CN1247569A - Transgenic plants with modified sterol biosynthetic pathways - Google Patents

Transgenic plants with modified sterol biosynthetic pathways Download PDF

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CN1247569A
CN1247569A CN97181954A CN97181954A CN1247569A CN 1247569 A CN1247569 A CN 1247569A CN 97181954 A CN97181954 A CN 97181954A CN 97181954 A CN97181954 A CN 97181954A CN 1247569 A CN1247569 A CN 1247569A
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smt
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W·D·内斯
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Pharmacia and Upjohn Co
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    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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Abstract

Plant phytosterol compositions are modulated in transgenic plants to confer resistance to insects, nematodes, fungi, and/or other environmental stresses, and/or to improve the nutritional value of the plants. Recombinant DNA molecules of the invention encode RNA or protein sequences capable of altering plant sterol profiles by affecting the expression or activity of sterol biosynthetic enzymes. The DNA molecules are transformed into plant cells and plants having altered sterol compositions are regenerated therefrom.

Description

The transgenic plant of sterol biosynthetic pathway have been changed
The present invention broadly relates to plant genetic engineering.More particularly, relate to the level and/or the activity of endogenous plant sterol component are operated, and the farm crop disaster that causes because of plant insect or other insects is reduced to bottom line, and/or improve plant nutrition and be worth as strategy.
Background of invention
Arid, severe cold, weeds and the various rugged environment factors such as biology of engulfing farm crop can both work the mischief to agriculture production.The traditional method of killing weeds and Parasites almost completely depends on chemical herbicide, agricultural chemicals and mycocide.Yet the widespread use of these agricultural chemicalses has caused drug-fast development.In fact, according to present report, insect can have been resisted the sterilant of the main kind of great majority, comprising organophosphate, chlorinated hydrocarbon and carbamate.
Sterol comprises the compound that a class is natural basically, and this compounds is essential by all eukaryotes to a certain extent.As shown in figure below, they have an identical tetracyclic sterid nuclei and a side chain.Some sterols play a part structural in cytolemma, and remaining is indispensable in the process of growing then.
Plant materials can produce the different plant sterol of kind more than 250 (Akisha etc., 1992).In single species (Zeamays (corn)), identified nearly 60 kinds sterol, (Guo etc., 1995).Yet insect, fungi and nematode and many other do not produce not synthetic again their required whole sterols of Parasites of sterol.On the contrary, they are by being that food satisfies self nutritional needs to sterol with the plant.This fact has been used to develop commercial agricultural chemicals, for example triazole, pyrimidine and diazonium sterol, and they play a role by the generation of disturbing sterol in the Parasites body.
Figure A9718195400061
Recently, molecular biological progress makes people to import the means of favourable proterties by genetic engineering in the plant materials.The pest-resistant proterties of existing several forms has been imported in the plant by genetic engineering method.For example, the transgenic plant of expressing the endotoxic foreign gene of coding bacillus thuringiensis (Bt) to have obtained to kill with it be the performance of the insect of food.Unfortunately, these class methods can only be resisted the specific insect to the intracellular toxin sensitivity effectively.Need alternate insect control strategy in agricultural industry, specifically these strategies can extensively be resisted numerous insect/pathogenic agent effectively always.
Summary of the invention
The present invention broadly relates to by plant being carried out genetically engineered and changes component, level and/or the metabolic method of sterol.This method can strengthen the natural insect-resistance of plant, drought resisting and winter resistance, and/or can improve nutrition/health care value of plant.
According to an aspect of the present invention, the recombinant DNA molecules that is provided comprises:
A promotor, it causes the generation of RNA sequence in the plant materials, this promotor is operably connected to
The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to
One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic.
Coding is in conjunction with elementary sterol and produce the dna encoding sequence of the enzyme of secondary sterol, can be that justice or antisense orientation are arranged.Therefore, can encode a kind of untranslatable rna molecule (for example, antisense or common inhibition) or a kind of protein molecule of dna molecular of the present invention.RNA of Chan Shenging or protein are target with the expression and/or the activity of sterol biosynthetic enzyme optionally like this, thereby influence endoxylophyte sterol component spectrum, produce desired change.
Therefore, according to a further aspect in the invention, thereby provide a kind of plant sterol component that changes to strengthen the method for plant to insect, nematode and rotten mould fungi resistant power.This aspect of the present invention is component and/or the distribution profile by change certain plants sterol, thereby improves insect-resistance and the disease resistance of plant.The many defectives of inherent when the method for this transgenic plant has overcome the use agricultural chemicals finally can be selected new pest-resistant mechanism because import the intravital allogenic material of plant.The present invention has not only kept the advantage in the agricultural chemicals use, and has overcome many weak points of agricultural chemicals simultaneously.At insect and the existing Basic Ways of pathogenic agent, the present invention has reduced to block the possibility that this pathway mechanisms is evolved.
In this respect, by increasing the following content that can not utilize sterol, change the sterol component in the plant: 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ 7-sterol, Δ 8-sterol, 14 Alpha-Methyl sterols, Δ 23 (24)-24-alkyl sterol, Δ 24 (25), 24-alkyl sterol or Δ 25 (27), 24-alkyl sterol.The alternate method is that change sterol component makes and wherein contains Δ 5The level of the sterol of group is lower.
Another aspect of the present invention relates to producing in plant can give plant drought resistance and cold resistant sterol.
Another aspect of the present invention relates to the sterol component spectrum that changes in the plant, improves the wherein level of norcholesterol sterol.
Typically, above-mentioned aspect of the present invention all is by expression that changes the enzyme relevant with sterol and/or active realization the, preferred S-adenosine-L-methionine(Met)-Δ 24-sterol methyltransgerase (SMT IAnd SMT II), C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase (cycloeucalenolto obtusifoliol isomerase), 14 Alpha-Methyl demethylases, Δ 8To Δ 7-isomerase, Δ 7-sterol-C-5-desaturase or 24, the 25-reductase enzyme.
Another aspect of the present invention relates to the transgenic plant that changed selected sterol levels, and the preparation of this plant comprises in the genome of recombinant DNA molecules importing vegetable cell of the present invention, selects to express the cell of this molecule then.Transgenic plant are born again from the vegetable cell that has transformed, and then contain the development of plants maturation of this recombinant DNA.Then the plant of expressing recombinant DNA is differentiated that selecting wherein, sterol component spectrum satisfies the plant of needs of the present invention and breeds.
The accompanying drawing summary
The following drawings constitutes a part of this specification sheets, writes at this to be described further aspect some of the present invention.One or more with reference to the following drawings, and the detailed description of the particular that provides in conjunction with this paper can have better understanding to the present invention.
The isotopic labeling that is to use that Fig. 1 provides is crossed the cofactor of substrate, the SMT enzyme is carried out the result of HPLC radiation counting B group and mass spectroscopy A group mensuration gained;
What Fig. 2 provided is six kinds of inhibitor that are used to measure the SMT enzyme;
What Fig. 3 provided is the activity of SMT in the seedling development process;
What Fig. 4 provided is the approach of sterol end product in the seedling development process;
What Fig. 5 provided is yeast SMT gene order (B group; SEQ ID NO:1) and the aminoacid sequence of deriving according to the expectation conservative region of highlighted demonstration (A group; SEQ ID NO:2);
What Fig. 6 provided is Arabidopsis SMT gene (B group; SEQ ID NO:3) and the aminoacid sequence of deriving (A group; SEQ ID NO:4);
What Fig. 7 provided is the ERG6 construct of expressing the cartridge clip preparation with pUC18cpexp;
What Fig. 8 provided is the sequence (SEQ ID NO:5) of yeast SMT gene.The sequence of underscore is as the primer of screening transgenic Fructus Lycopersici esculenti genomic dna; And
Fig. 9 provides is the resulting structures of measuring mealie noctuid plant sterol, and they are found to be available or can not utilize.
What Figure 10 (SEQ ID NO:6) provided is the nucleotide sequence and the aminoacid sequence of corn SMT gene.
The description plant sterol of illustrative embodiment
The plant sterol pathways metabolism is made up of the enzyme on ring-type steroid nucleus that acts on the Fourth Ring structure and the side chain.In high vascular plant, main path is from cycloartenol (I):
Figure A9718195400091
With Δ 5-24-alkyl sterol finishes, and what wherein be in the ascendance is Sitosterol (II), Stigmasterol (III) and campesterol (IV):
The number of alternative route is very big, is enough to make just to produce nearly 60 kinds or more kinds of different sterols in single plant.These alternative route are different because of the genetic program of tissue-specific and development-specific.
The biosynthetic inhibitor of sterol has been used to study the metabolism of sterol.These inhibitor comprise several commercial mycocides, the intravital sterol pathways metabolism of these mycocide blocking-up plant pathogenic fungis, thus suppress fungi growth.The step of following main metabolic pathway is determined by using metabolic poison.Main path is made up of 12 chemical conversions as described below.
In reaction 1, S-adenosine-L-methionine(Met)-sterol-C-24 methyltransgerase (SMT I) methyl group of catalysis is transformed into the C-24 of sterol side chain in the heart from cofactor S-adenosine-L-methionine(Met).The sterol of Cheng Huanhou is characterised in that the functional group of bearing conversion.
This is in two methyl conversion reactions first, and is the specificity step and the rate-limiting step of producing in the plant in the sterol approach.Different SMT enzymes, i.e. SMT II, then the catalysis cycloartenol is converted into Δ 23 (24)-24-alkyl sterol, cyclosadol (Guo etc., 1996).
Reaction 2 demethylating reactions that comprise on the C-4 position.This is first in several demethylating reactions on the steroid nucleus.
Figure A9718195400111
Reaction 3 comprises with cycloeucalenol-obtusifoliol isomerase (COI) opens cyclopropyl rings on C-9 (10) position, simultaneously formation pair keys on the C-8 position.
Figure A9718195400112
Reaction 4 is included in demethylation on the C-14 position, sloughs the methyl on the C-14 position and form two keys on the C-14 position.
Figure A9718195400113
Reaction 5 is by Δ 14Reductase enzyme catalysis.
Figure A9718195400121
Reaction 6 comprises Δ 8-to Δ 7The catalytic product Δ of-isomerase 7The reaction of group
Figure A9718195400122
Reaction 7 is to methylate for the second time on the sterol side chain C-24 position.Reaction is by SMT ICatalysis, identical with the enzyme that starts main path.
Figure A9718195400123
Reaction 8 comprises generation 4, the C-4 demethylase of 4-norsteroid.
Figure A9718195400131
Reaction 9 comprises Δ 5Desaturase generates a two key on the C-5 position of the ring of Fourth Ring structure.
React then 9 product in reaction 10 by Δ 7-reductase enzyme is removed the two keys on the C-7 position by catalyzed conversion.
Figure A9718195400141
Reaction 11 comprises the Δ that changes side-chain structure 24 (28)-to Δ 24 (25)-isomerase.(can believe if more favourable on the kinetics, this reaction will be directly just from reacting 5 product.)
Figure A9718195400142
Δ on the reaction 12:C-24 position 24 (25)Two keys produce Sitosterol (II) after being reduced by Stereoselective. Except the biosynthetic main path of sterol, have now found that a development program regulating the expression of SMT enzyme.Enzymology shows, exists two kinds of different SMT enzyme (SMT in corn IAnd SMT II), their expression depends on the stage of tissue and differentiation.Mainly contain 24-ethyl sterol in the blade (because of SMT IActivity produce), and mainly contain the 24-methylsterol in the leaf sheath (by SMT IIActivity produce).These sterols are that these two kinds different SMT enzymes and same initial substance are the product of cycloartenol reaction.
First kind of enzyme SMT IProduce Δ 24 (28)-methylene radical sterol, second kind of enzyme produces Δ 23 (24)-methylsterol (V).First kind of isomer produces a kind of sterol (being that insect, rotten mould fungi and nematode can utilize them to finish the life cycle of oneself) that utilizes.Second kind of isomer produces and a kind ofly can not utilize sterol (being that insect, rotten mould fungi and nematode can't utilize them to finish the life cycle of oneself).Therefore, it can suppress the expression of first kind of isomer so that cause the Δ that can not utilize 23 (24)-methylsterol is accumulated.
Figure A9718195400151
Therefore, the sterol of accumulating in the tissue contains two keys on the C-23 position (VI) and the methyl on C-24 position.
Figure A9718195400152
Recombinant DNA molecules:
In order on the sterol component, to obtain needed variation, the invention provides a kind of recombinant DNA molecules that can be used in the preparation transgenic plant.Recombinant DNA molecules of the present invention generally includes and can cause the promoter region that the RNA sequence produces in the plant materials, structural dna sequence dna and 3 ' end non-translational region.
The gene region that is called as " promotor " is regulated the process that DNA is transcribed into mRNA.One section base sequence is contained in this promoter region, and its guiding RNA polymerase is attached on DNA sense strand or the antisense strand, and is that template generates the corresponding mRNA chain of this sense strand complementary with the DNA sense strand.With DNA " expression " or " transcribing " that process that template generates mRNA is commonly called gene.
In recombinant DNA molecules of the present invention, be the allos reorganization usually between preferred promoter and the dna encoding sequence.With regard to promotor, term " allos " means that the dna encoding sequence in the recombinant DNA molecules of the present invention is not to derive from the same gene that this promotor is being connected originally.
The wide material sources of promotor, for example plant and plant virus.Selected concrete promotor should preferably such promotor in embodiment of the present invention, and promptly its caused expression amount is enough to make the variation of sterol distribution profile in the plant to be satisfied the demand.
Described many active promotors that have in plant materials in the document, they can be used for dna molecular of the present invention.These promotors comprise, for example, cauliflower mosaic virus (CaMV) 35S promoter (Odell etc., 1985), Scrophularia mosaic virus (FMV) 35S (Sanger etc. 1990), sugarcane bacilliform virus promoter (Bouhida etc., 1993), commelina yellow mottle virus promotor (Medberry and Olsewski 1993), come from ribulose-1,5-bisphosphate, photoinduction promoter (the Coruzzi etc. of the subunit that 5-bisphosphate carboxylase (ssRUBISCO) is little, 1984), paddy rice kytoplasm triose-phosphate isomerase (TPI) promotor (Xu etc. 1994), Arabidopsis adenine phosphoribosyltransferase (APRT) promotor (Moffatt etc., 1994), rice actin 1 gene promoter (Zhong etc. 1996), mannopine synthase and octopine synthase promoter (Ni etc. 1995).Various types of DNA construct of utilizing all these promotors to create are all expressed in plant materials.
Typically, recombinant DNA molecules also contains 5 ' end untranslated leader.This sequence can come from the selected promotor that is used for expressing gene, and if necessary, can modify so that increase the translation of mRNA this leader sequence.This 5 ' end non-translational region also can come from viral RNA s, suitable eukaryotic gene or synthetic gene order.
