CN1413257A - Trans-species transfer of apoptotic genes and transgenic plants developed thereby - Google Patents

Abstract

The invention relates to trans-species transfer of apoptotic genes to plant cells, transgenic plants developed therefrom, and screening assays using these plants. The invention also relates to drug discovery screening methods utilizing transgenic plant cells. In addition, the invention relates to methods of identifying plant apoptotic pathway components utilizing non-plant proteins and nucleic acids.

Description

Apoptosis gene stride kind of transfer and consequent transgenic plant

Technical field

The present invention relates generally to modulate the apoptosis of plant, relate more specifically to new transgenosis, be about to the proteinic gene of Codocyte apoptosis pathway and be transferred to plant by the non-plant species, thereby produce multiple biology and abiotic attack are had the plant of higher resistance, and other relevant advantage is provided.

Background of invention

Recently, people have deepened the understanding to the sophisticated signal pathway of induced animal cell apoptosis gradually, begin to be devoted to the organism of identifying that the evolution level is different, as the similar approach in the plant.It is believed that apoptosis-mode of plant the only specified point in growth course takes place, plant can cause apoptosis to the reaction of pathogenic agent, and (example is seen Woodson etc., plant physiology, 99:526-532,1992; O ' Neill etc., vegetable cell, 5:419-432,1993; Chasan, vegetable cell, 6:917-919,1994; Smart, new plant is learned, 126:419-448,1994; Keen, 24:447-463,1990 are commented in heredity academic year; Lamb, cell, 76:419-422,1994; Mittler etc., cell, 7:29-42,1995).In that be considered to play decisive role in necrocytosis and situation disease-relevant, super quick resistance-relevant (HR) necrocytosis is the feature of several inconsistent plant-microorganism interactions.Although confirmation comprises morphological evidence (Wyllie, Curr.Opin.Gen.Dev, the 5:97-104 of the apoptosis characteristics of dna ladder and apoptotic bodies, 1995), report (Wang etc., vegetable cell in disease-related death and the allergy to some extent at development of plants, 8:375-391,1996; Ryerson and Heath, vegetable cell, 8:393-402,1996), but the gene and the corresponding proteins matter of the death of relevant controlling vegetable cell are known little about it, and whether the also unclear known apoptotic animal gene of zooblast (vertebrates and non-vertebrates) of controlling exists homologue in plant.

In animal tissues, by apoptotic process, promptly the normal physiological processes of apoptosis is kept stable state.For the pathogeny of disease, prevent or postpone that changes of cell apoptosis and Cycle Regulation that normal cell upgrades are of equal importance unusually.Cell fission is controlled by complex interactions between the cyclin, and similarly, apoptosis is under normal circumstances also by preventing or the interaction of the gene product of inducing cell death is regulated.

Such as fetal development, in a series of physiological processs that immunocyte is regulated and normal cell upgrades, apoptotic effect is to keep tissue homeostasis, therefore, and the dysfunction of modulated ganglion cell apoptosis or lose and can cause multiple pathological conditions state.For example, apoptotic loss can cause self-accumulation of the pathologic of reactive lymphocyte, the consequent is the various autoimmune disease.Unsuitable loss of apoptosis or inhibition also can cause virus infected cell and hyper-proliferative sexual cell, as the accumulation of neoplastic cell or tumour cell.Similarly, the unsuitable activation of apoptosis also can cause multiple pathological conditions state, comprises for example acquired immunodeficiency syndrome (AIDS), neurodegenerative disease and ischemic injury.For these and other pathological conditions, the methods of treatment that the special design of warp can be modulated its apoptotic pathways can change the normal procedure of multiple this type of disease.Therefore, if apoptosis is generally acknowledged the importance of animal, it is reported its growth and also very important (Mittler of pathogen resistance to plant, when necrocytosis, Lockshin etc. (volume), p147-174,1998), the apoptotic pathways of understanding so in the plant biological body also will be valuable.

