquor.Carrier 1.0ml EtOH: MeCN: H ₂O/3: washing in 1: 1 three times, vortex is added to supernatant liquor in first portion of supernatant liquor after stirring again.Be dried to white powder to what contain oligoribonucleotide in conjunction with supernatant liquor.

The oligoribonucleotide of base deprotection is suspended in the anhydrous TEAHF/NMP solution (the 1.5mL N-Methyl pyrrolidone solution of 250 μ l, the TEA of 750 μ l and 1.0 mLTEA3HF (1.4 M HF concentration are provided)) again, and is heated to 65 ℃ of maintenances 1.5 hours.Before the anionresin desalination, handle the oligomer of the whole deprotections that generate with 50 mM TEAB (9ml).

In the anionresin desalination of deprotection oligomer, TEAB solution is loaded on the Qiagen 500  anionresin cylinders (Qiagen company) of using 50 mM TEAB (10ml) washing in advance.Behind the cylinder that loads with 50 mM TEAB (10ml) washing,, and be dried into white powder with 2M TEAB (10ml) eluted rna.

By replacing G with U ₅Replace A with U ₁₄The hammerhead ribozyme of (numbering comes from Hertel, K.J. etc. (1992, nucleic acids research 20,3252)) synthetic non-activity.

As the above-mentioned synthetic hair clip ribozyme of method that tup RNA is described.

Utilize the phage t7 RNA polymerase also to synthesize ribozyme (Milligan and Uhlenbeck, 1989, Enzymology method 180,51) by dna profiling.Use common method by gel electrophoresis purifying ribozyme or by high pressure liquid chromatography (HPLC) method (HPLC; Referring to above Wincott etc., 1996, its full text in the lump with reference to) the purifying ribozyme, and ribozyme is suspended in the water again.The sequence that has shown the chemical process synthetic ribozyme that is used for this research among Table VII below and the VIII.Embodiment 6: the long substrate test of Δ 9 desaturase ribozymes

Be used for long 1621 nt of target RNA of this research, and contain at all HH of Δ 9 desaturation ribozymes and the cleavage site of HP ribozyme.The template that contains the sub-upstream of T7 rna polymerase promoter of Δ 9 desaturase target sequences is passed through pcr amplification by the cDNA clone.Use T7 RNA polymerase from then on pcr amplification template is transcribed target RNA.In transcription, pass through to add [α- ³²P] CTP carries out inner marker as one of 4 kinds of triphosphoric acid ribonucleotides with transcript.After transcribing 2 hours under 37 ℃, with the DNA enzyme-mixture is transcribed in I processing so that the dna profiling that digestion is used to transcribe.Separate in denaturing polyacrylamide gel transcribing mixture.In gel film, isolate band, and go out RNA, throw out is stored in 4 ℃ with isopropanol precipitating corresponding to full-length RNA.

At the excessive (k of ribozyme _CatCarry out under/KM) the condition ribozyme cleavage reaction (Herschlag and Cech, 1990, biological chemistry 29,10159-10171).In brief, at 50 mM Tris-HCl (pH7.5) and 10 mM MgCl ₂Exist down, by be heated to 65 ℃ and keep 2 minutes respectively with the 1mM ribozyme and＜the target RNA sex change of the inner marker of 10nM.By being cooled to temperature of reaction (37 ℃, 26 ℃ or 20 ℃) 10-20 minute with the RNA renaturation.Under suitable reaction temperature,, ribozyme begins cleavage reaction by being mixed with target RNA.In certain time interval, take out aliquots containig, and reaction is stopped by adding isopyknic damping fluid that stops.Sample is separated on 4% sequencing gel.

Summed up the ribozyme cleavage reaction result under 26 ℃ or 20 ℃ among Table I X, Figure 15 and 16.In the ribozyme of all tests, 7 tups and 2 obvious cuttings (Figure 15 and 16) that hair clip shows Δ 9 desaturation ribozymes.Ribozyme shows the activity level of variation to other site.Embodiment 7: with the multinuclear enzyme combination cutting target RNA of Δ 9 desaturases

Above-mentioned several ribozymes are combined into a polymer ribozyme construct that contains the ribozyme in two or more complementary RNA sections that are embedded into adjacency.Figure 17,18,19 and 23 has shown the non-limitative example of polymer ribozyme.By with annealed complementary oligonucleotide and be cloned into and contain cauliflower mosaic virus 35S and strengthen promotor (Franck etc., 1985 cells 21,285), corn Adh 1 intron (Dennis etc., 1984, nucleic acids research 12,3983), in the expression vector of Nos polyadenylation signal (DePicker etc., 1982 molecular application genetics magazines 1,561) and produce said ribozyme.In Figure 20 and 21, shown to use and contained the cutting analysis that T7 transcript that polymeric transcriptional units obtains carries out from these.Embodiment 8: the structure of the Δ 9 desaturase transcriptional units of ribozyme expression

Be used for cutting ribozyme endogenous expression in plant materials of Δ 9 desaturase mRNA.Perhaps express (stable conversion), perhaps express (transient expression) by the episome transcriptional units that belongs to a plasmid vector or a virus sequence part by the gene that is inserted on the Plant Genome.These ribozymes can pass through rna plymerase i, II or III, plant or plant virus promoters (as CaMV) expresses.Promotor or composing type, tissue-specific, or express in the growth course.Δ 9259 monomer ribozyme constructs (RPA 114,115)

These are Δ 9 desaturases 259 monomer hammerhead ribozymes clones.Design these ribozymes, make it to have long stem II district of 3bp and the long substrate brachium conjunctivum of 20bp (all) at site 259.Activity form is RPA 114, and the form of non-activity is RPA 115.With the Not I digestion pDAB367 of parental plasmid, and mend flat so that produce the flush end acceptor site with Klenow.Digest this carrier with Hind III restriction enzyme then.Cut the plasmid that contains ribozyme with Eco RI, cut again with flat this plasmid of Klenow benefit and with Hind III.Contain the insertion fragment of whole ribozyme transcriptional units with gel-purified, and it is connected in pDAB 367 carriers.Check this construct by digesting, and confirmed by order-checking with SgfI/Hind III and Xba I/SstI.Δ 9453 polymer ribozyme constructs (RPA 118,119)

These are Δ 9 desaturases 453 polymer hammerhead ribozymes clones (referring to Figure 17).Design these ribozymes, make it to have the long stem II district of 3bp.The total length of the substrate brachium conjunctivum of polymer construct is 42bp.Activity form is RPA 118, and the form of non-activity is RPA 119.Produce construct according to above-mentioned 259 monomer methods.Designed the polymer construct, made it to have 4 hammerhead ribozymes at the site 453,464,475,484 in the Δ 9 desaturation ribozymes.Δ 9 252 polymer ribozyme constructs (RPA 85,113)

These are the Δ 9 desaturases 252 polymer ribozymes clones that are positioned at 3 of bar (phosphoinothricin Transacetylase, Thompson etc., 1987 EMBO are J.6:2519-2523) open reading frame ' end.Design these polymer constructs, make it to have the long stem II district of 3bp.The total length of the substrate brachium conjunctivum of polymer construct is 91 bp.Active ribozyme is RPA 85, and RPA113 is a non-activity.Carrier construction as follows: partly digest the pDAB of parental plasmid 367 with Bgl II, and with the single cutting of gel-purified plasmid.Again cut this plasmid with Eco RI, and again with gel-purified to isolate correct Bgl II/Eco RI cutting fragment.From the ribozyme construct, go out Bam HI/Eco RI and insert fragment (containing ribozyme and NOS poly A district), and be connected in 367 carriers with gel separation.Identify positive plasmid by Nco I/SstI digestion and order-checking.

Mensuration by standard can identify useful transgenic plant.Can estimate transgenic plant Δ 9 desaturases by the method for discussing in the following examples expresses and the active minimizing of Δ 9 desaturases.The evaluation of potential ribozyme cleavage site among the embodiment 9:GBSS RNA

241, the, hammerhead, ribozyme, sites, (referring, to, Table, III, A), in, corn, GBSS, mRNA, polypeptid, coding, area, have, been, identified.A, hammerhead, ribozyme, site, is, made, up, of, uridine, and, any, Nucleotide, (except, the, guanine, (UH)) .It, below, is, the, sequence, (SEQ.I.D.No:25), of, the, GBSS, coding, region, of, corn. numbering system starts from having following sequence, the GBSS of (5 '-3 '), 1 of cDNA clone's 5 ' end: GACCGATCGATCGCCACAGCCAACACCACCCGCCGAGGCGACGCGACAGCCGCCAG GAGGAAGGAATAAACT73, 144CACTGCCAGCCAGTGAAGGGGGAGAAGTGTACTGCTCCGTCCACCAGTGCGCG CACCGCCCGGCAGGGCTGC145, 216TCATCTCGTCGACGACCAGTGGATTAATCGGCATGGCGGCTCTAGCCACGTCG CAGCTCGTCGCAACGCGCG217, 288CCGGCCTGGGCGTCCCGGACGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGC CTGAGGGGGGGCCGGACGG289, 360CGTCGGCGGCGGACACGCTCAGCATTCGGACCAGCGCGCGCGCGGCGCCCAGG CTCCAGCACCAGCAGCAGC361, 432AGCAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCC GGCATGAACGTCGTCTTCG433, 504TCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGCGGCCTCGGCGACGTCCTC GGCGGCCTGCCGCCGGCCA505, 576TGGCCGCGAATGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTAC AAGGACGCCTGGGACACCA577, 648GCGTCGTGTCCGAGATCAAGATGGGAGACAGGTACGAGACGGTCAGGTTCTTC CACTGCTACAAGCGCGGAG649, 720TGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAG ACCGAGGAGAAGATCTACG721, 792GGCCTGACGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTA TGCCAGGCAGCACTTGAAG793, 864CTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGG GAGGACGTCGTGTTCGTCT865, 936GCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGCAACTACCAG TCCCACGGCATCTACAGGG937, 1008ACGCAAAGACCGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGGTTCGC CTTCTCCGACTACCCGGAGC1009, 1080TGAACCTCCCGGAGAGATTCAAGTCGTCCTTCGATTTCATCGACGGCTACGA GAAGCCCGTGGAAGGCCGGA1081, 1152AGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGT CAGCCCCTACTACGCCGAGG1153, 1224AGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCT CACCGGCATCACCGGCATCG1225, 1296TCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGC CGTGAAGTACGACGTGTCGA1297, 1368CGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGG GCTCCCGGTGGACCGGAACA1369, 1440TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGT CATGGCGGCCGCCATCCCGC1441, 1512AGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAA GAAGAAGTTCGAGCGCATGC1513, 1584TCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTT CAACGCGGCGCTGGCGCACC1585, 1656ACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTG CGGCCTCATCCAGCTGCAGG1657, 1728GGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGA CACCATCATCGAAGGCAAGA1729, 1800CCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGC GGACGTCAAGAAGGTGGCCA1801, 1872CCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGAT GGTGAGGAACTGCATGATCC1873, 1944AGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGTGCTGCTCAG CCTCGGGGTCGCCGGCGGCG1945, 2016AGCCAGGGGTCGAAGGGGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGC CGCGCCCTGAAGAGTTCGGC2017, 2088CTGCAGGCCGCCTGATCTCGCGGGTGGTGCAAACATGTTGGGACATCTTCTT ATATATGCTGTTTCGTTTAT2089, 2160GTGATATGGACAAGTATGTGTAGCTGCTTGCTTGTGCTAGTGTAATATAGTG TAGTGGTGGCCAGTGGCACA2161, 2232ACCTAATAAGCGCATGAACTAATTGCTTGCGTGTGTAGTTAAGTACCGATCG GTAATTTTATATTGCGAGTA2233AATAAATGGACCTGTAGTGGTGGAAAAAAAAA AAA, (SEQ, I.D.NO.25).

Nearly 53 potential hair clip ribozyme sites in GBSS mRNA.Ribozyme and target sequence in Table V, have been listed.

According to the above, at these sites be easy to the design and synthetic ribozyme, with 5 to 100 or more the polybase base as substrate brachium conjunctivum (referring to Fig. 1-5).The selection of embodiment 10:GBSS ribozyme cleavage site

By Computer Analysis utilize folding algorithm (as the algorithm of M.Zuker development (Zuker, M., 1989 science, 244,48-52) assessed the secondary structure of GBSS mRNA.Identified and do not formed secondary pleated sheet structure with the RNA/RNA stem that surpasses 8 Nucleotide and the mRNA district of containing potential hammerhead ribozyme cleavage site.

Utilize RNA enzyme H measuring method to assess the oligonucleotide accessibility (referring to Fig. 6) in these sites then.58 DNA oligonucleotide (each long 21 Nucleotide) in these determination and analysis, have been used.These oligonucleotide have covered 85 sites.The position of these oligonucleotide and name are 195,205,240,307,390,424,472,481,539,592,625,636,678,725,741,811,859,891,897,912,918,928,951,958,969,993,999,1015,1027,1032,1056,1084,1105,1156,1168,1186,1195,1204,1213,1222,1240,1269,1284,1293,1345,1351,1420,1471,1533,1563,1714,1750,1786,1806,1819,1921,1954, with 1978.Also covering and comprised the secondary site, is 202,394,384,385,484,624,627,628,679,862,901,930,950,952,967,990,991,1026,1035,1108,1159,1225,1273,1534,1564,1558 and 1717.The RNA enzyme H of embodiment 11:GBSS measures

Use the total length transcript of GBSS coding region, 3 ' non-coding region and part 5 ' non-coding region to carry out RNA enzyme H mensuration (Fig. 7).General method by descriptions such as Draper above (this paper in the lump with reference to) has screened GBSS RNA and can reach cleavage site.In brief, synthesized the DNA oligonucleotide of representing the hammerhead ribozyme cleavage site.Use polymerase chain reaction to produce T7 rna polymerase transcribe substrate from corn cDNA clone.By the rna transcription thing of these templates at external complex sign.With the annealing of the transcript of oligonucleotide and mark, add RNA enzyme H and under 37 ℃ with mixture incubation 10 minutes.Termination reaction and the order-checking polyacrylamide gel on isolation of RNA.Use the phosphorescent substance imaging system to measure the per-cent (Fig. 7) of the substrate of cutting by the radioautograph quantitative analysis method.The hammerhead ribozyme of embodiment 12:GBSS

Designed hammerhead ribozyme with 10/10 (promptly can form 10 base pairs on each arm at ribozyme) Nucleotide brachium conjunctivum at the site that oligonucleotide covered of fullest cutting in RNA enzyme H measures.Analyze these ribozymes by the computer folding algorithm then, and eliminate ribozyme with obvious secondary structure.The result of screening process is 23 ribozymes that designed at open base area among the GBSS mRNA, has shown the sequence of these ribozymes in the Table IV.

Use the synthetic ribozyme of chemical process.That the synthetic method of using adopts is above-mentioned (Usman etc., above; Scaringe etc.; Wincott etc., above, this paper in the lump with reference to) the standard rna synthetic method, and used the protection of common nucleic acid and the coupling group phosphamide of dimethoxy trimethylphenyl and 3 ' end of holding (as 5 ').Average substep coupling productive rate＞98%.By replacing G with U ₅Replace A with U ₁₄The ribozyme of (numbering comes from Hertel mentioned above etc.) synthetic non-activity.Divide two portions to synthesize the hair clip ribozyme, and annealing with rebuild active ribozyme (Chowira and Burke, 1992, nucleic acids research, 20,2835-).By with 2 '-the O-methyl group modifies 5 ' 5 ribonucleotides terminal and 3 ' end and improved all ribozymes to increase stability.Use common method by gel electrophoresis (Ausubel etc., the scheme Wiley ﹠amp that 1990 molecular biology are present; Sons, NY) purifying ribozyme is resuspended in the water perhaps by above-described high pressure liquid chromatography (HPLC) method purifying ribozyme, and with ribozyme.The long substrate test of embodiment 13:GBSS

Be used for long 900 nt of target RNA of this research, and comprise cleavage site at whole 23 HH ribozymes of GBSS RNA.The template that contains T7 rna polymerase promoter of GBSS target sequence upstream by the cDNA clone by pcr amplification.Use the T7 RNA polymerase to transcribe target RNA by the template of pcr amplification.In transcription, pass through to add [c- ³²P] CTP carries out inner marker as one of 4 kinds of triphosphoric acid ribonucleotides with transcript.After transcribing 2 hours under 37 ℃, transcribe the dna profiling that mixture is used to transcribe with digestion with DNA enzyme-1 processing.Separate in denaturing polyacrylamide gel transcribing mixture.In gel slice, isolate band, and go out RNA, throw out is stored under 4 ℃ the condition with isopropanol precipitating corresponding to full-length RNA.

At the excessive (k of ribozyme _Cat/ K _M) carry out ribozyme cleavage reaction (above-mentioned Herschlag and Cech) under the condition.In brief, at 50 mM Tris-HCl (pH7.5) and 10 mM MgCl ₂Exist down by being heated to 90 ℃ and keep 2 minutes respectively with the target RNA sex change of the inner marker of 1000 nM ribozymes and＜10 nM.By being cooled to temperature of reaction (37 ℃, 26 ℃ and 20 ℃) and continuing 10-20 minute with the RNA renaturation.Under suitable reaction temperature,, ribozyme begins cleavage reaction by being mixed with target RNA.In certain time interval, take out aliquots containig, and reaction is stopped by adding isopyknic damping fluid that stops.Sample is separated on 4% sequencing gel.

Fig. 8 has summed up the ribozyme cleavage reaction result under three kinds of differing tempss.Select 7 leading (lead) ribozymes (425,892,919,959,968,1241 and 1787).One of active ribozyme (811) has produced unusual cleaved products pattern, and it is not chosen as a kind of leading ribozyme as a result.Embodiment 14: use multinuclear enzyme combination cutting GBSS RNA

With four leading ribozymes (892,919,959,1241) target RNA incubation with inner marker in following combination: 892,892+919,892+919+959,892+919+959+1241.RNA cutting share increases (Fig. 9) in the additivity mode along with the quantity increase of used ribozyme in the cleavage reaction.The ribozyme cleavage reaction carries out under 20 ℃ as mentioned above.These data show decides target aggravates target RNA in the additivity mode to the multinuclear enzyme of the last different loci of same mRNA minimizing.Embodiment 15: the GBSS transcriptional units clone GBSS polymer ribozyme RPA63 (activity) and the RPA64 (non-activity) of construction expression ribozyme

Structure contains four the hammerhead ribozyme target sites 892,919,959 of GBSS mRNA and 968 polymer ribozyme.Buy two have 18 Nucleotide eclipsed DNA oligonucleotide (Macromolecular Resourses, Fort Collins, CO).Sequence is as follows:

Oligonucleotide 1:CGC GGA TCC TGG TAG GAC TGA TGA GGCCGA AAG GCC GAA ATG TTG TGC TGA TGA GGC CGA AAGGCC GAA ATG CAG AAA GCG GTC TTT GCG TCC CTG TAG ATGCCG TGG C

Oligonucleotide 2:CGC GAG CTC GGC CCT CTC TTT CGG CCTTTC GGC CTC ATC AGG TGC TAC CTC AAG AGC AAC TAC CAGTTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC TAC AGGG

Replace G by catalytic center with T at each enzyme primitive ₅Replace A with T ₁₄Obtain the ribozyme of above-mentioned non-activity.

Under 90 ℃ these enzymes were annealed 5 minutes in 1X Klenow damping fluid (Gibco/BRL), then cool to room temperature (22 ℃) slowly.Adding NTP is 0.2mM to concentration, and 37 ℃ are extended oligonucleotide 1 hour with 1 unit/μ l with the Klenow enzyme down.With phenol/chloroform extraction, ethanol sedimentation, resuspension in 1X React 3 damping fluids (Gibco/BRL) is used Bam HI and Sst I digestion 1 hour down for 37 ℃ then.Use the Qiagen gel extraction kit on 2% sepharose, said DNA to be carried out gel-purified.

Dna fragmentation is connected among the pDAB353 of Bam HI/Sst I digestion.Be transformed in competence DH5 α F ' bacterium (Gibco/BRL) with this connector.By cloning with Bam HI/Eco RI digestion screening potential.Confirm these clones by order-checking.With target sequence homologous total length be 96 Nucleotide.919 monomer ribozymes (RPA66)

Use 10/10 arm as described below makes up the monokaryon enzyme at 919 sites of GBSS mRNA.Buy following two DNA oligonucleotide:

Oligonucleotide 1:GAT CCG ATG CCG TGG CTG ATG AGG CCGAAA GGC CGA AAC TGG TAG TT

Oligonucleotide 2:AAC TAC CAG TTT CGG CCT TTC GGC CTC ATCAGC CAC GGC ATC G

These two oligonucleotide are carried out phosphorylation respectively in 1X kinase buffer liquid (Gibco/BRL), thermally denature, 90 ℃ are mixed down and made it annealing in 10 minutes, then cool to room temperature (22 ℃) slowly.By also terminal polishing being prepared carrier with the T4 archaeal dna polymerase with Sst I digestion pDAB 353.This carrier is digested with Bam HI again, and carry out gel-purified as mentioned above.Connect in the damping fluid (Gibco/BRL) the annealed oligonucleotide spent the night at 1X under 16 ℃ and be connected in the carrier.Use Bam HI/Eco RI digestion potential clone, and confirm by order-checking.Embodiment 16: be used for the Plant Transformation plasmid pDAB 367 of Δ 9 ribozymes experiment and be used for the pDAB353 part A that the GBSS ribozyme is tested: pDAB367

Plasmid pDAB367 has following dna structure: to be positioned at pUC19 (441 bases, base behind last C residue in Sph I site reference 1) begins, and with the contiguous chain of LacZ genes encoding chain on read, joint sequence CTGCAGGCCGGCCTTAATTAAGCGGCCGCGTTTAAACGCCCGGGCATTTAAATGGC GCGCCGCGATCGCTTGCAGATCTGCATGGGTG, the 7093-7344 Nucleotide (2) of CaMV DNA, joint sequence CATCGATG, the 7093-7439 Nucleotide of CaMV, joint sequence GGGGACTCTAGAGGATCCAG, the 167-186 Nucleotide (3) of MSV, the 188-277 Nucleotide (3) of MSV, the C residue, be the Nucleotide 119-209 (4) that contains the corn Adh 1S of exons 1 and introne 1 part afterwards, the Nucleotide 555-672 (4) that contains Adh 1S introne 1 and exon 2 part, joint sequence GACGGATCTG, the Nucleotide 278-317 of MSV.Be the pIJ4104 BAR coding region of modifying (5) afterwards, wherein with the AGC Serine codon on the GCC L-Ala codon replacement second position, 546 Nucleotide of coding region change over A by G, so that remove Bgl II site.Next be joint sequence TGAGATCTGAGCTCGAATTTCCCC, the 1298-1554 Nucleotide (6) of Nos, the G residue is the other parts (comprising Eco RI site) of pUC 19 sequences afterwards.Part B:pDAB353

Plasmid pDAB353 has following dna structure: to be positioned at pUC19 (441 bases, base behind last C residue in Sph I site reference 1) begins, and with the contiguous chain of LacZ genes encoding chain on read, joint sequence CTGCAGATCTGCATGGGTG, the 7093-7344 Nucleotide (2) of CaMV DNA, joint sequence CATCGATG, the 7093-7439 Nucleotide of CaMV, joint sequence GGGGACTCTAGAG, the Nucleotide 119-209 (4) that contains the corn Adb 1S of exons 1 and introne 1 part, the Nucleotide 555-672 (4) that contains Adh 1S introne 1 and exon 2 part, joint sequence GACGGATCCGTCGACC, GGATCC wherein represents the recognition sequence of BamH I restriction enzyme.Be beta-Glucuronidase (GUS) gene (7) (as Nco I/Sac I fragment cloning) of pRAJ275 afterwards, joint sequence GAATTTCCCC, the poly A district (6) of Nos Nucleotide 1298-1554, the G residue is the other parts (comprising Eco RI site) of pUC 19 sequences afterwards.Below with reference to document this paper reference in the lump: 1.Messing, J. (1983) " Enzymology method " (Wu., editors such as R.) 101:20-78.2.Franck, A., H.Guilley, G.Jonard, K.Richards, and the nucleotide sequence of L.Hirth (1980) cauliflower mosaic virus DNA, cell 21:285-294.3.Mullineaux, P.M., J.Donson, B.A.M.Morris-Krsinich, the nucleotide sequence of M.I.Boulton and J.W.Davies (1984) corn virus 2 DNA, EMBO is J.3:3063-3068.4.Dennis, E.S., W.L.Gerlach, A.J.Pryor, J.L.Bennetzen, A.Inglis, D.Llewellyn, M.M.Sachs, R.J.Ferl, and the analysis of molecules of W.J.Peacock (1984) maize alcohol dehydrogenase (Adhl) gene, nucleic acids research 12:3983-4000.5.White, J., S-Y Chang, M.J.Bibb, and M.J.Bibb (1990) contains the box of streptomyces hygroscopicus bar gene: be used for the selective marker of Plant Transformation, nucleic acids research 18:1062.6.Depicker, A., S.Stachel, P.Dhaese, P.Zambryski, and H.M.Goodman (1982) nopaline synthetic enzyme: the transcript collection of illustrative plates is measured and dna sequence dna, molecular application genetics magazine 1:561-573.7.Jefferson R.A. (1987) analyzes the mosaic gene in the plant: gus gene emerging system, molecular biology of plants circular 5:387-405.Embodiment 17: plasmid pDAB359: the Plant Transformation plasmid that contains γ-zein promotor, antisense GBSS and Nos polyadenylic acid sequence

Plasmid pDAB359 is the double-stranded cyclic DNA of one 6702 bp, this plasmid contains the Nucleotide 1-404 of following sequences composition: pUC18, the lactose operon sequence that wherein contains the from the 238th to 404 base, and finish (1,2) with the HindIII site of M13mp18 polylinker; The Nos polyadenylic acid sequence (3) of Nucleotide 412 to 668; The synthetic adapter sequence of the 679-690 of Nucleotide, this sequence makes Sac I site change Xho I site into by GAGCTC being become GAGCTT and adding CTCGAG; The corn particle of from 691 to 2953 bases is in conjunction with starch synthase cDNA, corresponding to the Nucleotide 1-2255 of SEQ.I.D.NO.25.By original cDNA by increase by 5 ' Xho I and 3 ' Nco I site and make inner Nco I and Xho I site disappearance modify GBSS sequence among the plasmid pDAB359 by using Klenow to fill enzyme recognition sequence.Base 2971 to 4453 is 5 ' non-translated sequences of the 27kD γ-zein spirit-soluble gene of corn, corresponding to 1078 to 2565 Nucleotide of disclosed sequence (4).Modify γ-zein sequence and made it to contain 5 ' Kpn I site and 3 ' BamH/SalI/Nco I site.With respect to disclosed sequence, in γ-zein sequence other change be included in the T disappearance of Nucleotide 104, in the TACA of Nucleotide 613 disappearance, in the change of the C to T of Nucleotide 812, insert in the A of Nucleotide 1165 disappearance with at the A of Nucleotide 1353.At last, the Nucleotide 4454 to 6720 of pDAB359 and the base 456 to 2686 of pUC18 (the KpnI/EcoRI/Sac I site, from 4471 to 4697 lac operon fragment and from 5642 to 6433 the β-Nei Xiananmei gene (1,2) that wherein comprise from 4454 to 4471 M13/mp18 polylinker) are identical.Following reference this paper reference in the lump: pUCI8-Norrander, J., Kempe, T., Messing, gene magazine (1983) 26:101-106; Pouwels, P.H., Enger-Valk, B.E., Brammar, W.J. cloning vector, Elsevier1985 and supplementary issue.NosA-Depicker, A., Staehel, S., Dhaese, P., Zambryski.P., and Goodman, H.M. (1982) nopaline synthase: the transcript collection of illustrative plates is measured and dna sequence dna, molecular application genetics magazine 1:561-573.Corn 27kD γ-zein-Das, O.P., Poliak, E.L., Ward, K., Messing, nucleic acids research magazine 19,3325-3330 (1991).Embodiment 18: make up the plasmid pDAB430 part A that contains antisense Δ 9 desaturases of being expressed by ubiquitin promotor/intron (Ubil): the structure of plasmid pDAB421

Plasmid pDAB421 contains unique flush end SrfI cloning site and nopaline synthase polyadenylic acid sequence by corn ubiquitin promotor/intron side joint.Prepare pDAB421 by the following method: digest pDAB355 to downcut the R coding region on the 2.2kB fragment with restriction enzyme KpnI and BamHI.After the gel-purified, this carrier is connected on the adapter of being made up of two annealed oligonucleotide OF235 and OF236.OF235 has 5 '-sequence of GAT CCG CCCGGG GCC CGG GCG GTA C-3 ', OF236 has 5 '-sequence of CGC CCGGGC CCC GGG CG-3 '.By using in adapter and do not identify the clone who contains adapter in the enzyme SrfI of other sites cuttings of plasmid and SmaI digestion and linearization plasmid DNA.The representational clone that checked order has only an adapter to be inserted in the plasmid with checking.In the structure of subsequently Δ 9 desaturase antisense plasmid pDAB430, use the plasmid pDAB421 that generates.Part B: make up plasmid pDAB430 (antisense Δ 9 desaturases)

The amplified production of the flush end cloning site by being cloned in plasmid pDAB421 produces the antisense Δ 9 desaturase constructs that are present among the plasmid pDAB430.Produce two constructs simultaneously by same experiment.First construct contains Δ 9 delta 8 desaturase genes in the ubiquitin promotor back of sense orientation, and contains the c-myc tag of the C-terminal fusions proteinic immunology detection and Δ 9 desaturase open reading frame that are useful on excessive generation in the transgenic lines.This construct is used for detecting ribozyme in the system that can not express corn Δ 9 desaturases.Never used this construct, but the primer that is used to increase with making up Δ 9 desaturase inverted defined genes is identical.The Δ 9 desaturase cDNA sequences described herein of having used two kinds of primer amplifications.N-terminal primer OF279 has following sequence: 5 '-GTG CCC ACA ATG GCG CTC CGC CTC AACGAC-3 '.Nucleotide 146-166 with the base of underscore and cDNA sequence is corresponding.C-terminal primer OF280 has following sequence: 5 '-TCA TCA CAG GTC CTC CTCGCT GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACCGTA-3 ', and be sequence 5 '-reverse complementary sequence of TAC GGT AGG GAC GTC CAA CTG GAGGAG AAG CTG ATC AGC GAG GAG GAC CTG TGA TGA-3 '.The Nucleotide 1304-1324 that has the base of underscore and cDNA sequence in this sequence is corresponding, and the base of italic is corresponding with c-myc epitope sequence.By using the amplified production of 1.0% sepharose purifying, 1179 bp, and it is connected to on the linearizing plasmid pDAB421 of restriction enzyme SrfI.Use colony hybridization to select to contain the pDAB421 plasmid clone of (have and insert fragment).Measure the segmental direction of insertion by the restrictive diges-tion of carrying out plasmid DNA with diagnostic enzyme KpnI and BamHI.Reclaimed that to contain with respect to ubiquitin promotor/intron be the clone that the Δ 9 desaturase encoding sequences of justice orientation are arranged, and with its called after pDAB429.Containing with respect to promotor is another clone's called after pDAB430 of the Δ 9 desaturase encoding sequences of antisense orientation.Plasmid pDAB430 is carried out sequential analysis, determined the mistake (comparing) that this sequence contains three PCR and causes with desired sequence.With the corresponding sequence of primer OF280 in found a mistake, found that in encoding sequence inside two Nucleotide change.Do not proofread and correct these mistakes, because the negative adjusting of antisense need be in antisense transcript and negative 100% the sequence identity of regulating between the target.Embodiment 19: the subsequent regeneration part A of the helium bombardment of embryogenetic corn culture and transgenosis filial generation: the foundation of embryogenetic corn culture

The tissue culture that transformation experiment uses is from the immature zygotic embryo of genotype ＂ Hi-II ＂.Hi-II is 2 hybrids (Armstrong etc. 1990) that R3 system hands over gained mutually that derive from B73 x A188 hybridization.When cultivating, this genotype produces the callus that is called Type II.The Type II callus is fragile, and growth is very fast, and demonstrates and keep high-level embryo that active ability takes place for a long time.