As following further discussion, the structural dna sequence dna of recombinant DNA molecules of the present invention will cause that sterol component spectrum produces the variation that suits the requirements in the plant.
3 ' end non-translational region of recombinant DNA molecules of the present invention can obtain from the range gene that vegetable cell can be expressed.For example, 3 ' of the nopaline synthase of often using is held non-translational region (Fraley etc. 1983), is derived from 3 ' the end non-translational region (Coruzzi etc. 1994) of pea ssRUBISCO and derive from 3 ' of soybean 7S seed storage protein gene and hold non-translational region (Schuler etc. 1982).3 ' end non-translational region of recombinant DNA molecules contains one section polyadenylation signal sequence, and it is to add adenylic acid (AMP) Nucleotide 3 ' terminal the going up of RNA in the intravital function of plant.
Recombinant DNA molecules of the present invention can comprise other adjusting sequence or its binding substancess of satisfying the demand well known by persons skilled in the art.For example, often be used to the production transgenic plant so that the intron sequences in the recombinant DNA molecules of raising expression level.The example that is suitable for the plant introne of expression of plants comprises: corn hsp70 intron, rice actin 1 intron, corn ADH 1 intron, Arabidopsis SSU intron, green winter Solanum EPSPS intron and other introns well known by persons skilled in the art.Conversion of plant and regeneration
Use any one suitable method to be inserted into double chain DNA molecule of the present invention in the genome of plant.Many kind of plant method for transformation have now been reported, comprising agrobacterium mediation converted, utilize liposome conversion, electroporation, can increase chemical conversion that dissociative DNA takes in, discharge dissociative DNA, utilize virus or pollen to transform or the like by microparticle bombardment.
After cell (protoplastis) transforms, selection for this step method of regeneration is not strict, for the host who comes from pulse family (clover, soybean, trifolium etc.), umbellate form section (Radix Dauci Sativae, celery, Selinum pastinaca etc.), Cruciferae (Caulis et Folium Brassicae capitatae, radish, Semen Brassicae campestris etc.), Curcurbitaceae (muskmelon and cucumber), Gramineae (wheat, paddy rice, corn etc.) and Solanaceae (potato, tobacco leaf, tomato, pepper), these methods all have suitable operation scheme.Dicotyledons transforms and the regenerated method mainly is to obtain transgenic plant by the use agrobacterium tumefaciens, and these methods have been used in many plant varieties, comprising cotton (United States Patent (USP) 5,004,863; United States Patent (USP) 5,159,135; United States Patent (USP) 5,518,908), soybean (United States Patent (USP) 5,569,834; United States Patent (USP) 5,416,011; Christou etc. (1988)), Btassica (United States Patent (USP) 5,463,174), peanut (Cheng etc. (1996)); Papaya belongs to (Yang etc. (1996)) and pea (Schroeder etc. (1993); (1990) such as De Kathen and Jacobsen) and the plant of other kinds.
In addition, the report that utilizes electroporation, particle bombardment and Agrobacterium to carry out the monocotyledons conversion has been arranged now.
Conversion and plant regeneration have been obtained success in following plant: Asparagus (Bytebier etc. (1987)), barley (Wan and Lemaux (1994)), corn (Rhodes etc. (1988); Gordon-Kamm etc. (1990); Fromm etc. (1990); Koziel etc. (1993); Armstrong etc. (1995)), oat (Somers etc. (1992); ), orchardgrass (Horn etc. (1988)), paddy rice (Toriyama etc. (1988); Battraw and Hall (1990); Christou etc. (1991); ), rye (Bryant etc. (1987)), sugarcane (Bower and Brich (1992)), fescue (tall fescue) (Wang etc. (1992)) and wheat (Vasil etc. (1992); Weeks etc. (1993)).
About the summary of Plant Transformation and/or regenerated method referring to following document: Richie and (1994) such as Hodges (1993) or Hinchee.Strengthen plant resistance to insect by changing the plant sterol component
In insect body, a series of plant sterols are tested the back find, the wherein many growths that can not support insect, promptly they can not utilize.These can not utilize sterol to comprise 9,19-cyclopropyl sterol.Further also confirmed new Δ 23 (24)-and Δ 24 (25)-alkene and Δ 25 (27)-alkyl sterol can not be supported growth and the maturation of insect.These experiments are carried out in bollworm (mealie noctuid) body, and these mealie noctuids are to remove experiment and what cultivate in the synthetic medium of his sterol with the sterol local official and form not containing.Have now found that: if can utilize sterol and can not utilize ratio between the sterol is 1: 9 or less than 1: 9, insect just can not normal development.In fact, even when ratio is 1: 1, also can be to the growth generation injurious effects of insect.
The metabolism of insect, nematode and rotten mould fungi is subjected to the restriction of main plant sterol utilizability.These insects can not utilize the sterol that has following groups: C-4 methyl, 9 β, 19-cyclopropyl or Δ 8Base.Say that further nematode and insect can not utilize 14-α methyl-sterol, and comprise some insects of lepidopteran, Diptera and Coleoptera, owing to the reason of machine-processed aspect in the body can not be utilized and has Δ 24 (25), Δ 23 (24)Or Δ 25 (27)C-24 alkyl sterol Deng group.Some insects can not utilize the disappearance Δ 5The sterol of group.As a result, the increase of these sterols provides harmful sterol from the channel of ingesting for insect in the plant.
When being expressed in dna molecular of the present invention in the transgenic plant, component/distribution that it will cause containing sterol in the plant changes.In a preferred embodiment, dna molecular causes accumulating of those sterols that can not be utilized by insect and other insects, resists these biological abilities thereby strengthened plant.This point can realize by many methods, for example, overexpression, antisense, suppresses etc. altogether.Typically, the target gene of dna molecular is the endogenous gene of those codases among the present invention, and these enzymes are selected from sterol biosynthetic pathway favourable on the kinetics.
In this embodiment preferably, the target inhibition is carried out in the biosynthetic enzyme genes expression and/or the translation of sterol.For example, this restraining effect can realize by following thinking: prepare antisense molecule, ribozyme or suppress the RNA molecule altogether with endogenous target gene complementary by making up dna molecular of the present invention.The working method that this genetic expression target suppresses be those skilled in the art well-known (overall understanding, referring to Bird etc., 1991; Schuch, 1991; Gibson etc., 1997).
A preferred inhibition target is S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase (SMT).By the construct orientation of an antisense or inhibition altogether is attached on the gene, suppress the expression of SMT effectively, thereby cause to utilize accumulating of sterol.
In order to change the component spectrum of sterol in the plant, also can be in this or other embodiment of the present invention other genes in the plant sterol path for transformation as target gene.Though preferred target gene depends on applicable cases and decides that method is the same, promptly expresses the RNA or a protein molecule that can change the sterol component by the expection mode.
Therefore, except SMT, the interior target of other preferred cells that can also cause sterol to change comprises:
(i) C-4 demethylase: this kind of enzyme participates in sloughing two methyl on the C-4 position and appears in the reaction 2 and reaction 8 of specification sheets part.Single protein is responsible for these two reactions.The blocking-up this kind of enzyme will cause cycloartenol, 24 (28)-methylene radical cycloartenols etc. 4, the accumulating of 4-dimethyl sterol or 24-lanostenol new sterols such as (structural formula 18 among Fig. 9).Can realize this point by this gene that suppresses in the plant.
(ii) cycloeucalenol-obtusifoliol isomerase (COI) and Δ 8To Δ 7-isomerase: these two kinds of enzymes appear in the reaction 3 and reaction 6 of route of synthesis.Known some mycocide can be blocked this two kinds of enzymes, causes 9 β, and 19-cyclopropyl sterol is accumulated.Known is the locust heteroplasia of food with these plants, cholesterol and ecdysteroids exhaustion in its body.If any one in these two kinds of enzymes of this explanation is disturbed or be suppressed, plant sterol all can change so that they do not support the growth (Coste etc., 1987) of insect.
(iii) C-14 demethylase: this is the reaction 4 in the route of synthesis.There are several mycocides and plant-growth regulator can block this reactions steps in fungi and the plant.In plant, this blocking-up causes normal Δ 5The exhaustion of-sterol and 9 β, 19-cyclopropyl sterol, 14 Alpha-Methyl sterol and Δs 8Accumulating of-sterol.These accumulate the intermediate that the sterol that gets off is main sterol approach.They also are to utilize sterol.Studies show that of chemical inhibitor, the plant of having accumulated these intermediates has tolerance to water and cold.Therefore, the activity by genetic manipulation inhibition this kind of enzyme also is a kind of useful strategy.
(iv) Δ 7-sterol-C-5-desaturase: this is the reaction 9 in the route of synthesis.Suppress this kind of enzyme and cause Δ 5Exhaustion of-sterol and Δ 7The increase of-sterol.Known some insect can not be Δ 7-sterol is metabolized to ecdysteroids.Therefore, Δ 7-sterol also becomes the approach that a generation can not utilize sterol intravital the accumulating of plant.And, Δ 7-sterol replaces the Δ in the plant membrane 5-sterol can't cause any morphologic change to development of plants.
(v) C-24 reductase enzyme: this is the final step that plant sterol transforms in the process that Sitosterol forms (reaction 12), and Sitosterol is a main Δ in the plant materials 5-sterol.The gene of interference or inhibition coding this kind of enzyme will cause
Δ 24 (25)Accumulating of-24-alkyl sterol, this sterol also can not utilize.
From yeast, be separated to now the gene of these preferred plant sterol biosynthetic enzymes of many codings, simultaneously those target genes just of the present invention (overall understanding, referring to Lees etc., 1997).Some such genes from plant, also have been separated to.For example, the SMT gene that from following plant, is separated to: soybean (Shi etc., 1996), Arabidopsis (Husselstein etc., 1996; Bouvier-Nave etc., 1997), tobacco and castor-oil plant (Bouvier-Nave etc., 1997) and corn (Grabenok etc., 1997).The plant sterol biosynthesis gene that other have been separated to comprises the Δ from Arabidopsis 7-sterol-C-5-desaturase (Gachotte etc., 1996) and from the cycloartenol synthase (Corey etc., 1993) of Arabidopsis.
Except existing these genes, use method known to those skilled in the art, can easily obtain the gene of coding sterol biosynthetic enzyme from the source of expection.For example, separate the homologous sequence in other sources as hybridization probe obtain the gene or the cDNA that are separated to from a certain source.Yet it should be noted, source regardless of the sterol biosynthesis gene in the construct with targetting, dna molecular of the present invention all should be to have actively in the plant of numerous species, and this paper has provided a successful real example that changes sterol component spectrum in the tomato with yeast ERG6 antisense constructs.
Preferably, be target with the enzyme in the following sterol metabolism: S-adenosine-L-methionine(Met)-Δ 24-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ 8-to Δ 7-isomerase, Δ 7-sterol-C-5-desaturase or 24, the 25-reductase enzyme.
Preferably, in the plant that produces according to the present embodiment, some can not utilize the content of sterol to increase, specifically, i.e. and 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ 7-sterol, Δ 8-sterol, 14 Alpha-Methyl sterols, Δ 23 (24)-24-alkyl sterol, Δ 24 (25), 24-alkyl sterol or Δ 25 (27), 24-alkyl sterol.The alternate method is to change the reduction of sterol component and wherein contain Δ 5The level of the sterol of group.
According to this embodiment of the present invention, being preferably it provides the farm crop of anti-insect performance to comprise: corn (Pyrausta nubilalis (Hubern)., mealie noctuid, meadow mythimna separata), paddy rice, Chinese sorghum, forest, potato, tomato (tomato hawkmoth) and brassica vegetable.
According to this embodiment of the present invention, being preferably it provides the farm crop of nematicide performance to comprise: soybean (soybean cyst nematode), tomato (root-knot eel-worm), sugar beet and pumpkin.
According to this embodiment of the present invention, being preferably it provides the farm crop of anti-fungal property to comprise: corn, paddy rice, wheat, Chinese sorghum, soybean (the root-rot epidemic disease is mould), Sunflower Receptacle, forest, fruit and berry, potato (late blight), tomato (late blight), sugar beet, pumpkin and brassica vegetable.As the plant sterol that reduces the cholesterol agent
Animal and human's class be studies show that plant sterol can reduce serum/or the content of total cholesterol of blood plasma and low-density lipoprotein (LDL) cholesterol (Ling and Jone, 1995).In this, the transgenic plant that changed sterol component spectrum help setting up a kind of sitotherapy and control cholesterol and preventing cardiovascular disease.
Structure specificity Research on effect to single plant sterol shows, is reducing on the cholesterol, and saturated plant sterol such as sitostanol is more more effective than unsaturated compounds such as Sitosterol.As if another constitutional features in action is the esterification of plant sterol.Some studies show that, are reducing on the serum ornitrol, and the effect of the forulic acid of Sitosterol, sitostanol or cycloartenol (ferrulate) ester is than corresponding free state sterol stronger (Meittinen and Vanhanen, 1994).
In diet, some natural origins of plant sterol are Rice pollard oil, zein fiber oil and soya-bean oil.Up to the present, rice bran and zein fiber are the abundantest sources of plant sterol.Soybean phytosterol is the byproduct of olein refining process.Allow these plants and other plant generate more nutritious plant sterol, these technology help to develop the emerging food that improves human health and trophic structure.
Therefore, in another embodiment, the present invention relates in transgenic plant, increase the sterol of norcholesterol.For example, use recombinant DNA molecules of the present invention, by antisense, suppress altogether or the ribozyme mediation suppresses the conversion of the cycloartenol in the seed that the expression of SMT suppresses growing, thereby cause this sterol to be accumulated in the seed oil.Otherwise, surpass (amount) for the content that increases Sitosterol and express the SMT gene.
The preferred farm crop that are used for this embodiment of the present invention comprise: Sunflower Receptacle, corn, soybean, Btassica oil grain and cotton.Strengthen the tolerance of plant by changing plant sterol to severe environment
Another embodiment of the invention derives from the following fact: some sterol is with to reduce cytolemma moisture permeability-related.Therefore,, should provide a kind of efficient ways to prevent the disaster that arid is brought, perhaps at least it is reduced to bottom line by operation to sterol.
Several of the biosynthetic chemical inhibitor of sterol studies show that: the plant after the processing shows the physiological response of secondary, comprising the tolerance (Fletcher, 1988) to severe environment such as arid and frosts.The generation of these physiological responses mainly causes owing to hormonal readinesses such as dormin raise.Yet the variation of after birth flowability also is considered to tolerate the reason (Steponkus, 1984) of severe environment.