Although apoptosis is by multiple signal and the mediation of cytogene product complex interactions, these results of interaction finally are summed up as the necrocytosis approach, and this approach between people and the non-vertebrates is guarded on evolving.This approach self comprises a succession of proteolysis proenzyme activation incident similar with the blood coagulation cascade.What is interesting is that identify that in plant the effort of similar approach does not bear fruit as yet, the relation of Mammals and non-vertebrate cells apoptosis and plant apoptosis approach also is uncertain at most.Yet, the aging feature consistent (Drake etc., molecular biology of plants, 30:755-767,1996) of having illustrated of allergy and plant with the apoptosis approach.Therefore, the apoptosis approach of understanding plant is understood the method for this approach of adjusting incidentally, thereby obtain to carry the transgenic plant of necrocytosis modulator, described plant has unique phenotypic characteristic, as resistance to multiple biology and abiotic attack, and the plant of gathering, the prolongation of fruit and vegetables shelf-lives.

Therefore, this area needs the method for plant identification necrocytosis pathway component, and the method for modulation plant apoptosis.In addition, this area also needs to produce multiple biology and abiotic attack is had the plant of resistance with improvement crop yield and viability.This area also needs to produce postponement aged plant and plant product.The present invention is by striding kind of the proteinic gene of transfer Codocyte apoptosis pathway, modulation plant apoptosis approach is to prevent vegetable cell pathology and aging, the inhibition of detection compound is provided and strengthens the method and composition of the ability of apoptosis, and other relevant advantage further is provided and has satisfied the demand.

The invention summary

Briefly, the invention provides the method for modulation vegetable cell apoptosis, described method is to realize by the proteinic nucleotide sequence of apoptotic pathways that uses coding to have biological function.

On the one hand, the invention provides transgenic plant, it contains vegetable cell, and described cell contains at least one can encode apoptotic pathways protein with biological function or heterologous nucleotide sequence of its functional variant.The nucleotide sequence of anti--apoptosis protein matter that in certain embodiments, transgenic plant have coding.In a plurality of embodiments, nucleotide sequence codified Ced-9, Bcl-2, IAP, E1B 19K and functional variant thereof.In another embodiment, the apoptotic pathways protein expression is subjected to tissue specificity, the control of induction type or constitutive promoter.In other the embodiment, plant has pathogen resistance or energy is anti-aging at some.As hereinafter will be discussed in detail, others of the present invention provide the plant of multiple biology and abiotic resistance.

The present invention also provides vegetable cell and seed, and it contains can encode the apoptotic pathways protein with biological function or the heterologous nucleotide sequence of its functional variant.In other a plurality of embodiments, provide the vegetable cell that has same characteristic features with above-mentioned transgenic plant.

As mentioned above, the invention provides the transgenic plant that biological attack had resistance.This plant contains the apoptotic pathways protein that coding has biological function, is preferably the proteinic heterologous nucleic acid sequence of anti-apoptotic.In certain embodiments, anti-apoptotic protein is selected from Ced-9, Bcl-2, IAP, E1B 19K and homologue thereof.In a plurality of embodiments, biological attack is that pathogenic agent causes.

At related aspect, the transgenic plant that abiotic attack had resistance are provided.This plant contains the apoptotic pathways protein that coding has biological function, is preferably the proteinic heterologous nucleic acid sequence of anti-apoptotic.In certain embodiments, anti-apoptotic protein is selected from Ced-9, Bcl-2, IAP, E1B 19K and functional variant thereof.In addition, the present invention also provides to produce thisly has the method for the transgenic plant of resistance to biological or abiotic attack, described method comprises: use the carrier transformed plant cells, described carrier contains at least one the proteinic heterologous nucleic acid sequence of apoptotic pathways that can encode and have biological function, this nucleotide sequence can be operated with promotor and link to each other, by producing plant through the plant transformed cell, and select the plant that biological or abiotic attack are had resistance through transforming.

In others of the present invention, the method of modulation vegetable cell apoptosis is provided, described method comprises: use the carrier transformed plant cells, described carrier contains can encode by the normal generation of vegetable cell, apoptotic pathways nucleic acid sequences to proteins with biological function, this nucleotide sequence can be operated with inducible promoter and link to each other, cultivates through the plant transformed cell being suitable for forming under the condition of plant, and condition and time culturing plants to be enough to induce nucleotide sequence to transcribe.

The present invention also provides and has identified to have the method for the active plant gene of apoptotic pathways, described method comprises: with plant cDNA library transformed animal cell, wherein each member in cDNA library can operate with promotor and link to each other, transformant is contacted with cell death inducer, detect the apoptosis activity in the cell, and compare wherein active increase or reduce the proteinic existence of apoptotic pathways in the expression plant cDNA library with the activity of control cells system.