The Type II culture is from the immature embryo of 1.5-3.0mm, and these embryos are to carry out controlled pollination by the Hi-II plant to greenhouse cultivation to obtain.Used initial substratum is to contain 1.0mg/l 2,4-D, 25mM L-proline(Pro), 100mg/l casein hydrolysate, 10mg/lAgNO ₃, 2.5g/L gelrite and 2% sucrose N6 substratum (Chu, 1978), pH regulator to 5.8.Approximately 2-8 selects the Type II callus after week, removes non-embryogenetic and/or type i callus.In case selected the Type II callus, just it transferred to and do not contain AgNO ₃, the L-proline content is reduced on the maintenance substratum of 6mM.

Succeeding transfer culture (has wherein increased the quantity and the quality of embryo's generation culture) after about 3 months, and this culture is regarded as can be used in the transformation experiment.Part B: the preparation of plasmid DNA

Before being used for transformation experiment, plasmid DNA is adsorbed onto the surface of gold grain.Used gold grain in GBSS target experiment, these particles be sphere, diameter between the 1.5-3.0 micron (Aldrich chemical company, Milwaukee, WI).The transformation experiment of Δ 9 desaturase targets used 1.0 microns spherical gold grains (Bio-Rad, Hercules, CA).Before the use, all gold grains are carried out surface sterilization with ethanol.Finish absorption by adding 74 μ l 2.5M calcium chloride and 30 μ l 0.1M spermidines in 300 μ l plasmid DNA and sterilized water.The concentration of plasmid DNA is 140 μ g.To coat the gold grain vortex of DNA immediately, and make it from suspension, be precipitated out.Remove the clarifying supernatant liquor of gained, and particle is suspended in 1ml 100% ethanol again.The final weaker concn that is used for the suspension of helium bombardment is that every milliliter of ethanol contains the 7.5mgDNA/ gold.Portion C: prepare tissue target and it is carried out helium bombardment

The about 600mg embryo of every target generation callus is tiled in contains the Type II callus and keep substratum to add the culture dish surface of 0.2 M sorbyl alcohol and 0.2 M mannitol (as permeate agent).After pre-treatment in about 4 hours, all tissues are transferred on the culture dish that contains 2% agar bombardment substratum (keeping substratum+permeate agent+2% agar).

Helium bombardment relates to the gold grain guiding that scribbles DNA that promotes suspension and enters in the tissue target of preparation.Used instrument is the early stage prototype of the instrument of DowElanco United States Patent (USP) (#5,111,131) (this paper is reference in the lump) description, but the functional similarity of the two.This instrument comprises the high pressure helium source, contain the syringe of DNA/ bronze suspension and pneumatic multi-functional valve (to connecting control between the helium source and the DNA/ bronze suspension loop of having loaded).

Before the bombardment, tissue target is covered with aseptic 104 microns Stainless Steel Screens, this screen can guarantee that in the bombardment process tissue location is constant.Next under the vacuum target is placed in the main chamber of this instrument, the gold grain that uses 1500 psi helium to be pressed in will to scribble DNA on the tissue target quickens 4 times.Each bombardment discharges 20 μ lDNA/ gold suspension.After the bombardment, immediately target is put back in the maintenance substratum that is added with permeate agent and cultivates 16 to 24 hours (decubation).Part D: the tissue that screening transforms and by transgenosis culture regeneration plant

Bombard after 12-24 hour, tissue segmentation is become fritter and transfer to the selection substratum (to keep substratum+30 mg/l Basta ^TM).Whenever all around tissue block is not had and selectively transfer in the fresh selection substratum 3 totally months.After 8 thoughtful 24 weeks, remove and separate any part of breeding of organizing with respect to growth-inhibiting of being found.Organize succeeding transfer culture in fresh selection substratum with what infer conversion.Set up the transgenosis culture behind the succeeding transfer culture 1 to 3 time again.

In case Basta ^TMResistant calli is built into a system, begin plant regeneration by callus being transferred on the culture dish that contains based on the inducing culture of phytokinin, then it is placed a week down according to (125 ft-c) at low light, place a week down in high illumination (325 ft-c) afterwards.Inducing culture is by MS salt and VITAMIN (Murashige and Skoog, 1962), 30g/l sucrose, 100mg/l flesh inositol, 5mg/l 6-aminotoluene base purine, 0.025mg/l 2, and 4-D, 2.5mg/l gelrite form, pH regulator to 5.7.After inducing through two weeks, will tissue do not have and selectively transfer in the regeneration culture medium of no hormone, and remain under the high illumination.Regeneration culture medium is formed pH regulator to 5.7 by MS salt and VITAMIN, 30g/l sucrose and 2.5mg/l gelrite.Induce with regeneration culture medium and all comprise 30mg/l Basta ^TMOrganize after week at 2-4 and to begin to differentiate stem and root.Take off plantlet (1.5-3cm) and place it in the test tube that contains the SH substratum.The SH substratum is by SH salt and VITAMIN (Schenk and Hildebrandt, 1972), 10g/l sucrose, 100mg/l flesh inositol, 5ml/l FeEDTA and 7g/l agar or 2.5g/l gelrite composition, pH regulator to 5.8.The concurrent enough root systems (1-2 week) that bring out in case plantlet begins to grow, it is transferred to about 0.1 kg Metro-Mix  360 that is equipped with in the greenhouse, and (The Scotts company, Marysville is in 10cm flowerpot OH).In the 3-5 leaf phase, plant is transferred in 5 gallons of flowerpots that about 4 kg Metro-Mix  360 are housed, cultivate to ripe.With these R ₀The plant selfing and/or with the non-transgenic inbred line cross, to obtain genetically modified filial generation.In the transgenic plant for the generation of GBSS target, planted by R again ₀The R1 seed that pollination produces.The R1 plant cultivation to ripe, is produced enough quantitative analyses R2 seed through pollination.Embodiment 20: the generation of Δ 9 transgenic lines and regeneration section A: transform and separate embryo's generation callus

Above-described six ribozyme constructs at Δ 9 desaturases are transformed in the reproducible Type II callus culture thing described herein.These 6 constructs by 3 activity/non-activities to forming, i.e. RPA85/RPA113, RPA114/RPA115 and RPA118/RPA119.Prepare altogether and bombarded 1621 tissue target, and it is transferred to select in the substratum.In these bombardment experiments, by selecting to have separated 334 independently Basta  resistance transforming tissues (" being "), with DNA PCR or GC/FAME as detection means which is determined this regenerate and which this discard, analyzed about 50% be.Because remaining 50% can not regenerate embryo or polluted and do not analyze.Part B: by transgenic calli regeneration Δ 9 plant

After having analyzed transgenic calli, each ribozyme construct selects 12 systems to be used for regeneration, and each is with 15 R of generation like this ₀Plant.These are generally to be made up of+2 negative controls of 10 positive systems, yet because the regenerative power of some cultures is poor, construct RPA113, RPA115, RPA118 and RPA119 are to produce plant by being less than 12.854 R have altogether regenerated from 66 independently are ₀Plant (referring to Table X).When plant is ripe, preferentially carries out from flower or sib-pollination, yet in the time can not carrying out, use inbred lines CQ806, CS716, OQ414 or HO from flower or sib-pollination ₁Carry out hybridization pollination as pollen donor (sometimes also as the pollen acceptor).Carried out the controlled pollination above 715, wherein majority is from flower or sib-pollination (55%), and minority is F1 hybridization.Gathered in the crops R after pollinating about 45 days ₁Seed.The generation of embodiment 21:GBSS transgenic corns and regeneration section A: the selection and the foundation of the conversion of embryogenetic maize calli and follow-up transgenosis culture

To select plasmid pDAB308 to be inserted in the Type II callus described herein RPA63 and RPA64 with bar at the polymeric activity/non-activity of the ribozyme of GBSS.From the selection of the tissue target of 590 bombardments, 115 Basta have altogether been reclaimed ^TMThe independent transforming tissue of resistance.The culture callus sample of setting up transforming from all has been carried out Southern to be analyzed to determine the state of interest genes.Part B: regeneration plant and produce R from use the culture that transforms at the ribozyme of GBSS ₂Generation

From Southern " positive " transgenosis culture regeneration plant, and in the greenhouse, cultivate to ripe.Main regeneration plant is pollinated to produce R ₁Seed.Pollinate and gather in the crops seed after 30 to 45 days, seed drying is planted to correct water capacity and with seed.752 R altogether of 16 original systems will be represented ₁Plant cultivating is to sexual maturity and pollination.Gather in the crops the wheat head about pollination after 19 to 22 days greatly, from each fringe, take 30 grain randomly, and freezing to be used for later analysis.Embodiment 22: the test section A of the ribozyme of target GBSS in corn Black Mexican Sweet (BMS) the stable conversion callus: the generation of the BMS callus of usefulness GBSS stable conversion and the ribozyme of target GBSS

BMS can not produce the mRNA with the endogenous GBSS mRNA of corn homologous GBSS.Therefore two conversion systems have been worked out to produce the transformant of expressing target and ribozyme.At succeeding transfer culture after four days, by transferring to 100 * 200mm culture dish (Fisher Scientific, Pittsburgh, PA) and remove the partially liq medium preparation ＂ ZM ＂ BMS suspension (obtain from JackWidholm (Illinois university), also referring to W.F.Sheridan, " Black MexicanSweet corn: be used for tissue culture " (corn biological study, W.F.Sheridan edits, university press, North Dakokta university, Grand Forks, ND, 1982, pp.385-388)) be used to carry out helium bombardment, form the cell water gruel.Target is to be grown in by 100-125mg (fresh weight) to place bombardment 1/2 on substratum " microbiotic dish (Schleicher and Schuell; Keene; the cell NH), DN6[N6 salt and VITAMIN (Chu etc.; 1978), 20g/l sucrose, 1.5mg/l 2; 4-dichlorophenoxyacetic acid (2; 4-D), 25 mM L-proline(Pro), 121 ℃ of following autoclavings before 20 minutes with pH regulator to 5.8] form, with 2%TC agar (JRH bio-science, Lenexa, Kansas) solidify, be layered on the 60 X 20mm flat boards.DNA is deposited on the gold grain.In transforming for the first time, use pDAB 426 (Ubi/GBSS) and pDAB 308 (35T/Bar).Use DowElanco helium bombardment instrument I respectively target to be bombarded.Pressing in vacuum is 650 mm Hg, and target is under the 15.5cm to the distance of instrument spout, bombards each sample once with 500psi with DNA/ gold mixture.After the bombardment, immediately the microbiotic dish is transferred on the DN6 substratum of being made up of 0.8%TC agar, made target tissue recover a week.After the recovery, each target is tiled in to place do not contain proline(Pro) and contain on 5.5cmWhatman # 4 filter membrane of 3mg/l Basta  (Hoechst, Frankfort, Germany).After two weeks, filter membrane is transferred on the fresh selection substratum that contains 6mg/l Basta .Once shift in per two weeks later.Picking isolate and being placed on from the filter membrane with 0.8%TC agar (containing 6mg/l Basta ) solidified AMCF-ARM substratum (N6 salt and VITAMIN, 20g/l sucrose, 30g/l mannitol, 100mg/l acid casein hydrolyzate, 1mg/l 2,4-D, 24mM L-proline(Pro); 121 ℃ of following autoclavings before 20 minutes with pH regulator to 5.8) on.Per two weeks isolate is transferred to and carried out succeeding transfer culture on the fresh substratum, thereby keep isolate.

The Basta  resistance isolate of expressing GBSS is carried out transforming the second time.As the BMS suspension, at succeeding transfer culture after 4 days, by tissue being tiled in the target of preparation transgenic calli on the 1/2 ＂ filter membrane.Yet, because remaining on AMCF-ARM, transformant selects on the substratum, use the AMCF-ARM that contains 2%TC agar to be used for bombardment.Cover each sample with aseptic 104 μ m purposes screen, under 1500 psi, bombard.With pDAB 319 (35S-ALS; 35T-GUS) and RPA63 (the many bodies of active ribozyme) or pDAB319 and RPA64 (the many bodies of the ribozyme of non-activity) bombardment or use pDAB 319 bombardment target tissues separately altogether.After the bombardment, all targets are transferred in the non-selection substratum (AMCF-ARM) immediately, recovered for 1 week.Next, said target is placed in the AMCF-ARM substratum that contains two kinds of selective reagents 6mg/L Basta  and 2 μ g/L chlorsulfurons (CSN).After 2 weeks, the level of CSN increases to 4ug/L.Shift filter membrane continuously and produce isolate according to the description that transforms for the first time, isolate is maintained in the AMCF-ARM substratum that contains 6mg/LBasta and 4 μ g/L CSN.Part B: the BMS stable conversion body of analyzing the ribozyme of expressing GBSS and target GBSS

The isolate that transforms for the first time by the evaluation of Northern engram analysis method, with detection functionality target gene (GBSS), and definite relative expression's level.In 25 isolates of being analyzed, there are 12 to detect the GBSS transcript.Observed the expression scope, shown that conversion process is independently.Transform the isolate of generation for the second time by the evaluation of Northern engram analysis method, express, whether exist by RT-PCR screening ribozyme transcript to detect successive GBSS.In 19 isolates being tested from a system of conversion originally, 18 expression activity ribozymes (RPA63), all isolates are all expressed GBSS.In 6 vehicle Control, all detected GBSS; Ribozyme is not expressed in these samples.As described herein, in the tissue of ribozyme expression and vehicle Control tissue, carried out the RNA enzyme protection and measured (RPA) and Northern engram analysis, so that relatively there is and do not exist the level of GBSS transcript under the situation at active ribozyme.Make the relative internal reference of GBSS value (Δ 9 desaturases) stdn.Northern trace data are shown in figure (25).The Northern trace is the result show, compares with vehicle Control, and the level of GBSS obviously reduces when ribozyme exists.The RPA data show that the independent sample (＂ L ＂ and ＂ O ＂) of some expression activity ribozymes is obviously different with vehicle Control, and similar to non-conversion contrast.Embodiment 23: the analysis of plant and callus material

At callus level and R0 horizontal analysis vegetable material, and select system in the F1 horizontal analysis with one of pDAB308 and following carrier that contains ribozyme (pRPA63, pRPA64, pRPA85, pRPA113, pRPA114, pRPA115, pRPA118 or pRPA119) cotransformation.When plantlet reaches the 6-8 leaf during phase, gather in the crops blade material.The DNA that from freeze dried tissue, prepares plant and callus material as the description (seeing above) of Saghai-Maroof etc.(Bethesda research laboratory, Gaithersburg MD) digest each DNA (8 μ g) down, and separate by agarose gel electrophoresis in the condition of manufacturer's suggestion to the specific Restriction Enzyme of each construct in use.As Southern, E. the ＂ of (1975) detection specificity sequence ＂ (molecular biology magazine 98:503) and Southern in by gel electrophoresis separated DNA fragment, the description of the gel electrophoresis ＂ of the ＂ restriction fragment of E. (1980) (Enzymology method 69:152) (this paper in the lump with reference to) with said southern blotting technique to nylon membrane.

With ribozyme coding region specific probe and the hybridization of said film.Said dna probe is joined have 50 microcurie α- ³²P-dCTP (Amersham life science, Arlington Heights, Ready-To-Go dna marker pearl (Pharmacia LKB IL), Piscataway, NJ) before, boiled 10 minutes, prepare dna probe in cooled on ice fast then by dna probe with 50ng.On nylon membrane, probe and genomic dna are hybridized.Under 60 ℃, this film was washed 45 minutes in 0.25X SSC and 0.2% SDS, blots, and with two strengthen screen to XAR-5 exposure spend the night.

Digest the DNA of RPA63 and RPA64 with Restriction Enzyme HindIII and EcoRI, and will contain the trace and the RPA63 probe hybridization of these samples.The RPA63 probe is made up of RPA63 ribozyme polymer coding region, will produce single 1.3kb hybridization product when with RPA63 or the hybridization of RPA64 material.This 1.3kb hybridization product should contain enhanced 35S promoter, AdhI intron, ribozyme coding region and nopaline synthase poly A 3 ' end.Digest the DNA of RPA85 and RPA113 with restriction enzyme HindIII and EcoRI, and will contain the trace and the RPA122 probe hybridization of these samples.RPA122 is the 252 polymer ribozymes that replace the GUS reporter gene in pDAB353.The RPA122 probe is made up of 3 of RPA122 ribozyme polymer coding region and nopaline synthase ' end, should be able to produce a single 2.1kb hybridization product when with RPA85 or the hybridization of RPA113 material.This 2.1kb hybridization product should contain enhanced 35S promoter, AdhI intron, bar gene, ribozyme coding region and nopaline synthase poly A3 ' end.Digest the DNA of RPA114 and RPA115 with Restriction Enzyme HindIII and SmaI, and will contain the trace and the RPA115 probe hybridization of these samples.The RPA115 probe is made up of RPA115 ribozyme coding region, should produce single 1.2 kb hybridization product when with RPA114 or the hybridization of RPA115 material.This 1.2 kb hybridization product should contain enhanced 35S promoter, AdhI intron, ribozyme coding region and nopaline synthase poly A 3 ' end.Digest the DNA of RPA118 and RPA119 with Restriction Enzyme HindIII and SmaI, and will contain the trace and the RPA118 probe hybridization of these samples.The RPA118 probe is made up of RPA118 ribozyme coding region, should produce single 1.3 kb hybridization product when with RPA118 or the hybridization of RPA119 material.This 1.3 kb hybridization product should contain enhanced 35S promoter, AdhI intron, ribozyme coding region and nopaline synthase poly A 3 ' end.Embodiment 24: the extraction of transgenic calli genomic dna

Be the callus of 300 mg vigorous growths freezing rapidly on ice.With cold Bessman tissue powder millstone (Spectrum, Houston, TX) callus is ground to form fine powder, and extract with 400 μ l 2X CTAB damping fluids (2% 6 decyl trimethylammonium bromide, 100 mMTris pH, 8.0,20 mM EDTA, 1.4 M NaCl, 1% polyvinylpyrrolidone).With suspension dissolving 25 minutes, use isopyknic trichloromethane then: isoamyl alcohol extracting under 60 ℃.The 10%CTAB damping fluid (10% 6 decyl trimethylammonium bromide, 0.7M NaCl) that adds 0.1 times of volume to aqueous phase.Use isopyknic trichloromethane: behind the isoamyl alcohol extracting, add the cold isopropanol of 0.6 times of volume to aqueous phase, and under-20 ℃, placed 30 minutes.14, under the 000rpm after centrifugal 5 minutes, with the precipitation vacuum-drying of gained 10 minutes.Under 65 ℃ it is resuspended in 200 μ l TE (10mM Tris, 1mM EDTA, pH8.0) in 20 minutes.Adding 20%Chelex (Biorad) in DNA reaches under 5%, 56 ℃ with its incubation 15-30 minute so that remove impurity its ultimate density.Go up measurement DNA concentration at Hoefer photofluorometer (Hoefer, San Francisco).Embodiment 25: the pcr analysis of genome callus DNA

Use polymerase chain reaction (PCR) to prove that ribozyme gene stably is inserted on the karyomit(e) of transgenic corns callus.Part A: the method that is used to detect ribozyme DNA

(GeneAmp PCR test kit, Perkin Elmer Cetus) carry out polymerase chain reaction (PCR) as the description in manufacturer's method to use the AmpliTaq archaeal dna polymerase.With 300ng genome callus DNA, 1 μ l, 50 μ M downstream primers (5 ' CGC AAG ACC GGC AACAGG 3 '), 1ml upstream primer, 1 μ l Perfect Match (Stratagene, Ca) mix with reagent constituents by the sample aliquot of PCR enhanser.Use following parameters to carry out 40 round-robin PCR reactions: 1 minute, 55 ℃ annealing of 94 ℃ of following sex change were extended 3 minutes down for 2 minutes, 72 ℃.Getting the sample aliquot of 0.2 times of volume in each PCR reactant used the TEA sepharose condition of standard to carry out electrophoresis at 2%3: 1 on agarose (FMC) gel.Part B: the upstream primer that is used to detect Δ 9 desaturase ribozyme genes is fused to the many body 5 ' TGG of RPA85/RPA body more than the 113 251 RPA 1,14/,RPA,115 258 ribozyme monomer RPA 1,18/,RPA,119 452 ribozymes ATT GAT GTG ATA TCT CCA C3 ' of BAR 3 ' ORF

In 35S promoter, this primer is used for the amplification across Eco RV site.The oligonucleotide synthetic schemes of use standard prepares primer on applying biological system 394 type DNA/RNA synthesizers.Embodiment 26: the total RNA part A of preparation from transgenic corns callus and plant: the total RNA of preparation from the non-renewable and reproducible callus of transgenosis

The callus of 300mg vigorous growth is freezing rapidly on dry ice.With cold Bessman tissue powder millstone (Spectrum, Houston, TX) callus is ground to form fine powder, and extract damping fluid (50 mM Tris-HCl pH8.0,4% para-aminosalicylic acid, 1% triisopropyl naphthene sulfonic acid, 10 mM dithiothreitol (DTT) and 10 mM meta-acid formula S-WATs) with RNA and extract by fierce vortex.Then with isopyknic phenol extraction homogenate that contains 0.1% oxine.After centrifugal, water layer is with isopyknic trichloromethane that contains: the phenol extraction of primary isoamyl alcohol (24: 1), use trichloromethane afterwards: octanol (24: 1) extracts.Next adding 7.5 M ammonium acetates, to make its ultimate density be 2.5M, 4 ℃ following precipitated rna 1-3 hour.With 14, after 000rpm is centrifugal RNA is resuspended in the sterilized water under 4 ℃, with 2.5 M NH ₄100% ethanol sedimentation of OAc and 2 times of volumes, incubation spends the night under-20 ℃.The RNA that collects with 70% washing with alcohol precipitates, and dry under vacuum.RNA is resuspended in the sterilized water, and is stored in-80 ℃.Part B: prepare total RNA from rotaring gene corn plant

Downcut the maize leaf tissue slice of (being approximately 150mg) vigorous growth of 5cm size, freezing rapidly on dry ice.In cold mortar, blade is ground to form fine powder.Use the total RNA test kit of Qaigen RNeasy plant (Qiagen company, Chatsworth, CA) the total RNA of purifying from powder according to the explanation of manufacturer.Discharge total RNA by the twice serial wash-out rotation that preheats (50 ℃) sterilized water (each 30 μ l) from the RNeasy post, and be kept at-80 ℃.Embodiment 27: use RT-PCR to analyze and confirm the expression part A of ribozyme rna in transgenic corns callus and plant: the method that is used to detect ribozyme rna

Use heat-resisting rTth archaeal dna polymerase (rTth archaeal dna polymerase RNA PCR test kit, Perkin Elmer Cetus) to carry out reverse transcription-polymerase chain reaction (RT-PCR) as the description of provider scenario.Aliquots containig and 1 μ l, the 15 μ M downstream primers (5 ' CGC AAG ACC GGC AAC AGG3 ') of the total RNA of 300ng (blade or callus) are mixed with the RT component of test kit.Divided for three steps carried out reverse transcription reaction in 5 minutes respectively at 60 ℃, 65 ℃ and 70 ℃ of following incubations.In order to carry out the PCR reaction, 1 μ l is had specific upstream primer to the RNA that is analyzed join in the RT reaction of PCR component.Carry out 35 round-robin PCR reaction with following parameters: 96 ℃ of following incubations 1 minute, 94 ℃ of following sex change 30 seconds, 50 ℃ of annealing 30 seconds down, 72 ℃ were extended 3 minutes down.With 0.2 times of volume aliquots containig of each RT-PCR reactant with the TAE sepharose condition of standard electrophoresis on 2% 3: 1 agarose (FMC) gels.Part B: the special upstream primer that is used to detect the GBSS ribozyme

Active and the many body 5 ' CAG of the non-activity ATC AAG TGC AAA GCT GCGGAC GGA TCT G3 ' of GBSS, this primer covers the Adh I intron footprint upstream of first ribozyme arm.GBSS 918 intron (-) monomers: 5 ' ATC CGA TGC CGT GGC TGA TG3 ', this primer covers the ribozyme arm of 10 base pairs and preceding 6 bases in ribozyme catalysis territory.Confirmed the expression of GBSS ribozyme in transgenic calli and plant by RT-PCR.

Also confirmed the expression of many bodies of GBSS ribozyme in the stable conversion callus by rnase protection analysis.Portion C: the special upstream primer that is used to detect Δ 9 desaturase ribozymes

Be fused to the RPA85/RPA113 body more than 252 among BAR 3 ' ORF: 5 ' GAT GAGATC CGG TGG CAT TG3 ', this primer is crossed over the joint of BAR gene and RPA85/113 ribozyme.RPA 1,14/,RPA,115 259 ribozyme monomers: 5 ' ATC CCC TTG GTGGAC TGA TG3 ', this primer covers the ribozyme arm of 10 base pairs and preceding 6 bases in ribozyme catalysis territory.The many bodies of RPA 1,18/,RPA,119 453 ribozymes: 5 ' CAG ATC AAG TGCAAA GCT GCG GAC GGA TCT G3 '.This primer covers the AdhI intron footprint upstream of first ribozyme arm.Confirmed that by RT-PCR Δ 9 desaturase ribozymes are expression among 85-06,113-06 and the 85-15 transgenic plant.