The after birth flowability is subjected to the control of Several Factors, such as, there are ratio in the kind of sterol and lipid acid and the two in after birth.Up to now, the kind of sterol is a most important factor in these factors.The major function of sterol is the mobile sudden change that takes place of buffering after birth.They can more specifically influence the activity with membrane-bound enzyme.By using inhibitor can destroy the biosynthesizing of sterol, cause the exhaustion of last sterol and accumulating of intermediate eventually, thereby the after birth function is changed.
Evidence suggests that the biosynthesizing meeting that suppresses sterol in the plant materials causes the closure (Hareuser, C. etc. 1990 " plant physiology magazine " are 137:201-207 (J.PlantPhysiol.)) of rising of dormin level and pore.How not clear this approach is mediated at present.But many documents prove, change plant sterol and can cause plant to tolerate several severe environment, this point maximum may be that rising by the dormin level mediates.In addition, use in the research of sterol biosynthesizing chemical inhibitor at all these, those are confirmed as the sterol that can not utilize by the present invention to the sterol of accumulating just, just, and 9 β, 19-cyclopropyl sterol, 14 Alpha-Methyl sterol and Δs 8-sterol.Therefore, the present invention generates by the range gene operation strategy can not utilize sterol, not only can avoid the infringement of insect and pathogenic agent by protective plant, and plant is resisted such as severe environment such as arid and severe cold.At this on the one hand, the preferred sterol that increases comprises Δ 5-24 alkyl sterols, 24-methyl courage steroid-5 for example, 23-dienol and cycloartenol.
The preferred farm crop that are used for this embodiment of the present invention comprise: corn, wheat, paddy rice, Chinese sorghum, soybean, Btassica oil grain (Semen Brassicae campestris, canola), Sunflower Receptacle, palm, peanut, cotton, forest, fruit, berry, nut, potato, tomato, sugar beet, sugarcane, cucurbit (pumpkin, muskmelon, cucumber, watermelon, summer squash), brassica vegetable, clover, ornamental crop, turfgrass, peanut, tealeaves and coffee.
The following examples are used for illustrating the preferred embodiments of the invention.One skilled in the art would recognize that the technology that on behalf of the inventor, disclosed technology can disclose in the following example, doing like this is in order to implement the present invention better, thereby can be considered to implement optimal way of the present invention.Yet, content disclosed according to the present invention, those skilled in the art should reckon with, can do many changes and still not broken away from the similar or identical result of the spirit and scope of the invention disclosed particular.Except that specified otherwise is arranged, all technology of using in that discussed in the above-mentioned part of specification sheets and the following example can both operate according to the general method in standard molecular biology well known to those skilled in the art and the biological chemistry (example of particular content referring to, Sambrook etc., 1989).
EXAMPLE Example 1. plant sterols
Extracting goes out the sterol isomer from cotton, and is separated into the form of homogeneity with chromatography.With not identifying new plant sterol for the side chain that insect utilizes.
By mass spectroscopy and 1H and 13C nucleus magnetic resonance (NMR) experiment finds that sterol has structure specificity (table 1) (Guo etc., 1995).
Initial studies show that, 4 age in days corns can produce monoalkylation on the C-24 position or two alkylations sterol.Why corn can generate those sterols, is because isolating 24 (28)-methylene radical and 24 (28)-ethylidene sterols are to obtain from the seedling tissue of corn, and the nuclear magnetic resonance spectrometry of its structure by quality and proton obtained definite.
Table 1
The sterol component of corn
MS aTLC aSterol BdPlant origin c
(M +) (Rf) cycloartenol 426 0.29 st, c, g, r, sh, b,
P24 (28)-methylene radical-ring wood 440 0.29 st, c, g, r, sh, b, pineapple alkanol pCyclosadol 440 0.29 st, g, sh Cyclolaudenol 440 0.29 st cycloartanes alcohol * 428 0.29 sh24-methyl cycloartanol 442 0.29 g24 (28)-methylene radical 440 0.29 shparkeol* alpha-amyrins (triterpene) 426 0.29 st, c, g, r, sh, b beta-amyrin (triterpene) 424 0.29 st, c, g, r, sh, b4 α, 14 alpha-alpha-dimethyl ergots, 424 0.25 st, c, g, r, sh, b steroid-7,24 (28)-dienol lophenol 400 0.25 g, sh24-methylene radical 4-methyl-7-alkene 412 0.25 c, g, r, sh, b, p, Dihydrocholesterol I24-methyl 4-methyl-7-alkene courage 414 0.25 g, sh stanols 24-ethyl group 4-methyl-7-alkene 428 0.25 g Dihydrocholesterol cycloeucalenols 426 0.25 c, g, r, sh obtusifoliol 426 0.25 c, g, r, sh, b, lanosterol falls in p dihydro obtusifoliol 428 0.25 sh31-
412 0.25 sh (norlanosterol) *, 4 Alpha-Methyl ergot steroids-
412 0.25 b8,24 (28)-dienol *, 4 Alpha-Methyl ergot steroid-412 0.25 c, g, sh7 (E)-23-dienol 4 Alpha-Methyl ergot steroid-7 (Z)-412 0.25 sh23-dienol * α 1-Sitosterol 426 0.25 c, g, r, sh, b α 1-different Sitosterol * 426 0.25 sh4 α, 14 alpha-alpha-dimethyl ergots, 426 0.25 c, sh steroid-8 (E)-23-dienol 4 α, 14 alpha-alpha-dimethyls-Mai 426 0.25 sh angle steroids-8 (Z)-23-dienol * 4 α, 14 alpha-alpha-dimethyls-24-442 0.25 sh ethyl-courage steroid-8-enol * 4 α, 14 alpha-alpha-dimethyls-9,19-ring ergot steroid-23-alkene 426 0.25 c, sh alcohol 4 Alpha-Methyls-courage steroid-8 (9), 14 (15), 24 (28)-410 0.25 sh, three enol * courage steroids-5,22-dienol * 384 0.18 sh cholest-7-enol * 386 0.16 b courage steroids-8 (9)-enol * 386 0.18 b cholesterol 386 0.18 st, c, g, sh, b, p Dihydrocholesterol 388 0.16 st brassicasterols 398 0.18 st, sh
St, c, g, sh, b, r, 24-methylene radical-cholesterol 398 0.18 t, p ergot steroid-5 (E)-23-diene 398 0.18 st, c, g, sh, b, r alcohol Codisterol 398 0.18 st, sh ergot steroid-7 (E)-23-two-alkene 398 0.16 st, c, sh alcohol 24-methylene radical-courage steroid-7-alkene 398 0.16 st, c, sh, p alcohol 24-methylene radical-zymosterol 398 0.18 p campesterols 400 0.18 st, c, g, sh, b, r;
T, p24-table campesterol 400 0.18 st, c, g, sh, b, r, (epicampesterol) p ergot steroid-(E)-23-enol * * 400 0.16 sh14 Alpha-Methyl-courage steroid-7-alkene 400 0.16 sh alcohol * ergot steroid-7-enol 400 0.16 st, c ergot steroid-8 (9)-enol * 400 0.18 sh ergostanols 402 0.16 st, c, sh24 β-ethyl courage steroid-410 0.18 sh5,22,25-three enols 14 Alpha-Methyl ergot steroid-412 0.18 sh8,25-dienol * 14 Alpha-Methyl ergot steroid-412 0.18 sh8,24 (28)-dienol * beans steroids-7,25-dienol 412 0.16 sh beans steroids-8,25-dienol * 412 0.18 sh24 β-ethyls-courage steroid-5,25-412 0.18 st, sh dienol beans steroid-5,23-dienol 412 0.18 sh fucosterol 412 0.18 st, g, sh isofucosterol 412 0.18 st, c, g, sh, b, r;
T, p24-ethyl courage steroid-412 0.18 st, sh5,24 (25)-dienol avenasterols, 412 0.16 st, c, sh25-methyl-24-methylene radical-courage 412 0.18 sh sterol * Stigmasterol 412 0.18 st, c, g, sh, b, r,
T, p 7-Stigmastenol 412 0.16 c 22-Stigmastenols 414 0.16 st, sh14 Alpha-Methyl ergot steroid-8 414 0.18 sh (9)-dienol Sitosterol 414 0.18 st, c, g, sh, b, r,
t、 p
Stigmastanol 416 0.16 st, sh
aMS, mass spectroscopy; TLC, thin-layer chromatography
B*New corn sterol; The natural sterol that * is new
cSt, sprouting; C, cloeoptile; G, germ oil (germ oil);
Sh, sheath; B, blade r, root; T, fringe; P, pollen
dWhat provide is habitual title or systematic name
The biosynthetic process of sterol is analyzed with the precursor of definite sterol and the relation between product.Detecting the metabolic growth of sterol by more different corn tissues regulates.The result shows that the sterol in the blade mainly comprises 24-ethyl sterol, for example, and Sitosterol, and mainly contain the 24-methylsterol in the leaf sheath, for example, 24-methyl-courage steroid-5,23-dienol.
Four kinds of [3-that go out with the leaf sheath tissue culture that bleaches from 8 ages in days 3H] 24-methylsterol isomer absorb-catches experiment, and the result shows: Δ 24 (28)-methylene radical sterol and Δ 24 (25)-24-methylsterol is the precursor of 24 Alpha-Methyl sterols and 24 Beta-methyl sterols, and Δ 23 (24)-24-methylsterol and Δ 25 (27)-24-methylsterol is the end product of sterol approach.
The result shows, single S MT IEnzyme is responsible for that two ground beetles glycosylation reaction of catalysis, cycloartenol (initial substance of approach) and Δ 5The long response time step of the key the between-24-alkyl plant sterol (end product of approach) is exactly the methylation reaction step, and this reactions steps is subjected to the feedback regulation of 24-ethyl sterol.In plant-growth and the ripening process, SMT IEnzyme is being regulated kind and the amount that generates plant sterol from cycloartenol.This finds to contradict with the received viewpoint about 3-hydroxy-3-methylglutaric acid list acyl coenzyme A reductase enzyme (HMGR) usually.This enzyme catalysis step occurs in the initial stage of the isoprenoid approach that produces sterol, and this step is considered to the rate-limiting reaction in the plant sterol biosynthesizing.This discovery shows that the effect of HMGR only is that control carbon flows to into the sterol approach.
In the seedling development process after the seed imbibition, the research (Fig. 3 C and 3D) that microsome HMGR is active and microsome SMT enzymic activity is expressed shows: (1) SMT activity is synthetic with sterol and plant-growth is relevant; (2) when concentration is 100mM, Sitosterol and 24 (28)-methylene radical-cycloartanol all do not influence the activity of HMGR, and the activity of this explanation HMGR and the generation of plant-growth or sterol are irrelevant; (3) speed and the SMT of plant sterol conversion IEnzyme catalysis methylates for the first time and for the second time the activity of methylation reaction is relevant, and irrelevant with the activity of HMGR.
These results show, the sprouting early development after the seed imbibition, and the biosynthesizing of sterol is regulated by negative.The sterol of accumulating in the sprouting of 3 ages in days is by the sterol transhipment that originates from the seed.The seedling development of corn causes the plant sterol synthetic just to be regulated subsequently.The directed plant sterol approach that flows into of carbon stream: Δ 5-24-alkyl sterol synthetic speed should satisfy the synthetic ever-increasing needs of after birth.Cycloartenol and the relevant C-4 sterol that methylates is converted to Δ 5-end product.Guan Jian long response time step is methylating of cycloartenol during this time, first step of converting during just plant sterol synthesizes.
Fig. 4 has summarized under dark growth (dark-grown) condition seedling development and has become to generate favourable end product Δ on the kinetics in blade and the leaf sheath process 5The approach of-24-alkyl sterol.Generating the early stage of blade and leaf sheath process, the expression of SMT enzymic activity and the data of sterol-specific show, synthetic at least two kinds of different SMT enzymes: the SMT of corn IThe ensuing methyl of catalysis transforms and generates Δ 24 (28)-methylene radical sterol and Δ 24 (28)-ethylidene sterol, and SMT IIThen the catalysis methyl transforms and generates Δ 23 (24)-24-methylsterol.The evaluation of the required sterol of embodiment 2 plant-growths
The plant sterol that identifies among the embodiment 1 is tested the ability that it supports growth respectively.Owing to lack the plant sterol mutant that can be used for this research, thus under the condition that the sterol that embodiment 1 identifies exists culturing yeast sterol auxotroph GL-7, referring to above-mentioned (Li, 1996).Why this yeast mutants is used as model system, is because it can absorb sterol from substratum, the survey sterol is mixed the double-layer of lipoid and the propagation of after birth.The ergosterol of hormonal readiness exist and the condition that lacks under, measure the cell proliferation amount, ergosterol is main zymosterol.
Sterol can be classified according to the influence to process of growth.The sterol of activation growth comprises ergosterol.Do not influence under the prerequisite of cell growth rate, the sterol of being moved in after birth and the cellularstructure composition comprises cholesterol and Sitosterol (Nes etc., 1993).The enzymology of embodiment 3 sterol saccharases
For the zymetology basis of the sterol that identifies among the embodiment 1 is described, the microsome from 4 age in days corn seedlings is measured in conjunction with the sterol-specific of solubility SMT enzyme.We find that cycloartenol is preferred sterol acceptor when using microsome bonded enzyme system, and 24 (28)-methylene radical-lophenol is methylated generates 24 (28)-ethylidene-lophenol.Table 2 has been summed up from the solubility SMT enzyme of the corn seedling specificity to various sterol substrates.
Table 2
(S)-sterol-specific of adenosine-L-methionine(Met): D 24-sterol methyltransgerase substrates enzymes vigor is with respect to the cycloartenol methyl
(dpm/min) the % vigor cycloartenol 37 of Huaing, 515 100 (C1) lanosterol 24,384 65 (C1) parkeol 6, lanosterol 18 falls in 002 16 (C1) 31-, 757 50 (C1) 24-dehydrogenation pollinastanol 8,253 22 (C1) zymosterol 5,252 14 (C1) 4 Alpha-Methyl zymosterols 10,504 28 (C1) 14 Alpha-Methyl zymosterols 3,376 9 (C1) 3-deoxidation zymosterol BG 0 (C1) courage steroid-8-enol BG 0 (C1) 24 (28) methylene radical-4-methyl-7-3,800 10 (C2) alkene Dihydrocholesterol, 4 Alpha-Methyl ergot steroids-1,500 4 (C2) 8,24 (28)-dienol obtusifoliol BG 0 (C2) cycloeucalenol BG 0 (C2) ergot steroids-8,24 (28)-dienol BG 0 (C2) ergot steroids-7,24 (28)-dienol BG 0 (C2) ergot steroids-5,24 (28)-dienol BG, 0 (C2), 24 (28)-methylene radical-wooden spinach BG 0 of ring (C2) trailing plants alkanol
In the microsome bonded lyoenzyme system that is studied, sterol is at relative joint efficiency (K m) on almost do not have difference.Apparent V at each substrate MaxBetween exist difference, but this point in the contemplation because the level of protein and total endogenous sterol changes at the enzyme dissolution process.Be similar to from the microsome of Sunflower Receptacle characteristic from the property class of the solubility SMT enzyme of 4 age in days corns in conjunction with the SMT enzyme.