At related aspect, the invention provides the method for the apoptosis gene that evaluation works in plant, described method comprises: transform one or more vegetable cells with at least one heterologous nucleic acids molecule, wherein nucleic acid molecule can be operated with promotor and link to each other, transformant is contacted with cell death inducer, detect the apoptosis activity in the cell, and compare with the activity of control cells system, wherein active increase or reduction are illustrated in the existence of the apoptotic pathways gene that works in the plant.In certain embodiments, the heterologous nucleic acids molecule comprises allos cDNA library, and wherein each member in cDNA library can operate with promotor and link to each other.

Aforesaid transgenic plant of the present invention, vegetable cell and method generally all are applicable to the plant apoptosis.Therefore, the present invention also provides the method for plant identification necrocytosis pathway component, and the method for modulation plant apoptosis.As mentioned above, the present invention also provides the plant that biological and abiotic attack are had resistance to improve crop yield, improves viability.In addition, the present invention also provides aged plant and the plant product that reduces, its can cause gathering shelf life extension of fruits and vegetables and cut-flower etc.In addition, the present invention can be applicable to the inhibition of detection compound and strengthens the method and composition of the ability of apoptosis, and other relevant advantage further is provided.

With reference to following the detailed description and the accompanying drawings, these and other aspect of the present invention will be conspicuous.In addition, hereinafter listed many pieces of reference, described some method or composition (as plasmid etc.) in more detail, therefore, these reference are all listed this paper in as a reference in full.

The accompanying drawing summary

Figure 1A and 1B illustrate the plasmid of the nucleic acid molecule that carries coding IAP.

Fig. 2 A and 2B illustrate the plasmid of the nucleic acid molecule that carries coding Bcl-2.

Fig. 3 A and 3B illustrate the plasmid of the nucleic acid molecule that carries coding Ced-9.

Fig. 4 A-4C illustrates the plasmid of the nucleic acid molecule that carries coding E1B-19K.

Fig. 5 is a scanning image, and it has shown the Northern engram analysis of transgene expression in the transformation of tobacco plant.Swimming lane 1-3 represents that the IAP in the tobacco plant independently expresses, the expression in the tobacco plant that swimming lane 4 expressions are transformed by control vector, and swimming lane 5-6 represents that the Ced-9 in the tobacco plant independently expresses.

Fig. 6 be with 3 transgene tobacco strains of sclerotinite (Sclerotinia sclerotiorum) inoculation (A, C, D) and check clone (B) leaf attitude photo afterwards.

Fig. 7 is that (A, B is C) with 2 transgene tobacco strain (D and E) leaf attitude photos afterwards of independently expressing IAP with the contrast of tobacco mosaic virus (TMV) (TMV) inoculation wild-type.

Fig. 8 is a scanning image, and it has shown the Northern engram analysis of the elementary tobacco transformant (Glurk kind) that carries Bcl-2. Swimming lane 2,3,5 and 7 have illustrated the Bcl-2 expression, and the contrast swimming lane is represented to transform with the carrier (G115) that does not contain the Bcl-2 inset.

Fig. 9 is a scanning image, and it has shown the Northern engram analysis of the elementary tobacco transformant that carries Ced-9. Swimming lane 4 and 8 has been illustrated expression.

Figure 10 A and 10B are the tobacco leaf attitude photos afterwards that carries Bcl-2 with the TMV inoculation.The intermediary leaf is a wild-type, and remaining leaf is derived from the tobacco plant that independently carries Bcl-2.

Figure 11 A and 11B are scanning images, and it has shown the Western engram analysis that TMV expresses in the tobacco strain (carrying Bcl-2 or Ced-9 gene) that contains N gene (A) and do not contain N gene (B).B is Bcl-2, and C is Ced-9, and T is TMV, and A represents to take from the leaf sample of inoculation site adjacent area.

Figure 12 is the tobacco plant leaf attitude photo afterwards that carries Bcl-2 and Ced-9 with tomato spotted wilf virus (TSWV) inoculation.Upper right side and bottom-right leaf are contrasts, and upper left leaf is the Glurk kind that carries Ced-9, and the leaf of lower left is the Turkish kind that carries Ced-9.

Figure 13 is a scanning image, and it has shown the Northern engram analysis that autophilous rotaring gene tobacco plant Ced-9 expresses.Swimming lane 1-4 is each progeny plants.