The oligonucleotide synthetic schemes of use standard prepares primer on applying biological system 394 type DNA/RNA synthesizers.Embodiment 28: the minimizing part A that proves the said target mrna level of ribozyme mediation in transgenic corns callus and the plant: use the minimizing of Northern analytical procedure proof said target mrna level

Total RNA of 5 μ g is dry under vacuum, be resuspended in sample-loading buffer (20mM phosphoric acid buffer pH 6.8,5mM EDTA; 50% methane amide: 16% formaldehyde: 10% glycerine), and 65 ℃ of following sex change 10 minutes.In 20mM phosphoric acid buffer (pH 6.8) (damping fluid recirculation), carry out electrophoresis under the 50V through 1% sepharose.(Gibco/BRL, Gaithersburg MD) dye to BRL0.24-9.5 Kb RNA series ladder with ethidium bromide on gel.By carry out capillary transfer with sterilized water RNA is transferred on the Gene Screen filter membrane (DuPontNEN, Boston MA).Hybridize under 42 ℃ as being described in of (1983) such as DeLeon, 55 ℃ are washed filter membrane down so that remove the probe that does not have hybridization.Use the cDNA fragment and the internal rna crt gene of target gene to survey trace successively.Use to mark in the RNA and distinguish the said target mrna level because the error that the RNA with real ribozyme mediation that last sample or wrong-way cause reduces.For each sample, the contrast mRNA level in the sample is relatively therewith with the level of said target mrna.By Qiaex resin (Qaigen company, Chatsworth, CA) purifying fragment from the 1x TAE sepharose.Use contain α- ³²(Amersham company, Arlington Heights III.) carry out nick translation to the Amersham nick translation test kit of P dCTP.It is big to use down intensifying screens (DuPont, Wilmington DE) to carry out radioautograph 1-3 at-70 ℃.Detect the radioautograph signal of each probe after 24 hours by densometer in exposure, and calculate the ratio of target/internal control rna level.

The RNA enzyme protection is measured following carrying out: use the total RNA test kit of Qiagen RNeasy plant by BMS protoplastis or callus material preparation RNA.Use Ambion Maxiscript test kit to prepare probe, probe is generally 10 ⁸Cpm/ μ g or higher.Prepared probe on the same day of using.These probes are carried out gel-purified, be resuspended among the 10mM Tris (pH8) of no RNA enzyme, and be kept on ice.Face with preceding probe dilution to 5 * 10 ⁵Cpm/ μ l.With 5 μ g derive from the RNA of callus or RNA that 20 μ g derive from protoplastis in 4M guanidine damping fluid with 5 * 10 ⁵The cpm probe is incubation together.[4M guanidine damping fluid: 4M guanidine thiocyanate/0.5% sarcosyl/25mM Trisodium Citrate (pH 7.4)].40 μ l PCR mineral oil are joined in each pipe avoid evaporating.Sample is heated to 95 ℃, kept 3 minutes, put into 45 ℃ water-bath immediately.Incubation spends the night.In each sample, add 600 μ l RNA enzymes and handle mixed solution, and 37 ℃ of following incubations 30 minutes.(the RNA enzyme is handled mixed solution: 400 mM NaCl, 40 units per ml RNA enzyme A and T1).Add 12 μ l, 20% SDS in each pipe, in each pipe, add 12 μ l (20mg/ml) Proteinase Ks afterwards immediately.These are managed vortex leniently, 37 ℃ of following incubations 30 minutes.The Virahol that under the room temperature 750 μ l is not had the RNA enzyme joins in each pipe, puts upside down mixing up and down repeatedly, and SDS is entered in the solution.Then under the room temperature with little whizzer with the most at a high speed with centrifugal 20 minutes of these samples.Gained be deposited in air drying 45 minutes.The RNA electrophoretic buffer of 15 μ l is joined in each pipe fierce vortex 30 seconds.(RNA electrophoretic buffer: 95% methane amide/20mM EDTA/0.1% tetrabromophenol sulfonphthalein/0.1% xylidene(s)).Sample is heated to 95 ℃, kept 3 minutes, then with sample on it to 8% denaturing acrylamide gel.This gel is carried out vacuum-drying, and on the phosphorescence imaging screen, exposed 4-12 hour.Part B: prove the result that GBSS mRNA level reduces in the non-renewable callus of the ribozyme rna of expressing GBSS and target GBSS

Produce the RNA of expression GBSS target gene and the non-renewable callus of the fixed active many bodies ribozyme to GBSS mRNA of target.Also produced the transgenosis body of expression GBSS and ribozyme (-) contrast RNA.The total RNA of preparation from transgenic lines.7 ribozymes (-) contrast transformant and 8 active RPA63 are carried out Northern to analyze.The probe that is used for this analysis is total length corn GBSS cDNA and corn Δ 9 cDNA fragments.In order to distinguish GBSS mRNA level because the error that the RNA with real ribozyme mediation that last sample or wrong-way cause reduces, with the comparison of the 9 mRNA levels of the Δ in GBSSmRNA level and this sample.Ribozyme expression and do not have total length GBSS transcript level in the callus of ribozyme relatively, so as to identify that target RNA with ribozyme mediation reduces be.By carrying out the error between parallel analysis minimizing trace twice.

In the transgenosis body of ribozyme (-), observe the ratio range of GBSS/ Δ 9.Produce said target mrna by transgenosis, it is compared with endogenous Δ 9 mRNA on expressing may have more difference.GBSS/ Δ 9 levels of proof active system (RPA 63) AA, EE, KK and JJ reduce the most obvious, compare with ribozyme (-) contrast transgenosis body as shown in figure 25 and reduce nearly 10 times.As described hereinly show that by RT-PCR these active systems express the fixed ribozymes to GBSS of targets.

RNA enzyme protection mensuration also shows with Δ 9 mRNA to be compared, and GBSS mRNA reduces.Portion C: the evidence that Δ 9 desaturase levels reduce in the transgenic plant of expressing the fixed ribozyme to Δ 9 desaturase mRNA of target

Transgenosis body RPA85-06 that stearic acid content is high and RPA85-15 contain the complete copy that merges many bodies of ribozyme gene.In each is, by the existence of ribozyme rna in the RT-PCR screening plant.Use the scheme of describing among the embodiment 27.In blade, contain the expression that has proved the RPA85 ribozyme in the plant RPA85-06 of high stearic acid and the RPA85-15 system.6 high stearic acid plants in to each being and non-conversion (NT) and contrast (TC) plant that transforms carry out Northern and analyze.The probe that is used for this analysis is cDNA fragment and the maize actin cDNA of corn Δ 9 desaturase cDNA.In order to distinguish Δ 9 mRNA levels because the error that the RNA with real ribozyme mediation that last sample or wrong-way cause reduces, with the comparison of the Actin muscle mRNA level in Δ 9 mRNA levels and this sample.Use Densitometer Readings to calculate the ratio of each sample as mentioned above.The Δ 9/ Actin muscle ratio that calculates the 85-06 plant is between 0.55-0.88.The mean value of contrast Δ 9/ Actin muscle of non-conversion is 2.7.The moving egg ratio of Δ 9/ flesh in the blade of 85-06 and NT has obviously reduced 4 times.Compare the Δ 9/ Actin muscle ratio between 85-06 high stearic acid plant and the TC plant, 3 times of the value decreased average of Δ 9/ Actin muscle of discovery 85-06 plant.These data form with chart in Figure 26 shows.Calculated the Δ 9/ Actin muscle ratio of RPA85-15 high stearic acid transfer-gen plant, its scope is between 0.35 to 0.53, and mean value is 0.43.In this test, the mean value of Δ 9/ Actin muscle of NT plant is 1.7.The mean value of Δ 9/ Actin muscle of NT contrast and 85-15 high stearic acid plant relatively, the minimizing of Δ 9 mRNA of discovery 85-15 3.9 times.Relatively the Δ 9/ Actin muscle value of 85-15 high stearic acid plant and normal stearic acid (TC) plant is found obviously to have reduced 3 times in the Δ 9 mRNA levels of RPA85-15 high stearic acid transfer-gen plant.These data form with chart in Figure 27 shows.These data show is expressing the RPA85 ribozyme and the minimizing of the ribozyme mediation of Δ 9 desaturase mRNA in the stearic transfer-gen plant of generation higher level in blade.Embodiment 29: there is the negative evidence of regulating of Δ 9 desaturases in the activity owing to active ribozyme in the maize leaf

Δ 9 desaturase ribozyme gene plants transformed have been produced with inactive form.Be given in the data of stearic control level in non-activity Δ 9 ribozyme transgenic lines RPA113-06 and the 113-17 blade.RPA113-06 system ribozyme expression and Northern analysis have been carried out.Measured RPA113-17 and be the Δ 9 desaturase protein levels in the plant.Measured the expression of ribozyme as the description of this paper.But plant 113-06-04,113-06-07 and 113-06-10 have expressed RPA113 non-activity Δ 9 ribozymes of detection level.5 plant to RPA113-06 system have carried out the Northern analysis, and the stearic acid in its blade is all in the scope of contrast, all between 1.8-3.9%.As shown in figure 28, compare, in the RPA113-06 plant, do not have to find to express minimizing or the stearic increase of blade of relevant Δ 9 desaturase mRNA with ribozyme with contrast.Protein analysis shows any minimizing of Δ 9 desaturase protein levels not relevant with the blade stearic acid that improves in the RPA113-17 plant.These data form with chart in Figure 29 (a) shows.In a word, the data of two RPA113 non-activity transgenic lines show ribozyme activity and observed high stearic acid phenotypic correlation in RPA85 system.The RPA85 ribozyme is the activity form of RPA113 ribozyme.Embodiment 30: the proof part A that stearyl-ACP Δ 9 desaturase levels reduce in the maize leaf (R0) of ribozyme mediation: the partial purification of stearyl in the maize leaf-ACP Δ 9 desaturases

Unless specified otherwise, all processes are all carried out under 4 ℃.Collect maize leaf (50mg), and with mortar and pestle with it at liquid N ₂In be ground into fine powder.In isopyknic buffer A of forming by 25mM sodium phosphate (pH6.5), 1mM disodium salt, 1mM dithiothreitol (DTT), 10mM phenylmethylsulfonyl fluoride, 5mM leupeptin and 5mM antipapin, extract protein.10,000xg is down with centrifugal 5 minutes of rough homogenate.By Bio-Rad protein analysis test kit (Bio-Rad laboratory, Hercules, CA) total protein concentration in the mensuration supernatant liquor.The total protein of 100 μ g is joined in the buffer A that final volume is 500 μ l, and (Pharmacia biotech company, Piscataway NJ), and pass through simple vortex resuspension to join 50 μ l blended SP-sepharose 4Bs again.Make protein and sepharose 4B in conjunction with 10 minutes on ice.In conjunction with after, centrifugal (10, in 000xg) 10 seconds, decant with buffer A (500 μ l) washing three times, and washs once with 200mM sodium-chlor (500 μ l) with Δ 9 desaturases-sepharose 4B material.Came elute protein in 5 minutes by handling at 50 μ l to boil in the damping fluid (125mM Tris-HCl pH6.8,4% sodium lauryl sulphate, 20% glycerine, 10%2-mercaptoethanol).Sample centrifugal (10,000x g) 5 minutes.Preserve supernatant liquor and analyze, and discard the precipitation that contains sepharose 4B to be used for Western.Part B: use the minimizing of the just bright stearyl of Western analytical procedure-ACP Δ 9 desaturases

As Laemmli, U.K. (1970), during assembling phage T4 head to the cutting of structural protein, nature 227,660-685 is described in the protein that sodium lauryl sulphate (SDS)-polyacrylamide gel (10%PAGE) is gone up the separate part purifying.In order to distinguish the error of Δ 9 desaturase levels, on each trace, add Δ 9 desaturases of purified and quantitative intestinal bacteria overexpression as a reference.(Pharmacia biotech company, Piscataway NJ) use Towbin damping fluid (Towbin etc., 1979) that protein transduction is moved on to ECL to utilize Pharmacia partial desiccation trace paper by electrophoresis ^TMNitrocellulose filter (the Amerham life science, ArlingtonHeights, Illinois) on.In phosphoric acid buffer, nonspecific binding site was sealed 1 hour with 10% dried milk.Use the rabbit anti-serum of corn Δ 9 desaturases of Chinese People's Anti-Japanese Military and Political College's enterobacteria expression, utilize ECL ^TM(Amerham life science, Arlington Heights Illinois) detect the immunoreactivity polypeptide to Western trace detection reagent.Standard scheme according to Berkeley antibody company produces antibody.Second antibody is the goat-anti rabbit anteserum that is connected on the horseradish peroxidase (BioRad).With densometer scanned autoradiogram(ARGM) and with the relative quantity of intestinal bacteria Δ 9 desaturases of purifying serve as the basis quantitatively.Repeat the mean value of these tests and record reduction.Portion C: the proof that Δ 9 desaturase levels reduce in the R0 maize leaf of the fixed ribozyme to Δ 9 desaturase mRNA of expression target

High stearic acid transgenic lines RPA85-15 contains the complete copy that merges many bodies gene.Δ 9 desaturases in the R0 maize leaf that utilized scheme partial purification as herein described.As described in part B, active ribozyme plant (RPA85-15), non-activity ribozyme plant RPA113-17 and non-transformed plant (HiII) are carried out Western analyze.By the Western analyzing and testing natural variation (referring to Figure 29 A) of Δ 9 desaturases of non-conversion system (HiII).At the non-activity ribozyme is to find among the RPA113-17 that Δ 9 desaturases do not reduce, more than all plant all in non-conversion system (HiII) scope.Compare with contrast, find that 6 plant Δ 9 desaturases of RPA85-15 system reduce by 50% (referring to Figure 29 B) significantly.The stearic acid of these six plant things has also increased simultaneously, and Δ 9 desaturase mRNA have reduced (as described in embodiment 28 and 32).Yet, to compare with non-activity ribozyme system (RPA113-17) with non-conversion system (HiII), Δ 9 desaturases of 9 active ribozyme plant of RPA85-15 system do not find obviously to reduce (Figure 29 A and B).In a word, these results show that Δ 9 desaturases of the ribozyme activity of 6 plant of RPA85-15 system and minimizing are relevant.Embodiment 31: the escherichia coli expression of corn Δ 9 desaturases and purification part A: the mature protein coding region of corn Δ 9 desaturase cDNA be inserted into bacterium T7 expression vector pET9D (Novagen company, Madison, WI) in.

The mature protein coding region is inferred from ripe Semen Ricini peptide sequence.With 32 L-Ala (nt239-241 of cDNA) as first residue.This finds in sequence A la.Val.Ala.Ser.Met.Thr..By PCR restriction enzyme Nhe I site is incorporated in the sequence of corn through engineered method, the result becomes GCTAGC with GCCTCC, and the BamHI site is joined 3 ' end.This does not change proteinic aminoacid sequence.Use Nhe I and BamHI site with the cDNA sequence clone in the pET9d carrier.With plasmid recombinant called after pDAB428.Corn Δ 9 desaturase proteins of expressing in bacterium have other methionine residues at 5 ' end.With the pDAB428 plasmid be transformed into bacterial isolates BL21 (Novagen company, Madison, WI) in and be seeded on LB/ kantlex (25mg/ml) culture dish.Bacterium colony is resuspended among the 10ml LB of have kantlex (25mg/ml) and IPTG (1mM), and 37 ℃ of following shaking culture 3 hours.By under 4 ℃ at centrifugal 10 minutes harvested cells of 1000xg.By freezing and thawing cell precipitation 2X dissolved cell, then add the dissolving damping fluid (10 mM Tris-HCl pH, 8.0,1 mM EDTA, 150 mM NaCl, 0.1%Triton X100,100 μ g/ml DNA enzyme I, 100 μ g/ml RNA enzyme A and 1 mg/ml N,O-Diacetylmuramidase) of 1ml.Under 37 ℃, mixture was cultivated 15 minutes, then 4 ℃ under 1000x g centrifugal 10 minutes.Supernatant liquor partly uses as soluble protein.

Supernatant liquor is adjusted to 25mM sodium phosphate buffer (pH6.0) and cooled on ice 1 hour.Afterwards by the centrifugal flocculation sediment of removing generation.When solution keeps clear, repeat ice bath step twice again.After this solution still keeps clarification.Clear soln is loaded into on 25mM sodium phosphate buffer (pH6.0) the equilibrated Mono S HR10/10 post (Pharmacia).Use 0-500mM NaCl gradient elution to be attached to the basic protein 1 hour (2ml/ minute of base for post matter; With 2ml is a component).The interest albumen of inferring carry out SDS-PAGE, inhale print on the pvdf membrane, Coomassie blue colour developing, excision, and deliver to Harvard Microchem and carry out the N-terminal sequential analysis.The protein of protein amino end sequence and cDNA clones coding compared show that this protein is real Δ 9.Two iron oxygen component (Fox etc. with the expressed proteins qualitative correlation, 1993 Proc. Natl. Acad. Sci.USAs 90, spectrophotometric analysis 2486-2490) and use specific nonheme iron staining agent to identify (Leong etc., 1992 biological chemistry yearbooks 207,317-320) protein of all confirming this purifying is Δ 9.Part B: the generation of polyclonal antiserum

Δ 9 protein that the intestinal bacteria of identifying by N-terminal order-checking produce are through the SDS-PAGE gel-purified, excision, and be placed into gel matrix (Berkeley antibody company, Richmond, CA) in so that in the rabbit body, produce polyclonal serum.Analyzing use ECL detection system (Amersham company) by Western carries out measuring at the antibody titer of Δ 9.Portion C: the purifying of corn grain Δ 9 desaturases

Protein precipitation: use Warring agitator Δ 9 of purifying corn grain after the homogenate in containing the 25mM sodium phosphate buffer (pH7.0) of 25mM sodium bisulfite and 2.5% polyvinylpyrrolidone.By the rough homogenate of filtered through gauze, centrifugal (10,000x g) 0.25 hour and use the filtered through gauze supernatant liquor again.Sometimes, through saturated ammonium sulphate (first 20%v/v is the 80%v/v precipitation then) fractional separation supernatant liquor.By add 50% polyglycol solution (mw=8000) to final concentration be 5 and 25%v/v come fractional separation by the extract that obtains in the oily germplasm of height.In all cases, go out Δ 9 albumen, at 25mM sodium phosphate buffer (pH 6.0) precipitation of gained is fully dialysed then with 80% ammonium sulfate or 25% polyethylene glycol precipitation.

Cation-exchange chromatography:, and join in 25mM sodium phosphate buffer (pH 6.0) on the equilibrated Mono S HR10/10 post by the above-mentioned sedimentable matter of dissolved of centrifugal clarification.Behind the thorough washing post, be combined in basic protein 1 hour (2ml/ minute: with 2 ml was a component) on the base for post matter with 0-500 mM NaCl gradient elution.In general, Δ 9 albumen that wash-out between 260 and 350 mM NaCl goes out by enzyme and Western assay determination.After the dialysis, by acyl carrier protein (ACP)-agarose and phenyl superose chromatography with the further fractional separation of this material.

Acyl carrier protein-agarose chromatography: ACP buys from sigma chemical company, and before being connected on the globule in pH 4.1 times through deposition and purification (Rock and Cronan.1981, journal of biological chemistry, 254,7116-7122).In the packing inset, the ACP-agarose prepares by 100 mg ACP are covalently bound on the cyanogen bromide activated sepharose 4B globule described in the specification sheets of Inc. basically as Pharmacia.After connecting and blockading the residue site with glycine, with ACP-agarose material load into the HR5/5 post (Pharmacia, Inc.) in, and in 25mM sodium phosphate buffer (pH 7.0) balance.Dialyzed component with above discriminating installs to (McKeon and Stumpf, 1982 journal of biological chemistry 257,12141-12147 on the post then; Thompson etc., 1991 Proc. Natl. Acad. Sci.USAs 88,2578-2582).Behind the thorough washing post, adopt 1MNaCl wash-out ACP conjugated protein.Enzyme analysis and western analyze and the order-checking of ensuing N-terminal shows that effluent liquid contains Δ 9 protein.Analyze the molecular weight (Hames, 1981 proteinic gel electrophoresises: practical approach, editor Hames BD and Rickwood, D., IRL press, Oxford) of determining to have about 38 kDa through SDS-PAGE from Δ 9 protein of corn purifying.

The phenyl sepharose chromatography: the component that will contain the Δ 9 that obtains from the ACP-agarose column is adjusted to 0.4 M ammonium sulfate (25mM sodium phosphate, pH 7.0), and is added on the Phamacia phenyl sepharose post (HR10/10).Protein is through 1 hour gradient (0.4-0.0 M ammonium sulfate) wash-out of 2 ml/min.During by enzyme analysis and western assay determination, Δ 9 protein are wash-out between 60-30 mM ammonium sulfate typically.Embodiment 32: as the evidence of stearic acid increase in the blade as a result that transforms plant with Δ 9 desaturase ribozymes

Part A: the method that is used for measuring plant tissue stearic acid level.From the method for plant tissue extracting and esterified fatty acid from a kind of method improvement of describing (Browse etc., 1986, analytical biochemistry, 152,141-145).1 to 20 milligram plant tissue is placed in the Pyrex 13mm screw plug test tube.Add 1 milliliter of HCl methanol solution (Supelco, Bellefonte) after, blow test tube and sealing with nitrogen.Test tube was descended heating 1 hour and made its cooling at 80 ℃.Heating in the presence of the HCl methanol solution causes the extracting and the esterification of lipid acid.By with the hexane extracting from reaction mixture weeding of grease fatty acid methyl esters.Add 1 milliliter of hexane and 0.9% (W/V) NaCl, then the thermal agitation test tube.After 5 minutes, shift out the top hexane layer at centrifuge tube under 2000 rpm, be used for the fatty acid methyl ester analysis.By 1 microlitre sample is expelled to HewlettPackard (Wilmington, DE) II analyzes in the 5890 type gas chromatographs, this chromatograph is equipped with flame ionization detector and J﹠amp; W Scientific (Folsom, CA) DB-23 post.Furnace temperature is 150 ℃ in operating process, and carrier gas (helium) flow velocity is 80cm/ second, and the operating time is 20 minutes.This condition makes can separate 5 kinds of interest fatty acid methyl ester: C16:0, Uniphat A60; C18:0, methyl stearate; C18:1, Witconol 2301; C18:2, methyl linoleate; And C18:3, Linolenic acid methylester.With Hewlett Packard II is that (CT) data gathering system is carried out the Collection and analysis of data for Perkin Elmer, Norwalk for 3396 type totalizing instruments and PENelson.The per-cent of each lipid acid is directly obtained from the reading of data gathering system in the sample.The quantity of each lipid acid standard substance (Matreya, Pleasant Gap, calculated by peak area PA), said standard substance is made up of five kinds of known fatty acid methyl esters of known quantity.The amount of being calculated is used for estimating in sample the per-cent by total fresh weight of five kinds of lipid acid representatives.During extracting and esterification process, do not adjust the lipid acid of forfeiture.After carrying out extracting and esterification process (tissue does not exist), the rate of recovery of standard model depends on sample primary amount in 90% to 100% scope.The existence of plant tissue in the extracting mixture to the same receipts of the standard substance of known quantity without any influence.

Part B: in blade, increase stearic proof owing to introduce Δ 9 desaturase ribozymes.Stearic acid as test bion leaf tissue as described in the part A.428 kind of plant of 35 systems that transform with active Δ 9 desaturase ribozymes (RPA85, RPA114, RPA118) have been tested and with 406 kind of plant of 31 systems of the nonactive ribozyme of Δ 9 desaturases (RPA113, RPA115, RPA 119) conversion.Table X I has summed up the result of the stearic acid level in these plants that obtain.The plant of 7% activity system has and is higher than 3% hard fatty acids level, and 2% has and is higher than 5% level.Only there is the plant of 3% nonactive system to have and is higher than 3% hard fatty acids level.The blade of 2% control plant has and is higher than 3% hard fatty acids level.Contrast comprise 49 kinds of non-plant transformed and 73 kinds with the plant of the incoherent gene transformation of Δ 9 desaturases.Not having nonactive is that plant and control plant have and be higher than 4% stearic acid.Two with active Δ 9 desaturase ribozyme RPA85 conversion is to produce many plants, and the stearic acid that they demonstrate blade increases.RPA85-06 is that 6 in 15 plants record the stearic acid level that has between 3 and 4%, is 2 times (Figure 30) of contrast mean value approximately.The average hard fatty acids content of control plant (122 kind of plant) is 1.69% (SD+/-0.49%).The average stearic acid content of RPA85-06 system is 2.86% (+/-0.57%).RPA85-15 is that 6 in 15 plants record to have and are higher than contrast mean level (ML) about 4 times stearic acid level (Figure 31).RPA85-15 is that average blade hard fatty acids content is 3.83% (+/-2.53).When repeating that the RPA85-15 plant carried out foliar analysis, proved in the past that the stearic acid level in the plant leaf with normal stearic acid level still kept normally, the plant leaf with high stearic acid is found to have high stearic acid content (Figure 31) once more.In Figure 32 and 33, shown the stearic acid level in the plant leaf of two systems that transform with inactive Δ 9 desaturase ribozyme RPA113.It is 3% or 3 higher kind of plant that RPA 113-06 has stearic acid content.The average stearic acid content level of the blade of RPA 113-06 system is 2.26% (+/-0.65).RPA113-17 does not have the blade stearic acid content greater than 3% plant.The average blade stearic acid content of RPA 113-17 system is 1.76% (+/-0.29%).Figure 34 has shown the stearic acid content of the blade of 15 kinds of control plants.The average hard fatty acids content of these 15 kinds of control plants is 1.70% (+/-0.6%).When comparing with contrast and inactive Δ 9 desaturase ribozyme data, the presentation of results of the hard fatty acids content that obtains in RPA85-06 and RPA85-15 has increased stearic acid content owing to introduce Δ 9 desaturase ribozymes.Embodiment 33: the heredity of high stearic acid proterties in blade

Part A: the result of high stearic acid plant offspring's stearic acid level.Describe as this paper, making RPA85-15 is plant pollination.In pollination back 20 days, downcut zygotic embryo from the prematurity grain of these RPA85-15 plants, and place on the pipe substratum growth for regrowth as the description of this paper.After plant is transferred to the greenhouse, leaf tissue is carried out fatty acid analysis.Figure 35 has shown the stearic acid level of 10 kinds of different plant leafs a kind of hybridization, autogamous RPA85-15.07.50% plant has high blade stearic acid, 50% have a normal blade stearic acid.Table X II has shown the result of the RPA85-15 plant of 5 kinds of different hybridization.The quantity of plant with high stearic acid is in 20 to 50% scope.

Part B: the result that Δ 9 desaturase levels reduce in expressing at the next generation (R1) maize leaf of the ribozyme of Δ 9 desaturase mRNA is described.In the maize plant of future generation that shows high stearic acid content (seeing above part A), adopt scheme described herein from R1 maize leaf partial purification Δ 9 desaturases.Several high stearic acid plants are carried out Western to be analyzed.In the blade of plant of future generation, in plant, observe Δ 9 desaturases and reduce 40-50% (Figure 36) with high stearic acid content.This reduces with the R0 maize leaf similar.This is reduced in the RPA85-15 plant with the QQ414 plant of RPA85-15 pollen hybridization or selfing or inbreeding and observes.Therefore, the gene of the described ribozyme of this explanation coding is heritable.Embodiment 34: adopt antisense Δ 9 desaturases to increase stearic acid in plant tissue

Part A: the method that is used to cultivate corn body embryo.This paper has described the generation and the regeneration of maize generation callus.Most of this class embryo generation callus of body embryogeny.The body embryo forms in callus continuously, because per two weeks of callus shift once.The body embryo is bred the commitment that still remains on fetal development usually continuously in embryo generation callus, this is owing to contain 2 in substratum, 4-D.Somatic embryo regeneration is a seedling, because callus experiences regenerative process described herein.The body embryo forms root and bud at regeneration period, and goes up embryonic development eventually.Handle by defined medium, make the body embryo, that is, surmount the early development stage and the no longer regeneration that see embryo's generation callus as the seed embryonic development.This substratum is handled to comprise embryo's generation callus is transferred to Murashige and Skoog substratum (MS; Murashige and Skoog described in 1962) in, this substratum contains 6% (W/V) sucrose and does not contain plant hormone.Callus was cultivated 7 days having on the MS substratum of 6% sucrose, the body embryo is individually transferred in the MS substratum with 6% sucrose and 10 μ M dormins (ABA) then.After cultivating 3 to 7 days on the ABA substratum, measure the lipid acid of body embryo as the description of this paper and form.The lipid acid ratio of components of the corn zygotic embryo that the lipid acid composition of the body embryo that will cultivate on above-mentioned substratum and embryo generation callus and pollination are back 12 days is (Table X III).The lipid acid of body embryo is formed the lipid acid that is different from embryo generation callus and is formed.Embryo generation callus has the C16:0 and the C18:3 of higher percent, and the C18:1 and the C18:2 of low per-cent.When comparing with the body embryo, embryo generation callus is by the lipid per-cent difference of fresh weight representative; 0.4% pair 4.0%.The lipid acid of zygotic embryo and body embryo is formed closely similar, they by the lipid per-cent of fresh weight representative much at one.The conclusion that obtains is, body embryo culture described above system is used for testing the useful vitro system that some gene pairs corn is grown the synthetic influence of embryo lipid.