Use from the lyoenzyme incubation cycloartenols of 4 age in days sproutings and [methyl- 3H]-AdoMet, and first methyl conversion reaction is described in proper order.In to corn, methylate in the research of mechanism operation [27 13C]-lanosterol is used to determine the mechanism that methylates (Guo etc., 1996) that generates 24 (28)-methylene radical sterols in the 4 age in days sproutings.
In the experiment of incubation cycloartenol or lanosterol, not that the sterol acceptor molecule is methylated and becomes second methyl product (Nes etc., 1991; Venkatramesh etc., 1996).If SMT is the protein of single kind, on this kind of enzyme, have two binding sites so.
Corn SMT protein is the tetramer that the subunit that is 39kDa by 4 sizes forms.With the following step a kind of bifunctional sterol of partial purification from the corn sprouting that 4 ages in days bleach (SMT) enzyme that methylates:
(i) non-ionic detergent soluble particles body bonded SMT enzyme;
Protein after the (ii) gel-filtration fractional separation dissolving generates the active fraction of the about 156kd of apparent natural molecule amount; And
(iii) active fraction is carried out hydroxyapatite chromatography.
Two kinds of all about 200 times of copurifications of vigor that methylate.
Fig. 1 demonstrates HPLC radiation counting (Figure 1B) and mass spectroscopy (Figure 1A) 50 reaction product of collecting in the sample is measured the results of gained, collects samples for these 50 and comes from experiment with 24 (28)-methylene radical-lophenol analysis solubility SMT enzyme (4 age in days seedling).In this incubation process, proved from second methyl of 24 (28)-methylene radical-lophenol and transformed to 24 (28)-ethylidene-lophenol.Therefore, carry out successive first and second methyl conversion reaction from the suitable sterol acceptor molecule of the SMT enzyme catalysis of 4 age in days corn sproutings.
What table 3 provided is, a series of substrates and transition state analog are to the influence of first and second methyl conversion reaction.
Table 3
Substrate and transition state analog inhibitor are right
(S)-and adenosine-L-methionine(Met): Δ 24The influence of-sterol Methyl transporters enzyme activity.
Entry site transforms the K that methyl transforms with respect to first with respect to the individual methyl of second inhibitor (entry) i
The K of no.* i Campesterol 1 NA NA24 (28)-methylene radical-ring 2 20 μ M NA jackfruit alkanols 26,27-ring propylidene ring Ah 3 25 μ M NA collect pure 24-(R, S)-25-epimino-lanosterol 4 55nM 55 μ MZ-24 (28)-ethylidene-lophenol 5 NA 75 μ M Sitosterol 6 NA 100 μ M
* the structure among digitized representation Fig. 2
Use solubility SMT enzyme to detect various inhibitor (Fig. 2) from 4 age in days corn seedlings.Some inhibitor can not influence the vigor that methylates of two kinds of sterol substrates, and this statement of facts SMT enzyme has two binding sites.
Two successive of SMT catalysis are generated the transmethylase reaction of different substrates by coenzyme (S)-adenosine-L-methionine(Met): generate cycloartenol (Δ 24-4,4-dimethyl sterol) Km is that 20mM and Vmax are 4pmol/min/mg protein the time, and generates 24 (28)-methylene radical-lophenol (Δ 7, 24 (28)-4-monomethyl sterols) time Km and Vmax be respectively 11 μ M and 1pmol/min/mg protein.Therefore, cycloartenol is the preferred substrate of first methylation reaction, and 24 (28)-methylene radical-lophenol is the preferred substrate of second methylation reaction.Zymosterol (Δ 8,24-4-demethylation sterol) be the preferred sterol substrate of yeast SMT enzyme, it also is the rare sterol substrate of first methylation reaction simultaneously.
Substrate specificity and restraining effect studies show that in two binding sites on the SMT enzyme, I catalysis first methyl in site transforms and generates 24 (28)-methylene radical sterols; And II catalysis second methyl in site transforms generation 24 (28)-ethylidene sterols.
For example, Sitosterol (24 α-ethyl cholesterol) is the main end product that corn sterol is produced in the maize leaf tissue, and it suppresses second methyl and transforms (100 μ M K i), do not transform and do not influence first methyl; Campesterol (24 Alpha-Methyl cholesterol) all can not suppress first and second methyl conversion; The product of 24 (28)-methylene radical cycloartanols, a kind of cycloartanol transmethylaseization is not methylated; 24 (28)-methylene radical-cycloartanol suppresses first methyl and transforms (20 μ M K i), do not transform but do not suppress second methyl.26,27-ring propylidene cycloartenol not can be incorporated on the yeast SMT enzyme, and it is the strong competitive inhibitor of first methyl conversion reaction, and it can not influence second methyl conversion.
The inhibition that second alkylated reaction suppressed by the product from 24 (28)-ethylidene-lophenol, but that first methyl transforms is unaffected.Transition state analog 24-(R, S)-25-epimino-lanosterol is the similar K of 55nM with a size iValue suppresses first and second methylation reaction, demonstrates a kind of noncompetitive kinetics model.In initial enzyme-substrate interacted, it was the typical case of other plant SMT enzyme that the sterol characteristic of substrate appears, and promptly needs a nucleophilic group on C-3 and C-24 position.Coenzyme K mThis value that equals 5 μ M for first with second methylation reaction all be the same.Embodiment 4 is from zymic SMT gene
Zymic SMT gene ERG6 is from zymic ERG6 genomic fragment pRG 458/erg6 (Fig. 5 B; SEQ ID NO:1).
ERG6 gene with the escherichia coli expression clone.Enzymology shows that recombinant protein is a sterol biological methylation enzyme, and this research proves that also this recombinant protein is similar to naturally occurring enzyme in the yeast body aspect dynamics.Different with the preferred cycloartenol of plant SMT, yeast SMT is a kind of Δ as the zymosterol of substrate preferably 24-4-demethylation sterol.
In intestinal bacteria successfully behind this active protein of overexpression, the monomeric molecular weight of yeast SMT is confirmed as 43KD with PET23a (+) carrier that has the T7 promotor.The protein of testing overexpression with Coomassie blue stain and western blotting is presented on the SDS-PAGE gel, and what use in the western blotting experiment is yeast SMT polyclonal antibody.This recombinant protein of now having used the said system purifying.
From yeast SMT aminoacid sequence (Fig. 5 A that derives; SEQ IDNO:2), potential AdoMet binding motif is presumably first conserved regions (YEYGWGS) that is identified among Fig. 5 A, and according to the mechanism analysis of the biological methylation of describing among the embodiment 3, it is binding site that tryptophane (W) is confirmed as.By to the ERG6 gene site-directed mutagenesis, and replace tryptophane with L-Ala.The sudden change DNA be cloned into PET23a (+) equally and in intestinal bacteria overexpression.Have under the active condition at wild-type protein, this mutein does not have activity.
Above-mentioned strategy provides a kind of SMT protein that imports inactivation in plant to change the method for plant sterol.The non-functional SMT monomer that imports can suppress the vigor of SMT, for example, influences cell and forms functional SMT enzyme complex, thereby cause utilizing the formation of sterol.For example, suppress first SMT IReactive behavior causes SMT IIActive catalytic generates the product Δ 23 (24)-24-alkyl sterol.On the contrary, suppress second SMTI reaction, will cause Δ 24 (25)The generation of-24-alkyl sterol.Embodiment 5 is from the SMT gene of Arabidopsis
The SMT gene of clone's Arabidopsis and order-checking (Fig. 6; SEQ ID NO:3).This gene of overexpression in intestinal bacteria, partial purification Arabidopsis SMT is also qualitative to it with concrete stereochemistry.
SMT gene by pcr amplification Arabidopsis from the cDNA library.The primer designs according to the full length cDNA sequence (registration number X89867) that extracts from gene pool.Amplified production is a total length Arabidopsis SMT gene, and then this gene product subclone is also checked order in the T/A cloning vector.Determine open reading frame (ORF) from this sequence data.On the ATG initiator codon, create a Nde I site by the directed mutagenesis of PCR mediation.According to the method for clone's ERG6 gene among the embodiment 4, initiating terminal is contained the total length ORF that a Nde I site and whole end contain a BamHI site be cloned in PET23a (+) carrier.The activity of recombinant protein can the two all change into alkylate (Tong etc., 1997) separately to cycloartenol and 24 (28)-methylene radical-lophenol respectively.When substrate is cycloartenol, only generate a product, i.e. SMT in 24 (28)-methylene radical-cycloartanol, Here it is Fig. 4 ICatalytic reaction.Because two kinds of sterol substrates of single gene product energy metabolism, this has just further determined the zymetology data among the embodiment 3.Say that further because the catalytic cycloartenol metabolism of recombinant plant SMT only generates a product, this product is SMT just IProduct, this illustrates that another product cyclosadol (structural formula 6 among Fig. 4) is the isomer (SMT by the different genes encoding of another one II) the catalysis generation.Embodiment 6 is from the SMT gene of corn
Separation corn sterol methyltransgerase (SMT) gene from commercial corn cDNA library (Stratagene, La Jolla, CA).(be equal to 5 * 10 with 5 milliliters of corn cDNA 7Pfu) make template by polymerase chain reaction (PCR) amplification SMT gene.Because the cDNA library makes up (Stratagene) in carrier Uni-ZapXR, thus the T7 sequence in the carrier as one in a pair of primer of pcr amplification reaction (3 ' end primer).5 ' end primer (2650-1) is to design according to 2～20 nucleotide sequences in disclosed one section SMT fragment of inferring in the gene pool (T23297).Requirement to specifications adds the Taq polymerase that 5 Promega companies of unit produce in 100 milliliters of cumulative volumes, 30 circulations are carried out in reaction.The PCR product of getting 1 milliliter of above-mentioned reaction carries out second as template and takes turns PCR, and wherein the primer is a T7 primer and according to the primers of 250～268 nucleotide sequences design among the T23297.During with the final reaction product of 1% agarose gel analysis, see that a size is the band of 1.3kb.Then this PCR band subclone is also checked order in plasmid pGEM-T (Promega).
In order to obtain 5 ' end SMT gene, use a pair of primer to carry out pcr amplification according to 2～20 and 366～349 nucleotide sequence designs among the T23297.Nucleotide fragments that to obtain one section size be 366bp and order-checking.This 366bp fragments sequence and above-mentioned 1.3kb clone overlapping 116 Nucleotide.These two fragment assemblies are in the same place by PCR with primer 2 650-1 and 3082-2.The primer in back is to design according to segmental 20 Nucleotide of the 1.3kb before the polyA sequence.These two PCR fragments of 366bp and 1.3kb as dna profiling.This SMT gene that rebuilds is connected among the PCR cloning vector pGEM-T and carries out two-way order-checking with ABI Prism automatic dna sequencer (Model 310).
Clone's SMT cDNA is 1497 Nucleotide, wherein has the coding region of 1032 Nucleotide, and 344 amino acid (Figure 10 can encode; SEQ ID No:6).Initiator codon ATG is positioned at the 66th～68 of nucleotide sequence.A terminator codon that is positioned at the 42nd～44 arranged in that this initiator codon (ATG) is preceding, the SMT gene that this explanation rebuilds contains 5 ' complete end.Have at 371 the Nucleotide place in terminator codon downstream one long be the polyA tail of 28 Nucleotide, the segmental 3 ' end of this explanation cDNA is complete.Therefore, this cDNA clone is the clone of the cDNA of total length.
Contain 344 amino acid, the polypeptide that size is 38.8KD of encoding from this cDNA clone deduced amino acid.Institute's deduced amino acid contains the conserved regions (Kagan and Clarke, 1994. " biological chemistry and biological physiology documents " are 310:417-427 (Arch.Biochem.biophys.)) of whole 3 identifications of methyltransgerase: the LDVGCGIGGP of 104-114 position (aminoacid sequence), the TLLDAVYA of 167-174 position and the VLKPGQ of 194-199 position.In addition, another conserved regions (SFYEYGWGESFHFA, Guo etc., 1997. " antimycotic sterol biosynthesis inhibitors " of assert by Nes of sterol methyltransgerase.In " subcellular biochemistry " 28 volumes: cholesterol: " its function and metabolism in biology and medical science ", Robert Bittman writes.Plenum Press, New York) is positioned at the 60-73 position.
With GCG system (Gap and Bestfit) the known SMT gene of corn SMT aminoacid sequence and other of being derived is compared.The corn SMT sequence (gene pool U79669) of SMT aminoacid sequence of deriving and independent separate has 93.6% similarity, has 88.1% homology and 78.8% identity with soybean SMT (gene pool U43683), has 93.9% homology and 88.3% identity with part wheat SMT sequence (gene pool U60754), have 58.8% homology and 39% identity with mouseearcress (gene pool X89867), have 66.5% homology and 50.4% identity with yeast SMT (gene pool X74249).High similarity between this cDNA clone and other kind plant SMT genes has confirmed that this cDNA clone is the full length cDNA clone of corn SMT.Say further, because people such as Grabenok have functionally expressed corn SMT gene in yeast expression system, and find to have only this a kind of 24-alkyl sterol of ergosterol, this laboratory isolating corn SMT gene catalysis that I am described is with a kind of stereoselective C-generation Δ that methylates 24 (28)Thereby, supported the viewpoint of the synthetic several different SMT enzymes of corn.
Can separate coding SMT with similar strategy IIThe cDNA of isomer.In fact, the cDNA fragment that is separated to aforesaid method should be SMT IAnd SMT IIThe representative of the two, this is because the conservative region between them can be used for designing primer.Embodiment 7 is from the SMT gene of Prototheca wickerhamii
The example of another preferred SMT gene is the SMT gene of Prototheca wickerhamii.This kind yeast algae produces Δ 25 (27)-24-methylsterol is as the primary product of transmethylase reaction.Preferred substrate is a cycloartenol.
P.Wickerhamii microsome preparation research has shown that the preferred substrate of SMT is a cycloartenol.But preferred product is not 24 (28)-methylene radical-cycloartenol but Cyclolaudenol (VII), and it is a kind of Δ 25 (27)-24-alkyl sterol, and be a kind ofly can not utilize sterol.