Figure 14 A to 14C is the leaf attitude photo of self-pollination rotaring gene tobacco plant.14A has shown the plant that the carries Bcl-2 resistance to TMV, the positive contrast of intermediary leaf, and 14B has shown the plant that the carries Ced-9 resistance to TMV, 14C has shown the plant that the carries Ced-9 resistance to TSWV.

Figure 15 is the form photo with the leaf of sclerotinite inoculation.Following leaf is the wild-type contrast, and top leaf carries Ced-9.

Figure 16 A and 16B are with the leaf attitude photo after the transgenic plant of sclerotinite inoculation autophilous Bcl-2 of carrying (A) and Ced-9 (B).

Figure 17 A-17C inoculates tobacco strain (B and C) the leaf attitude photo afterwards that carries the tobacco strain (A) of Bcl-2 and carry Ced-9 with gray botrytis.

Figure 18 is a scanning image, and it has shown the Northern engram analysis that Ced-9 expresses in sclerotinite resistance plant (swimming lane 5-7) and the non--resistance plant (swimming lane 1-4).

Figure 19 is respectively with after the inoculation of tobacco tail spore from left to right, Bcl-2, IAP, Ced-9, the leaf attitude photo of E1B 19K and contrast (only being carrier).

Figure 20 A and 20B are the leaf attitude photos that carries the tobacco plant of Bcl-xL.In 20A, top leaf has the G138A sudden change in Bcl-xL, and following leaf carries wild-type Bcl-xL.In 20B, the leaf of left-hand side has the G138A sudden change in Bcl-xL, and dexter leaf carries wild-type Bcl-xL.

Detailed Description Of The Invention

Before describing the present invention, the definition that provides some term that hereinafter will use will help to understand the present invention.

" downright bad nutrition " used herein refers to cell killing to finish its life cycle and obtain the pathogen (for example a lot of fungies) of nutrition from the dead cell material.

" biological attack " used herein refers to the plant that is caused by live body or biologic product (biotic factor) and attacks. Therefore, cause the biotic factor of biological attack to comprise for example insect, fungi, bacterium, virus, nematode, viroid, mycoplasma etc.

" abiotic attack " used herein refers to the plant that the factor (abiotic component) by non-living body or non--survival causes and attacks. Therefore, cause the abiotic component of abiotic attack to comprise for example environmental factor, for example low humidity (arid), high humility (floods), nutritional deficiency, radiation level, air pollution (ozone, acid rain, sulfur dioxide etc.), temperature (overheated and excessively cold), and soil toxicity, and the herbicide infringement, the pesticide infringement, or other agricultural operation (for example fertilising is excessive, and the chemicals sprinkling is improper etc.).

" apoptotic pathways protein " used herein refers to any protein that participates in the programmed cell death approach, illustrated this proteinoid in the multiple organism, comprise the people, mouse, nematode (C.elegans), the protein of fruit bat (Drosophila Melanogaster) and baculoviral, but except the p35 gene of baculoviral. In addition, term " gene of Codocyte apoptosis pathway protein " or " apoptotic pathways gene " in this article can Alternates. Known apoptosis of many kinds pathway protein can use these protein in the context of the present invention. The molecule that exemplifies comprises caspase molecule (having identified at present 14 kinds of these quasi-molecules), and the bcl-2 family member (such as bcl-2, bcl-x_L，bcl-x _s, bcl-W, McL-1, A1, NR-13, Ced-9, E1B 19K, BHRF1, KSHV ORF 16, LMW5-HL, KS-bcl-2 etc.), Bax, Bak, Bok, Bad, Bik, Bid, Blk, Hrk, BNIP3, Bim_L, EGL-1, apoptosis inhibitor (such as the IAP-baculoviral, DIAP 1 ﹠ 2-fruit bat, c-IAP 1 ﹠ 2-people, X-IAP-people, NIAP-people, survivin-people etc.) etc. The sequence of these and other gene can derive from the Genbank database, also can be referring to the multi-section publication, comprise " when the cell death " of being write by Lockshin etc., Wiley-Liss, New York, 1998.

" rev-caspase " used herein refers to the cysteine proteinase in the back of Asp residue specificity crack protein matter, this protease is expressed with the form of proenzyme, and its medium and small subunit is positioned at the N-terminal (example is seen PCT WO 99/00632) of large subunit.