Part B: as result's stearic increase in corn body embryo of introducing antisense Δ 9 delta 8 desaturase genes.Adopt method described herein to produce the body embryo from the embryo generation callus that transforms with pDAB308/pDAB430.Measuring the lipid acid of 16 not homologous body embryos forms.Find that two is that 308/430-12 and 308/430-15 generation have high-caliber stearic body embryo.It is that the stearic acid of body embryo contains the stearic acid content with control series body embryo that Figure 37 and 38 has compared these two.Except they were transformed, control series was from being identical to be with what transform that system originated, and for 308/430-12 system, the stearic acid scope in the body embryo is 1 to 23%, and the scope of contrast is 0.5 to 3%.For 308/430-15 system, the stearic acid scope in the body embryo is 2 to 15%, and the scope of contrast is 0.5 to 3%.Transform in the system at two kinds, the body embryo more than 50% has the stearic acid level that is higher than illumination range.These presentation of results antisense Δ 9 delta 8 desaturase genes can be used for improving the stearic acid level in corn body embryo.

Portion C: in blade, increase stearic proof owing to introduce antisense Δ 9 delta 8 desaturase genes.The embryogenesis culture of 308/430-12 and 308/430-15 system is used for aftergrowth.The lipid acid of these plant leafs of methods analyst of describing before adopting is formed.Only obtain 4 plants from the 308/430-15 culture, the stearic acid level of the blade of these plants is normal, is 1-2%.308/430-12 is that the stearic acid level in the blade of plant is presented among Figure 39.At 308/430-12 is in the plant, and the stearic acid level in the blade is in 1 to 13% scope.308/430-12 is that about 30% plant has the stearic acid level that surpasses viewed scope (1-2%) in contrast.These results show by introducing antisense Δ 9 delta 8 desaturase genes can improve stearic acid level in the maize leaf.

＂ antisense ＂ means by RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm etc., 1993, nature, 365,566) interacting is attached to the non-enzymatic nucleic acid molecule that RNA (target RNA) went up and changed target RNA activity (summary is seen Stein and Cheng, 1993, science, 261,1004).Embodiment 35: corn compiles the mensuration of starch sample and single grain amylose content

Method (potato research 31:241-246) with the Hovenkamp-Hermelink that revises etc. is measured amylose content.For compiling starch sample, with 10mg to the 100mg starch dissolution in 45% perchloric acid of the 5ml in the plastic culture pipe.Eddy current mixes this solution frequently.After 1 hour, water is diluted to 10 milliliters with 0.2 milliliter of starch solution.The solution of 0.4 milliliter of dilution is mixed in 1 milliliter of Xiao Chi with Lugol ' the s solution (Sigma) of 0.5 milliliter of dilution.Obtain reading immediately, and calculate R ratio (618 nm/550 nm) at 618nm and 550nm.Employing is from potato amylose starch and corn amylopectin (Sigma, St.Louis) the equation P of the standard of Chan Shenging (per-cent of amylose starch)=(4.5R-2.6)/(7.3-3R), the content of mensuration amylose starch.For freezing single grain sample, except extracting in 45% perchloric acid 20 minutes rather than 1 hour, adopt above identical method.Embodiment 36: fecula purifying and particle are measured in conjunction with starch synthase (GBSS)

The method of the purifying of starch and ensuing GBSS determination of activity is revised from the method for (plant physiology, 62:383-386,1978) such as Shure etc. (cell, 35:225-233,1983) and Nelson.At (v/w) of 2 times of volumes 50mM Tris-HCl, pH 8.0 with corn grain, homogenate in the 10mM ethylenediamine tetraacetic acid (EDTA), and filter through 120 μ m nylon membranes.Then with this material under 5000g centrifugal 2 minutes.Abandoning supernatant.By being resuspended in the water and centrifugally removing the supernatant liquor washing precipitation 3 times.After washing, starch filters by 20 μ m nylon membranes and is centrifugal.Lyophilize precipitates and is stored under-20 ℃ up to being used for determination of activity then.

The GBSS reaction mixture of standard is included in 0.2 M Tricine among the cumulative volume 200 μ l, pH8.5,25 mM gsh, 5mM ethylenediamine tetraacetic acid (EDTA), 1mM ¹⁴C ADPG (6nci/ μ mol) and 10 mg starch.Be reflected at and carried out under 37 ℃ 5 minutes, and by adding 70% ethanol (V/V) termination reaction among the 200 μ l 0.1M KCl.Centrifugal this material, and remove uncorporated ADPG in supernatant liquor.Use 1 ml water washing precipitation 4 times in an identical manner.After washing, precipitation is suspended in 500 microliters of water, and places scintillation vial, (Fullerton, CA) scintillometer is counted the ADPG that is mixed through Beckman.The picomole number that is incorporated into the ADPG in every milligram of starch with per minute provides specific activity.Embodiment 37: the analysis of antisense GBSS plant

Because the separation of R2 seed, the amylose content that should analyze single grain is to differentiate phenotype.Owing in this research, produce a large amount of samples, therefore use two step screening strategies.In first step, obtain 30 grain at random from identical fringe, lyophilize also all changes into the starch face.This starch face is carried out amylose starch to be measured.What analyze to differentiate amylose content with reduction by statistics is.In second step, further analyze the amylose content with reduction be in the amylose content (25 to 50 grain of every fringe) of single grain.Two groups of contrasts are used for screening, and one group is the unconverted system with identical genetic background, and another group is that not carried genetically modified conversion because of separation is (Southern analyze negative system).

Measure 81 systems that represent 16 transformation events compiling on the starch level.In these are, identify 6 systems that analyze amylose content by statistics and supply further single grain analysis with obvious reduction.It is 308/425-12.2.1 that a system is arranged, and demonstrates the amylose content (Figure 40) of obvious reduction.

Analyze 25 kinds of single grain of CQ806 (a kind of corn inbred lines of routine).The amylose content of CQ806 in 24.4% to 32.2% scope, average out to 29.1%.The single grain of amylose content distributes a little to tilting than low amylose content.49 single grain of 308/425-12.2.1.1 have been analyzed.If 308/425-12.2.1.1 is from oneself's pollination of hemizygote individuality, then Yu Qi distribution will be made up of 4 visibly different hereditary classifications that exist with the frequency that equates, because endosperm is the triploid tissue.These 4 hereditary classifications are made up of the individuality of the antisense constructs of carrying 0,1,2 and 3 copy.If there is the effect of genetically modified heavy dose, then the distribution of amylose content will be four kinds of forms.A kind of form of the distribution that forms is should right and wrong transgenosis parent indistinguishable.If there is not transgenosis dosage effect (the genetically modified individuality that carries 1,2 or 3 copy is to be equal on the phenotype), then distributing be two kinds of forms, and one of them is identical with the parent.The quantity that is included in the individuality in these forms should be transgenosis: parent 3: 1.308/425-12.2.1.1 distribution obviously be three kinds of forms.The center form approximately is the twice of arbitrary other form.Two kinds of distally form sizes approximately equate.Checked the goodness of fit to 1: 2: 1 ratio, match is good.

Illustrate that the form further evidence identical with the non-transgenic parent with high amylose content is obtainable.This adopts discriminant analysis to carry out.CQ806 and 308/425-12.2.1.1 data set combined carry out this analysis.The distance measure that is used to analyze only adopts amylose content to calculate.The estimation variance that in all checks, all adopts ontoanalysis and drawn.Do not adopt and compile variance.The check raw data is for classification again.According to discriminant analysis, have the whole form that the 308/425-12.2.1.1 of high amylose content distributes and more suitably to be categorized as the parent.This has confirmed that effectively this distribution form belongs to the parent.In two kinds of remaining forms, the center form is the twice of the size of minimum amylose content form approximately.If the center form comprises two kinds of hereditary forms (individuality with antisense constructs of 1 or 2 copy), then it is desired.Therefore, the form with minimum amylose content is represented fully isozygoty those individualities of (3 copies) of antisense constructs.Check 2: 1 ratios, can not get rid of this ratio according to data.

The antisense gbss gene that this analysis revealed works in 308/425-12.2.1.1 makes corn grain amylose content reach the dose-dependently reduction.Embodiment 38: the analysis of ribozyme GBSS plant

Be used for the two step screening strategies identical and analyze ribozyme GBSS plant with antisense research (embodiment 37).Measure 160 systems that represent 11 transformation events compiling on the starch level.In control series (unconverted system and Southern feminine gender are both), amylose content changes in 28% to 19%.Be not observe any tangible reduction in (the positive system of Southern) in all of carrying ribozyme gene.The selection of further analysis more than 20 is on single grain level, does not find that tangible amylose starch reduces and clastotype.Clearly, ribozyme does not cause any change of phenotypic level.

Further measure the GBSS activity (as the description of embodiment 36) that transforms system.For respectively being, obtain 30 grain from the refrigerated fringe, and purifying starch.Table X IV is presented at the result who compiles 9 plants representing the active a kind of transformation event of GBSS in the starch sample, the result that the result of amylose content and Southern analyze in compiling starch sample.With three kinds of Southern feminine genders is that RPA63.0283, RPA63.0236 and RPA63.0219 are with comparing.

The GBSS activity of control series RPA63.0283, RPA63.0236 and RPA63.0219 is approximately 300 units/mg starch.In RPA63.0211, RPA63.0218, RPA63.0209 and RPA63.0210 system, observe the active reduction of GBSS and surpass 30%.The GBSS activity that the active dependency explanation of analyzing with Southern of the GBSS that changes in this group (Table X IV from RPA63.0314 to RPA63.0210) reduces is caused by the expression that is incorporated into the ribozyme gene in the corn gene group.

Adopt RPA63.0306 (Southern feminine gender in compiling starch, GBSS is active normal) in contrast, further measure the GBSS activity on the single grain level of RPA63.0218 system (the Southern positive in compiling starch, GBSS is active to be reduced).Obtain about 30 grain from every system, difference purifying starch sample from each grain.Figure 41 clearlys show that the GBSS activity that compares in the RPA63.0218 system with RPA63.0306 reduces.

Other embodiments within the scope of the appended claims.The feature I group intron of the naturally occurring ribozyme of table 1 ^*Size :～150 arrive greater than 1000 Nucleotide. ^*Needing has a U near cleavage site 5 ' in the target sequence. ^*In cleavage site 5 '-side in conjunction with 4-6 Nucleotide. ^*Reaction mechanism: by 3 ' of guanosine-OH attack, generation has the cleaved products of 3 '-OH and 5 '-guanosine. ^*In some cases, need additional protein cofactor with help folding and keep active structure [ ¹]. ^*300 known members of surpassing are arranged in this class.In Tetrahymena thermophilarRNA, fungi plastosome, chloroplast(id), phage T4, blue-green algae etc., be found as a kind of intervening sequence. ^*Main constitutional features to a great extent by kind of a system take place a comparison, mutagenesis and biochemical research [ ^2,3] set up. ^*To a kind of ribozyme set up completely dynamical frame [ ^4,5,6,7]. ^*Ribozyme is folding to carry out with substrate stop research [ ^8,9,10]. ^*The chemically modified research foundation fully of important residue [ ^11,12]. ^*Little (4-6nt) binding site can be so that this ribozyme be poor excessively to target RNA cleavage specificity.Yet, Tetrahymena I group intron be used for repairing " defective " beta galactosidase enzyme information (by new beta-galactosidase enzymes sequence is connected to defect information) [ ¹³].RNA enzyme P RNA (M1 RNA) ^*Size :～290 to 400 Nucleotide. ^*A kind of RNA part of omnipresence ribonucleoprotein enzyme. ^*Cutting tRNA precursor forms sophisticated tRNA[ ¹⁴]. ^*Reaction mechanism: may be by M ²⁺-OH attack, generation has the cleaved products of 3 '-OH and 5 '-phosphoric acid. ^*All find RNA enzyme P in all prokaryotic organism and the eukaryote.RNA subunit is from bacterium, yeast, rodent and primate order-checking. ^*By external guide sequence (EGS) is hybridized on the target RNA, the therepic use of endogenous RNA enzyme P be possible [ ^15,16]. ^*Important phosphoric acid and 2 '-OH contact recently and be determined [ ^17,18].II organizes intron ^*Size: greater than 1000 Nucleotide. ^*The trans cutting of target RNA illustrated recently [ ^19,20]. ^*Sequence requires not definite fully. ^*Reaction mechanism: 2 ' of inner adenosine-OH produces has the cleaved products of 3 '-OH and ＂ lasso trick ＂ RNA (containing 3 '-5 ' and 2 '-5 ' branch point). ^*Only have natural nuclear enzyme be proved to be except that RNA cutting with is connected participation DNA cut [ ^21,22]. ^*Main constitutional features to a great extent by kind of a system take place relatively to set up [ ²³]. ^*Important 2 ' OH contact beginning quilt discriminating [ ²⁴]. ^*The kinetics framework in foundation [ ²⁵].Neurospora VS RNA ^*Size :～144 Nucleotide. ^*The trans cutting of hair clip target RNA illustrated recently [ ²⁶]. ^*Sequence requires not definite fully. ^*Reaction mechanism: by 5 ' of the scissile key of 2 '-OH attack, generation has the cleaved products of 2 ', 3 '-ring-type phosphoric acid and 5 '-OH end. ^*Binding site and structural requirement are not definite fully. ^*This group is 1 known member only.In neurospora VS RNA, find.Hammerhead ribozyme (reference is seen text) ^*Size :～13 to 40 Nucleotide. ^*Requirement is having target sequence UH near cleavage site 5 '. ^*In the both sides of cleavage site in conjunction with the Nucleotide of variable number. ^*Reaction mechanism: by 5 ' of the scissile key of 2 '-OH attack, generation has the cleaved products of 2 ', 3 '-ring-type phosphoric acid and 5 '-OH end. ^*Known 14 members of this group.Some with RNA as the phytopathogen (virusoid) of infectosome in the discovery. ^*Important structural performance major part is definite, comprises 2 kinds of crystalline structure []. ^*Illustrated minimum connection active (so that processing) [] by external selection. ^*Two or more ribozymes have been set up kinetics framework [] completely. ^*[] set up in chemically modified research to important residue fully.The hair clip ribozyme ^*Size :～50 Nucleotide. ^*Require to have target sequence GUC near cleavage site 3 '. ^*In 5 ' side of cleavage site in conjunction with 4-6 Nucleotide, at 3 ' side of cleavage site Nucleotide in conjunction with variable number. ^*Reaction mechanism: by the scissile key of 2 '-OH attack, generation has the cleaved products of 2 ', 3 '-ring-type phosphoric acid and 5 '-OH end. ^*The known 3 kinds of members of this group.In the phytopathogen (satellite RNA of nepovirus, arabis mosaic virus and witloof yellow color and luster mottle virus) of some usefulness RNA, find as infectosome. ^*Important structure feature major part be defined [ ^27,28,29,30]. ^*Connect active (except nicking activity) make ribozyme be suitable for through external selection process [ ³¹]. ^*To a kind of ribozyme set up completely the kinetics framework [ ³²]. ^*Begun important residue chemically modified research [ ^33,34].Hepatitis Δ virus (HDV) ribozyme ^*Size :～60 Nucleotide. ^*Illustrated target RNA trans cutting [ ³⁵]. ^*Though cleavage site 5 ' is without any need for sequence, binding site and structural requirement are not definite fully.Folding ribozyme comprise the pseudokeratin structure [ ³⁶]. ^*Reaction mechanism: by 5 ' of the scissile key of 2 '-OH attack, generation has the cleaved products of 2 ', 3 '-ring-type phosphoric acid and 5 '-OH end. ^*This group is 2 kinds of known members only.In human HDV, find. ^*The loop type of HDV is active, and demonstrates the nuclease stability [37] of increase.1.???????Mohr.G.；Caprara.M.G.；Guo，Q.；Lambowitz.A.M.Nature，370，147-150(1994).2.???????Michel，Francois；Westhof，Eric.Slippery?substrates.Nat.Struct.Biol.(1994)，1(1)，5-7.3.???????Lisacek，Frederique；Diaz，Yolande；Michel，Francois.Automatic?identification?of?group?I?introncoresin?genomic?DNA?sequences.J.?Mol.Biol.(1994)，235(4).1206-17.4.???????Herschlag，Daniel；Cech.Thomas?R.，Catalysis?of?RNA?cleavage?by?the?Tetrahvmena?thermophilaribozyme.1.Kinetic?description?of?the?reaction?of?an?RNA?substrate?complementary?to?the?active?site.Biochemistry(1990)，29(44)，10159-71.5.???????Herschlag.Daniel；Cech.Thomas?R.，Catalysis?of?RNA?cleavage?by?the?Tetrahymena?thermophilaribozyme.2.Kinetic?description?of?the?reaction?of?an?RNA?substrate?that?forms?a?mismatch?at?the?active?site.Biochemistry(1990)，29(44)，10172-80.6.???????Knitt.Deborah?S.；Herschlag.Daniel.pH?Dependencies?of?the?Tetrahymena?Ribozyme?Reveal?anUnconventional?Origin?of?an?Apparent?pKa.Biochemistry(1996).?35(5).1560-70.7.???????Bevilacqua，Philip?C.；Sugimoto.Naoki；Turner，Douglas?H.，A?mechanistic?framework?for?the?secondstep?of?splicing?catalyzed?by?the?Tetrahymena?ribozyme.Biochemistry(1996)，35(2)，648-58.8.???????Li，Yi；Bevilacqua，Philip?C.；Mathews.David；Turner.Douglas?H.，Thermodynamic?and?activationparameters?for?binding?of?a?pyrene-labeled?substrate?by?the?Tetrahymena?ribozyme：docking?is?notdiffusion-controlled?and?is?driven?by?a?favorable?entropy?change.Biochemistry(1995)，34(44)，14394-9.9.???????Banerjee，Aloke?Raj；Tumer.Douglas?H.，The?time?dependence?of?chemical?modification?reveals?slowsteps?in?the?folding?of?a?group?I?ribozyme.?Biochemistry(1995)，34(19)，6504-12.10.??????Zarrinkar，Patrick?P.；Williamson.James?R.，The?P9.1-P9.2?peripheral?extension?helps?guide?folding?ofthe?Tetrahymena?ribozyme.?Nucleic?Acids?Res.(1996)，24(5)，854-8.11.??????StrobeL?Scott?A.；Cech.Thomas?R.，Minor?groove?recognition?of?the?conserved?G.cntdot.U?pair?at?theTetrahymena?ribozyme?reaction?site.?Science(Washington.D.C.)(1995)，267(5198)，675-9.12.Strobel.Scott?A.；Cech，Thomas?R.，Exocyclic?Amine?of?the?Conserved?G.cntdot.?U?Pair?at?theCleavage?Site?of?the?Tetrahymena?Ribozyme?Contributes?to?5′-Splice?Site?Selection?and?Transition?StateStabilization.Biochemistry(1996)，35(4)，1201-11.13.??????Sullenger，Bruce?A.；Cech，Thomas?C.Ribozyme-mediated?repair?of?defective?mRNA?by?targetedtrans-splicing.Nature(London)(1994).371(6498)，619-22.14.??????Robertson.H.D.；Altman，S.；Smith，J.D.J.Biol.Chem.，247，5243-5251(1972).15.??????Forster，?Anthony?C.；?Alrman.?Sidney.?External?guide?sequences?for?an?RNA?enzyme.?Science(Washington，D.C.，1883-)(1990)，249(4970)，783-6.16.??????Yuan，Y.；Hwang，E.S.；Altman.S.Targeted?cleavage?of?mRNA?by?human?RNase?P.Proc.Natl.Acad.Sci.USA(1992)89，8006-10.17.??????Harris，Michael?E.；Pace.Norman?R.，Identification?of?phosphates?involved?in?catalysis?by?theribozyme?RNase?P?RNA.RNA(1995)，1(2)，210-18.18.??????Pan，Tao；Loria.Andrew；Zhong.Kun.Probing?of?tertiary?interactions?in?RNA：2′-hydroxyl-basecontacts?between?the?RNase?P?RNA?and?pre-tRNA.Proc.Natl.Acad.Sci.U.S.A.(1995).92(26).12510-14.19.??????Pyle，Anna?Marie；Green.Justin?B..Building?a?Kinetic?Framework?for?Group?II?Intron?RibozymeActivity：Quantitation?of?Interdomain?Binding?and?Reaction?Rate.?Biochemistry(1994)，33(9)，2716-25.20.??????Michels.William?J.Jr.；Pyle，Anna?Marie.Conversion?of?a?Group?II?lntron?into?a?NewMultiple-Turnover?Ribozyme?that?Selectively?Cleaves?Oligonucleotides：Elucidation?of?Reaction?Mechanismand?Structure/Function?Relationships.Biochemistry(1995)，34(9)，2965-77.21.??????Zimmerly，Steven；Guo.Huarao；Eskes.Robert；Yang.Jian；Periman.Philip?S.；Lambowittz.Alan?M..A?group?II?intron?RNA?is?a?catalytic?component?of?a?DNA?endonuclease?involved?in?intron?mobiliry.Cell(Cambridge.?Mass.)(1995)，83(4)，529-38.22.??????Griffin.Edmund?A.，Jr.；Qin，Zhifeng；Michels.Williams?J.，Jr.；Pyle.Anna?Marie.Group?II?intronribozymes?that?cleave?DNA?and?RNA?linkages?with?similar?efficiency.and?lack?contacts?with?substrate2’-hydroxyl?groups.Chem.Biol.(1995)，2(11).761-70.23.??????Michel.Francois；Ferat.Jean?Luc.Structure?and?activities?of?group?II?introns.Annu.Rev.Biochem.(1995)，64，435-61.24.??????Abramovitz.Dana?L.；Friedman.Richard?A.；Pyle.Anna?Marie.?Catalytic?role?of?2’-hydroxyl?groupswithin?a?group?II?intron?active?site.Science(Washington，D.C.)(1996)，271(5254)，1410-13.25.??????Daniels.Danette?L.；Michels.William?J.，Jr.；Pyle.Anna?Marie.Two?competing?pathways?forself-splicing?by?group?II?introns；a?quantitative?analysis?of?in?vitro?reaction?rates?and?products.J.Mol.Biol.(1996)，256(1)，31-49.26.??????Guo.?Hans?C.T.；Collins，Richard?A.Efficient?trans-cleavage?of?a?stem-loop?RNA?substrate?by?aribozyme?derived?from?Neurospora?VS?RNA.EMBO?J.(1995)，14(2)，368-76.27.??????Hampel.Arnold；Tritz?Richard；Hicks.Margaret；Cruz.Phillip.′Hairpin′catalytic?RNA?model：evidence?for?helixes?and?sequence?requitement?for?substrate?RNA.Nucleic?Acids?Res.(1990)，18(2)，299-304.28.??????Chowrira，Bharat?M.；Berzal-Herranz，Alfredo；Burke，John?M.，Novel?guanosine?requirement?forcatalysis?by?the?hairpin?ribozyme.Nature(London)(1991)，354(6351)，320-2.29，?????Berzal-Herranz，Alfredo；Joseph，Simpson；Chowrira.Bharat?M.；Butcher.Samuel?E.；Burke，John?M.，Essential?nucleotide?sequences?and?secondary?structure?elements?of?the?hairpin?ribozyme.?EMBO?1.(1993)，12(6)，2567-73.30.??????Joseph，Simpson；Berzal-Herranz，Alfredo；Chowrira，Bharat?M.；Butcher.Samuel?E.，Substrateselection?rules?for?the?hairpin?ribozyme?determined?by?in?vitro?selection.mutation.and?analysis?of?mismatchedsubstrates.Genes?Dev.(1993)，7(1)，130-8.31.??????Berzal-Herranz，Alfredo；Joseph，Simpson；Burke.John?M.，In?vitro?selection?of?active?hairpinribo?zymes?by?sequential?RNA-catalyzed?cleavage?and?ligation?reactions.Genes?Dev.(1992)，6(1)，129-34.32.??????Hegg.Lisa?A.；Fedor，Martha?J.，Kinetics?and?Thermodynamics?of?Intermolecular?Catalysis?by?HairpinRibozymes.Biochemisuy(1995)，34(48)，15813-28.33.??????Grasby，Jane?A.；Mersmann，Karin；Singh，Mohinder；Gait.Michael?J.，Purine?Functional?Groups?inEssential?Residues?of?the?Hairpin?Ribozyme?Required?for?Catalytic?Cleavage?of?RNA.Biochemistry(1995)，34(12)，4068-76.34.??????Schmidt.Sabine；Beigelman，Leonid；Karpeisky，Alexander；Usman.Nassim；Sorensen，Ulrik?S.；Gait，Michael?J.，Base?and?sugar?requirements?for?RNA?cleavage?of?essential?nucleoside?residues?in?internal?loop?B?ofthe?hairpin?ribozyme：implications?for?secondary?structure.Nucleic?Acids?Res.(1996)，24(4)，573-81.35.??????Perrotta.Anne?T.；Been.Michael?D.，Cleavage?of?oligoribonucleotides?by?a?ribozyme?derived?from?thehepatitis.delta.virus?RNA?sequence.Biochemistry(1992)，31(1)，16-21.36.??????Perrotta，Anne?T.；Been，Michael?D.，A?pseudoknot-like?structure?required?for?efficient?self-cleavage?ofhepatitis?delta?virus?RNA.Nature(London)(1991)，350(6317)，434-6.37.??????Puttaraju.M.；Perrotta.Anne?T.；Been，Michael?D.，A?circular?trans-acring?hepatitis?delta?virusribozyme.Nucleic?Acids?Res.(1993)，21(18)，4253-8.

Table II: 2.5 μ mol RNA synthesis cycle

Reagent	Equivalent	Quantity	Waiting time
				Phosphoramidite	6.5	?163μL	?2.5
S-ethyl tetrazolium	23.8	?238μL	?2.5
				Diacetyl oxide	100	?233μL	5 seconds
The N-Methylimidazole	186	?233μL	5 seconds
				TCA	83.2	?1.73ml	21 seconds
Iodine	8.0	?1.18ml	45 seconds
				Acetonitrile	NA	?6.67ml	?NA

^*Waiting time does not comprise the duration of contact of deenergized period.