Generate the product Cyclolaudenol for this plant sterols of cycloartenol is transformed, clone SMT gene can promote this gene transformation in plant, and the generation of Cyclolaudenol can cause utilizing accumulating of sterol, i.e. Δ 25 (27)Accumulating of-24-alkyl sterol. The clone of Prototheca SMT
In being rich in the substratum of YPD (yeast extract-peptone-glucose) the Protothecawickerhamii cell cultures to logarithmic growth mid-term.Under the condition that adds Tri reagent, (be the product of Biospec, Bartlesville, OK) the granular sedimentary cell that breaks with the granulated glass sphere of 0.5mm and small-sized-microballon agitator (mini-Beadbeater).Require to separate high-quality whole-cell rna to specifications.
The reagent and the method that are provided in the test kit with GibcoBRL production are carried out 3 ' RACE (random amplification cDNA end) and 5 ' RACE to whole-cell rna.For 3 ' RACE, after the primer annealing that contains olig (dT) is attached to the RNAs that has poly (A) tail among the full RNA of fractional separation not and goes up, synthetic total cDNA under the effect of ThermoScript II.The above-mentioned RNA template of degrading is that template is carried out polymerase chain reaction (PCR) amplification with synthetic cDNA.The primer that the user provides " YEYGWG " (referring to following design of primers theory) is annealed on the cDNA, and 3 ' end towards gene extends under the guiding of Taq polymerase.Test kit provides limits the terminal primer annealing to that extends from 3 ' end to " YEYGWG " and section contains on the sequence of 3 restriction enzyme recognition site, and the sequence that this section contains restriction enzyme recognition site is a part that contains the initial primer of olig (dT).Second to take turns pcr amplification be for the cDNAs of enrichment corresponding to SMT 3 ' end half long sequence, wherein used primer to be second " nested " formula primer (GCGVGG) and test kit provide 3 ' hold primer.Another nested primers (" ATCHAP ") also is used in a similar fashion.
Whole-cell rna is carried out 5 ' RACE again.Synthesize cDNA with antisense primer " EWVMTDas " by ThermoScript II.Change cDNA by adding poly-Deoxyribose cytidine " tail " at 3 ' end with terminal deoxynucleotidyl transferase (TdT).Make template with this cDNA that has poly C tail, the primer that contains poly-G that provides with primer " EWVMTDas " and test kit carries out initial p CR reaction.On the basis of initial p CR reaction product, carry out second and take turns the PCR reaction, current the primer be nested primers " ATCHAPas " and test kit provide can be annealed to primer on the part poly-G sequence, contain restriction enzyme recognition site in that a part of poly-G sequence of institute's bonded.Current second purpose of taking turns the PCR reaction is enrichment SMT a 5 ' end cDNA sequence.
From gel, separate above-mentioned 3 ' RACE and 5 ' RACE pcr amplification product, and be connected among the plasmid pPCRII (Invitrogen).Change the clone who obtains behind the intestinal bacteria over to by the order-checking evaluation.In all DNA of plants with all contained an Apa I restriction site in the cerevisiae dna that checks order in the GCGVGG motif, this restriction site is cloned all existence in the two 3 ' cDNA clone and 5 ' cDNA.So just can constitute a complete full length coding region to 3 ' end, the half long sequence of SMT gene and the half long sequence montage of 5 ' end together.The design of primers theory
The first step of the primer that provides of design user is, the plant SMT that checked order and the peptide chain motif of the several extreme high conservatives among the yeast SMT are checked.Finding wherein to have can be by the short aminoacid sequence that extends of the dna sequence encoding of minimum number, and these codons only change the 3rd (degeneracy) base usually.The codon of finding according to people such as Wada uses table (" nucleic acids research " (Nucleic Acids Res.), the 19th volume, p1981,1991), be that three kinds of preferred codons of different yeast are present in the mixture of each amino acid whose degenerate codon, this point also meets the desired.Therefore, each primer of user's design all is the mixture of the deoxynucleotide of definition PCR product interior extremity.A requirement in addition is, 4 or 5 preferred all kinds of coupling are arranged in 63 ' the end deoxynucleotides of each primer, and wherein the content of G and/or C is greater than 50%.
Preceding 3 in the primer given below is sense orientation, can be annealed on the antisense DNA (with initial cDNA).The 4th and the 5th primer are the antisense primers that is annealed on the DNA sense strand of SMT gene.
YE[Y/F/W] GWG (yeast sequence the 81st～86 amino acids; Amino acid in the bracket is variable residue) be a part of the big conserved regions of SMT, primer " YEYGWG " is exactly according to this section sequences Design:
5’-TA[T/C]GA[A/G]T[A/G/T][T/G]GG[T/A/C]TGGGG-3’
(in the bracket is the position of degenerate core thuja acid)
The dna sequence dna of a part that is positioned at second conserved regions (GCG[V/I] GG) of the 129th～134 yeast amino acid residue by coding designs primer " GCGVGG ".The sequence of primer " GCGVGG " is:
5’-GGATG[T/C]GG[T/A][G/A]T[T/C]GG[G/C]GG-3’。
Primer " ATCHAP " is to design on the basis of the dna sequence dna of the 3rd conserved regions (yeast the 196th～203 amino-acid residue) of coding high conservative.Primer sequence is:
5’-GCCAC[A/G/T]TG[T/C]CA[C/T]GC[T/G/A]CC-3’。
Primer " EWVMDas " is the antisense primer that is used for synthetic article one cDNA chain in 5 ' the RACE experiment.It is to design on the basis of the little conserved regions that is positioned at yeast the 225th～231 amino-acid residue.The sequence of this primer is:
5’-TC[A/C/G]GTC[G/A]T[T/A/G][C/A][C/A]CCA[C/T]TC-3’。
Primer " ATCHAPas " is the nested antisense primer that is used for 5 ' RACE experiment, and its sequence is:
5 '-GG[T/C/A] GC[A/G] TG[G/A] CA[A/C/T] GTGGC-3 ' embodiment 8 is from the SMT gene of other plant
, as probe the cDNA library of interested any farm crop is screened with the SMT sequence of Arabidopsis cDNA or another kind of plant, isolate sizeable corresponding clone and order-checking.CDNA library construction and method for screening are that those skilled in the art are well-known.Just as described in example 6 above, according to the information of the conservative region of the known array of various SMT genes, can design suitable combination of primers at an easy rate.In order to determine the identity of this method institute cloned sequence, can and/or carry out external translation and biochemical Evaluation with them and known plants SMT enzyme sequence comparison.Embodiment 9 usefulness ERG6 DNA transform plant
For the transgenic plant of the sterol component spectrum that acquired change, a dna fragmentation that contains the open reading-frame (ORF) of isolated yeast SMT ERG6 gene from a genomic clone is identified (embodiment 4).Modifying ERG6 DNA with PCR method makes its arbitrary smearing at open reading-frame (ORF) contain the NcoI restriction site in the terminal sequence.This PCR method provides a kind of thinking for the phase shift mutation that realizes gene.This sudden change causes being transformed into endophytic ERG6 gene becomes the untranslated gene, but it can suppress endogenous tomato SMT by antisense or inhibition mechanism altogether, specifically adopts which kind of mechanism to decide on the character of construct.
ERG6 dna fragmentation after modifying is cloned into pUC18cpexp expresses in the cartridge clip carrier.It is the clone (Fig. 7) that the ERG6 DNA of justice and antisense strand is arranged that propagation has with respect to 35S promoter.
Digest these clones with HindIII and produce the ERG6 construct, this construct comprises 35S promoter and the terminator sequence that is positioned at ERG6 open reading-frame (ORF) both sides.To binary vector pJTS246, this carrier contains T-DNA edge recognition sequence and has the NPTII gene of kalamycin resistance HindIII digestion gained fragment cloning.
There is the clone binary vector of justice or antisense ERG6 construct to be transformed in the agrobacterium tumefaciens of cultivating altogether with the cotyledon of tomato (Solanum lycoperisum), the vegetable cell after obtaining to transform.From the calli that the selective medium that contains kantlex forms, produce transgenic plant.
Control plant (not having the fragment of insertion) and the blade of transgenic plant (the insertion fragment is arranged) are carried out transgenic analysis.From the leaf sample of the tomato plant of each transformant and the non-conversion of another one, extract DNA.With the A260 absorbancy DNA extraction liquid is carried out quantitative analysis.
The sample aliquot that contains 200ng DNA with each sample is made template, uses the Oligonucleolide primers pcr amplification ERG6 fragment corresponding to ERG6 sequence (the underscore part among Fig. 8).The control sample of PCR reaction comprises a sample that justice and antisense ERG6 are arranged that does not contain the sample of template DNA and contain binary plasmid.PCR is reflected under the nonstringent condition (annealing 2 minutes for 55 ℃ in each circulation) and carries out 20 circulations, then with each sample aliquot electrophoresis on 0.8% sepharose.
Select can the increase fragment (Fig. 8) of a 1100bp among the ERG6 DNA of such primer.The same with control plasmid, all regenerated transgenic Fructus Lycopersici esculenti plant (R 0) all have an above-mentioned fragment.Also exist non-specific amplification in the above-mentioned reaction, this is because nonstringent condition causes in the plant after non-target DNA chain appears at conversion and in the reference substance of non-conversion.Yet the level of these amplifications is well below the segmental level of amplification of target.This has further determined to exist ERG6 DNA in the tomato dna group.
Nonsaponifying lipid fraction in the blade material that the regeneration plant that regeneration plant that adopted construct transforms and antisense constructs transform is arranged is carried out Analysis of Sterol.Gained the results are shown in table 4.
Table 4
Sterol component in the tomato plant
(shared % in total sterol)
Contain ERG6 the anti-sterol control group of the ERG6 of containing is arranged
Insertion fragment cholesterol 29 18 20 cholest-7-enols of the insertion fragment justice of justice do not have 21 13 Stigmasterol, 25 22 24 Sitosterol, 26 27 24 isofucosterols, 20 12 19mg sterol/g fr.wt. 16 150 380
The result has confirmed that the ERG6 gene has been incorporated in the transgenic plant, and change has taken place the sterol component of transgenic plant.Detect and qualitative a kind of new sterol with mass spectroscopy, i.e. cholest-7-enol, this sterol does not exist in the tomato plant blade of contrast usefulness.
The scheme that imports new way by insertion yeast ERG6 gene in tomato plant is as described below:
Because the ERG6 gene has justice to insert fragment and antisense to insert fragment the two has all caused accumulating of cholest-7-enol (VIII), so endogenous SMT activity all may be suppressed in both cases.This will cause the carbon diverting flow to enter the metabolic substituting minor path of plant sterol, and the metabolic the first step of cycloartenol is by the two keys on the reductase enzyme reduction C-24 position in this approach.The sterol that generates is cycloartanol (IV), and cycloartanol will be as the form that generates cholest-7-enol in the main path through common demethylation, isomery, desaturation and reduction reaction then.This is a Δ 7-sterol, the two keys on its C-5 position disappear, and this just means that some insects can not utilize this sterol to finish their life cycle.
Regeneration plant (R 0) yield positive results.The results seed, and cultivated successfully plant (R of future generation 1).By the proteic ELISA experiment of NPT2 the single plant that above-mentioned seed grows up to is analyzed, detected alternative mark (NPT2) and whether exist.To from 6 R 153 plant of filial generation and non-transgenic plant carry out the check analysis of sterol component.The sterol component spectrum of these plant can be divided into 4 different groups or phenotype:
The R of the 4 class filial generations that table 5 is identified 1The mean value and the standard deviation (Std) of sterol in the plant (shared per-cent in total sterol).Phenotype 1234
Average
Std Std Std Std
The wooden spinach 1.14 1.46 6.80 6.61 2.17 2.12 2.00 2.00 trailing plants alkanols of value value value value sterol cholesterine 7.62 2.54 6.20 2.77 4.93 1.14 8.60 2.97 campesterols 4.17 3.15 16.60 11.24 4.50 1.95 6.60 4.83 stigmasterol, 13.14 3.13 12.80 5.26 8.86 1.41 22.60 1.14 sitosterol, 11.48 2.86 11.60 2.19 9.57 1.87 16.60 3.91 isofucosterols, 13.14 2.08 7.60 3.71 9.86 2.32 14.40 4.98b-armomadendrins, 12.52 3.90 9.75 5.91 10.36 3.95 8.80 1.79 cycloartenols, 31.76 5.67 31.60 4.72 49.36 4.91 28.80 6.9824 (28)-methylene-rings
Except that the non-transgenic plant is used in contrast, all negative R of NPT2 labeled test result 1Plant (so they also are not genetically modified segregants) also shows normal phenotype (phenotype 1).The R that NPT2 labeled test result is positive 1Plant (so they are genetically modified segregants) belongs in all 4 classes.Every kind of sterol is carried out statistics relatively (with the inverse sine conversion of sterol level of percent; Student-Neuman-Keuls experiment, 5% significance level), provided result's qualitative summary below:
The normal low high isofucosterol of the normal high normal stigmasterol of the normal normal low normal campesterol of comparison between each phenotype of table 6 sterol ( phenotype 2,3 and 4 is compared with normal phenotype 1) sterol phenotype 1 phenotype 2 phenotypes 3 phenotypes 4 cholesterine normally normal high normal 24 (28)-methylene of the normal cycloartenol of low normal beta-amyrin normal-the cycloartanol sitosterol is normal high
The distribution (be non-transgenic adjoining tree only be distributed in normal category, and transfer-gen plant be present in all 4 classifications) of plant in various classifications with antisense or suppress construct altogether to transform the predicated value obtain plant be consistent.Therefore, the inhibition of different levels causes a kind of expression of different levels of altered sterol phenotype between filial generation or in the filial generation.Therefore, these results with transform that the result of inhibiting ERG6 gene is arranged is consistent.More specifically, phenotype 2 and 3 has been accumulated intermediate, in this and the biosynthetic pathway sterol methyltransgerase for the first time or for the second time the part of methylated vigor be suppressed and match.Sitosterol and Stigmasterol (normal dead end product) level raises and contradicts with restraining effect, and this needs further research to explain.
Independent studies to a subgroup of these progeny plants has further been supported following hypothesis, promptly can observe the inhibition of SMT gene in the transgenosis pedigree.Below table 7 provides is the sterol component of non-conversion segregant and non-transgenic segregant.