In the context of the present invention, should be appreciated that apoptotic pathways protein comprises the wild-type protein sequence, and other variant (comprising allele variant) of native protein sequence. Briefly, described variant can be due to the natural polymorphism, perhaps also can learn by recombination method syntheticly, and the difference of described variant and wild-type protein is one or more amino acid whose replacements, inserts, and lacks etc. In general, when carrying out when genetic engineering modified, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is guarded, and is polarity, and is non--polarity, aromatic series, the charged 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that waits in the amino acid group. With the zone of native sequences homology in, variant should preferably have at least 90% amino acid sequence identity, in certain embodiments, for greater than 92%, 95% or 97% homogeneity. Can measure by the methodology of standard the homogeneity of this amino acid sequence and nucleotide sequence, comprise that use can derive from the BLAST of the NCBI search method of www.ncbi.nlm.nih.gov. Preferred homogeneity methodology is the BLAST algorithm without the room. Yet, also can use United States Patent (USP) 5691179 and Altschul etc., nucleic acids research, the described homogeneity algorithm of 25:3389-3402, these two pieces of documents are all listed this paper in as a reference. Therefore, if utilize the BLAST 2.0 that has vacant position, so also should use simultaneously default setting. In addition, nucleotide variants generally can have enough similar sequence with under tight hybridization conditions with canonical sequence hybridization, for surpassing the nucleic acid molecules of 500bp, stringent condition comprises and contains the 1M Na that has an appointment⁺Solution, be lower than 25 ℃ to 30 ℃ of Tm; For example, 5 * SSP_E, 0.5%SDS, 65 ℃; Example is seen Ausubel etc., up-to-date molecular biology method, Greene Publishing, 1995; Sambrook etc., molecular cloning: laboratory manual, cold spring port publishing house, 1989. Some variants can because of the degeneracy (degeneracy that for example imports for the codon optimization that makes in the specific host) of codon with canonical sequence hybridization, wherein can assess variant and similitude with reference to protein with amino acid homogeneity.

" nucleic acid molecules of separation " refers to the form of isolated fragment or the polynucleotide molecule that exists as the component of larger nucleic acid construct, and this molecule separates from its derived cell (chromosome that comprises its normal residence) with basically pure form at least one times. Nucleic acid molecules can be comprised of multiple nucleotides, comprises DNA, RNA, nucleotide analog or its combination.

Term " external " refers to and contains the system that is less than an intact cell.

Term " ex vivo " refers to the cell culture system, and it comprises for example former generation and subculture cell culture, whole organ cultures and similar system.

Term " in the body " refers to whole organism.

" genetic modification " used herein refers to one or more heterologous nucleic acid sequence imported one or more vegetable cells, and described cell can produce complete, and the survival plant of sexuality is arranged.Term " transgenosis " and " genetic modification " can exchange use in this article, mean the plant that produces by aforesaid method.Transgenic plant of the present invention can with other plant self-pollination or cross-pollination of same species, thereby will plant the plant variety that the foreign gene that carries in the system inserted or imported agriculturally useful.

Term used herein " vegetable cell " refers to protoplastis, produces the cell of gamete and the cell of the complete plant of energy regeneration.Therefore, contain a plurality of can regenerations the seed of vegetable cell of complete plant be included in the definition of " vegetable cell ".

" plant tissue " used herein comprises differentiation and undifferentiated plant tissue, includes but not limited to root, stem, and branch, leaf, pollen, seed, the cell of root nodule tissue and various ways and culture, as unicellular, protoplastis, embryo and callus.

Term used herein " plant " refers to complete plant, and plant part, or one group of vegetable cell are as plant tissue.Also comprise plantlet in the definition of " plant ".Be used for suitable plant of the present invention and comprise any plant that is applicable to transformation technology, comprise unifacial leaf and dicotyledons.

Monocotyledonous example includes but not limited to asparagus, wild corn and sweet corn, barley, wheat, paddy rice, Chinese sorghum, onion, pearl millet, rye and oat.The example of dicotyledons includes but not limited to tomato, potato, Arabidopis thaliana, tobacco, cotton, coleseed, wild beans, soybean, pepper, lettuce, pea, clover, trifolium, rape crop or wild cabbage (wild cabbage for example, asparagus broccoli, Cauliflower, the soup dish), capsicum, Radix Dauci Sativae, beet, eggplant, spinach, cucumber, pumpkin, muskmelon, cantaloupe, Sunflower Receptacle and multiple ornamental plant.Tobacco plant is used as the example of plant in this article.