Table III A

Table IIIA: GBSS hammerhead substrate sequence nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 12 CGAUCGAUC GCCACAGC 26 538 GGUCGUCUC UCCCCGCU 27 68 GAAGGAAUA AACUCACU 28 540 UCGUCUCUC CCCGCUAC 29 73 AAUAAACUC ACUGCCAG 30 547 UCCCCGCUA CGACCAGU 31 103 AGAAGUGUA CUGCUCCG 32 556 CGACCAGUA CAAGGACG 33 109 GUACUGCUC CGUCCACC 34 581 ACCAGCGUC GUGUCCGA 35 113 UGCUCCGUC CACCAGUG 36 586 CGUCGUGUC CGAGAUCA 37 146 GGGCUGCUC AUCUCGUC 38 593 UCCGAGAUC AAGAUGGG 39 149 CUGCUCAUC UCGUCGAC 40 610 AGACAGGUA CGAGACGG 41 151 GCUCAUCUC GUCGACGA 42 620 GAGACGGUC AGGUUCUU 43 154 CAUCUCGUC GACGACCA 44 625 GGUCAGGUU CUUCCACU 45 169 CAGUGGAUU AAUCGGCA 46 626 GUCAGGUUC UUCCACUG 47 170 AGUGGAUUA AUCGGCAU 48 628 CAGGUUCUU CCACUGCU 49 173 GGAUUAAUC GGCAUGGC 50 629 AGGUUCUUC CACUGCUA 51 186 UGGCGGCUC UAGCCACG 52 637 CCACUGCUA CAAGCGCG 53 188 GCGGCUCUA GCCACGUC 54 661 CCGCGUGUU CGUUGACC 55 196 AGCCACGUC GCAGCUCG 56 662 CGCGUGUUC GUUGACCA 57 203 UCGCAGCUC GUCGCAAC 58 665 GUGUUCGUU GACCACCC 59 206 CAGCUCGUC GCAACGCG 60 679 CCCACUGUU CCUGGAGA 61 230 CUGGGCGUC CCGGACGC 62 680 CCACUGUUC CUGGAGAG 63 241 GGACGCGUC CACGUUCC 64 692 GAGAGGGUU UGGGGAAA 65 247 GUCCACGUU CCGCCGCG 66 693 AGAGGGUUU GGGGAAAG 67 248 UCCACGUUC CGCCGCGG 68 716 GAGAAGAUC UACGGGCC 69 292 GACGGCGUC GGCGGCGG 70 718 GAAGAUCUA CGGGCCUG 71 308 GACACGCUC AGCAUUCG 72 742 AACGGACUA CAGGGACA 73 314 CUCAGCAUU CGGACCAG 74 763 GCUGCGGUU CAGCCUGC 75 315 UCAGCAUUC GGACCAGC 78 764 CUGCGGUUC AGCCUGCU 77 344 CCCAGGCUC CAGCACCA 78 773 AGCCUGCUA UGCCAGGC 79 385 GGCCAGGUU CCCGUCGC 80 788 GCAGCACUU GAAGCUCC 81 386 GCCAGGUUC CCGUCGCU 82 795 UUGAAGCUC CAAGGAUC 83 391 GUUCCCGUC GCUCGUCG 84 803 CCAAGGAUC CUGAGCCU 85 395 CCGUCGCUC GUCGUGUG 86 812 CUGAGCCUC AACAACAA 87 398 UCGCUCGUC GUGUGCGC 88 826 CAACCCAUA CUUCUCCG 89 425 AUGAACGUC GUCUUCGU 90 829 CCCAUACUU CUCCGGAC 91 428 AACGUCGUC UUCGUCGG 92 830 CCAUACUUC UCCGGACC 93 430 CGUCGUCUU CGUCGGCG 94 832 AUACUUCUC CGGACCAU 95 431 GUCGUCUUC GUCGGCGC 96 841 CGGACCAUA CGGGGAGG 97 434 GUCUUCGUC GGCGCCGA 98 854 GAGGACGUC GUGUUCGU 99 473 GGCGGCCUC GGCGACGU 100 859 CGUCGUGUU CGUCUGCA 101 482 GGCGACGUC CUCGGCGG 102 850 GUCGUGUUC GUCUGCAA 103 485 GACGUCCUC GGCGGCCU 104 863 GUGUUCGUC UGCAACGA 105 527 CACCGUGUC AUGGUCGU 106 888 CCGGCCCUC UCUCGUGC 107 533 GUCAUGGUC GUCUCUCC 108 890 GGCCCUCUC UCGUGCUA 109 536 AUGGUCGUC UCUCCCCG 110 892 CCCUCUCUC GUGCUACC 111 898 CUCGUGCUA CCUCAAGA 112 1241 AUGGACGUC AGCGAGUG 113 902 UGCUACCUC AAGAGCAA 114 1270 GGACAAGUA CAUCGCCG 115 913 GAGCAACUA CCAGUCCC 116 1274 AAGUACAUC GCCGUGAA 117 919 CUACCAGUC CCACGGCA 118 1285 CGUGAAGUA CGACGUGU 119 929 CACGGCAUC UACAGGGA 120 1294 CGACGUGUC GACGGCCG 121 931 CGGCAUCUA CAGGGACG 122 1346 GCGGAGGUC GGGCUCCC 123 951 AGACCGCUU UCUGCAUC 124 1352 GUCGGGCUC CCGGUGGA 125 952 GACCGCUUU CUGCAUCC 126 1370 CGGAACAUC CCGCUGGU 127 953 ACCGCUUUC UGCAUCCA 128 1384 GGUGGCGUU CAUCGGCA 129 Table IIIA nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 959 UUCUGCAUC CACAACAU 130 1385 GUGGCGUUC AUCGGCAG 131 968 CACAACAUC UCCUACCA 132 1388 GCGUUCAUC GGCAGGCU 133 970 CAACAUCUC CUACCAGG 134 1421 CCCGACGUC AUGGCGGC 135 973 CAUCUCCUA CCAGGGCC 136 1436 GCCGCCAUC CCGCAGCU 137 985 GGGCCGGUU CGCCUUCU 138 1445 CCGCAGCUC AUGGAGAU 139 986 GGCCGGUUC GCCUUCUC 140 1472 GUGCAGAUC GUUCUGCU 141 991 GUUCGCCUU CUCCGACU 142 1475 CAGAUCGUU CUGCUGGG 143 992 UUCGCCUUC UCCGACUA 144 1476 AGAUCGUUC UGCUGGGC 145 994 CGCCUUCUC CGACUACC 146 1501 GAAGAAGUU CGAGCGCA 147 1000 CUCCGACUA CCCGGAGC 148 1502 AAGAAGUUC GAGCGCAU 149 1016 CUGAACCUC CCGGAGAG 150 1514 CGCAUGCUC AUGAGCGC 151 1027 GGAGAGAUU CAAGUCGU 152 1534 GGAGAAGUU CCCAGGCA 153 1028 GAGAGAUUC AAGUCGUC 154 1535 GAGAAGUUC CCAGGCAA 155 1033 AUUCAAGUC GUCCUUCG 156 1559 GCCGUGGUC AAGUUCAA 157 1036 CAAGUCGUC CUUCGAUU 158 1564 GGUCAAGUU CAACGCGC 159 1039 GUCGUCCUU CGAUUUCA 160 1565 GUCAAGUUC AACGCGGC 161 1040 UCGUCCUUC GAUUUCAU 162 1589 CACCACAUC AUGGCCGG 163 1044 CCUUCGAUU UCAUCGAC 164 1610 GACGUGCUC GCCGUCAC 165 1045 CUUCGAUUU CAUCGACG 166 1616 CUCGCCGUC ACCAGCCG 167 1046 UUCGAUUUC AUCGACGG 168 1627 CAGCCGCUU CGAGCCCU 169 1049 GAUUUCAUC GACGGCUA 170 1628 AGCCGCUUC GAGCCCUG 171 1057 CGACGGCUA CGAGAAGC 172 1643 UGCGGCCUC AUCCAGCU 173 1085 CGGAAGAUC AACUGGAU 174 1646 GGCCUCAUC CAGCUGCA 175 1106 GCCGGGAUC CUCGAGGC 176 1886 GAUGCGAUA CGGAACGC 177 1109 GGGAUCCUC GAGGCCGA 178 1690 CUGCGCGUC CACCGGUG 179 1124 GACAGGGUC CUCACCGU 180 1703 GGUGGACUC GUCGACAC 181 1127 AGGGUCCUC ACCGUCAG 182 1706 GGACUCGUC GACACCAU 183 1133 CUCACCGUC AGCCCCUA 184 1715 GACACCAUC AUCGAAGG 185 1141 CAGCCCCUA CUACGCCG 186 1718 ACCAUCAUC GAAGGCAA 187 1144 CCCCUACUA CGCCGAGG 188 1735 GACCGGGUU CCACAUGG 189 1157 GAGGAGCUC AUCUCCGG 190 1736 ACCGGGUUC CACAUGGG 191 1160 GAGCUCAUC UCCGGCAU 192 1751 GGCCGCCUC AGCGUCGA 193 1162 GCUCAUCUC CGGCAUCG 194 1757 CUCAGCGUC GACUGCAA 195 1169 UCCGGCAUC GCCAGGGG 196 1769 UGCAACGUC GUGGAGCC 197 1187 UGCGAGCUC GACAACAU 198 1787 GCGGACGUC AAGAAGGU 199 1196 GACAACAUC AUGCGCCU 200 1807 CACCACCUU GCAGCGCG 201 1205 AUGCGCCUC ACCGGCAU 202 1820 CGCGCCAUC AAGGUGGU 203 1214 ACCGGCAUC ACCGGCAU 204 1829 AAGGUGGUC GGCACGCC 205 1223 ACCGGCAUC GUCAACGG 206 1843 GCCGGCGUA CGAGGAGA 207 1226 GGCAUCGUC AACGGCAU 208 1871 UGCAUGAUC CAGGAUCU 209 Table IIIA nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 1878 UCCAGGAUC UCUCCUGG 210 2219 CGGUAAUUU UAUAUUGC 211 1880 CAGGAUCUC UCCUGGAA 212 2220 GGUAAUUUU AUAUUGCG 213 1882 GGAUCUCUC CUGGAAGG 214 2221 GUAAUUUUA UAUUGCGA 215 1922 GUGCUGCUC AGCCUCGG 216 2223 AAUUUUAUA UUGCGAGU 217 1928 CUCAGCCUC GGGGUCGC 218 2225 UUUUAUAUU GCGAGUAA 219 1934 CUCGGGGUC GCCGGCGG 220 2232 UUGCGAGUA AAUAAAUG 221 1955 CCAGGGGUC GAAGGCGA 222 2236 GAGUAAAUA AAUGGACC 223 1970 GAGGAGAUC GCGCCGCU 224 2248 GGACCUGUA GUGGUGGA 225 1979 GCGCCGCUC GCCAAGGA 226 2012 UGAAGAGUU CGGCCUGC 227 2013 GAAGAGUUC GGCCUGCA 228 2033 CCCCUGAUC UCGCGCGU 229 2035 CCUGAUCUC GCGCGUGG 230 2055 AAACAUGUU GGGACAUC 231 2063 UGGGACAUC UUCUUAUA 232 2065 GGACAUCUU CUUAUAUA 233 2066 GACAUCUUC UUAUAUAU 234 2068 CAUCUUCUU AUAUAUGC 235 2069 AUCUUCUUA UAUAUGCU 236 2071 CUUCUUAUA UAUGCUGU 237 2073 UCUUAUAUA UGCUGUUU 238 2080 UAUGCUGUU UCGUUUAU 239 2061 AUGCUGUUU CGUUUAUG 240 2082 UGCUGUUUC GUUUAUGU 241 2085 UGUUUCGUU UAUGUGAU 242 2086 GUUUCGUUU AUGUGAUA 243 2087 UUUCGUUUA UGUGAUAU 244 2094 UAUGUGAUA UGGACAAG 245 2104 GGACAAGUA UGUGUAGC 246 2110 GUAUGUGUA GCUGCUUG 247 2117 UAGCUGCUU GCUUGUGC 248 2121 UGCUUGCUU GUGCUAGU 249 2127 CUUGUGCUA GUGUAAUA 250 2132 GCUAGUGUA AUAUAGUG 251 2135 AGUGUAAUA UAGUGUAG 252 2137 UGUAAUAUA GUGUAGUG 253 2142 UAUAGUGUA GUGGUGGC 254 2165 CACAACCUA AUAAGCGC 255 2168 AACCUAAUA AGCGCAUG 256 2181 CAUGAACUA AUUGCUUG 257 2184 GAACUAAUU GCUUGCGU 258 2188 UAAUUGCUU GCGUGUGU 259 2197 GCGUGUGUA GUUAAGUA 260 2200 UGUGUAGUU AAGUACCG 261 2201 GUGUAGUUA AGUACCGA 262 2205 AGUUAAGUA CCGAUCGG 263 2211 GUACCGAUC GGUAAUUU 264 2215 CGAUCGGUA AUUUUAUA 265 2218 UCGGUAAUU UUAUAUUG 266 Table IIIB Table IIIB: The hammerhead ribozyme against GBSS mRNA sequence nt. positions HH ribozyme sequence Seq.ID ...

No.12, UGGCUGUGGC, CUGAUGA, X, GAA, AUCGAUCGGU, 26768, GCAGUGAGUU, CUGAUGA, X, GAA, AUUCCUUCCU, 26873, GGCUGGCAGU, CUGAUGA, X, GAA, AGUUUAUUCC, 269103, GACGGAGCAG, CUGAUGA, X, GAA, ACACUUCUCC, 270109, CUGGUGGACG, CUGAUGA, X, GAA, AGCAGUACAC, 271113, CGCACUGGUG, CUGAUGA, X, GAA, ACGGAGCAGU, 272146, UCGACGAGAU, CUGAUGA, X, GAA, AGCAGCCCUG, 273149, UCGUCGACGA, CUGAUGA, X, GAA, AUGAGCAGCC, 274151, GGUCGUCGAC, CUGAUGA, X, GAA, AGAUGAGCAG, 275154, ACUGGUCGUC, CUGAUGA, X, GAA, ACGAGAUGAG, 276169, CAUGCCGAUU, CUGAUGA, X, GAA, AUCCACUGGU, 277170, CCAUGCCGAU, CUGAUGA, X, GAA, AAUCCACUGG, 278173, CCGCCAUGCC, CUGAUGA, X, GAA, AUUAAUCCAC, 279186, GACGUGGCUA, CUGAUGA, X, GAA, AGCCGCCAUG, 280188, GCGACGUGGC, CUGAUGA, X, GAA, AGAGCCGCCA, 281196, GACGAGCUGC, CUGAUGA, X, GAA, ACGUGGCUAG, 282203, GCGUUGCGAC, CUGAUGA, X, GAA, AGCUGCGACG, 283206, CGCGCGUUGC, CUGAUGA, X, GAA, ACGAGCUGCG, 284230, ACGCGUCCGG, CUGAUGA, X, GAA, ACGCCCAGGC, 285241, GCGGAACGUG, CUGAUGA, X, GAA, ACGCGUCCGG, 286247, GCCGCGGCGG, CUGAUGA, X, GAA, ACGUGGACGC, 287248, CGCCGCGGCG, CUGAUGA, X, GAA, AACGUGGACG, 288292, GUCCGCCGCC, CUGAUGA, X, GAA, ACGCCGUCCG, 289308, UCCGAAUGCU, CUGAUGA, X, GAA, AGCGUGUCCG, 290314, CGCUGGUCCG, CUGAUGA, X, GAA, AUGCUGAGCG, 291315, GCGCUGGUCC, CUGAUGA, X, GAA, AAUGCUGAGC, 292344, GCUGGUGCUG, CUGAUGA, X, GAA, AGCCUGGGCG, 293385, GAGCGACGGG, CUGAUGA, X, GAA, ACCUGGCCCC, 294386, CGAGCGACGG, CUGAUGA, X, GAA, AACCUGGCCC, 295391, CACGACGAGC, CUGAUGA, X, GAA, ACGGGAACCU, 296395, CGCACACGAC, CUGAUGA, X, GAA, AGCGACGGGA, 297398, UGGCGCACAC, CUGAUGA, X, GAA, ACGAGCGACG, 298425, CGACGAAGAC, CUGAUGA, X, GAA, ACGUUCAUGC, 299428, CGCCGACGAA, CUGAUGA, X, GAA, ACGACGUUCA, 300430, GGCGCCGACG, CUGAUGA, X, GAA, AGACGACGUU, 301431, CGGCGCCGAC, CUGAUGA, X, GAA, AAGACGACGU, 302434, UCUCGGCGCC, CUGAUGA, X, GAA, ACGAAGACGA, 303473, GGACGUCGCC, CUGAUGA, X, GAA, AGGCCGCCGG, 304482, GGCCGCCGAG, CUGAUGA, X, GAA, ACGUCGCCGA, 305485, GCAGGCCGCC, CUGAUGA, X, GAA, AGGACGUCGC, 306527, AGACGACCAU, CUGAUGA, X, GAA, ACACGGUGCC, 307533, GGGGAGAGAC, CUGAUGA, X, GAA, ACCAUGACAC, 308536, AGCGGGGAGA, CUGAUGA, X, GAA, ACGACCAUGA, 309538, GUAGCGGGGA, CUGAUGA, X, GAA, AGACGACCAU, 310540, UCGUAGCGGG, CUGAUGA, X, GAA, AGAGACGACC, 311 Table III Bnt. positions, the HH ribozyme sequence, Seq.ID

No.547, GUACUGGUCG, CUGAUGA, X, GAA, AGCGGGGAGA, 312556, GGCGUCCUUG, CUGAUGA, X, GAA, ACUGGUCGUA, 313581, UCUCGGACAC, CUGAUGA, X, GAA, ACGCUGGUGU, 314586, CUUGAUCUCG, CUGAUGA, X, GAA, ACACGACGCU, 315593, CUCCCAUCUU, CUGAUGA, X, GAA, AUCUCGGACA, 316610, GACCGUCUCG, CUGAUGA, X, GAA, ACCUGUCUCC, 317620, GGAAGAACCU, CUGAUGA, X, GAA, ACCGUCUCGU, 318625, GCAGUGGAAG, CUGAUGA, X, GAA, ACCUGACCGU, 319626, AGCAGUGGAA, CUGAUGA, X, GAA, AACCUGACCG, 320628, GUAGCAGUGG, CUGAUGA, X, GAA, AGAACCUGAC, 321629, UGUAGCAGUG, CUGAUGA, X, GAA, AAGAACCUGA, 322637, UCCGCGCUUG, CUGAUGA, X, GAA, AGCAGUGGAA, 323661, GUGGUCAACG, CUGAUGA, X, GAA, ACACGCGGUC, 324662, GGUGGUCAAC, CUGAUGA, X, GAA, AACACGCGGU, 325665, GUGGGUGGUC, CUGAUGA, X, GAA, ACGAACACGC, 326679, CCUCUCCAGG, CUGAUGA, X, GAA, ACAGUGGGUG, 327680, CCCUCUCCAG, CUGAUGA, X, GAA, AACAGUGGGU, 328692, UCUUUCCCCA, CUGAUGA, X, GAA, ACCCUCUCCA, 329693, GUCUUUCCCC, CUGAUGA, X, GAA, AACCCUCUCC, 330716, CAGGCCCGUA, CUGAUGA, X, GAA, AUCUUCUCCU, 331718, GUCAGGCCCG, CUGAUGA, X, GAA, AGAUCUUCUC, 332742, GUUGUCCCUG, CUGAUGA, X, GAA, AGUCCGUUCC, 333763, UAGCAGGCUG, CUGAUGA, X, GAA, ACCGCAGCUG, 334764, AUAGCAGGCU, CUGAUGA, X, GAA, AACCGCAGCU, 335773, CUGCCUGGCA, CUGAUGA, X, GAA, AGCAGGCUGA, 336788, UUGGAGCUUC, CUGAUGA, X, GAA, AGUGCUGCCU, 337795, AGGAUCCUUG, CUGAUGA, X, GAA, AGCUUCAAGU, 338803, UGAGGCUCAG, CUGAUGA, X, GAA, AUCCUUGGAG, 339812, GGUUGUUGUU, CUGAUGA, X, GAA, AGGCUCAGGA, 340826, UCCGGAGAAG, CUGAUGA, X, GAA, AUGGGUUGUU, 341829, UGGUCCGGAG, CUGAUGA, X, GAA, AGUAUGGGUU, 342830, AUGGUCCGGA, CUGAUGA, X, GAA, AAGUAUGGGU, 343832, GUAUGGUCCG, CUGAUGA, X, GAA, AGAAGUAUGG, 344841, GUCCUCCCCG, CUGAUGA, X, GAA, AUGGUCCGGA, 345854, AGACGAACAC, CUGAUGA, X, GAA, ACGUCCUCCC, 346859, GUUGCAGACG, CUGAUGA, X, GAA, ACACGACGUC, 347860, CGUUGCAGAC, CUGAUGA, X, GAA, AACACGACGU, 348863, AGUCGUUGCA, CUGAUGA, X, GAA, ACGAACACGA, 349888, UAGCACGAGA, CUGAUGA, X, GAA, AGGGCCGGUG, 350890, GGUAGCACGA, CUGAUGA, X, GAA, AGAGGGCCGG, 351892, GAGGUAGCAC, CUGAUGA, X, GAA, AGAGAGGGCC, 352898, GCUCUUGAGG, CUGAUGA, X, GAA, AGCACGAGAG, 353902, AGUUGCUCUU, CUGAUGA, X, GAA, AGGUAGCACG, 354913, GUGGGACUGG, CUGAUGA, X, GAA, AGUUGCUCUU, 355919, GAUGCCGUGG, CUGAUGA, X, GAA, ACUGGUAGUU, 356929, CGUCCCUGUA, CUGAUGA, X, GAA, AUGCCGUGGG, 357931, UGCGUCCCUG, CUGAUGA, X, GAA, AGAUGCCGUG, 358951, UGGAUGCAGA, CUGAUGA, X, GAA, AGCGGUCUUU, 359952, GUGGAUGCAG, CUGAUGA, X, GAA, AAGCGGUCUU, 360953, UGUGGAUGCA, CUGAUGA, X, GAA, AAAGCGGUCU, 361959, AGAUGUUGUG, CUGAUGA, X, GAA, AUGCAGAAAG, 362968, CCUGGUAGGA, CUGAUGA, X, GAA, AUGUUGUGGA, 363 Table III Bnt. positions, the HH ribozyme sequence, Seq.ID

No.970, GCCCUGGUAG, CUGAUGA, X, GAA, AGAUGUUGUG, 364973, CCGGCCCUGG, CUGAUGA, X, GAA, AGGAGAUGUU, 365985, GGAGAAGGCG, CUGAUGA, X, GAA, ACCGGCCCUG, 366986, CGGAGAAGGC, CUGAUGA, X, GAA, AACCGGCCCU, 367991, GUAGUCGGAG, CUGAUGA, X, GAA, AGGCGAACCG, 368992, GGUAGUCGGA, CUGAUGA, X, GAA, AAGGCGAACC, 369994, CGGGUAGUCG, CUGAUGA, X, GAA, AGAAGGCGAA, 3701000, CAGCUCCGGG, CUGAUGA, X, GAA, AGUCGGAGAA, 3711016, AUCUCUCCGG, CUGAUGA, X, GAA, AGGUUCAGCU, 3721027, GGACGACUUG, CUGAUGA, X, GAA, AUCUCUCCGG, 3731028, AGGACGACUU, CUGAUGA, X, GAA, AAUCUCUCCG, 3741033, AUCGAAGGAC, CUGAUGA, X, GAA, ACUUGAAUCU, 3751036, GAAAUCGAAG, CUGAUGA, X, GAA, ACGACUUGAA, 3761039, GAUGAAAUCG, CUGAUGA, X, GAA, AGGACGACUU, 3771040, CGAUGAAAUC, CUGAUGA, X, GAA, AAGGACGACU, 3781044, CCGUCGAUGA, CUGAUGA, X, GAA, AUCGAAGGAC, 3791045, GCCGUCGAUG, CUGAUGA, X, GAA, AAUCGAAGGA, 3801046, AGCCGUCGAU, CUGAUGA, X, GAA, AAAUCGAAGG, 3811049, CGUAGCCGUC, CUGAUGA, X, GAA, AUGAAAUCGA, 3821057, GGGCUUCUCG, CUGAUGA, X, GAA, AGCCGUCGAU, 3831085, UCAUCCAGUU, CUGAUGA, X, GAA, AUCUUCCGGC, 3641106, CGGCCUCGAG, CUGAUGA, X, GAA, AUCCCGGCCU, 3851109, UGUCGGCCUC, CUGAUGA, X, GAA, AGGAUCCCGG, 3861124, UGACGGUGAG, CUGAUGA, X, GAA, ACCCUGUCGG, 3871127, GGCUGACGGU, CUGAUGA, X, GAA, AGGACCCUGU, 3881133, AGUAGGGGCU, CUGAUGA, X, GAA, ACGGUGAGGA, 3891141, CUCGGCGUAG, CUGAUGA, X, GAA, AGGGGCUGAC, 3901144, CUCCUCGGCG, CUGAUGA, X, GAA, AGUAGGGGCU, 3911157, UGCCGGAGAU, CUGAUGA, X, GAA, AGCUCCUCGG, 3921160, CGAUGCCGGA, CUGAUGA, X, GAA, AUGAGCUCCU, 3931162, GGCGAUGCCG, CUGAUGA, X, GAA, AGAUGAGCUC, 3941169, AGCCCCUGGC, CUGAUGA, X, GAA, AUGCCGGAGA, 3951187, UGAUGUUGUC, CUGAUGA, X, GAA, AGCUCGCAGC, 3961196, UGAGGCGCAU, CUGAUGA, X, GAA, AUGUUGUCGA, 3971205, UGAUGCCGGU, CUGAUGA, X, GAA, AGGCGCAUGA, 3981214, CGAUGCCGGU, CUGAUGA, X, GAA, AUGCCGGUGA, 3991223, UGCCGUUGAC, CUGAUGA, X, GAA, AUGCCGGUGA, 4001226, CCAUGCCGUU, CUGAUGA, X, GAA, ACGAUGCCGG, 4011241, CCCACUCGCU, CUGAUGA, X, GAA, ACGUCCAUGC, 4021270, CACGGCGAUG, CUGAUGA, X, GAA, ACUUGUCCCU, 4031274, ACUUCACGGC, CUGAUGA, X, GAA, AUGUACUUGU, 4041285, CGACACGUCG, CUGAUGA, X, GAA, ACUUCACGGC, 4051294, CACGGCCGUC, CUGAUGA, X, GAA, ACACGUCGUA, 4061346, CCGGGAGCCC, CUGAUGA, X, GAA, ACCUCCGCCU, 4071352, GGUCCACCGG, CUGAUGA, X, GAA, AGCCCGACCU, 4081370, CCACCAGCGG, CUGAUGA, X, GAA, AUGUUCCGGU, 4091384, CCUGCCGAUG, CUGAUGA, X, GAA, ACGCCACCAG, 4101385, GCCUGCCGAU, CUGAUGA, X, GAA, AACGCCACCA, 4111388, CCAGCCUGCC, CUGAUGA, X, GAA, AUGAACGCCA, 4121421, CGGCCGCCAU, CUGAUGA, X, GAA, ACGUCGGGUC, 4131436, UGAGCUGCGG, CUGAUGA, X, GAA, AUGGCGGCCG, 4141445, CCAUCUCCAU, CUGAUGA, X, GAA, AGCUGCGGGA, 415 Table III Bnt. positions, the HH ribozyme sequence, Seq.ID