Table 7
The sterol component of adjoining tree (non-transformed plant and non-transgenic segregant)
G55 (non-commentaries on classics G62 (non-commentaries on classics
The branch of the branch gene of the non-transforming gene of the inclined to one side plant sterol of average
Value difference
Ion) sterol cholesterol 18 13 13 14.7 2.9 Δs ion) 0-cholesterol-tr. 1 1.014-Alpha-Methyl-Δ 7--5 5 5.0 0.0 cholesterol Δ 7-cholesterol---14-Alpha-Methyl-Δ 8-311 1.7 1.2 cholesterol zymosterol 18--18.0 Δs 7,24-zymosterol 5--pure campesterol 283 4.3 3.2 desmosterols of 5.024-methylene radical-courage steroid-19 1 10.0 12.7 2-2.0 Δs 0-campesterol--1 1.0 Stigmasterol 18 20 25 21.0 3.6 Δs 0-Stigmasterol-tr. 1 1.0 Sitosterol 7 13 18 12.7 5.5 Δs 0-sitosterol--tr. isofucosterol 422 2.7 1.2 cycloartenols 7 19 29 18.3 11.024-methylene-ring Ah 14-tr. 14.0 village pure 24-methylene-4-first 1-tr. 1.0 bases-lathosterol obtusifoliol 1-tr. 1.0-representative does not detect; Tr. represent trace; N.D. represent uncertain.NSF at first carries out chromatography on the thin plate of thin-layer chromatography, wash-out goes out to correspond respectively to 4-demethylation, 4-monomethyl and 4 from the thin plate, and 3 bands of 4-dimethyl sterol index zone are done further detection by 3% SE-30 column chromatography and GC-MS then.The limit that detects is for containing the 0.1mg sterol in each leaf sample.
These sterols can compare with transgenic plant, and the sterol component of transgenic plant is listed in the table 8,9 and 10.
Table 8
Sterol component plant sterol G31 G32 G34 G35 G37 G38 G39 sterol cholesterol 12 10 8 10 8 11 8 Δs from the transgenic plant of G3 pedigree 0-cholesterine 1 tr. 111 tr. 114-Alpha-Methyls-Δ 7-cholesterine 3-----3 Δ 7-cholesterine-8 6 13 11 1-14-Alpha-Methyl-Δ 8-cholesterine 122----kryptosterol 10 5 12---8 Δs7,24-zymosterol 2-1---124-methylene radical-cholesterol-------campesterol 42 3--11 desmosterol------Δ 0-campesterol-------Stigmasterol 16 14 12 20 16 16 6 Δs 0-Stigmasterol 1--tr.---Sitosterol 15 9 12 10 8 16 6 Δs 0-sitosterol 1 tr.-----isofucosterol 4222211 cycloartenols 26 41 36 40 44 41 4124-methylene-cycloartenols 133444 424-methylene-4-methyl-7-alkene 232 tr. 46 tr. cholestanol obtusifoliols 111 tr. 23 tr.-representative does not detect; Tr. represent trace; N.D. represent uncertain.NSF at first carries out chromatography on the thin plate of thin-layer chromatography, wash-out goes out to correspond respectively to 4-demethylation, 4-monomethyl and 4 from the thin plate, and 3 bands of 4-dimethyl sterol index zone are done further detection by 3% SE-30 column chromatography and CC-MS then.The limit that detects is for containing the 0.1mg sterol in each leaf sample.
Table 9
Sterol component plant sterol G51 G52 G53 G54 G56 G57 G58 G59 G510 sterol cholesterol 13 56 11 16 11 4 15 5 Δs from the transgenic plant of G5 pedigree 0-cholesterine 11111 tr. tr. 1 114-Alpha-Methyl-Δ 7-131265241 cholesterine Δ 7-cholesterine---------14-Alpha-Methyl-Δ 8--1 tr. tr. 1 tr. tr. 11 cholesterine kryptosterols---------Δ7,24-kryptosterol---------24-methylene-cholesterine--3 4-1 6 6-campesterol, 8 15 421223 19 desmosterols---2----Δ0-campesterol-1------1 Stigmasterol 20 6 10 13 20 17 4 11 6 Δs 0-Stigmasterol--tr. tr.-tr.--1 Sitosterol 21 11 79983 11 1 Δs 0-sitosterol-tr. 1 tr.-tr. tr. 11 isofucosterols 111118121 cycloartenols 34 48 58 52 41 47 49 35 4324-methylene-ring Ah village 146511 28 10 14 pure 24-methylene-4-methyl 131 tr.-tr.-tr. 4-7-alkene cholestanol obtusifoliol tr. 11 tr.-tr. tr. tr. 1
-representative does not detect; Tr. represent trace; N.D. represent uncertain.NSF at first carries out chromatography on the thin plate of thin-layer chromatography, wash-out goes out to correspond respectively to 4-demethylation, 4-monomethyl and 4 from the thin plate, and 3 bands of 4-dimethyl sterol index zone are done further detection by 3% SE-30 column chromatography and GC-MS then.The limit that detects is for containing the 0.1mg sterol in each leaf sample.
Table 10
Sterol component plant sterol G63 G65 G66 G67 G68 G69 G610 sterol cholesterol 7798567 Δs from the transgenic plant of G3 pedigree 0-cholesterol tr. 111 tr. tr. 114-Alpha-Methyl-Δs 7-2251131 cholesterol Δs 7-cholesterol-------14-Alpha-Methyl-Δ 8-1111 tr. 11 cholesterol zymosterols-------Δ 7,24-zymosterol-------24-methylene radical-courage steroid 2 tr. tr.----campesterol 18 313 20 13 desmosterols-------Δ 0-campesterol tr.-tr. tr. 1--Stigmasterol 10 7 11 8567 Δs 0-Stigmasterol tr. tr. 1 tr. tr. tr. tr. Sitosterol 13 779847 Δs 0-sitosterol tr. 1 tr. 1 tr. tr. tr. isofucosterol 22111 tr. 2 cycloartenols 30 61 61 61 39 72 7024-methylene-ring Ah 12 836 20 71 village pure 24-methylene-4-first 2------base-lathosterol obtusifoliol 1------
-representative does not detect; Tr. represent trace; N.D. represent uncertain.NSF at first carries out chromatography on the thin plate of thin-layer chromatography, wash-out goes out to correspond respectively to 4-demethylation, 4-monomethyl and 4 from the thin plate, and 3 bands of 4-dimethyl sterol index zone are done further detection by 3% SE-30 column chromatography and GC-MS then.The limit that detects is for containing the 0.1mg sterol in each leaf sample.
These analysis revealeds are compared with control group, and the level of cycloartenol significantly raises in many transgenic plant.Following described as embodiment 10, the level of cycloartenol is met or exceeded the required level that can not utilize sterol of insect generation harmful effect.In addition, successfully suppress the true consistent of catalytic first methylation reaction of SMT in this result and the body.Embodiment 10 Heliothis zeas are to the utilization and the metabolism of sterol
With the sterol of several natural separation or the synthetic insect of feeding.A kind of body internal schema that is used for studying Heliothis zea is not contain sterol in the synthetic medium of cultivating Heliothis zea, and only add the experiment sterol in foodstuff.In this body internal schema, find that cycloartenol and several 24-methylsterols and 24-ethyl sterol isomer can suppress the growth of insect.
Two kinds of important corn sterols, i.e. 24-methylcholestane-5,23-dienol and 24-methyl courage steroid-5,25 (27)-dienol are to utilize sterol.With Δ 23 (24)-24-alkene sterol and Δ 25 (27)-24-alkene sterol isomer is the same, and 9,19-cyclopropyl sterol also is to utilize sterol.
With adding artificial diet that different sterols the add material Heliothis zea of feeding, study sterol structure and its with this and in insect body, utilize the relation of situation between the two.Set up an anosis colony of storing up with the Heliothis zea ovum.
In order to pinto is the main aseptic above-mentioned insect of storing up of feeding of foodstuff.Feed 10% sucrose of moth class.Culture temperature remains 27 ± 1 ℃, and other conditions comprise that Light To Dark Ratio is 14: 10 photoperiod, 40 ± 10% relative humidity and add the artificial foodstuff that different sterols add material.Be infected with the micro-cholesteric agar except that containing, experiment is not contain sterol with foodstuff.
Sterol is dissolved in the acetone.To not contain in the foodstuff of sterol in the equal portions solution adding mortar then, the material in the thorough mixing foodstuff, and allow organic solvent vapor away.With the concentration of 200ppm sterol is joined in the substratum and (to be equivalent to each experiment that an insect is housed) with adding the 1mg sterol in container.
During by 20 days, the Heliothis zea larvae development is to the final stage (the 6th age) of larvae development phase, and after this insect just can pupate.Single newborn larvae is put into experiment with culturing bottle, allow it grow 20 days.The record 20 days larvas fresh weight, length and slough off the phase.
In some experiments, allow larva continued growth 4 days again, with determine its whether fully nymphosis develop into moth.
When no longer adding sterol in the foodstuff, the newborn larvae of Heliothis zea just can not be casted off a skin and be developed into the 2nd instar larvae.Part in these insects can tolerate more than 15 days.
Sterols separated from the nonsaponifying lipidic component of being extracted by larva comprises long chain aliphatic alcohol in this sterol.In the chromatography of some forms, thereby these Fatty Alcohol(C12-C14 and C12-C18) can move the quantitative of interference sterol altogether with sterol, particularly to cholesteric quantitative.Therefore, in order to determine cholesteric identity and content in the insect, and need in the post of HPLC, inject the NSF of equal portions, and to carry out GC-MS mensuration with respect to cholesteric fraction.
Larva is failure to thrive on the substratum of sterol-free.Δ after being replaced by groups such as hydrogen, methylene radical, E-ethylidene or Z-ethylidene or a-ethyl or b-ethyls on the C-24 position of side chain 5-sterol, cholesterol, 24 (28)-methylenecholesterols, Sitosterol, isofucosterol, fucosterol, clionasterol and Stigmasterol are supported 6 later stages in age of larvae development to the.These sterols are defined as " can utilize " sterol (table 11 and Fig. 9).In each incubation period, the main sterol that reclaims from larva all is a cholesterol, and this shows that Heliothis zea is moving the typical 24-dealkylation sterol approach that insect has.
On the contrary, have the 3 β-cholesteric derivative of following groups: 9 β, paired dimethyl group on 19-cyclopropyl group, the C-4 position (for example, cycloartenol and lanosterol), Δ 8-key, the side chain that perhaps has following groups changes derivative: Δ 23 (24)-24-methyl or 24-ethyl, Δ 24 (25)-24-methyl or 24-ethyl or Δ 25 (27)-24 β-ethyl, they all can not satisfy the demand of Heliothis zea to sterol.These sterols are defined as " can not utilize " sterol (table 11 and Fig. 9).
The main sterol that is recovered in relying on the larva body that can not utilize sterol and grow is the experiment sterol that joins the substratum.With the cholesterol and 24 that is not listed as on year-on-year basis, 25-lanostenol (9/1～1/9 sterol mixture) is done competitive assay and is shown, when utilizing sterol in the sterol mixture and can not utilizing ratio (table 12) between the sterol less than 1 to 1 the time, the growth of Heliothis zea will be unusual.The absorption of sterol is relevant with the utilization of sterol and metabolic degree.
Table 11
Sterol is to Heliothis zea growth and metabolic influence
The sterol component 3(total
Enter portion growth total sterol when imitating 20 days
Percentage sterol source in the sterol
The position 1Should 2Mg/ insect in the length of time
Than) can utilize the inferior ts/ cholesterol of sterol cholesterol 1 100 6 56 cholesterol 24-(28) methyl-cholesterol 2 100 6 59 ((16/84)
Ts/ cholesterol fucosterol 4 100 6 71 ((10/90)
Ts/ desmosterol/courage isofucosterol 3 100 6 52 sterols (8/14/78) Sitosterol 5 100 6 66 ts/ cholesterol
((20/80) clionasterol ts/ cholesterol (50/50) 6 100 6 43 ((14/84) (75/2
5)
Ts/ desmosterol/courage stigmasterol 7 100 6 27 sterols (15/1/84) can not utilize the sterol courage to consolidate-8-enol 13 53 ND ND24-dehydrogenation pollen 14 53 0.6 ts/ cholesterine alkane sterol (86/14) 24-methyl courage steroid-ts/ cholesterine 5; 23-dienol 10 50 56 (80/20) 24-base courage steroid-ts/ cholesterine 5; 23-dienol 12 20 33 (86/14) 24-methyl courage steroid-ts/ cholesterine 5; 24-dienol 9531 (65/35) 24-ethyl cholestane-5,23-2 11 10 3 ND ND enol ts/ cholesterine clereosterol 8 20 33 (80/20)
Ts/7-dehydrogenation courage steroid ergosterol 15 30 35 alcohol/cholesterol
(36/41/23) structure of cycloartenol 17 53 ND ND lanosterol 16 53 ND ND24-dihydro wools 18 53 ND ND sterols 1 sterol is disclosed in Fig. 9.2 by long 30mm on average 20 cholesterol that day old larva is produced of heavy 323mg support the growth.
Usually, there are 16 to rely on cholesterol to survive to pupate and develop in 20 larvas
The moth that grows up.The growth effect of the sterol of surveying is exactly, when the journey that the cholesterol support is grown
Metric is decided to be at 100% o'clock, the degree of the sterol support of surveying growth and the relative value between it.
The formation of moth of pupating and grow up of 24-methyl courage steroid-5, the support of 23-dienol.Yet, this
A little insects show inborn deformity.Usually, can not utilize the insect that grows in the sterol
Weight is less than 100mg, and length is 2～15mm, lives for 6 in 12 larvas always and arrives
The 20th day.Behind 3 contents of removing in the insect gut, organize from insect with RP-HPLC and GC-MS analysis
The sterol that is separated in knitting.ND does not survey the total sterol of ts
The top efficiency that sterol is absorbed and mixes in the tissue is every insect 27～36mg, and minimum efficiency is every insect 0.6～6mg.These study explanation: (i) Heliothis zea has reduced the structural changes on sterol steroid nucleus and the side chain, (ii) plant sterol take off alkyl generate cholesteric approach have the regioselectivity of height and spatial selectivity and (iii) corn produced several and as herein describedly can not utilize sterol.
Table 12
Compare Heliothis zea to 24-lanostenol (can not utilize) with cholesterol (can utilize)
Utilize situation
Sterol component (total sterol when the growth of total sterol mixture portion of entering is imitated 20 days
The mg/ insect in the length of time that percentage in the sterol (ratio) position * answers
Than) can utilize sterol cholesterol 1 100 6 56 cholesterol (100%) (100%) cholesterol/24,25 2 cholesterol // 24,25 hydrogen lanosterol, 1,/18 100 6 45 lanostenol (90: 10) (93: 7) cholesterol/24,25 2 cholesterol // 24,25
1,/18 100 6 36 lanostenol hydrogen lanosterol (88: 12) (70: 30) cholesterol
Cholesterol/24,25/24,25 2 1,/18 70 6 25 lanostenol hydrogen lanosterol (75: 25) (50: 50) cholesterol cholesterol // 24,25/24,25 2 1,/18 30 3 12 lanostenol hydrogen lanosterol (50: 50) (30: 70) cholesterol/24,25 2 1,/18 10 3 ND ND hydrogen lanosterol (10: 90)
* the structure in the representative graph 9.