Term used herein " heterologous nucleic acid sequence " refers at least one structure gene, and this gene generally links to each other with can operating such as the adjusting sequence of promotor.Nucleotide sequence comes from different kinds, perhaps is derived from identical kind, but is to be modified by its primary form basically.For example, term " heterologous nucleic acid sequence " comprises and comes from nucleic acid mutually of the same race that wherein this sequence can be operated with the promotor that is different from natural or wild-type promotor and be linked to each other.

" structure gene " is to be transcribed into messenger RNA(mRNA) (mRNA), then is translated into the dna sequence dna of the characteristic aminoacid sequence of specific polypeptide.Term " expression " refers to the biosynthesizing of gene product.

Term " can operate " to refer to promoter sequence continuous and the structure gene of regulating by nucleic acid sequence of promoter between function connect.Can operate the polypeptide expression of continuous promotor control by structural gene coding.

" promotor " used herein refers to the dna sequence dna that mediation structure gene is transcribed.Promotor generally is positioned at 5 ' zone of gene, near the transcription initiation site of structure gene.

" cloning vector " used herein refer to can self-replacation in host cell dna molecular, as plasmid, phasmid or phage.Cloning vector generally contains one or a few limitations endonuclease enzyme recognition site, can exogenous DNA array be inserted these sites in decidable mode, and can not lose the biological function of carrier necessity, also contain marker gene in the cloning vector, it is applicable to evaluation and selects by the cloning vector cell transformed.

" expression vector " used herein refers to dna molecular, and it contains the gene of desiring to express in host cell.Genetic expression generally is under the control of some regulatory element, and described regulatory element comprises promotor, tissue specificity regulatory element and enhanser.As mentioned above, described gene " can be operated and link to each other " with regulatory element.

" pathogenic agent " used herein refers to any factor that causes plant disease or morbid state, includes but not limited to virus, fungi, bacterium, the microorganism that nematode is relevant with other.

" modulation " used herein refers to the ability that changes basal level.

" inhibition " used herein refers in the significant mode of statistics and blocks, and postpones or reduce the ability of activity or result's seriousness.A. apoptotic pathways gene and gene product

As mentioned above, the invention provides transgenic plant and plant tissue, it contains the proteinic gene of Codocyte apoptosis pathway, thereby can express this gene.The proteinic gene of a plurality of Codocyte apoptosis pathway is known, and can be used for context of the present invention.The molecule that exemplifies comprises caspase molecule (having identified 14 kinds of these quasi-molecules at present), Apaf-1 (GenbankNM 001160 and AF013263), the bcl-2 family member (as bcl-2, bcl-x _L, bcl-x _s, bcl-W, McL-1, A1, NR-13, Ced-9, E1B 19K, BHRF1, KSHV ORF 16, LMW5-HL, KS-bcl-2 etc.), Bax, Bak, Bok, Bad, Bik, Bid (GenbankNM 001196), B1k, Hrk, BNIP3, Bim _L, EGL-1, and apoptosis inhibitor (as baculovirus OpIAP, DIAP 1 ﹠amp; The 2-fruit bat, c-IAP 1 ﹠amp; 2-people, X-IAP-people, NIAP-people, survivin-people etc.) or the like (example that the I that sees the following form lists and Genbank registration number).According to content provided herein, can from the various kinds of cell type, isolate required apoptotic pathways gene, and be inserted in the expression vector that is applicable to plant.

Table I

Anti--and preceding-apoptosis protein and Genbank registration number thereof
		Anti--apoptosis protein	The Genbank registration number
		? Bcl-2Family
The people
		?Bcl-2	?M14745
?Bcl-xL	?Z23115，L20121
		?Mcl-1	?L08246
?Bfl-1(A1)	?U27467
		?Bcl-w	?U59747