No.1472, CCAGCAGAAC, CUGAUGA, X, GAA, AUCUGCACGU, 4161475, UGCCCAGCAG, CUGAUGA, X, GAA, ACGAUCUGCA, 4171476, GUGCCCAGCA, CUGAUGA, X, GAA, AACGAUCUGC, 4181501, CAUGCGCUCG, CUGAUGA, X, GAA, ACUUCUUCUU, 4191502, GCAUGCGCUC, CUGAUGA, X, GAA, AACUUCUUCU, 4201514, CGGCGCUCAU, CUGAUGA, X, GAA, AGCAUGCGCU, 4211534, CUUGCCUGGG, CUGAUGA, X, GAA, ACUUCUCCUC, 4221535, CCUUGCCUGG, CUGAUGA, X, GAA, AACUUCUCCU, 4231559, CGUUGAACUU, CUGAUGA, X, GAA, ACCACGGCGC, 4241564, CGCCGCGUUG, CUGAUGA, X, GAA, ACUUGACCAC, 4251565, GCGCCGCGUU, CUGAUGA, X, GAA, AACUUGACCA, 4261589, CGCCGGCCAU, CUGAUGA, X, GAA, AUGUGGUGCG, 4271610, UGGUGACGGC, CUGAUGA, X, GAA, AGCACGUCGG, 4281616, AGCGGCUGGU, CUGAUGA, X, GAA, ACGGCGAGCA, 4291627, GCAGGGCUCG, CUGAUGA, X, GAA, AGCGGCUGGU, 4301628, CGCAGGGCUC, CUGAUGA, X, GAA, AAGCGGCUGG, 4311643, GCAGCUGGAU, CUGAUGA, X, GAA, AGGCCGCAGG, 4321646, CCUGCAGCUG, CUGAUGA, X, GAA, AUGAGGCCGC, 4331666, GGGCGUUCCG, CUGAUGA, X, GAA, AUCGCAUCCC, 4341690, UCCACCGGUG, CUGAUGA, X, GAA, ACGCGCAGGC, 4351703, UGGUGUCGAC, CUGAUGA, X, GAA, AGUCCACCGG, 4361706, UGAUGGUGUC, CUGAUGA, X, GAA, ACGAGUCCAC, 4371715, UGCCUUCGAU, CUGAUGA, X, GAA, AUGGUGUCGA, 4381718, UCUUGCCUUC, CUGAUGA, X, GAA, AUGAUGGUGU, 4391735, GCCCAUGUGG, CUGAUGA, X, GAA, ACCCGGUCUU, 4401736, GGCCCAUGUG, CUGAUGA, X, GAA, AACCCGGUCU, 4411751, AGUCGACGCU, CUGAUGA, X, GAA, AGGCGGCCCA, 4421757, CGUUGCAGUC, CUGAUGA, X, GAA, ACGCUGAGGC, 4431769, CCGGCUCCAC, CUGAUGA, X, GAA, ACGUUGCAGU, 4441787, CCACCUUCUU, CUGAUGA, X, GAA, ACGUCCGCCG, 4451807, GGCGCGCUGC, CUGAUGA, X, GAA, AGGUGGUGGC, 4461820, CGACCACCUU, CUGAUGA, X, GAA, AUGGCGCGCU, 4471829, CCGGCGUGCC, CUGAUGA, X, GAA, ACCACCUUGA, 4481843, CAUCUCCUCG, CUGAUGA, X, GAA, ACGCCGGCGU, 4491871, AGAGAUCCUG, CUGAUGA, X, GAA, AUCAUGCAGU, 4501878, UUCCAGGAGA, CUGAUGA, X, GAA, AUCCUGGAUC, 4511880, CCUUCCAGGA, CUGAUGA, X, GAA, AGAUCCUGGA, 4521882, GCCCUUCCAG, CUGAUGA, X, GAA, AGAGAUCCUG, 4531922, CCCCGAGGCU, CUGAUGA, X, GAA, AGCAGCACGU, 4541928, CGGCGACCCC, CUGAUGA, X, GAA, AGGCUGAGCA, 4551934, CGCCGCCGGC, CUGAUGA, X, GAA, ACCCCGAGGC, 4561955, CCUCGCCUUC, CUGAUGA, X, GAA, ACCCCUGGCU, 4571970, CGAGCGGCGC, CUGAUGA, X, GAA, AUCUCCUCGC, 4581979, UCUCCUUGGC, CUGAUGA, X, GAA, AGCGGCGCGA, 4592012, CUGCAGGCCG, CUGAUGA, X, GAA, ACUCUUCAGG, 4602013, CCUGCAGGCC, CUGAUGA, X, GAA, AACUCUUCAG, 4612033, CCACGCGCGA, CUGAUGA, X, GAA, AUCAGGGGGC, 4622035, CACCACGCGC, CUGAUGA, X, GAA, AGAUCAGGGG, 4632055, AAGAUGUCCC, CUGAUGA, X, GAA, ACAUGUUUGC, 4642063, UAUAUAAGAA, CUGAUGA, X, GAA, AUGUCCCAAC, 4652065, CAUAUAUAAG, CUGAUGA, X, GAA, AGAUGUCCCA, 4662066, GCAUAUAUAA, CUGAUGA, X, GAA, AAGAUGUCCC, 457nt. position, the HH ribozyme sequence, Seq.ID

No.2068???????????????CAGCAUAUAU?CUGAUGA?X?GAA?AGAAGAUGUC???????????????4682069???????????????ACAGCAUAUA?CUGAUGA?X?GAA?AAGAAGAUGU???????????????4692071???????????????AAACAGCAUA?CUGAUGA?X?GAA?AUAAGAAGAU???????????????4702073???????????????CGAAACAGCA?CUGAUGA?X?GAA?AUAUAAGAAG???????????????4712080???????????????ACAUAAACGA?CUGAUGA?X?GAA?ACAGCAUAUA???????????????4722081???????????????CACAUAAACG?CUGAUGA?X?GAA?AACAGCAUAU???????????????4732082???????????????UCACAUAAAC?CUGAUGA?X?GAA?AAACAGCAUA???????????????4742085???????????????AUAUCACAUA?CUGAUGA?X?GAA?ACGAAACAGC???????????????4752086???????????????CAUAUCACAU?CUGAUGA?X?GAA?AACGAAACAG???????????????4762087???????????????CCAUAUCACA?CUGAUGA?X?GAA?AAACGAAACA???????????????4772094???????????????UACUUGUCCA?CUGAUGA?X?GAA?AUCACAUAAA???????????????4782104???????????????CAGCUACACA?CUGAUGA?X?GAA?ACUUGUCCAU???????????????4792110???????????????AGCAAGCAGC?CUGAUGA?X?GAA?ACACAUACUU???????????????4802117???????????????UAGCACAAGC?CUGAUGA?X?GAA?AGCAGCUACA???????????????4812121???????????????ACACUAGCAC?CUGAUGA?X?GAA?AGCAAGCAGC???????????????4822127???????????????UAUAUUACAC?CUGAUGA?X?GAA?AGCACAAGCA???????????????4832132???????????????UACACUAUAU?CUGAUGA?X?GAA?ACACUAGCAC???????????????4842135???????????????CACUACACUA?CUGAUGA?X?GAA?AUUACACUAG???????????????4852137???????????????ACCACUACAC?CUGAUGA?X?GAA?AUAUUACACU???????????????4862142???????????????UGGCCACCAC?CUGAUGA?X?GAA?ACACUAUAUU???????????????4872165???????????????AUGCGCUUAU?CUGAUGA?X?GAA?AGGUUGUGCC???????????????4882168???????????????UUCAUGCGCU?CUGAUGA?X?GAA?AUUAGGUUGU???????????????4892181???????????????CGCAAGCAAU?CUGAUGA?X?GAA?AGUUCAUGCG???????????????4902184???????????????ACACGCAAGC?CUGAUGA?X?GAA?AUUAGUUCAU???????????????4912188???????????????CUACACACGC?CUGAUGA?X?GAA?AGCAAUUAGU???????????????4922197???????????????GGUACUUAAC?CUGAUGA?X?GAA?ACACACGCAA???????????????4932200???????????????AUCGGUACUU?CUGAUGA?X?GAA?ACUACACACG???????????????4942201???????????????GAUCGGUACU?CUGAUGA?X?GAA?AACUACACAC???????????????4952205???????????????UACCGAUCGG?CUGAUGA?X?GAA?ACUUAACUAC???????????????4962211???????????????UAAAAUUACC?CUGAUGA?X?GAA?AUCGGUACUU???????????????4972215???????????????AAUAUAAAAU?CUGAUGA?X?GAA?ACCGAUCGGU???????????????4982218???????????????CGCAAUAUAA?CUGAUGA?X?GAA?AUUACCGAUC???????????????4992219???????????????UCGCAAUAUA?CUGAUGA?X?GAA?AAUUACCGAU???????????????5002220???????????????CUCGCAAUAU?CUGAUGA?X?GAA?AAAUUACCGA???????????????5012221???????????????ACUCGCAAUA?CUGAUGA?X?GAA?AAAAUUACCG???????????????5022223???????????????UUACUCGCAA?CUGAUGA?X?GAA?AUAAAAUUAC???????????????5032225???????????????AUUUACUCGC?CUGAUGA?X?GAA?AUAUAAAAUU???????????????5042232???????????????UCCAUUUAUU?CUGAUGA?X?GAA?ACUCGCAAUA???????????????505??2236???????????????CAGGUCCAUU?CUGAUGA?X?GAA?AUUUACUCGC???????????????5062248???????????????UUUCCACCAC?CUGAUGA?X?GAA?ACAGGUCCAU???????????????507

Wherein ＂ X ＂ represent the HH ribozyme stem II district (Hertel etc., 1992, nucleic acids research,

20?3252)。The length of stem II can 〉=2 base pairs.Table IV

Table IV: for GBSS, the HH ribozyme sequence nt. of mRNA test, the HH ribozyme sequence, sequence location, I.D.425, CGACGAAGAC, CUGAUGAGGCCGAAAGGCCGAA, ACGUUCAUGC, 2593, CUCCCAUCUU, CUGAUGAGGCCGAAAGGCCGAA, AUCUCGGACA, 3742, GUUGUCCCUG, CUGAUGAGGCCGAAAGGCCGAA, AGUCCGUUCC, 4812, GGUUGUUGUU, CUGAUGAGGCCGAAAGGCCGAA, AGGCUCAGGA, 5892, GAGGUAGCAC, CUGAUGAGGCCGAAAGGCCGAA, AGAGAGGGCC, 6913, GUGGGACUGG, CUGAUGAGGCCGAAAGGCCGAA, AGUUGCUCUU, 7919, GAUGCCGUGG, CUGAUGAGGCCGAAAGGCCGAA, ACUGGUAGUU, 8953, UGUGGAUGCA, CUGAUGAGGCCGAAAGGCCGAA, AAAGCGGUCU, 9959, AGAUGUUGUG, CUGAUGAGGCCGAAAGGCCGAA, AUGCAGAAAG, 10968, CCUGGUAGGA, CUGAUGAGGCCGAAAGGCCGAA, AUGUUGUGGA, 111016, AUCUCUCCGG, CUGAUGAGGCCGAAAGGCCGAA, AGGUUCAGCU, 121028, AGGACGACUU, CUGAUGAGGCCGAAAGGCCGAA, AAUCUCUCCG, 131085, UCAUCCAGUU, CUGAUGAGGCCGAAAGGCCGAA, AUCUUCCGGC, 141187, UGAUGUUGUC, CUGAUGAGGCCGAAAGGCCGAA, AGCUCGCAGC, 151196, UGAGGCGCAU, CUGAUGAGGCCGAAAGGCCGAA, AUGUUGUCGA, 161226, CCAUGCCGUU, CUGAUGAGGCCGAAAGGCCGAA, ACGAUGCCGG, 171241, CCCACUCGCU, CUGAUGAGGCCGAAAGGCCGAA, ACGUCCAUGC, 181270, CACGGCGAUG, CUGAUGAGGCCGAAAGGCCGAA, ACUUGUCCCU, 191352, GGUCCACCGG, CUGAUGAGGCCGAAAGGCCGAA, AGCCCGACCU, 201421, CGGCCGCCAU, CUGAUGAGGCCGAAAGGCCGAA, ACGUCGGGUC, 211534, CUUGCCUGGG, CUGAUGAGGCCGAAAGGCCGAA, ACUUCUCCUC, 221715, UGCCUUCGAU, CUGAUGAGGCCGAAAGGCCGAA, AUGGUGUCGA, 231787, CCACCUUCUU, CUGAUGAGGCCGAAAGGCCGAA, ACGUCCGCCG, 24 Table V A

Table V A:GBSS hairpin ribozyme and substrate sequence nt., the hairpin ribozyme sequence, Seq.ID, substrate, Seq.ID, No. position, No.48, CUCCUGGC, AGAA, GUCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 508, CGACA, GCC, GCCAGGAG, 509129, CCCUGCCG, AGAA, GUGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 510, GCACC, GCC, CGGCAGGG, 511468, GUCGCCGA, AGAA, GCCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 512, CGGCG, GCC, UCGGCGAC, 513489, CGGCGGCA, AGAA, GCCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 514, CGGCG, GCC, UGCCGCCG, 515496, CCAUGGCC, AGAA, GCAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 516, CUGCC, GCC, GGCCAUGG, 517676, UCUCCAGG, AGAA, GUGG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 518, CCACU, GUU, CCUGGAGA, 519737, UCCCUGUA, AGAA, GUUC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 520, GAACG, GAC, UACAGGGA, 521760, GCAGGCUG, AGAA, GCAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 522, CUGCG, GUU, CAGCCUGC, 5231298, GCCUCCAC, AGAA, GUCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 524, CGACG, GCC, GUGGAGGC, 5251427, GGGAUGGC, AGAA, GCCA, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 526, UGGCG, GCC, GCCAUCCC, 5271601, GCGAGCAC, AGAA, GCGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 528, GCGCC, GAC, GUGCUCGC, 5291638, CUGGAUGA, AGAA, GCAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 530, CUGCG, GCC, UCAUCCAG, 5311746, GACGCUGA, AGAA, GCCC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 532, GGGCC, GCC, UCAGCGUC, 5331781, UUCUUGAC, AGAA, GCCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 534, CGGCG, GAC, GUCAAGAA, 5352077, AUAAACGA, AGAA, GCAU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 536, AUGCU, GUU, UCGUUUAU, 537 Table V B

Table V B:GBSS hair clip ribozyme and substrate sequence t. position ribozyme sequence Seq.ID substrate Seq.ID No.

No.31, GUCGCCUC, AGAA, GGUGGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 538, ACCACCC, GCC, GAGGCGAC, 53948, CUCCUGGC, AGAA, GUCGCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 540, CGCGACA, GCC, GCCAGGAG, 541105, GUGGACGG, AGAA, GUACAC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 542, GUGUACU, GCU, CCGUCCAC, 543110, CACUGGUG, AGAA, GAGCAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 544, CUGCUCC, GUC, CACCAGUG, 545129, CCCUGCCG, AGAA, GUGCGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 546, GCGCACC, GCC, CGGCAGGG, 547142, ACGAGAUG, AGAA, GCCCUG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 548, CAGGGCU, GCU, CAUCUCGU, 549182, GUGGCUAG, AGAA, GCCAUG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 550, CAUGGCG, GCU, CUAGCCAC, 551199, UUGCGACG, AGAA, GCGACG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 552, CGUCGCA, GCU, CGUCGCAA, 553219, GACGCCCA, AGAA, GGCGCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 554, CGCGCCG, GCC, UGGGCGUC, 555233, GUGGACGC, AGAA, GGGACG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 556, CGUCCCG, GAC, GCGUCCAC, 557249, GGCGCCGC, AGAA, GAACGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 558, ACGUUCC, GCC, GCGGCGCC, 559283, CCGACGCC, AGAA, GGCCCC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 560, GGGGCCG, GAC, GGCGUCGG, 561316, GCGCGCUG, AGAA, GAAUGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 562, GCAUUCG, GAC, CAGCGCGC, 563388, CGACGAGC, AGAA, GGAACC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 564, GGUUCCC, GUC, GCUCGUCG, 565468, GUCGCCGA, AGAA, GCCGGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 566, ACCGGCG, GCC, UCGGCGAC, 567489, CGGCGGCA, AGAA, GCCGAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 568, CUCGGCG, GCC, UGCCGCCG, 569493, UGGCCGGC, AGAA, GGCCGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 570, GCGGCCU, GCC, GCCGGCCA, 571496, CCAUGGCC, AGAA, GCAGGC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 572, GCCUGCC, GCC, GGCCAUGG, 573676, UCUCCAGG, AGAA, GUGGGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 574, ACCCACU, GUU, CCUGGAGA, 575725, GUUCCAGC, AGAA, GGCCCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 576, CGGGCCU, GAC, GCUGGAAC, 577737, UCCCUGUA, AGAA, GUUCCA, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 578, UGGAACG, GAC, UACAGGGA, 579754, UGAACCGC, AGAA, GGUUGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 580, ACAACCA, GCU, GCGGUUCA, 581760, GCAGGCUG, AGAA, GCAGCU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 582, AGCUGCG, GUU, CAGCCUGC, 583765, GCAUAGCA, AGAA, GAACCG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 584, CGGUUCA, GCC, UGCUAUGC, 585834, CCCGUAUG, AGAA, GGAGAA, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 586, UUCUCCG, GAC, CAUACGGG, 587882, CGAGAGAG, AGAA, GGUGUG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 588, CACACCG, GCC, CUCUCUCG, 589916, UGCCGUGG, AGAA, GGUAGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 590, ACUACCA, GUC, CCACGGCA, 591947, AUGCAGAA, AGAA, GUCUUU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 592, AAAGACC, GCU, UUCUGCAU, 593982, AGAAGGCG, AGAA, GGCCCU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 594, AGGGCCG, GUU, CGCCUUCU, 595995, UCCGGGUA, AGAA, GAGAAG, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 596, CUUCUCC, GAC, UACCCGGA, 5971134, GUAGUAGG, AGAA, GACGGU, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 598, ACCGUCA, GCC, CCUACUAC, 5991298, GCCUCCAC, AGAA, GUCGAC, ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA, 600, GUCGACG, GCC, GUGGAGGC, 601 Table V B positions, ribozyme sequence, Seq.ID, substrate, Seq.ID, No.