The required cholesteric minimum of larval growth and nymphosis is ingested concentration for testing with 0.01% of foodstuff.The cholesterol of this level can not be supported casting off a skin fast of larva as the cholesterol of higher level.But when cholesterol proportion in the foodstuff reached or is higher than 0.015%, the development rate of larva will be accelerated.Therefore, recipe sterol amount higher slightly (0.02%) guarantees that the sterol (existing separately or with mixture) of unrestricted amount can perhaps not add any sterol in contrast in the foodstuff from experiment with obtaining the foodstuff.
Micro-cholesterol is arranged in the useful larva body that can not utilize sterol to handle, be every insect 80～350ng according to its content range of disposition.These cholesterol are very big may to derive from cholesterol entrained in the ovum (we detect about 80ng cholesterol from every ovum) and to originally being present in the cholesteric absorption of the minor levels the agar.
Along with the increase of insect polypide, insect can be accumulating from ever-increasing cholesterol in the agar foodstuff.The cholesterol that this mode obtains can become the precursor of ecdysteroids.The efficient of these two pairs of isomer of Sitosterol/clionasterol and isofucosterol/fucosterol in supporting growth and generating cholesteric active metabolism is different, and the operation of this explanation 24-demethylation approach has stereoselectivity.
The growth result who the Heliothis zea larva is generated the moth class compares research.With can utilizing (cholesterol processing) sterol larva of feeding, and with utilizing (24-methyl courage steroid-5, the 23-dienol is handled) sterol other larvas of feeding.
Can not utilize major part in the insect that sterol feeds not grow and surpass for the 3rd age (table 11), this illustrates that they are invalid cholesterol substitutes, and harmful to growing.Can not utilize some nymphosis in the insect that sterol feeds and develop into moth.But, wing of these moths and leg incomplete development.
Table 11 and Fig. 9 show that the determining positions of two keys the growth course of being controlled by sterol in sterol side chain and the steroid nucleus.Courage is solid-and the 8-enol can not support that the phenomenon explanation Heliothis zea of growing can not be 9 β, and 19-cyclopropyl sterol changes into Δ 5-sterol.Block the generation that this approach will cause utilizing sterol.These results show that for the first time it is inappropriate replacing cholesterol with several sterols of corn synthetic.
According to content disclosed by the invention, do not need further experiment just can finish and implement open and claimed all components and the method for this paper.Component and method among the present invention are described at preferred embodiment, and obviously, those skilled in the art can make the variation that does not break away from inventive concept, essence and scope.More specifically, with chemically with physiology on all relevant reagent replace the reagent of description herein, the gained result is same or analogous, this point also is conspicuous.Those skilled in the art believe that obviously all these are similarly replaced or change all should fall within thought of the present invention, essence and the scope that is defined by the following claims.
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Sequence table
(1) general information:
(i) applicant: NES, DAVID W.
(ii) denomination of invention: the transgenic plant that changed the sterol component
(iii) sequence number: 6
(iv) address:
(A) addressee: ARNOLD, WHITE ﹠amp; DURKEE
(B) street: P.O.BOX 4433
(C) city: HOUSTON
(D) state: TX
(E) country: USA
(F) postcode: 77210-4433
(v) computer-reader form:
(A) medium type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: Patent In Release#1.0, version #1.30
(vi) the application's data:
(A) application number: US 60/033,923
(B) applying date: 1996.12.26
(C) classification:
(viii) lawyer/proxy's information:
(A) name: KAMMERER,
PATRICIA?A.
(B) number of registration: 29,775
(C) reference/registration number: MOBT148 (ix) telecommunications information:
(A) phone: 713/787-1400
(B) information (i) sequence signature of fax: 713/787-1440 (2) SRQ ID NO:1
(A) length: 1320 base pairs
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( xi ) SRQ ID NO：1：TTACTTTCGA TTTAAGTTTT ACATAATTTA AAAAAACAAG AATAAAATAA TAATATAGTA60GGCAGCATAA GATGAGTGAA ACAGAATTGA GAAAAAGACA GGCCCAATTC ACTAGGGAGT120TACATGGTGA TGATATTGGT AAAAAGACAG GTTTGAGTGC ATTGATGTCG AAGAACAACT180CTGCCCAAAA GGAAGCCGTT CAGAAGTACT TGAGAAATTG GGATGGTAGA ACCGATAAAG240ATGCCGAAGA ACGTCGTCTT GAGGATTATA ATGAAGCCAC ACATTCCTAC TATAACGTCG300TTACAGATTT CTATGAATAT GGTTGGGGTT CCTCTTTCCA TTTCAGCAGA TTTTATAAAG360GTGAGAGTTT CGCTGCCTCG ATAGCAAGAC ATGAACATTA TTTAGCTTAC AAGGCTGGTA420TTCAAAGAGG CGATTTAGTT CTCGACGTTG GTTGTGGTGT TGGGGGCCCA GCAAGAGAGA480TTGCAAGATT TACCGGTTGT AACGTCATCG GTCTAAACAA TAACGATTAC CAAATTGCCA540AGGCAAAATA TTACGCTAAA AAATACAATT TGAGTGACCA AATGGACTTT GTAAAGGGTG600ATTTCATGAA AATGGATTTC GAAGAAAACA CTTTCGACAA AGTTTATGCA ATTGAGGCCA 660 CATGTCACGC TCCAAAATTA GAAGGTGTAT ACAGCGAAAT CTACAAGGTT TTGAAACCGG720GTGGTACCTT TGCTGTTTAC GAATGGGTAA TGACTGATAA ATATGACGAA AACAATCCTG780AACATAGAAA GATCGCTTAT GAAATTGAAC TAGGTGATGG TATCCCAAAG ATGTTCCATG840TCGACGTGGC TAGGAAAGCA TTGAAGAACT GTGGTTTCGA AGTCCTCGTT AGCGAAGACC900TGGCGGACAA TGATGATGAA ATCCCTTGGT ATTACCCATT AACTGGTGAG TGGAAGTACG960TTCAAAACTT AGCTAATTTG GCCACATTTT TCAGAACTTC TTACTTGGGT AGACAATTTA1020CTACAGCAAT GGTTACTGTA ATGGAAAAAT TAGGTCTAGC CCCAGAAGGT TCCAAGGAAG1080TTACTGCTGC TCTAGAAAAT GCTGCGGTTG GTTTAGTTGC CGGTGGTAAG TCCAAGTTAT1140TCACTCCAAT GATGCTTTTC GTCGCTAGGA AGCCAGAAAA CGCCGAAACC CCCTCCCAAA1200CTTCCCAAGA AGCAACTCAA TAAATTCACT AGATCAATAA GATTCAAATA AAGCGCACGA1260TATATACCTA TTTTCCTATA TATGCAGATA AAAAGATAGC ACGTTCATTG CTAGCAGGCC1320 ( 2 ) SRQ ID NO：2 ( i )
(A) length: 383 amino acid
(B) type: amino acid
(C) chain:
(D) topological framework: line style (xi) SRQ ID NO:2 sequence chart: Met Ser Glu Thr Glu Leu Arg Lys Arg Gln Ala Gln Phe Thr Arg Glu1 5 10 15Leu His Gly Asp Asp Ile Gly Lys Lys Thr Gly Leu Ser Ala Leu Met
20 25 30Ser?Lys?Asn?Asn?Ser?Ala?Gln?Lys?Glu?Ala?Val?Gln?Lys?Tyr?Leu?Arg
35 40 45Asn?Trp?Asp?Gly?Arg?Thr?Asp?Lys?Asp?Ala?Glu?Glu?Arg?Arg?Leu?Glu
50 55 60Asp?Tyr?Asn?Glu?Ala?Thr?His?Ser?Tyr?Tyr?Asn?Val?Val?Thr?Asp?Phe65 70 75 80Tyr?Glu?Tyr?Gly?Trp?Gly?Ser?Ser?Phe?His?Phe?Ser?Arg?Phe?Tyr?Lys
85 90 95Gly?Glu?Ser?Phe?Ala?Ala?Ser?Ile?Ala?Arg?His?Glu?His?Tyr?Leu?Ala
100 105 110Tyr?Lys?Ala?Gly?Ile?Gln?Arg?Gly?Asp?Leu?Val?Leu?Asp?Val?Gly?Cys
115 120 125Gly?Val?Gly?Gly?Pro?Ala?Arg?Glu?Ile?Ala?Arg?Phe?Thr?Gly?Cys?Asn
130 135 140Val?Ile?Gly?Leu?Asn?Asn?Asn?Asp?Tyr?Gln?Ile?Ala?Lys?Ala?Lys?Tyr145 150 155 160Tyr?Ala?Lys?Lys?Tyr?Asn?Leu?Ser?Asp?Gln?Met?Asp?Phe?Val?Lys?Gly
165 170 175Asp?Phe?Met?Lys?Met?Asp?Phe?Glu?Glu?Asn?Thr?Phe?Asp?Lys?Val?Tyr
180 185 190Ala?Ile?Glu?Ala?Thr?Cys?His?Ala?Pro?Lys?Leu?Glu?Gly?Val?Tyr?Ser
195 200 205Glu?Ile?Tyr?Lys?Val?Leu?Lys?Pro?Gly?Gly?Thr?Phe?Ala?Val?Tyr?Glu
210 215 220Trp?Val?Met?Thr?Asp?Lys?Tyr?Asp?Glu?Asn?Asn?Pro?Glu?His?Arg?Lys225 230 235 240Ile?Ala?Tyr?Glu?Ile?Glu?Leu?Gly?Asp?Gly?Ile?Pro?Lys?Met?Phe?His
245 250 255Val?Asp?Val?Ala?Arg?Lys?Ala?Leu?Lys?Asn?Cys?Gly?Phe?Glu?Val?Leu
260 265 270Val?Ser?Glu?Asp?Leu?Ala?Asp?Asn?Asp?Asp?Glu?Ile?Pro?Trp?Tyr?Tyr
275 280 285Pro?Leu?Thr?Gly?Glu?Trp?Lys?Tyr?Val?Gln?Asn?Leu?Ala?Asn?Leu?Ala
290 295 300Thr?Phe?Phe?Arg?Thr?Ser?Tyr?Leu?Gly?Arg?Gln?Phe?Thr?Thr?Ala?Met305 310 315 320Val?Thr?Val?Met?Glu?Lys?Leu?Gly?Leu?Ala?Pro?Glu?Gly?Ser?Lys?Glu
325 330 335Val?Thr?Ala?Ala?Leu?Glu?Asn?Ala?Ala?Val?Gly?Leu?Val?Ala?Gly?Gly
340 345 350Lys?Ser?Lys?Leu?Phe?Thr?Pro?Met?Met?Leu?Phe?Val?Ala?Arg?Lys?Pro
355 360 365Glu?Asn?Ala?Glu?Thr?Pro?Ser?Gln?Thr?Ser?Gln?Glu?Ala?Thr?Gln
Data (i) sequence signature of 370 375 380 (2) SRQ ID NO:3
(A) length: 1420 base pairs
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( xi ) SRQ ID NO：3：CTCTCTCTCT CTCTCTCTTG GTCTTCCTCA CTCTTAACGA AAATGGACTC TTTAACACTC60TTCTTCACCG GTGCACTCGT CGCCGTCGGT ATCTACTGGT TCCTCTGCGT TCTCGGTCCA120GCAGAGCGTA AAGGCAAACG AGCCGTAGAT CTCTCTGGTG GCTCAATCTC CGCCGAGAAA180GTCCAAGACA ACTACAAACA GTACTGGTCT TTCTTCCGCC GTCCAAAAGA AATCGAAACC240GCCGAGAAAG TTCCAGACTT CGTCGACACA TTCTACAATC TCGTCACCGA CATATACGAG300TGGGGATGGG GACAATCCTT CCACTTCTCA CCATCAATCC CCGGAAAATC TCACAAAGAC360GCCACGCGCC TCCACGAAGA GATGGCGGTA GATCTGATCC AAGTCAAACC TGGTCAAAAG420ATCCTAGACG TCGGATGCGG TGTCGGCGGT CCGATGCGAG CGATTGCATC TCACTCGCGA480GCAACGTAGT CGGGATTACA ATAAACGAGT ATCAGGTGAA CAGAGCTCGT CTCCACAATA540AGAAAGCTGG TCTCGACGCG CTTTGCGAGG TCGTGTGTGG TAACTTCCTC CAGATGCCGT600TCGATGACAA CAGTTTCGAC GGAGCTTATT CCATCGAAGC CACGTGTCAC GCGCCGAAGC660TGGAAGAAGT GTACGCAGAG ATCTACAGGG TGTTGAAACC CGGATCTATG TATGTGTCGT720ACGAGTGGGT TACGACGGAG AAATTTAAGG CGGAGGATGA CGAACACGTG GAGGTAATCC780AAGGGATTGA GAGAGGCGAT GCGTTACCAG GGCTTAGGGC TTACGTGGAT ATAGCTGAGA840CGGCTAAAAA GGTTGGGTTT GAGATAGTGA AGGAGAAGGA TCTGGCGAGT CCACCGGCTG900AGCCGTGGTG GACTAGGCTT AAGATGGGTA GGCTTGCTTA TTGGAGGAAT CACATTGTGG960TTCAGATTTT GTCAGCGGTT GGAGTTGCTC CTAAAGGAAC TGTTGATGTT CATGAGATGT1020TGTTTAAGAC TGCTGATTGT TTGACCAGAG GAGGTGAAAC CGGAATATTC TCTCCGATGC1080ATATGATTCT CTGCAGAAAA CCGGAGTCAC CGGAGGAGAG TTCTTGAGAA AGGTAGAAAG1140GAAACATCAC CGGAAAAAGT ATGGAGAATT TTCTCAATTT GTTTTTATTT TTAAGTTAAA1200TCAACTTGGT TATTGTACTA TTTTTGTGTT TTAATTTGGT TTGTGTTTCA AGAATTATTA1260GTTTTTTTTT GTTTTGTTGC ATATGAGAAT CTTACTCTTG ATTTCTCCGC CGTAGAGCCG1320GCGAGACATA GGGGATTATT AGTATTTTTA AGTGTGTTTA AGATTGATTA ACAAGTTAGT1380AAAATAAAAT GTACTTAGGT GTCGAAAAAA AAAGGAATTC1420 ( 2 ) SRQ ID NO：4 ( i )
(A) length: 361 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: line style (xi) SRQ ID NO:4 sequence chart: Met Asp Ser Leu Thr Leu Phe Phe Thr Gly Ala Leu Val Ala Val Gly1 5 10 15Ile Tyr Trp Phe Leu Cys Val Leu Gly Pro Ala Glu Arg Lys Gly Lys
20 25 30Arg?Ala?Val?Asp?Leu?Ser?Gly?Gly?Ser?Ile?Ser?Ala?Glu?Lys?Val?Gln
35 40 45Asp?Asn?Tyr?Lys?Gln?Tyr?Trp?Ser?Phe?Phe?Arg?Arg?Pro?Lys?Glu?Ile
50 55 60Glu?Thr?Ala?Glu?Lys?Val?Pro?Asp?Phe?Val?Asp?Thr?Phe?Tyr?Asn?Leu65 70 75 80Val?Thr?Asp?Ile?Tyr?Glu?Trp?Gly?Trp?Gly?Gln?Ser?Phe?His?Phe?Ser
85 90 95Pro?Ser?Ile?Pro?Gly?Lys?Ser?His?Lys?Asp?Ala?Thr?Arg?Leu?His?Glu
100 105 110Glu?Met?Ala?Val?Asp?Leu?Ile?Gln?Val?Lys?Pro?Gly?Gln?Lys?Ile?Leu
115 120 125Asp?Val?Gly?Cys?Gly?Val?Gly?Gly?Pro?Met?Arg?Ala?Ile?Ala?Ser?His