Anti--and preceding-apoptosis protein and Genbank registration number thereof
		Anti--apoptosis protein	The Genbank registration number
Other organism
		?NR-13	?X84418，Q90343
?Ced-9?C.elegans	?L26545
		?Ced-9?C.briggsae	?L26546
		Virus Bcl-2 homologue
?E1B?19K	?L19443，AF099665?etc.
		?EBV-BHRF1	?A22899
?KSHV?ORF?16	?U93872
		?LMW5-HL	?L09548
?LMH-5W	?Q07818，Q07819
		?EAR_ASFB7	?P42485
?A9	?AAC58120
Cell IAP
		The people
?x-1AP	?U32974，NM001167
		?cIAP-1	?U45878
?cIAP-2	?U45879，NP00?1157
		?NAIP	?Q13075
?Survivin	?U75285，NP001159
		Other organism
?dIAP-1D.melanogaster	?Q24306
		?dIAP-2D.melanogaster	?U45881，Q24307
?BRUCE	?CAA76720
Virus IAP
		?IAP3?NPVOP	?P41437
?CfMNPV?IAP	?AAD00537
		?GVCP?IAP	?P41436
?BSNPV-IAP	?AAC34373
		?NPVOP-IAP1	?O10296
?NPVAC-IAP1	?P41435
		?NPVEP-IAP	?AAD19698
?IAP1AcMNPV?orf27IAP1	?AAC63701
		?NPVLD-1AP	?AAC70325

Anti--and preceding-apoptosis protein and Genbank registration number thereof
		Anti--apoptosis protein	The Genbank registration number
FLIPS
		The people
FLICE-sample arrestin elongated	?U97074
		?Casper	?AF010127
?FLAME-1-γ	?AF009618
		?MRIT-α-1	?U85059
?I-FLICE	?AF041458
		?Usurpin-α	?AF015450
Other organism
		The FLICE-sample suppresses (mouse)	?U97076
CASH α albumen (mouse)	?Y14041
		CASH β albumen (mouse)	?Y14042
		Virus FLIPS
?MC160L	?U60315
		?MC159L	?U60315
False albuminoid E8	?U20824

Anti--and preceding-apoptosis protein and Genbank registration number thereof
		Proapoptosis albumen	The Genbank registration number
		?Bcl-xS	?Z23116，L20122
?Bax	?L22473
		?Bak	?X84213，U16811，U23765
?Bok	?AF027954，AF051093
		?Bad	?AF031523
?Bik	?U34584
		?Nbk	?U49730
?Bid	?AF042083，NP001187
		?Hrk	?U76376
?Blk	?AF048838
		?Bim	?AF032457，AF032458
?Nip-3	?AF002697
		?Egl-1	?AF057309
The Nip-3C.elegans homologue	?AAD32265
		?Boo/Diva	?AF102501，AF067660
?Apaf-1	?AF013263，AF149794

Anti--and preceding-apoptosis protein and Genbank registration number thereof
		Proapoptosis albumen	The Genbank registration number
		?Caspase-1	?X65019
?Caspase-2	?U13021
		?Caspase-3	?U13737
?Caspase-4	?U25804
		?Caspase-5	?U28015
?Caspase-6	?U20536
		?Caspase-7	?U37448
?Caspase-8	?U60520
		?Caspase-9	?U56390
?Caspase-10	?U60519
		?Caspase-11	?Y13089，P70343
?Caspase-12	?Y13090
		?Caspase-13	?NM?003723
?Caspase-14	?AF097874

As described herein, the invention provides vegetable cell and plant tissue, it expresses the proteinic heterologous gene of Codocyte apoptosis pathway.The apoptotic pathways gene is separable from genomic dna, and perhaps preferable separation is from the cDNA library.Can from genomic dna or cDNA, separate the proteinic gene of Codocyte apoptosis pathway as follows: at first, (example is seen Sambrook etc. by library construction technology known in the art, molecular cloning: laboratory manual, cold spring port press, 1989) produce suitable DNA library, perhaps (Clontech for example, PaloAlto CA) buy the library by commercial source.Briefly, can be in the phage vector that is suitable for screening (as λ ZAPII), construction cDNA library in plasmid or other carrier; and at the chromosome vector as YAC (yeast artificial chromosome); as λ EMBL3, the phage vector of λ gt11 makes up genome dna library in clay or the plasmid.