No. 1372 ACGCCACC AGAA GGAUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 602 ACAUCCC GCU GGUGGCGU 603 1415 GCCAUGAC AGAA GGUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 604 GGGACCC GAC GUCAUGGC 605 1427 GGGAUGGC AGAA GCCAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 606 CAUGGCG GCC GCCAUCCC 607 1441 UCUCCAUG AGAA GCGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 608 UCCCGCA GCU CAUGGAGA 609 1468 GCAGAACG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 610 ACGUGCA GAU CGUUCUGC 611 1477 CCGUGCCC AGAA GAACGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 612 UCGUUCU GCU GGGCACGG 613 1601 GCGAGCAC AGAA GCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 614 CGGCGCC GAC GUGCUCGC 615 1620 CUCGAAGC AGAA GGUGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 616 GUCACCA GCC GCUUCGAG 617 1623 GGGCUCGA AGAA GCUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 618 ACCAGCC GCU UCGAGCCC 619 1638 CUGGAUGA AGAA GCAGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 620 CCCUGCG GCC UCAUCCAG 621 1648 UCCCCUGC AGAA GGAUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 622 UCAUCCA GCU GCAGGGGA 623 1746 GACGCUGA AGAA GCCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 624 AUGGGCC GCC UCAGCGUC 625 1781 UUCUUGAC AGAA GCCGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 626 GCCGGCG GAC GUCAAGAA 627 1918 CGAGGCUG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 628 ACGUGCU GCU CAGCCUCG 629 1923 GACCCCGA AGAA GAGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 630 CUGCUCA GCC UCGGGGUC 631 1975 CCUUGGCG AGAA GCGCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 632 UCGCGCC GCU CGCCAAGG 633 2014 GGCCUGCA AGAA GAACUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 634 GAGUUCG GCC UGCAGGCC 635 2029 CGCGCGAG AGAA GGGGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 636 GCCCCCU GAU CUCGCGCG 637 2077 AUAAACGA AGAA GCAUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 638 AUAUGCU GUU UCGUUUAU 639 2113 CACAAGCA AGAA GCUACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 640 UGUAGCU GCU UGCUUGUG 641 2207 AAUUACCG AGAA GUACUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 642 AAGUACC GAU CGGUAAUU 643 Table VI Table VI: Δ-9 desaturase HH ribozyme target sequence nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 13 CGCGCCCUC UGCCGCUU 644 319 GUCCAGGUU ACACAUUC 645 21 CUGCCGCUU GUUCGUUC 646 320 UCCAGGUUA CACAUUCA 647 24 CCGCUUGUU CGUUCCUC 648 326 UUACACAUU CAAUGCCA 649 25 CGCUUGUUC GUUCCUCG 650 327 UACACAUUC AAUGCCAC 651 28 UUGUUCGUU CCUCGCGC 652 338 UGCCACCUC ACAAGAUU 653 29 UGUUCGUUC CUCGCGCU 654 346 CACAAGAUU GAAAUUUU 655 32 UCGUUCCUC GCGCUCGC 656 352 AUUGAAAUU UUCAAGUC 657 38 CUCGCGCUC GCCACCAG 658 353 UUGAAAUUU UCAAGUCG 659 63 ACACACAUC CCAAUCUC 660 354 UGAAAUUUU CAAGUCGC 661 69 AUCCCAAUC UCGCGAGG 662 355 GAAAUUUUC AAGUCGCU 663 71 CCCAAUCUC GCGAGGGC 664 360 UUUCAAGUC GCUUGAUG 665 92 AGCAGGGUC UGCGGCGG 666 364 AAGUCGCUU GAUGAUUG 667 117 GCCGCGCUU CCGGCUCC 668 371 UUGAUGAUU GGGCUAGA 669 118 CCGCGCUUC CGGCUCCC 670 377 AUUGGGCUA GAGAUAAU 671 124 UUCCGGCUC CCCUUCCC 672 383 CUAGAGAUA AUAUCUUG 673 129 GCUCCCCUU CCCAUUGG 674 386 GAGAUAAUA UCUUGACG 675 130 CUCCCCUUC CCAUUGGC 676 388 GAUAAUAUC UUGACGCA 677 135 CUUCCCAUU GGCCUCCA 678 390 UAAUAUCUU GACGCAUC 679 141 AUUGGCCUC CACGAUGG 680 398 UGACGCAUC UCAAGCCA 681 154 AUGGCGCUC CGCCUCAA 682 400 ACGCAUCUC AAGCCAGU 683 160 CUCCGCCUC AACGACGU 684 409 AAGCCAGUC GAGAAGUG 685 169 AACGACGUC GCGCUCUG 686 419 AGAAGUGUU GGCAGCCA 687 175 GUCGCGCUC UGCCUCUC 688 434 CACAGGAUU UCCUCCCG 689 181 CUCUGCCUC UCCCCGCC 690 435 ACAGGAUUU CCUCCCGG 691 183 CUGCCUCUC CCCGCCGC 692 436 CAGGAUUUC CUCCCGGA 693 193 CCGCCGCUC GCCGCCCG 694 439 GAUUUCCUC CCGGACCC 695 228 CGGCAGGUU CGUCGCCG 696 453 CCCAGCAUC UGAAGGAU 697 229 GGCAGGUUC GUCGCCGU 698 462 UGAAGGAUU UCAUGAUG 699 232 AGGUUCGUC GCCGUCGC 700 463 GAAGGAUUU CAUGAUGA 701 238 GUCGCCGUC GCCUCCAU 702 464 AAGGAUUUC AUGAUGAA 703 243 CGUCGCCUC CAUGACGU 704 475 GAUGAAGUU AAGGAGCU 705 252 CAUGACGUC CGCCGUCU 706 476 AUGAAGUUA AGGAGCUC 707 259 UCCGCCGUC UCCACCAA 708 484 AAGGAGCUC AGAGAACG 709 261 CGCCGUCUC CACCAAGG 710 505 AAGGAAAUC CCUGAUGA 711 271 ACCAAGGUC GAGAAUAA 712 515 CUGAUGAUU AUUUUGUU 713 278 UCGAGAAUA AGAAGCCA 714 516 UGAUGAUUA UUUUGUUU 715 288 GAAGCCAUU UGCUCCUC 716 518 AUGAUUAUU UUGUUUGU 717 289 AAGCCAUUU GCUCCUCC 718 519 UGAUUAUUU UGUUUGUU 719 293 CAUUUGCUC CUCCAAGG 720 520 GAUUAUUUU GUUUGUUU 721 296 UUGCUCCUC CAAGGGAG 722 523 UAUUUUGUU UGUUUGGU 723 307 AGGGAGGUA CAUGUCCA 724 524 AUUUUGUUU GUUUGGUG 725 313 GUACAUGUC CAGGUUAC 726 527 UUGUUUGUU UGGUGGGA 727 528 UGUUUGUUU GGUGGGAG 728 857 ACACUGCUC GUCACGCC 729 544 GACAUGAUU ACCGAGGA 730 860 CUGCUCGUC ACGCCAAG 731 545 ACAUGAUUA CCGAGGAA 732 873 CAAGGACUU UGGCGACU 733 557 AGGAAGCUC UACCAACA 734 874 AAGGACUUU GGCGACUU 735 559 GAAGCUCUA CCAACAUA 736 882 UGGCGACUU AAAGCUUG 737 567 ACCAACAUA CCAGACUA 738 883 GGCGACUUA AAGCUUGC 739 575 ACCAGACUA UGCUUAAC 740 889 UUAAAGCUU GCACAAAU 741 580 ACUAUGCUU AACACCCU 742 898 GCACAAAUC UGCGGCAU 743 581 CUAUGCUUA ACACCCUC 744 907 UGCGGCAUC AUCGCCUC 745 589 AACACCCUC GACGGUGU 746 910 GGCAUCAUC GCCUCAGA 747 598 GACGGUGUC AGAGAUGA 748 915 CAUCGCCUC AGAUGAGA 749 Table VI nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 637 UGGGCUGUU UGGACGAG 750 942 AACUGCGUA CACCAAGA 751 638 GGGCUGUUU GGACGAGG 752 952 ACCAAGAUC GUGGAGAA 753 680 AUGGUGAUC UGCUCAAC 754 966 GAAGCUGUU UGAGAUCG 755 685 GAUCUGCUC AACAAGUA 756 967 AAGCUGUUU GAGAUCGA 757 693 CAACAAGUA UAUGUACC 758 973 UUUGAGAUC GACCCUGA 759 695 ACAAGUAUA UGUACCUC 760 986 CUGAUGGUA CCGUGGUC 761 699 GUAUAUGUA CCUCACUG 762 994 ACCGUGGUC GCUCUGGC 763 703 AUGUACCUC ACUGGGAG 764 998 UGGUCGCUC UGGCUGAC 765 719 GGGUGGAUA UGAGGCAG 766 1024 AAGAAGAUC UCAAUGCC 767 730 AGGCAGAUU GAGAAGAC 768 1026 GAAGAUCUC AAUGCCUG 769 742 AAGACAAUU CAGUAUCU 770 1047 CCUGAUGUU UGACGGGC 771 743 AGACAAUUC AGUAUCUU 772 1048 CUGAUGUUU GACGGGCA 773 747 AAUUCAGUA UCUUAUUG 774 1071 CAAGCUGUU CGAGCACU 775 749 UUCAGUAUC UUAUUGGC 776 1072 AAGCUGUUC GAGCACUU 777 751 CAGUAUCUU AUUGGCUC 778 1080 CGAGCACUU CUCCAUGG 779 752 AGUAUCUUA UUGGCUCU 780 1081 GAGCACUUC UCCAUGGU 781 754 UAUCUUAUU GGCUCUGG 782 1083 GCACUUCUC CAUGGUCG 783 759 UAUUGGCUC UGGAAUGG 784 1090 UCCAUGGUC GCGCAGAG 785 770 GAAUGGAUC CUAGGACU 786 1102 CAGAGGCUU GGCGUUUA 787 773 UGGAUCCUA GGACUGAG 788 1108 CUUGGCGUU UACACCGC 789 785 CUGAGAAUA AUCCUUAU 790 1109 UUGGCGUUU ACACCGCC 791 788 AGAAUAAUC CUUAUCUU 792 1110 UGGCGUUUA CACCGCCA 793 791 AUAAUCCUU AUCUUGGU 794 1125 CAGGGACUA CGCCGACA 795 792 UAAUCCUUA UCUUGGUU 796 1135 GCCGACAUC CUCGAGUU 797 794 AUCCUUAUC UUGGUUUC 798 1138 GACAUCCUC GAGUUCCU 799 796 CCUUAUCUU GGUUUCAU 800 1143 CCUCGAGUU CCUCGUCG 801 800 AUCUUGGUU UCAUCUAC 802 1144 CUCGAGUUC CUCGUCGA 803 801 UCUUGGUUU CAUCUACA 804 1147 GAGUUCCUC GUCGACAG 805 802 CUUGGUUUC AUCUACAC 806 1150 UUCCUCGUC GACAGGUG 807 805 GGUUUCAUC UACACCUC 806 1181 UGACUGGUC UGUCGGGU 809 807 UUUCAUCUA CACCUCCU 810 1185 UGGUCUGUC GGGUGAAG 811 813 CUACACCUC CUUCCAAG 812 1212 GCAGGACUA CCUUUGCA 813 816 CACCUCCUU CCAAGAGC 814 1216 GACUACCUU UGCACCCU 815 817 ACCUCCUUC CAAGAGCG 816 1217 ACUACCUUU GCACCCUU 817 834 GGCGACCUU CAUCUCAC 818 1225 UGCACCCUU GCUUCAAG 819 835 GCGACCUUC AUCUCACA 820 1229 CCCUUGCUU CAAGAAUC 821 838 ACCUUCAUC UCACACGG 822 1230 CCUUGCUUC AAGAAUCA 823 840 CUUCAUCUC ACACGGGA 824 1237 UCAAGAAUC AGGAGGCU 825 1292 CGCUGCCUU UCAGCUGG 826 1494 UUUGAUGUA CAACCUGU 827 1293 GCUGCCUUU CAGCUGGG 828 1546 CAUGCCGUA CUUUGUCU 829 1294 CUGCCUUUC AGCUGGGU 830 1549 GCCGUACUU UGUCUGUC 831 1303 AGCUGGGUA UACGGUAG 832 1550 CCGUACUUU GUCUGUCG 833 1305 CUGGGUAUA CGGUAGGG 834 1553 UACUUUGUC UGUCGCUG 835 1310 UAUACGGUA GGGACGUC 836 1557 UUGUCUGUC GCUGGCGG 837 1318 AGGGACGUC CAACUGUG 838 1571 CGGUGUGUU UCGGUAUG 839 1331 UGUGAGAUC GGAAACCU 840 1572 GGUGUGUUU CGGUAUGU 841 1348 GCUGCGGUC UGCUUAGA 842 1573 GUGUGUUUC GGUAUGUU 843 1353 GGUCUGCUU AGACAAGA 844 1577 GUUUCGGUA UGUUAUUU 845 1354 GUCUGCUUA GACAAGAC 846 1581 CGGUAUGUU AUUUGAGU 847 1372 UGCUGUGUC UGCGUUAC 848 1582 GGUAUGUUA UUUGAGUU 849 1378 GUCUGCGUU ACAUAGGU 850 1584 UAUGUUAUU UGAGUUGC 851 1379 UCUGCGUUA CAUAGGUC 852 1585 AUGUUAUUU GAGUUGCU 853 1383 CGUUACAUA GGUCUCCA 854 1590 AUUUGAGUU GCUCAGAU 855 1387 ACAUAGGUC UCCAGGUU 856 1594 GAGUUGCUC AGAUCUGU 857 1389 AUAGGUCUC CAGGUUUU 858 1599 GCUCAGAUC UGUUAAAA 859 1395 CUCCAGGUU UUGAUCAA 860 1603 AGAUCUGUU AAAAAAAA 861 1396 UCCAGGUUU UGAUCAAA 862 1604 GAUCUGUUA AAAAAAAA 863 Table VI nt. substrate Seq.ID Position No. 1397 CCAGGUUUU GAUCAAAU 864 1401 GUUUUGAUC AAAUGGUC 865 1409 CAAAUGGUC CCGUGUCG 866 1416 UCCCGUGUC GUCUUAUA 867 1419 CGUGUCGUC UUAUAGAG 868 1421 UGUCGUCUU AUAGAGCG 869 1422 GUCGUCUUA UAGAGCGA 870 1424 CGUCUUAUA GAGCGAUA 871 1432 AGAGCGAUA GGAGAACG 872 1444 GAACGUGUU GGUCUGUG 873 1448 GUGUUGGUC UGUGGUGU 874 1457 UGUGGUGUA GCUUUGUU 875 1461 GUGUAGCUU UGUUUUUA 876 1462 UGUAGCUUU GUUUUUAU 877 1465 AGCUUUGUU UUUAUUUU 878 1466 GCUUUGUUU UUAUUUUG 879 1467 CUUUGUUUU UAUUUUGU 880 1468 UUUGUUUUU AUUUUGUA 881 1469 UUGUUUUUA UUUUGUAU 882 1471 GUUUUUAUU UUGUAUUU 883 1472 UUUUUAUUU UGUAUUUU 884 1473 UUUUAUUUU GUAUUUUU 885 1476 UAUUUUGUA UUUUUCUG 886 1478 UUUUGUAUU UUUCUGCU 887 1479 UUUGUAUUU UUCUGCUU 888 1480 UUGUAUUUU UCUGCUUU 889 1481 UGUAUUUUU CUGCUUUG 890 1482 GUAUUUUUC UGCUUUGA 891 1487 UUUCUGCUU UGAUGUAC 892 1488 UUCUGCUUU GAUGUACA 893 Table VII Table VII: Δ-9 desaturase HH ribozyme sequence nt. ribozyme sequences Seq.ID No. Location 13 AAGCGGCA CUGAUGA X GAA AGGGCGCG 894 21 GAACGAAC CUGAUGA X GAA AGCGGCAG 895 24 GAGGAACG CUGAUGA X GAA ACAAGCGG 896 25 CGAGGAAC CUGAUGA X GAA AACAAGCG 897 28 GCGCGAGG CUGAUGA X GAA ACGAACAA 898 29 AGCGCGAG CUGAUGA X GAA AACGAACA 899 32 GCGAGCGC CUGAUGA X GAA AGGAACGA 900 38 CUGGUGGC CUGAUGA X GAA AGCGCGAG 901 63 GAGAUUGG CUGAUGA X GAA AUGUGUGU 902 69 CCUCGCGA CUGAUGA X GAA AUUGGGAU 903 71 GCCCUCGC CUGAUGA X GAA AGAUUGGG 904 92 CCGCCGCA CUGAUGA X GAA ACCCUGCU 905 117 GGAGCCGG CUGAUGA X GAA AGCGCGGC 906 118 GGGAGCCG CUGAUGA X GAA AAGCGCGG 907 124 GGGAAGGG CUGAUGA X GAA AGCCGGAA 908 129 CCAAUGGG CUGAUGA X GAA AGGGGAGC 909 130 GCCAAUGG CUGAUGA X GAA AAGGGGAG 910 135 UGGAGGCC CUGAUGA X GAA AUGGGAAG 911 141 CCAUCGUG CUGAUGA X GAA AGGCCAAU 912 154 UUGAGGCG CUGAUGA X GAA AGCGCCAU 913 160 ACGUCGUU CUGAUGA X GAA AGGCGGAG 914 169 CAGAGCGC CUGAUGA X GAA ACGUCGUU 915 175 GAGAGGCA CUGAUGA X GAA AGCGCGAC 916 181 GGCGGGGA CUGAUGA X GAA AGGCAGAG 917 183 GCGGCGGG CUGAUGA X GAA AGAGGCAG 918 193 CGGGCGGC CUGAUGA X GAA AGCGGCGG 919 228 CGGCGACG CUGAUGA X GAA ACCUGCCG 920 229 ACGGCGAC CUGAUGA X GAA AACCUGCC 921 232 GCGACGGC CUGAUGA X GAA ACGAACCU 922 238 AUGGAGGC CUGAUGA X GAA ACGGCGAC 923 243 ACGUCAUG CUGAUGA X GAA AGGCGACG 924 252 AGACGGCG CUGAUGA X GAA ACGUCAUG 925 259 UUGGUGGA CUGAUGA X GAA ACGGCGGA 926 261 CCUUGGUG CUGAUGA X GAA AGACGGCG 927 271 UUAUUCUC CUGAUGA X GAA ACCUUGGU 928 278 UGGCUUCU CUGAUGA X GAA AUUCUCGA 929 288 GAGGAGCA CUGAUGA X GAA AUGGCUUC 930 289 GGAGGAGC CUGAUGA X GAA AAUGGCUU 931 293 CCUUGGAG CUGAUGA X GAA AGCAAAUG 932 296 CUCCCUUG CUGAUGA X GAA AGGAGCAA 933 307 UGGACAUG CUGAUGA X GAA ACCUCCCU 934 313 GUAACCUG CUGAUGA X GAA ACAUGUAC 935 319 GAAUGUGU CUGAUGA X GAA ACCUGGAC 936 320 UGAAUGUG CUGAUGA X GAA AACCUGGA 937 326 UGGCAUUG CUGAUGA X GAA AUGUGUAA 938 327 GUGGCAUU CUGAUGA X GAA AAUGUGUA 939 338 AAUCUUGU CUGAUGA X GAA AGGUGGCA 940 346 AAAAUUUC CUGAUGA X GAA AUCUUGUG 941 352 GACUUGAA CUGAUGA X GAA AUUUCAAU 942 353 CGACUUGA CUGAUGA X GAA AAUUUCAA 943 354 GCGACUUG CUGAUGA X GAA AAAUUUCA 944 355 AGCGACUU CUGAUGA X GAA AAAAUUUC 945 360 CAUCAAGC CUGAUGA X GAA ACUUGAAA 946 364 CAAUCAUC CUGAUGA X GAA AGCGACUU 947 Table VII nt. ribozyme sequences Seq.ID No. Location 371 UCUAGCCC CUGAUGA X GAA AUCAUCAA 948 377 AUUAUCUC CUGAUGA X GAA AGCCCAAU 949 383 CAAGAUAU CUGAUGA X GAA AUCUCUAG 950 386 CGUCAAGA CUGAUGA X GAA AUUAUCUC 951 388 UGCGUCAA CUGAUGA X GAA AUAUUAUC 952 390 GAUGCGUC CUGAUGA X GAA AGAUAUUA 953 398 UGGCUUGA CUGAUGA X GAA AUGCGUCA 954 400 ACUGGCUU CUGAUGA X GAA AGAUGCGU 955 409 CACUUCUC CUGAUGA X GAA ACUGGCUU 956 419 UGGCUGCC CUGAUGA X GAA ACACUUCU 957 434 CGGGAGGA CUGAUGA X GAA AUCCUGUG 958 435 CCGGGAGG CUGAUGA X GAA AAUCCUGU 959 436 UCCGGGAG CUGAUGA X GAA AAAUCCUG 960 439 GGGUCCGG CUGAUGA X GAA AGGAAAUC 961 453 AUCCUUCA CUGAUGA X GAA AUGCUGGG 962 462 CAUCAUGA CUGAUGA X GAA AUCCUUCA 963 463 UCAUCAUG CUGAUGA X GAA AAUCCUUC 964 464 UUCAUCAU CUGAUGA X GAA AAAUCCUU 965 475 AGCUCCUU CUGAUGA X GAA ACUUCAUC 966 476 GAGCUCCU CUGAUGA X GAA AACUUCAU 967 484 CGUUCUCU CUGAUGA X GAA AGCUCCUU 968 505 UCAUCAGG CUGAUGA X GAA AUUUCCUU 969 515 AACAAAAU CUGAUGA X GAA AUCAUCAG 970 516 AAACAAAA CUGAUGA X GAA AAUCAUCA 971 518 ACAAACAA CUGAUGA X GAA AUAAUCAU 972 519 AACAAACA CUGAUGA X GAA AAUAAUCA 973 520 AAACAAAC CUGAUGA X GAA AAAUAAUC 974 523 ACCAAACA CUGAUGA X GAA ACAAAAUA 975 524 CACCAAAC CUGAUGA X GAA AACAAAAU 976 527 UCCCACCA CUGAUGA X GAA ACAAACAA 977 528 CUCCCACC CUGAUGA X GAA AACAAACA 978 544 UCCUCGGU CUGAUGA X GAA AUCAUGUC 979 545 UUCCUCGG CUGAUGA X GAA AAUCAUGU 980 557 UGUUGGUA CUGAUGA X GAA AGCUUCCU 981 559 UAUGUUGG CUGAUGA X GAA AGAGCUUC 982 567 UAGUCUGG CUGAUGA X GAA AUGUUGGU 983 575 GUUAAGCA CUGAUGA X GAA AGUCUGGU 984 580 AGGGUGUU CUGAUGA X GAA AGCAUAGU 985 581 GAGGGUGU CUGAUGA X GAA AAGCAUAG 986 589 ACACCGUC CUGAUGA X GAA AGGGUGUU 987 598 UCAUCUCU CUGAUGA X GAA ACACCGUC 988 637 CUCGUCCA CUGAUGA X GAA ACAGCCCA 989 638 CCUCGUCC CUGAUGA X GAA AACAGCCC 990 680 GUUGAGCA CUGAUGA X GAA AUCACCAU 991 685 UACUUGUU CUGAUGA X GAA AGCAGAUC 992 693 GGUACAUA CUGAUGA X GAA ACUUGUUG 993 695 GAGGUACA CUGAUGA X GAA AUACUUGU 994 699 CAGUGAGG CUGAUGA X GAA ACAUAUAC 995 703 CUCCCAGU CUGAUGA X GAA AGGUACAU 996 719 CUGCCUCA CUGAUGA X GAA AUCCACCC 997 730 GUCUUCUC CUGAUGA X GAA AUCUGCCU 998 742 AGAUACUG CUGAUGA X GAA AUUGUCUU 999 743 AAGAUACU CUGAUGA X GAA AAUUGUCU 1000 747 CAAUAAGA CUGAUGA X GAA ACUGAAUU 1001 749 GCCAAUAA CUGAUGA X GAA AUACUGAA 1002 751 GAGCCAAU CUGAUGA X GAA AGAUACUG 1003 752 AGAGCCAA CUGAUGA X GAA AAGAUACU 1004 Table VII nt. ribozyme sequences Seq.ID No Location 754 CCAGAGCC CUGAUGA X GAA AUAAGAUA 1005 759 CCAUUCCA CUGAUGA X GAA AGCCAAUA 1006 770 AGUCCUAG CUGAUGA X GAA AUCCAUUC 1007 773 CUCAGUCC CUGAUGA X GAA AGGAUCCA 1008 785 AUAAGGAU CUGAUGA X GAA AUUCUCAG 1009 788 AAGAUAAG CUGAUGA X GAA AUUAUUCU 1010 791 ACCAAGAU CUGAUGA X GAA AGGAUUAU 1011 792 AACCAAGA CUGAUGA X GAA AAGGAUUA 1012 794 GAAACCAA CUGAUGA X GAA AUAAGGAU 1013 796 AUGAAACC CUGAUGA X GAA AGAUAAGG 1014 800 GUAGAUGA CUGAUGA X GAA ACCAAGAU 1015 801 UGUAGAUG CUGAUGA X GAA AACCAAGA 1016 802 GUGUAGAU CUGAUGA X GAA AAACCAAG 1017 805 GAGGUGUA CUGAUGA X GAA AUGAAACC 1018 807 AGGAGGUG CUGAUGA X GAA AGAUGAAA 1019 813 CUUGGAAG CUGAUGA X GAA AGGUGUAG 1020 816 GCUCUUGG CUGAUGA X GAA AGGAGGUG 1021 817 CGCUCUUG CUGAUGA X GAA AAGGAGGU 1022 834 GUGAGAUG CUGAUGA X GAA AGGUCGCC 1023 835 UGUGAGAU CUGAUGA X GAA AAGGUCGC 1024 838 CCGUGUGA CUGAUGA X GAA AUGAAGGU 1025 ...

Table I X

Table I X: the cutting of the Δ-9 desaturation ribozyme that produces by the HH ribozyme

Cutting per-cent

No. 1372 ACGCCACC AGAA GGAUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 602 ACAUCCC GCU GGUGGCGU 603 1415 GCCAUGAC AGAA GGUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 604 GGGACCC GAC GUCAUGGC 605 1427 GGGAUGGC AGAA GCCAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 606 CAUGGCG GCC GCCAUCCC 607 1441 UCUCCAUG AGAA GCGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 608 UCCCGCA GCU CAUGGAGA 609 1468 GCAGAACG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 610 ACGUGCA GAU CGUUCUGC 611 1477 CCGUGCCC AGAA GAACGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 612 UCGUUCU GCU GGGCACGG 613 1601 GCGAGCAC AGAA GCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 614 CGGCGCC GAC GUGCUCGC 615 1620 CUCGAAGC AGAA GGUGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 616 GUCACCA GCC GCUUCGAG 617 1623 GGGCUCGA AGAA GCUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 618 ACCAGCC GCU UCGAGCCC 619 1638 CUGGAUGA AGAA GCAGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 620 CCCUGCG GCC UCAUCCAG 621 1648 UCCCCUGC AGAA GGAUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 622 UCAUCCA GCU GCAGGGGA 623 1746 GACGCUGA AGAA GCCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 624 AUGGGCC GCC UCAGCGUC 625 1781 UUCUUGAC AGAA GCCGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 626 GCCGGCG GAC GUCAAGAA 627 1918 CGAGGCUG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 628 ACGUGCU GCU CAGCCUCG 629 1923 GACCCCGA AGAA GAGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 630 CUGCUCA GCC UCGGGGUC 631 1975 CCUUGGCG AGAA GCGCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 632 UCGCGCC GCU CGCCAAGG 633 2014 GGCCUGCA AGAA GAACUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 634 GAGUUCG GCC UGCAGGCC 635 2029 CGCGCGAG AGAA GGGGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 636 GCCCCCU GAU CUCGCGCG 637 2077 AUAAACGA AGAA GCAUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 638 AUAUGCU GUU UCGUUUAU 639 2113 CACAAGCA AGAA GCUACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 640 UGUAGCU GCU UGCUUGUG 641 2207 AAUUACCG AGAA GUACUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 642 AAGUACC GAU CGGUAAUU 643 Table VI Table VI: Δ-9 desaturase HH ribozyme target sequence nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 13 CGCGCCCUC UGCCGCUU 644 319 GUCCAGGUU ACACAUUC 645 21 CUGCCGCUU GUUCGUUC 646 320 UCCAGGUUA CACAUUCA 647 24 CCGCUUGUU CGUUCCUC 648 326 UUACACAUU CAAUGCCA 649 25 CGCUUGUUC GUUCCUCG 650 327 UACACAUUC AAUGCCAC 651 28 UUGUUCGUU CCUCGCGC 652 338 UGCCACCUC ACAAGAUU 653 29 UGUUCGUUC CUCGCGCU 654 346 CACAAGAUU GAAAUUUU 655 32 UCGUUCCUC GCGCUCGC 656 352 AUUGAAAUU UUCAAGUC 657 38 CUCGCGCUC GCCACCAG 658 353 UUGAAAUUU UCAAGUCG 659 63 ACACACAUC CCAAUCUC 660 354 UGAAAUUUU CAAGUCGC 661 69 AUCCCAAUC UCGCGAGG 662 355 GAAAUUUUC AAGUCGCU 663 71 CCCAAUCUC GCGAGGGC 664 360 UUUCAAGUC GCUUGAUG 665 92 AGCAGGGUC UGCGGCGG 666 364 AAGUCGCUU GAUGAUUG 667 117 GCCGCGCUU CCGGCUCC 668 371 UUGAUGAUU GGGCUAGA 669 118 CCGCGCUUC CGGCUCCC 670 377 AUUGGGCUA GAGAUAAU 671 124 UUCCGGCUC CCCUUCCC 672 383 CUAGAGAUA AUAUCUUG 673 129 GCUCCCCUU CCCAUUGG 674 386 GAGAUAAUA UCUUGACG 675 130 CUCCCCUUC CCAUUGGC 676 388 GAUAAUAUC UUGACGCA 677 135 CUUCCCAUU GGCCUCCA 678 390 UAAUAUCUU GACGCAUC 679 141 AUUGGCCUC CACGAUGG 680 398 UGACGCAUC UCAAGCCA 681 154 AUGGCGCUC CGCCUCAA 682 400 ACGCAUCUC AAGCCAGU 683 160 CUCCGCCUC AACGACGU 684 409 AAGCCAGUC GAGAAGUG 685 169 AACGACGUC GCGCUCUG 686 419 AGAAGUGUU GGCAGCCA 687 175 GUCGCGCUC UGCCUCUC 688 434 CACAGGAUU UCCUCCCG 689 181 CUCUGCCUC UCCCCGCC 690 435 ACAGGAUUU CCUCCCGG 691 183 CUGCCUCUC CCCGCCGC 692 436 CAGGAUUUC CUCCCGGA 693 193 CCGCCGCUC GCCGCCCG 694 439 GAUUUCCUC CCGGACCC 695 228 CGGCAGGUU CGUCGCCG 696 453 CCCAGCAUC UGAAGGAU 697 229 GGCAGGUUC GUCGCCGU 698 462 UGAAGGAUU UCAUGAUG 699 232 AGGUUCGUC GCCGUCGC 700 463 GAAGGAUUU CAUGAUGA 701 238 GUCGCCGUC GCCUCCAU 702 464 AAGGAUUUC AUGAUGAA 703 243 CGUCGCCUC CAUGACGU 704 475 GAUGAAGUU AAGGAGCU 705 252 CAUGACGUC CGCCGUCU 706 476 AUGAAGUUA AGGAGCUC 707 259 UCCGCCGUC UCCACCAA 708 484 AAGGAGCUC AGAGAACG 709 261 CGCCGUCUC CACCAAGG 710 505 AAGGAAAUC CCUGAUGA 711 271 ACCAAGGUC GAGAAUAA 712 515 CUGAUGAUU AUUUUGUU 713 278 UCGAGAAUA AGAAGCCA 714 516 UGAUGAUUA UUUUGUUU 715 288 GAAGCCAUU UGCUCCUC 716 518 AUGAUUAUU UUGUUUGU 717 289 AAGCCAUUU GCUCCUCC 718 519 UGAUUAUUU UGUUUGUU 719 293 CAUUUGCUC CUCCAAGG 720 520 GAUUAUUUU GUUUGUUU 721 296 UUGCUCCUC CAAGGGAG 722 523 UAUUUUGUU UGUUUGGU 723 307 AGGGAGGUA CAUGUCCA 724 524 AUUUUGUUU GUUUGGUG 725 313 GUACAUGUC CAGGUUAC 726 527 UUGUUUGUU UGGUGGGA 727 528 UGUUUGUUU GGUGGGAG 728 857 ACACUGCUC GUCACGCC 729 544 GACAUGAUU ACCGAGGA 730 860 CUGCUCGUC ACGCCAAG 731 545 ACAUGAUUA CCGAGGAA 732 873 CAAGGACUU UGGCGACU 733 557 AGGAAGCUC UACCAACA 734 874 AAGGACUUU GGCGACUU 735 559 GAAGCUCUA CCAACAUA 736 882 UGGCGACUU AAAGCUUG 737 567 ACCAACAUA CCAGACUA 738 883 GGCGACUUA AAGCUUGC 739 575 ACCAGACUA UGCUUAAC 740 889 UUAAAGCUU GCACAAAU 741 580 ACUAUGCUU AACACCCU 742 898 GCACAAAUC UGCGGCAU 743 581 CUAUGCUUA ACACCCUC 744 907 UGCGGCAUC AUCGCCUC 745 589 AACACCCUC GACGGUGU 746 910 GGCAUCAUC GCCUCAGA 747 598 GACGGUGUC AGAGAUGA 748 915 CAUCGCCUC AGAUGAGA 749 Table VI nt. substrate Seq.ID nt. substrate Seq.ID Position No. Position No. 637 UGGGCUGUU UGGACGAG 750 942 AACUGCGUA CACCAAGA 751 638 GGGCUGUUU GGACGAGG 752 952 ACCAAGAUC GUGGAGAA 753 680 AUGGUGAUC UGCUCAAC 754 966 GAAGCUGUU UGAGAUCG 755 685 GAUCUGCUC AACAAGUA 756 967 AAGCUGUUU GAGAUCGA 757 693 CAACAAGUA UAUGUACC 758 973 UUUGAGAUC GACCCUGA 759 695 ACAAGUAUA UGUACCUC 760 986 CUGAUGGUA CCGUGGUC 761 699 GUAUAUGUA CCUCACUG 762 994 ACCGUGGUC GCUCUGGC 763 703 AUGUACCUC ACUGGGAG 764 998 UGGUCGCUC UGGCUGAC 765 719 GGGUGGAUA UGAGGCAG 766 1024 AAGAAGAUC UCAAUGCC 767 730 AGGCAGAUU GAGAAGAC 768 1026 GAAGAUCUC AAUGCCUG 769 742 AAGACAAUU CAGUAUCU 770 1047 CCUGAUGUU UGACGGGC 771 743 AGACAAUUC AGUAUCUU 772 1048 CUGAUGUUU GACGGGCA 773 747 AAUUCAGUA UCUUAUUG 774 1071 CAAGCUGUU CGAGCACU 775 749 UUCAGUAUC UUAUUGGC 776 1072 AAGCUGUUC GAGCACUU 777 751 CAGUAUCUU AUUGGCUC 778 1080 CGAGCACUU CUCCAUGG 779 752 AGUAUCUUA UUGGCUCU 780 1081 GAGCACUUC UCCAUGGU 781 754 UAUCUUAUU GGCUCUGG 782 1083 GCACUUCUC CAUGGUCG 783 759 UAUUGGCUC UGGAAUGG 784 1090 UCCAUGGUC GCGCAGAG 785 770 GAAUGGAUC CUAGGACU 786 1102 CAGAGGCUU GGCGUUUA 787 773 UGGAUCCUA GGACUGAG 788 1108 CUUGGCGUU UACACCGC 789 785 CUGAGAAUA AUCCUUAU 790 1109 UUGGCGUUU ACACCGCC 791 788 AGAAUAAUC CUUAUCUU 792 1110 UGGCGUUUA CACCGCCA 793 791 AUAAUCCUU AUCUUGGU 794 1125 CAGGGACUA CGCCGACA 795 792 UAAUCCUUA UCUUGGUU 796 1135 GCCGACAUC CUCGAGUU 797 794 AUCCUUAUC UUGGUUUC 798 1138 GACAUCCUC GAGUUCCU 799 796 CCUUAUCUU GGUUUCAU 800 1143 CCUCGAGUU CCUCGUCG 801 800 AUCUUGGUU UCAUCUAC 802 1144 CUCGAGUUC CUCGUCGA 803 801 UCUUGGUUU CAUCUACA 804 1147 GAGUUCCUC GUCGACAG 805 802 CUUGGUUUC AUCUACAC 806 1150 UUCCUCGUC GACAGGUG 807 805 GGUUUCAUC UACACCUC 806 1181 UGACUGGUC UGUCGGGU 809 807 UUUCAUCUA CACCUCCU 810 1185 UGGUCUGUC GGGUGAAG 811 813 CUACACCUC CUUCCAAG 812 1212 GCAGGACUA CCUUUGCA 813 816 CACCUCCUU CCAAGAGC 814 1216 GACUACCUU UGCACCCU 815 817 ACCUCCUUC CAAGAGCG 816 1217 ACUACCUUU GCACCCUU 817 834 GGCGACCUU CAUCUCAC 818 1225 UGCACCCUU GCUUCAAG 819 835 GCGACCUUC AUCUCACA 820 1229 CCCUUGCUU CAAGAAUC 821 838 ACCUUCAUC UCACACGG 822 1230 CCUUGCUUC AAGAAUCA 823 840 CUUCAUCUC ACACGGGA 824 1237 UCAAGAAUC AGGAGGCU 825 1292 CGCUGCCUU UCAGCUGG 826 1494 UUUGAUGUA CAACCUGU 827 1293 GCUGCCUUU CAGCUGGG 828 1546 CAUGCCGUA CUUUGUCU 829 1294 CUGCCUUUC AGCUGGGU 830 1549 GCCGUACUU UGUCUGUC 831 1303 AGCUGGGUA UACGGUAG 832 1550 CCGUACUUU GUCUGUCG 833 1305 CUGGGUAUA CGGUAGGG 834 1553 UACUUUGUC UGUCGCUG 835 1310 UAUACGGUA GGGACGUC 836 1557 UUGUCUGUC GCUGGCGG 837 1318 AGGGACGUC CAACUGUG 838 1571 CGGUGUGUU UCGGUAUG 839 1331 UGUGAGAUC GGAAACCU 840 1572 GGUGUGUUU CGGUAUGU 841 1348 GCUGCGGUC UGCUUAGA 842 1573 GUGUGUUUC GGUAUGUU 843 1353 GGUCUGCUU AGACAAGA 844 1577 GUUUCGGUA UGUUAUUU 845 1354 GUCUGCUUA GACAAGAC 846 1581 CGGUAUGUU AUUUGAGU 847 1372 UGCUGUGUC UGCGUUAC 848 1582 GGUAUGUUA UUUGAGUU 849 1378 GUCUGCGUU ACAUAGGU 850 1584 UAUGUUAUU UGAGUUGC 851 1379 UCUGCGUUA CAUAGGUC 852 1585 AUGUUAUUU GAGUUGCU 853 1383 CGUUACAUA GGUCUCCA 854 1590 AUUUGAGUU GCUCAGAU 855 1387 ACAUAGGUC UCCAGGUU 856 1594 GAGUUGCUC AGAUCUGU 857 1389 AUAGGUCUC CAGGUUUU 858 1599 GCUCAGAUC UGUUAAAA 859 1395 CUCCAGGUU UUGAUCAA 860 1603 AGAUCUGUU AAAAAAAA 861 1396 UCCAGGUUU UGAUCAAA 862 1604 GAUCUGUUA AAAAAAAA 863 Table VI nt. substrate Seq.ID Position No. 1397 CCAGGUUUU GAUCAAAU 864 1401 GUUUUGAUC AAAUGGUC 865 1409 CAAAUGGUC CCGUGUCG 866 1416 UCCCGUGUC GUCUUAUA 867 1419 CGUGUCGUC UUAUAGAG 868 1421 UGUCGUCUU AUAGAGCG 869 1422 GUCGUCUUA UAGAGCGA 870 1424 CGUCUUAUA GAGCGAUA 871 1432 AGAGCGAUA GGAGAACG 872 1444 GAACGUGUU GGUCUGUG 873 1448 GUGUUGGUC UGUGGUGU 874 1457 UGUGGUGUA GCUUUGUU 875 1461 GUGUAGCUU UGUUUUUA 876 1462 UGUAGCUUU GUUUUUAU 877 1465 AGCUUUGUU UUUAUUUU 878 1466 GCUUUGUUU UUAUUUUG 879 1467 CUUUGUUUU UAUUUUGU 880 1468 UUUGUUUUU AUUUUGUA 881 1469 UUGUUUUUA UUUUGUAU 882 1471 GUUUUUAUU UUGUAUUU 883 1472 UUUUUAUUU UGUAUUUU 884 1473 UUUUAUUUU GUAUUUUU 885 1476 UAUUUUGUA UUUUUCUG 886 1478 UUUUGUAUU UUUCUGCU 887 1479 UUUGUAUUU UUCUGCUU 888 1480 UUGUAUUUU UCUGCUUU 889 1481 UGUAUUUUU CUGCUUUG 890 1482 GUAUUUUUC UGCUUUGA 891 1487 UUUCUGCUU UGAUGUAC 892 1488 UUCUGCUUU GAUGUACA 893 Table VII Table VII: Δ-9 desaturase HH ribozyme sequence nt. ribozyme sequences Seq.ID No. Location 13 AAGCGGCA CUGAUGA X GAA AGGGCGCG 894 21 GAACGAAC CUGAUGA X GAA AGCGGCAG 895 24 GAGGAACG CUGAUGA X GAA ACAAGCGG 896 25 CGAGGAAC CUGAUGA X GAA AACAAGCG 897 28 GCGCGAGG CUGAUGA X GAA ACGAACAA 898 29 AGCGCGAG CUGAUGA X GAA AACGAACA 899 32 GCGAGCGC CUGAUGA X GAA AGGAACGA 900 38 CUGGUGGC CUGAUGA X GAA AGCGCGAG 901 63 GAGAUUGG CUGAUGA X GAA AUGUGUGU 902 69 CCUCGCGA CUGAUGA X GAA AUUGGGAU 903 71 GCCCUCGC CUGAUGA X GAA AGAUUGGG 904 92 CCGCCGCA CUGAUGA X GAA ACCCUGCU 905 117 GGAGCCGG CUGAUGA X GAA AGCGCGGC 906 118 GGGAGCCG CUGAUGA X GAA AAGCGCGG 907 124 GGGAAGGG CUGAUGA X GAA AGCCGGAA 908 129 CCAAUGGG CUGAUGA X GAA AGGGGAGC 909 130 GCCAAUGG CUGAUGA X GAA AAGGGGAG 910 135 UGGAGGCC CUGAUGA X GAA AUGGGAAG 911 141 CCAUCGUG CUGAUGA X GAA AGGCCAAU 912 154 UUGAGGCG CUGAUGA X GAA AGCGCCAU 913 160 ACGUCGUU CUGAUGA X GAA AGGCGGAG 914 169 CAGAGCGC CUGAUGA X GAA ACGUCGUU 915 175 GAGAGGCA CUGAUGA X GAA AGCGCGAC 916 181 GGCGGGGA CUGAUGA X GAA AGGCAGAG 917 183 GCGGCGGG CUGAUGA X GAA AGAGGCAG 918 193 CGGGCGGC CUGAUGA X GAA AGCGGCGG 919 228 CGGCGACG CUGAUGA X GAA ACCUGCCG 920 229 ACGGCGAC CUGAUGA X GAA AACCUGCC 921 232 GCGACGGC CUGAUGA X GAA ACGAACCU 922 238 AUGGAGGC CUGAUGA X GAA ACGGCGAC 923 243 ACGUCAUG CUGAUGA X GAA AGGCGACG 924 252 AGACGGCG CUGAUGA X GAA ACGUCAUG 925 259 UUGGUGGA CUGAUGA X GAA ACGGCGGA 926 261 CCUUGGUG CUGAUGA X GAA AGACGGCG 927 271 UUAUUCUC CUGAUGA X GAA ACCUUGGU 928 278 UGGCUUCU CUGAUGA X GAA AUUCUCGA 929 288 GAGGAGCA CUGAUGA X GAA AUGGCUUC 930 289 GGAGGAGC CUGAUGA X GAA AAUGGCUU 931 293 CCUUGGAG CUGAUGA X GAA AGCAAAUG 932 296 CUCCCUUG CUGAUGA X GAA AGGAGCAA 933 307 UGGACAUG CUGAUGA X GAA ACCUCCCU 934 313 GUAACCUG CUGAUGA X GAA ACAUGUAC 935 319 GAAUGUGU CUGAUGA X GAA ACCUGGAC 936 320 UGAAUGUG CUGAUGA X GAA AACCUGGA 937 326 UGGCAUUG CUGAUGA X GAA AUGUGUAA 938 327 GUGGCAUU CUGAUGA X GAA AAUGUGUA 939 338 AAUCUUGU CUGAUGA X GAA AGGUGGCA 940 346 AAAAUUUC CUGAUGA X GAA AUCUUGUG 941 352 GACUUGAA CUGAUGA X GAA AUUUCAAU 942 353 CGACUUGA CUGAUGA X GAA AAUUUCAA 943 354 GCGACUUG CUGAUGA X GAA AAAUUUCA 944 355 AGCGACUU CUGAUGA X GAA AAAAUUUC 945 360 CAUCAAGC CUGAUGA X GAA ACUUGAAA 946 364 CAAUCAUC CUGAUGA X GAA AGCGACUU 947 Table VII nt. ribozyme sequences Seq.ID No. Location 371 UCUAGCCC CUGAUGA X GAA AUCAUCAA 948 377 AUUAUCUC CUGAUGA X GAA AGCCCAAU 949 383 CAAGAUAU CUGAUGA X GAA AUCUCUAG 950 386 CGUCAAGA CUGAUGA X GAA AUUAUCUC 951 388 UGCGUCAA CUGAUGA X GAA AUAUUAUC 952 390 GAUGCGUC CUGAUGA X GAA AGAUAUUA 953 398 UGGCUUGA CUGAUGA X GAA AUGCGUCA 954 400 ACUGGCUU CUGAUGA X GAA AGAUGCGU 955 409 CACUUCUC CUGAUGA X GAA ACUGGCUU 956 419 UGGCUGCC CUGAUGA X GAA ACACUUCU 957 434 CGGGAGGA CUGAUGA X GAA AUCCUGUG 958 435 CCGGGAGG CUGAUGA X GAA AAUCCUGU 959 436 UCCGGGAG CUGAUGA X GAA AAAUCCUG 960 439 GGGUCCGG CUGAUGA X GAA AGGAAAUC 961 453 AUCCUUCA CUGAUGA X GAA AUGCUGGG 962 462 CAUCAUGA CUGAUGA X GAA AUCCUUCA 963 463 UCAUCAUG CUGAUGA X GAA AAUCCUUC 964 464 UUCAUCAU CUGAUGA X GAA AAAUCCUU 965 475 AGCUCCUU CUGAUGA X GAA ACUUCAUC 966 476 GAGCUCCU CUGAUGA X GAA AACUUCAU 967 484 CGUUCUCU CUGAUGA X GAA AGCUCCUU 968 505 UCAUCAGG CUGAUGA X GAA AUUUCCUU 969 515 AACAAAAU CUGAUGA X GAA AUCAUCAG 970 516 AAACAAAA CUGAUGA X GAA AAUCAUCA 971 518 ACAAACAA CUGAUGA X GAA AUAAUCAU 972 519 AACAAACA CUGAUGA X GAA AAUAAUCA 973 520 AAACAAAC CUGAUGA X GAA AAAUAAUC 974 523 ACCAAACA CUGAUGA X GAA ACAAAAUA 975 524 CACCAAAC CUGAUGA X GAA AACAAAAU 976 527 UCCCACCA CUGAUGA X GAA ACAAACAA 977 528 CUCCCACC CUGAUGA X GAA AACAAACA 978 544 UCCUCGGU CUGAUGA X GAA AUCAUGUC 979 545 UUCCUCGG CUGAUGA X GAA AAUCAUGU 980 557 UGUUGGUA CUGAUGA X GAA AGCUUCCU 981 559 UAUGUUGG CUGAUGA X GAA AGAGCUUC 982 567 UAGUCUGG CUGAUGA X GAA AUGUUGGU 983 575 GUUAAGCA CUGAUGA X GAA AGUCUGGU 984 580 AGGGUGUU CUGAUGA X GAA AGCAUAGU 985 581 GAGGGUGU CUGAUGA X GAA AAGCAUAG 986 589 ACACCGUC CUGAUGA X GAA AGGGUGUU 987 598 UCAUCUCU CUGAUGA X GAA ACACCGUC 988 637 CUCGUCCA CUGAUGA X GAA ACAGCCCA 989 638 CCUCGUCC CUGAUGA X GAA AACAGCCC 990 680 GUUGAGCA CUGAUGA X GAA AUCACCAU 991 685 UACUUGUU CUGAUGA X GAA AGCAGAUC 992 693 GGUACAUA CUGAUGA X GAA ACUUGUUG 993 695 GAGGUACA CUGAUGA X GAA AUACUUGU 994 699 CAGUGAGG CUGAUGA X GAA ACAUAUAC 995 703 CUCCCAGU CUGAUGA X GAA AGGUACAU 996 719 CUGCCUCA CUGAUGA X GAA AUCCACCC 997 730 GUCUUCUC CUGAUGA X GAA AUCUGCCU 998 742 AGAUACUG CUGAUGA X GAA AUUGUCUU 999 743 AAGAUACU CUGAUGA X GAA AAUUGUCU 1000 747 CAAUAAGA CUGAUGA X GAA ACUGAAUU 1001 749 GCCAAUAA CUGAUGA X GAA AUACUGAA 1002 751 GAGCCAAU CUGAUGA X GAA AGAUACUG 1003 752 AGAGCCAA CUGAUGA X GAA AAGAUACU 1004 Table VII nt. ribozyme sequences Seq.ID No Location 754 CCAGAGCC CUGAUGA X GAA AUAAGAUA 1005 759 CCAUUCCA CUGAUGA X GAA AGCCAAUA 1006 770 AGUCCUAG CUGAUGA X GAA AUCCAUUC 1007 773 CUCAGUCC CUGAUGA X GAA AGGAUCCA 1008 785 AUAAGGAU CUGAUGA X GAA AUUCUCAG 1009 788 AAGAUAAG CUGAUGA X GAA AUUAUUCU 1010 791 ACCAAGAU CUGAUGA X GAA AGGAUUAU 1011 792 AACCAAGA CUGAUGA X GAA AAGGAUUA 1012 794 GAAACCAA CUGAUGA X GAA AUAAGGAU 1013 796 AUGAAACC CUGAUGA X GAA AGAUAAGG 1014 800 GUAGAUGA CUGAUGA X GAA ACCAAGAU 1015 801 UGUAGAUG CUGAUGA X GAA AACCAAGA 1016 802 GUGUAGAU CUGAUGA X GAA AAACCAAG 1017 805 GAGGUGUA CUGAUGA X GAA AUGAAACC 1018 807 AGGAGGUG CUGAUGA X GAA AGAUGAAA 1019 813 CUUGGAAG CUGAUGA X GAA AGGUGUAG 1020 816 GCUCUUGG CUGAUGA X GAA AGGAGGUG 1021 817 CGCUCUUG CUGAUGA X GAA AAGGAGGU 1022 834 GUGAGAUG CUGAUGA X GAA AGGUCGCC 1023 835 UGUGAGAU CUGAUGA X GAA AAGGUCGC 1024 838 CCGUGUGA CUGAUGA X GAA AUGAAGGU 1025 ...