130 135 140Ser?Arg?Ala?Asn?Val?Val?Gly?Ile?Thr?Ile?Asn?Glu?Tyr?Gln?Val?Asn145 150 155 160Arg?Ala?Arg?Leu?His?Asn?Lys?Lys?Ala?Gly?Leu?Asp?Ala?Leu?Cys?Glu
165 170 175Val?Val?Cys?Gly?Asn?Phe?Leu?Gln?Met?Pro?Phe?Asp?Asp?Asn?Ser?Phe
180 185 190Asp?Gly?Ala?Tyr?Ser?Ile?Glu?Ala?Thr?Cys?His?Ala?Pro?Lys?Leu?Glu
195 200 205Glu?Val?Tyr?Ala?Glu?Ile?Tyr?Arg?Val?Leu?Lys?Pro?Gly?Ser?Met?Tyr
210 215 220Val?Ser?Tyr?Glu?Trp?Val?Thr?Thr?Glu?Lys?Phe?Lys?Ala?Glu?Asp?Asp225 230 235 240Glu?His?Val?Glu?Val?Ile?Gln?Gly?Ile?Glu?Arg?Gly?Asp?Ala?Leu?Pro
245 250 255Gly?Leu?Arg?Ala?Tyr?Val?Asp?Ile?Ala?Glu?Thr?Ala?Lys?Lys?Val?Gly
260 265 270Phe?Glu?Ile?Val?Lys?Glu?Lys?Asp?Leu?Ala?Ser?Pro?Pro?Ala?Glu?Pro
275 280 285Trp?Trp?Thr?Arg?Leu?Lys?Met?Gly?Arg?Leu?Ala?Tyr?Trp?Arg?Asn?His
290 295 300Ile?Val?Val?Gln?Ile?Leu?Ser?Ala?Val?Gly?Val?Ala?Pro?Lys?Gly?Thr305 310 315 320Val?Asp?Val?His?Glu?Met?Leu?Phe?Lys?Thr?Ala?Asp?Cys?Leu?Thr?Arg
325 330 335Gly?Gly?Glu?Thr?Gly?Ile?Phe?Ser?Pro?Met?His?Met?Ile?Leu?Cys?Arg
340 345 350Lys?Pro?Glu?Ser?Pro?Glu?Glu?Ser?Ser
Information (i) sequence signature of 355 360 (2) SRQ ID NO:5
(A) length: 1320 base pairs
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( xi ) SRQ ID NO：5：TTACTTTCGA TTTAAGTTTT ACATAATTTA AAAAAACAAG AATAAAATAA TAATATAGTA60GGCAGCATAA GATGAGTGAA ACAGAATTGA GAAAAAGACA GGCCCAATTC ACTAGGGAGT120TACATGGTGA TGATATTGGT AAAAAGACAG GTTTGAGTGC ATTGATGTCG AAGAACAACT180CTGCCCAAAA GGAAGCCGTT CAGAAGTACT TGAGAAATTG GGATGGTAGA ACCGATAAAG240ATGCCGAAGA ACGTCGTCTT GAGGATTATA ATGAAGCCAC ACATTCCTAC TATAACGTCG300TTACAGATTT CTATGAATAT GGTTGGGGTT CCTCTTTCCA TTTCAGCAGA TTTTATAAAG360GTGAGAGTTT CGCTGCCTCG ATAGCAAGAC ATGAACATTA TTTAGCTTAC AAGGCTGGTA420TTCAAAGAGG CGATTTAGTT CTCGACGTTG GTTGTGGTGT TGGGGGCCCA GCAAGAGAGA480TTGCAAGATT TACCGGTTGT AACGTCATCG GTCTAAACAA TAACGATTAC CAAATTGCCA540AGGCAAAATA TTACGCTAAA AAATACAATT TGAGTGACCA AATGGACTTT GTAAAGGGTG600ATTTCATGAA AATGGATTTC GAAGAAAACA CTTTCGACAA AGTTTATGCA ATTGAGGCCA660CATGTCACGC TCCAAAATTA GAAGGTGTAT ACAGCGAAAT CTACAAGGTT TTGAAACCGG720GTGGTACCTT TGCTGTTTAC GAATGGGTAA TGACTGATAA ATATGACGAA AACAATCCTG780AACATAGAAA GATCGCTTAT GAAATTGAAC TAGGTGATGG TATCCCAAAG ATGTTCCATG840TCGACGTGGC TAGGAAAGCA TTGAAGAACT GTGGTTTCGA AGTCCTCGTT AGCGAAGACC900TGGCGGACAA TGATGATGAA ATCCCTTGGT ATTACCCATT AACTGGTGAG TGGAAGTACG960TTCAAAACTT AGCTAATTTG GCCACATTTT TCAGAACTTC TTACTTGGGT AGACAATTTA1020CTACAGCAAT GGTTACTGTA ATGGAAAAAT TAGGTCTAGC CCCAGAAGGT TCCAAGGAAG1080TTACTGCTGC TCTAGAAAAT GCTGCGGTTG GTTTAGTTGC CGGTGGTAAG TCCAAGTTAT1140TCACTCCAAT GATGCTTTTC GTCGCTAGGA AGCCAGAAAA CGCCGAAACC CCCTCCCAAA1200CTTCCCAAGA AGCAACTCAA TAAATTCACT AGATCAATAA GATTCAAATA AAGCGCACGA1260TATATACCTA TTTTCCTATA TATGCAGATA AAAAGATAGC ACGTTCATTG CTAGCAGGCC1320 ( 2 ) SRQ ID NO：6 ( i )
(A) length: 1497 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: line style (ix) feature:
(A) title/key: modification _ base
(B) position: 1419
(C) other information :/modify _ base=other/explain=" A or C
GT” ( xi ) SRQ ID NO：6：AGACTCTGGT TCTGACATGC AGCAATTATT GCAGGTGCAT TTGATCCGTC CCGGCCGCCT60ACACGATGTC CAAGTCGGGA GCGCTGGATC TTGCTTCTGG CCTCGGAGGG AAGATCAACA120AGGTGGAAGT CAAGTCGGCC GTCGATGAGT ATGAGAAATA TCATGGATAC TATGGAGGGA180AGGAGGAAGC AAGGAAGTCC AACTATACTG ATATGGTTAA TAAATACTAT GATCTTGCCA240CTAGCTTCTA TGAGTATGGT TGGGGTGAAT CCTTCCACTT TGCTCACAGA TGGAATGGAG300AATCCTTACG TGAAAGCATC AAGCGACATG AGCATTTTCT TGCCCTGCAA CTTGGTTTGA360AACCAGGAAT GAAGGTTTTA GATGTGGGCT GTGGAATAGG TGGACCACTG AGAGAAATTG420CAAGATTTAG CTCAACTTCA GTTACCGGAT TGAATAACCA CGAATACCAG ATAACCAGGG480GAAAGGAGCT CAACCGTTTA GCAGGAATTA GTGGAACATG TGATTTTGTC AAGGCGGACT540TCATGAAGAT GCCGTTCGAT GACACACTTT TGGATGCTGT TTACGCCATT GAGGCAACAT600GTCATGCACC TGATCCAGTT GGTTGCTACA AGGAGATATA TCGTGTGTTG AAGCCTGGCC660AGTGCTTTGC CGTGTACGAG TGGTGCGTTA CGGATCACTA TGATCCTAAC AATGCAACCC720ACAAAAGGAT CAAGGATGAA ATTGAGCTTG GCAATGGCCT GCCAGATATC AGAAGCACTC780CGCAATGTCT CCGGGCTCTA AAAGACGCCG GGTTTGACGT TGTTTGGGAT AAGGATCTTG840CTGAAGATTC TCCCTTGCCT TGGTACTTGC CCTTGGACTC CAGCCGATGC TCACTGAGTA900GCTTCCGTCG ACCTCCTGTC GGGACGCATG ATACCCGCAC AATGGTCAAG GCCCTGGAGT960ACGTTGGTCT TGCTCCGCAG GGCAGTGAGA GGTCTCTAGT TTTCCTGGAG AAGGCTGCAG1020AAGGGCTGGT AGAGGGCGGA AAGAAGGAGA TCTTCACGCC AATGTACTTC TTTTTTGTTC1080GGAAGCCTCT TCTGGAATGA GCTCTTGGAT CACCTTTTCA GAGAGAGAAG GCAAGTGGTC1140ATTTCGAAGA AGCCGAGGAG AGGGAACCTG GAATCAAGAA AACCTTCAGC TCTCCTGTGT1200AGGAGGAAAG TTAACGAACA GTGTAGTAAC TGTTCAGCTC TGTGTTTATT CAGTTGTTTT1260GCTGCTTGAG GTTATTCGTT TCTAGGTGGG GGTTGGAATC CTTTTCGCCA TAAACCTCTC1320AGTGGCATAA ATAAGATGGT TTGCATAAGA GTACTTCATG GATACCGTAA GGGCTACTAC1380TGAAAGAGAA ATGTTTAAGC AGCATGGTAT GTGAGCAANT AGTGATAATT ATTCCATCCT1440TTTTTTTAAT ATAAAGCAGG AGTTTTGTCA AAAAAAAAAA AAAAAAAAAA AAAAAAA1497

Claims (40)

1. a double chain DNA molecule comprises:
A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to
The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to
One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic.
2. the dna molecular in the claim 1, wherein said dna encoding sequence is a sense orientation.
3. the dna molecular in the claim 1, wherein said dna encoding sequence is an antisense orientation.
4. the dna molecular in the claim 1, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ 8-sterol, 14 Alpha-Methyl sterols, Δ 23, 24-alkyl sterol, Δ 24, 24-alkyl sterol and Δ 25 (27), 24-alkyl sterol.
5. the dna molecular in the claim 1, wherein said elementary sterol or secondary sterol lack Δ 5Group.
6. the dna molecular in the claim 1, the enzyme of wherein said dna encoding sequence encoding is selected from S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ 8To Δ 7-isomerase, Δ 7-C-5-desaturase and 24, the 25-reductase enzyme.
7. the dna molecular in the claim 1, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase (SMT).
8. according to the DNA in the claim 7, wherein said SMT is from plant or yeast.
9. according to the DNA in the claim 7, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.
10. according to the DNA in the claim 7, wherein said SMT is yeast ERG6.
11. transgenic plant that contain a double chain DNA molecule comprise:
A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to
The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to
One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic.
12. the plant in the claim 11, wherein said dna encoding sequence is a sense orientation.
13. the plant in the claim 11, wherein said dna encoding sequence is an antisense orientation.
14. the plant in the claim 11, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ 8-sterol, 14 Alpha-Methyl sterols, Δ 23, 24-alkyl sterol, Δ 24, 24-alkyl sterol and Δ 25 (27), 24-alkyl sterol.
15. the plant in the claim 11, wherein said elementary sterol or secondary sterol lack Δ 5Group.
16. the plant in the claim 11, the enzyme of wherein said dna encoding sequence encoding are selected from S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ 8To Δ 7-isomerase, Δ 7-C-5-desaturase and 24, the 25-reductase enzyme.
17. the plant in the claim 11, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase (SMT).
18. the plant in the claim 17, wherein said SMT is from plant or yeast.
19. the plant in the claim 17, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.
20. the plant in the claim 17, wherein said SMT is yeast ERG6.
21. the plant in the claim 11, this plant can be resisted insect, nematode or rotten mould fungi.
22. the plant in the claim 11, this plant has been improved the level of the sterol of norcholesterol.
23. the plant in the claim 22, wherein said sterol are cycloartenol or Sitosterol.
24. according to the plant in the claim 11, this plant can be resisted arid, high salt or severe cold.
25. according to the plant in the claim 11, this plant is tomato, corn or soybean.
26. a method of producing transgenic plant comprises:
(a) with the recombinant DNA molecules transformed plant cells that comprises following composition:
A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to
The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to
One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic;
(b) screening contain recombinant DNA molecules conversion vegetable cell; And
(c) become transgenic plant by the vegetable cell regeneration that has transformed.
27. the method in the claim 26, wherein said dna encoding sequence is a sense orientation.
28. the method in the claim 26, wherein said dna encoding sequence is an antisense orientation.
29. the method in the claim 26, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ 8-sterol, 14 Alpha-Methyl sterols, Δ 23, 24-alkyl sterol, Δ 24, 24-alkyl sterol and Δ 25 (27), 24-alkyl sterol.
30. the method in the claim 26, wherein said elementary sterol or secondary sterol lack Δ 5Group.
31. the method in the claim 26, the enzyme of wherein said dna encoding sequence encoding are selected from S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ 8To Δ 7-isomerase, Δ 7-C-5-desaturase and 24, the 25-reductase enzyme.
32. the method in the claim 26, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ 24 (25)-sterol methyltransgerase (SMT).
33. the method in the claim 32, wherein said SMT is from plant or yeast.
34. the method in the claim 32, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.
35. the method in the claim 32, wherein said SMT is yeast ERG6.
36. the method in the claim 26, wherein said plant can be resisted insect, nematode or rotten mould fungi.
37. the method in the claim 26, wherein said plant has been improved the level of the sterol of norcholesterol.
38. the method in the claim 37, wherein said sterol are cycloartenol or Sitosterol.
39. the method in the claim 26, wherein said plant can be resisted arid, high salt or severe cold.
40. the method in the claim 26, wherein said plant are tomato, corn or soybean.
CN97181954A 1996-12-26 1997-12-24 Transgenic plants with modified sterol biosynthetic pathways Pending CN1247569A (en)

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CN111500624A (en) * 2020-06-30 2020-08-07 中国农业科学院生物技术研究所 Use of CrSMT genes to increase resistance of plants to biotic and abiotic stress

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CN111500624A (en) * 2020-06-30 2020-08-07 中国农业科学院生物技术研究所 Use of CrSMT genes to increase resistance of plants to biotic and abiotic stress

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WO1998045457A1 (en) 1998-10-15

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