Table X

The construct numbering	The target that is bombarded	The isolate that reclaims	Greenhouse system	The plant that produces
					????RPA85	????231	??????70	????13	????161
????RPA113	????292	??????82	????9	????116
					????RPA114	????244	??????35	????12	????152
????RPA115	????285	??????42	????11	????165
					????RPA118	????268	??????38	????10	????125
????RPA119	????301	??????67	????11	????135
					Altogether	????1621	??????334	????66	????854

Table I X: compare with the contrast blade, with the stearic acid level in the active and inactive ribozyme plant transformed blade

With the stearic acid in the active and inactive ribozyme plant transformed blade (per-cent) with the stearic total plant of the blade of certain level
				Stearic acid	Ribozyme activity person (428 plants of 35 systems)	The not active person of ribozyme (406 plants of 31 systems)	Contrast (122 plants)
??＞3％ ??＞5％ ??＞10％	???????7％ ???????2％ ????????0	??????3％ ???????0 ???????0	?????2％ ??????0 ??????0

Table X II derives from the heredity of high stearic acid characteristic in blade of high stearic acid plant hybridization

The heredity of high stearic acid in blade
				Hybridization	Has the stearic R1 plant of normal blade	Has the stearic R1 plant of high blade	Per-cent with plant of high stearic acid
RPA85-15.06x RPA85-15.12 RPA85-15.07 selfing RPA85-15.10 selfing OQ414 x RPA85-15.06 OQ414 x RPA85-15.11	?????6 ? ?????5 ?????8 ?????5 ?????6	????3 ? ????5 ????2 ????3 ????4	????33％ ? ????50％ ????20％ ????38％ ????40％

Table X III: embryo's generation callus, the comparison that the lipid acid of body embryo and zygotic embryo is formed

Tissue and/or substratum are handled	Lipid acid is formed					The lipid % of fresh weight
							??C16：0	??C18：0	??C18：1	??C18：2	???C18：3
	Embryo's generation callus	????19.4 ????+/- ????0.9	????1.1 ????+/- ????0.1	????6.2 ????+/- ????2.0	????55.7 ????+/- ????3.1							????8.8 ????+/- ????2.0	????0.4 ????+/- ????0.1
The body embryo of cultivation on MS+6% sucrose+10mM ABA						????12.6 ????+/- ????0.7	????1.6 ????+/- ????0.8	????18.2 ????+/- ????4.9	????60.7 ????+/- ????5.1	????1.9 ????+/- ????0.3	????4.0 ????+/- ????1.1
	Pollinate zygotic embryo after 12 days	????14.5 ????+/- ????0.4	????1.1 ????+/- ????0.1	????18.5 ????+/- ????1.0	????60.2 ????+/- ????1.5							????1.4 ????+/- ????0.2	????3.9 ????+/- ????0.6

Table X IV: GBSS activity, amylose content and the Southern analytical results of the nucleic acid system of selection

Be the active amylose content Southern of GBSS

(unit/mg starch) (%) RPA63.0283 321.5 ± 31.2 23.3 ± 0.5-RPA63.0236 314.6 ± 9.2 27.4 ± 0.3-RPA63.0219 299.8 ± 10.4 21.5 ± 0.3-RPA63.0314 440.4 ± 17.1 19.1 ± 0.8-RPA63.0316 346.5 ± 8.5 17.9 ± 0.5-RPA63.0311 301.5 ± 17.4 19.5 ± 0.4-RPA63.0309 264.7 ± 19 21.7 ± 0.1+RPA63.0218 190.8 ± 7.8 21.0 ± 0.3+RPA63.0209 203 ± 2.4 22.6 ± 0.6+RPA63.0306 368.2 ± 7.5 19.0 ± 0.4-RPA63.0210 195.1 ± 7 22.1 ± 0.2+

Claims (95)

1. enzymatic nucleic acid molecule with RNA nicking activity, wherein said nucleic acid molecule is regulated the expression of plant gene.

2. the enzymatic nucleic acid molecule of claim 1, wherein said plant is a monocotyledons.

3. the enzymatic nucleic acid molecule of claim 1, wherein said plant is a dicotyledons.

4. the enzymatic nucleic acid molecule of claim 1, wherein said plant is a gymnosperm.

5. the enzymatic nucleic acid molecule of claim 1, wherein said plant is an angiosperm.

6. the enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is the tup configuration.

7. the enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is hairpin structure.

8. the enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid molecule are that hepatitis Δ virus, I group intron, II are organized intron, VS nucleic acid or RNA enzyme P nucleic acid configuration.

9. each enzymatic nucleic acid among the claim 1-8, wherein said nucleic acid comprises 12 to 100 bases of the RNA that is complementary to said gene.

10. each enzymatic nucleic acid among the claim 1-8, wherein said nucleic acid comprises 14 to 24 bases of the RNA that is complementary to said gene.

11. the enzymatic nucleic acid of claim 6, wherein said tup comprise the dried II district of length more than or equal to 2 base pairs.

12. the enzymatic nucleic acid of claim 7, wherein said hair clip comprise the dried II district of length between 3 and 7 base pairs.

13. the enzymatic nucleic acid of claim 7, wherein said hair clip comprise the dried IV district of length more than or equal to 2 base pairs.

14. the enzymatic nucleic acid of claim 2, wherein said monocotyledons is selected from corn, paddy rice, wheat and barley.

15. the enzymatic nucleic acid of claim 2, wherein said dicotyledons is selected from canola, Sunflower Receptacle, safflower, soybean, cotton, peanut, olive, sesame, sepal distance flower spp, flax, Jojoba genera and grape.

16. the enzymatic nucleic acid of claim 1, wherein said gene related gene during to be the fatty acid biological in said plant synthetic.

17. the enzymatic nucleic acid of claim 16, wherein said gene are Δ-9 desaturases.

18. the enzymatic nucleic acid of claim 16 or 17, wherein said plant is selected from corn, canola, flax, Sunflower Receptacle, cotton, peanut, safflower, soybean and paddy rice.

19. the enzymatic nucleic acid of claim 1, wherein said gene are related genes in the starch biosynthesizing in said plant.

20. the enzymatic nucleic acid of claim 19, wherein said gene is that particle is in conjunction with starch synthase.

21. the enzymatic nucleic acid of claim 19 or 20, wherein said plant is selected from corn, potato, wheat and cassava.

22. the enzymatic nucleic acid of claim 1, wherein said gene are related genes in caffeine is synthetic.

23. the enzymatic nucleic acid of claim 22, wherein said gene is selected from 7-methylguanosine and 3-methyltransgerase.

24. the enzymatic nucleic acid of claim 22 or 23, wherein said plant is a coffee plants.

25. the enzymatic nucleic acid of claim 1, wherein said gene are the Nicotines in said plant produce in related gene.

26. the enzymatic nucleic acid of claim 25, wherein said gene is selected from N-methyl putrescine oxidase and putrescine N-methyltransferase.

27. the enzymatic nucleic acid of claim 25 or 26, wherein said plant is a tobacco plant.

28. the enzymatic nucleic acid of claim 1, wherein said gene is a gene related in the ripening of fruits of said plant.

29. the enzymatic nucleic acid of claim 28, wherein said gene are selected from ethene and form enzyme, pectin methyl transferring enzyme, Rohapect MPE, polygalacturonase, 1-1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and acc oxidase.

30. the enzymatic nucleic acid of claim 28 or 29, wherein said plant is selected from apple, tomato, pears, plum and peach.

31. the enzymatic nucleic acid of claim 1, wherein said gene is a gene related in the cyanidin(e) of said plant forms.

32. the enzymatic nucleic acid of claim 31, wherein said gene are selected from chalcone synthase, phenyl styryl ketone flavanone isomerase, phenylalanine ammonia-lyase, dehydrogenation flavonol hydroxylase and dehydrogenation flavonol reductase enzyme.

33. the enzymatic nucleic acid of claim 31 or 32, wherein said plant is selected from rose, Petunia, Chrysanthemum and mary bush.

34. the enzymatic nucleic acid of claim 1, wherein said gene are related genes in the lignin of said plant produces.

35. the enzymatic nucleic acid of claim 34, wherein said gene is selected from O-methyltransgerase, cinnyl coenzyme A: NADPH reductase enzyme and cinnyl alcoholdehydrogenase.

36. the enzymatic nucleic acid of claim 34 or 35, wherein said plant is selected from tobacco, white poplar, willow and pine tree.

37. a nucleic acid fragment, this fragment comprise the cDNA sequence of coding corn Δ-9 desaturase, wherein said sequence is represented by SEQ ID NO:1.

38. the enzymatic nucleic acid molecule of claim 17, any sequence that limits in the wherein said nucleic acid specificity ground cutting Table VI, wherein said nucleic acid is the tup configuration.

39. the enzymatic nucleic acid molecule of claim 17, wherein said nucleic acid cut any sequence that limits in the Table VIII specifically, wherein said nucleic acid is hairpin structure.

40. the enzymatic nucleic acid molecule of claim 38 or 39, this molecule are made up of one or more sequences that are selected from the group shown in Table VII and the VIII basically.

41. any sequence defined in the enzymatic nucleic acid molecule of claim 20, wherein said nucleic acid specificity ground cutting Table III A, wherein said nucleic acid is the tup configuration.

42. any sequence defined in the enzymatic nucleic acid molecule of claim 20, wherein said nucleic acid specificity ground cutting Table V A and VB, wherein said nucleic acid is hairpin structure.

43. the enzymatic nucleic acid molecule of claim 41 or 42, this molecule are made up of one or more sequences that are selected from the group shown in Table III B, IV, VA and the VB basically.

44. the enzymatic nucleic acid molecule of claim 41, this molecule are made up of the sequence that any limited among the SEQ ID NO:2-24 basically.

45. a vegetable cell, this cell comprise among claim 1-8,11-17,19-20,22-23,25-26,28-29,31-32,34-35,37-39, the 41-42 or 44 each enzymatic nucleic acid molecule.

46. transgenic plant and its filial generation, it comprises among claim 1-8,11-17,19-20,22-23,25-26,28-29,31-32,34-35,37-39, the 41-42 or 44 each enzymatic nucleic acid molecule.

47. expression vector, it comprises the nucleic acid of each enzymatic nucleic acid molecule among coding claim 1-8,11-17,19-20,22-23,25-26,28-29,31-32,34-35,37-39, the 41-42 or 44, and its coded system allows to express and/or discharge this enzymatic nucleic acid molecule in vegetable cell.

48. expression vector, it comprises the nucleic acid of each many enzymatic nucleic acid molecule among coding claim 1-8,11-17,19-20,22-23,25-26,28-29,31-32,34-35,37-39, the 41-42 or 44, and its coded system allows to express and/or discharge said enzymatic nucleic acid molecule in vegetable cell.

49. vegetable cell that comprises the expression vector of claim 47.

50. vegetable cell that comprises the expression vector of claim 48.

51. transgenic plant and its filial generation, it comprises the expression vector of claim 47.

52. transgenic plant and its filial generation, it comprises the expression vector of claim 48.

53. a vegetable cell, it comprises the enzymatic nucleic acid of claim 16 or 17.

54. the vegetable cell of claim 53, wherein said cell is a maize cell.

55. the vegetable cell of claim 53, wherein said cell is the canola cell.

56. transgenic plant and its filial generation, it comprises the enzymatic nucleic acid of claim 16 or 17.

57. the transgenic plant of claim 56 and its filial generation, wherein said plant is a maize plant.

58. the transgenic plant of claim 56 and its filial generation, wherein said plant is the canola plant.

59. vegetable cell that comprises the enzymatic nucleic acid of claim 19 or 20.

60. the vegetable cell of claim 59, wherein said cell is a maize cell.

61. transgenic plant and its filial generation, it comprises the enzymatic nucleic acid of claim 19 or 20.

62. the transgenic plant of claim 61 and its filial generation, wherein said plant is a maize plant.

63. the method that regulatory gene is expressed in plant, this method comprise the enzymatic nucleic acid molecule of using among the claim 1-8 each to said plant.

64. the method for claim 63, wherein said plant is a monocotyledons.

65. the method for claim 63, wherein said plant is a dicotyledons.

66. the method for claim 63, wherein said plant is a gymnosperm.

67. the method for claim 63, wherein said plant is an angiosperm.

68. the method for claim 63, wherein said gene are Δ-9 desaturases.

69. the method for claim 68, wherein said plant is a maize plant.

70. the method for claim 68, wherein said plant is the canola plant.

71. the method for claim 68, wherein said gene is that particle is in conjunction with starch synthase.

72. the method for claim 71, wherein said plant is a maize plant.

73. the expression vector of claim 47, wherein said carrier comprises:

A) transcription initiation region;

B) transcription termination region;

C) gene of at least a said enzymatic nucleic acid molecule of coding;

Wherein said gene is operably connected on said initiator and the said terminator in the mode that allows to express in said vegetable cell and/or discharge said enzymatic molecule.

74. the expression vector of claim 47, wherein said carrier comprises:

A) transcription initiation region;

B) transcription termination region;

C) open reading frame;

D) encode at least a kind of gene of said enzymatic nucleic acid molecule, wherein said gene is operably connected on 3 ' of said open reading frame-end;

Wherein said gene is operably connected on said initiator, said open reading frame and the said terminator in the mode that allows to express in said vegetable cell and/or discharge said enzymatic molecule.

75. the expression vector of claim 47, wherein said carrier comprises:

A) transcription initiation region;

B) transcription termination region;

C) intron;

D) gene of at least a said enzymatic nucleic acid molecule of coding;

Wherein said gene is operably connected on said initiator, said intron and the said terminator in the mode that allows to express in said vegetable cell and/or discharge said enzymatic molecule.

76. the expression vector of claim 47, wherein said carrier comprises:

A) transcription initiation region;

B) transcription termination region;

C) intron;

D) open reading frame;

E) gene of at least a said enzymatic nucleic acid molecule of coding, wherein said gene is operably connected on 3 ' of said open reading frame-end, and wherein said gene is operably connected on said initiator, said intron, said open reading frame and the said terminator in the mode that allows to express in said vegetable cell and/or discharge said enzymatic molecule.

77. the enzymatic nucleic acid of claim 1, wherein said plant is selected from corn, paddy rice, soybean, canola, clover, cotton, wheat, barley, Sunflower Receptacle, flax and peanut.

78. transgenic plant, this plant comprise the enzymatic nucleic acid molecule with RNA nicking activity, the expression of wherein said nucleic acid molecule regulatory gene in said plant.

79. the transgenic plant of claim 78, wherein said plant is selected from corn, paddy rice, soybean, canola, clover, cotton, wheat, barley, Sunflower Receptacle, flax and peanut.

80. the transgenic plant of claim 78, wherein said gene are that particle is in conjunction with starch synthase (GBSS).

81. the transgenic plant of claim 78, wherein said gene is Δ-desaturase.

82. the transgenic plant of claim 78, wherein said plant is transformed by microparticle bombardment, whisker or electroporation with Agrobacterium, DNA bag.

83. the transgenic plant of claim 82, wherein said the bag with DNA finished with particle gun by microparticle bombardment.

84. the transgenic plant of claim 78 or 82, wherein said plant comprises the selected marker that is selected from chlorosulfuron, Totomycin, bar gene, bromoxynil and kantlex etc.

85. the transgenic plant of claim 78 or 82, wherein said nucleic acid is operably connected on the promotor, and said promotor is selected from octopine synthetic enzyme, nopaline synthase, manopine synthetic enzyme, Cauliflower mosaic virus (35S); Ribulose-1,5-bisphosphate, the little subunit of 6-bisphosphate (RUBP) carboxylase (ssu), β-conglycinin, phaseolin promotor, napin, γ zein, sphaeroprotein, ADH promotor, heat-shocked, Actin muscle and ubiquitin.

86. being tup, hair clip, hepatitis Δ virus, I group intron, II, the transgenic plant of claim 78, said enzymatic nucleic acid molecule organize intron, VS nucleic acid or RNA enzyme P nucleic acid configuration.

87. the transgenic plant of claim 86, the enzymatic nucleic acid of the wherein said RNA of having nicking activity is encoded with monomer.

88. the transgenic plant of claim 86, the enzymatic nucleic acid of the wherein said RNA of having nicking activity is encoded with many bodies.

89. the transgenic plant of claim 78, the nucleic acid of the enzymatic nucleic acid molecule of the said RNA of the having nicking activity of wherein said coding is operably connected on the 3 ' end of open reading frame.

90. the transgenic plant of claim 78, wherein said gene is a native gene.

91. a rotaring gene corn plant, this plant comprises with 5 ' to 3 ' transcriptional orientation:

The promotor that function is arranged in said plant;

Double-stranded DNA (dsDNA) sequence of coded delta 9 genes of SEQ ID NO:1, wherein said dsDNA's transcribes the endogenous RNA that chain is complementary to said plant;

The terminator that function is arranged in said plant;

92. a rotaring gene corn plant, this plant comprises with 5 ' to 3 ' transcriptional orientation:

The promotor that function is arranged in said plant;

The coding particle of SEQ ID NO:25 is in conjunction with double-stranded DNA (dsDNA) sequence of starch synthase (GBSS) gene, and wherein said dsDNA's transcribes the endogenous RNA that chain is complementary to said plant;

The terminator that function is arranged in said plant;

93. the enzymatic nucleic acid molecule of claim 1, wherein said gene is a native gene.

94. the method that the regulatory gene of claim 63 is expressed, wherein said gene is a native gene.

95. the carrier of Figure 42, wherein said carrier is used for transformed plant cells.

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