In one embodiment, can utilize known apoptotic pathways nucleotide sequence design to be suitable for the oligonucleotide hybridization probe in screening-gene group or cDNA library.The length of preferred described oligonucleotide probe is 20-30 base.Detect for the ease of hybridization, can use reporter molecule easily, as radionuclide (as ³²P), enzyme labelling, protein labeling, fluorescent mark or biology be labeled oligonucleotide usually, generally labeling be 5 ' end of oligonucleotide.According to used carrier, plate is paved as phage or bacterium colony in described library then.Subsequently, bacterium colony or phage are transferred to nitrocellulose or nylon membrane, contain the candidate clone of the gene of Codocyte apoptosis pathway protein with probe and the hybridization of described film with evaluation.Can confirm that described candidate clone contains Codocyte apoptosis pathway protein DNA by any in the several different methods, described method for example comprise dna sequence analysis or with second non--eclipsed probe hybridization.

Contain the proteinic gene of Codocyte apoptosis pathway in case identify certain library, can separate this gene by amplification.Briefly, when using the cDNA library DNA, can come design of amplification primers according to the proteinic gene order of known Codocyte apoptosis pathway (example sees Table I) as template.Preferred amplimer derives from the sequence of 5 ' and 3 ' non-translational region to isolate full-length cDNA.The GC content of preferred primer is about 50%, and contains restriction site so that the clone, described primer do not have self-the complementary sequence, its 3 ' end does not contain complementary sequence (to prevent to form primer-dimer) yet.With primer and cDNA or genomic dna annealing, carry out enough amplification cycles, be easy to by gel electrophoresis and the observed product of dyeing with generation.The fragment of purifying amplification is inserted into carrier, breeds as λ gt10 or pBS (M13+).By dna sequence analysis or indirectly the amino acid sequencing by coded protein confirm segmental feature.

Also can use other method to obtain the proteinic nucleic acid molecule of Codocyte apoptosis pathway.For example, can screen by use one or more antibody with the apoptotic pathways proteins react, (example is seen Sambrook etc. to obtain described nucleic acid molecule from expression library, molecular cloning: laboratory manual, the 2nd edition, press of cold spring harbor laboratory, New York, 1989; Ausubel etc., up-to-date molecular biology method, Greene Publishing Associates and Wiley-Interscience, New York, 1995).

Can use standard method from a plurality of species, to isolate the proteinic gene of Codocyte apoptosis pathway.For closely-related species, can be with human sequence or its part probe as genome or cDNA library.For example, for caspase, but mark comprises the fragment of catalytic site, used as by mouse, and primate, rat, dog or other vertebrates, the probe in the library that warm-blooded animal or Mammals kind make up.The hybridization of carrying out under normal stringent condition at first can produce candidate clone or fragment.If originally do not observe hybridization, can use different tight degree (example is seen Sambrook etc., the same and other well-known stringent condition source of document).Also can use the library hybridization of this probe species (as fruit bat) different with getting the self-evolution level, hybridization conditions may be looser.

Although use by the loose hybridization conditions of human sequence's designed probe and can identify species that the evolution level is different, the proteinic gene of Codocyte apoptosis pathway, but attempt using the method that does not need human sequence itself, it will be more favourable directly separating these genes from the library.These methods include but not limited to: use the primer derived from conservative region to increase, use the degenerated primer that derives from a plurality of zones to increase, survey expression library etc. with antibody.For example, (example is seen Enzymology method, 254:275,1995 in the amplification (as polymerase chain reaction) that can use at random-cause; Genetics trend, 11:242,1995; Liang and Pardee, science, 257:967,1992; Welsh etc., nucleic acids research, 20:4965,1992).In addition, also can use to some extent change at random-PCR that causes, particularly all the more so when needs specific gene or gene family.In this method, an amplimer is " anchor number (dT) (few (dT) dN) ", and other primer is based on the amino acid of genes involved or the degenerated primer of nucleotide sequence.By amino acid similarity, nucleic acid similarity and/or functional equivalent are accredited as apoptotic pathways protein with gene order.In addition, can be apoptotic pathways protein with gene identification according to computerized algorithm, described algorithm can compare the structural domain structure of functionality and secondary structure or correlation function and three grades of forms, and described algorithm is available at present.Express candidate gene, use the apoptosis test of standard and enzymic activity and/or other functionally active of test as herein described or other test that is equal to detection gene product.

Can go out the proteinic genetic mutation of Codocyte apoptosis pathway provided herein by natural variant (as polymorphism, splice variant, mutant) is engineered, also can synthesize or make up described genetic mutation.(generally referring to Sambrook etc., document is the same to have developed the method for multiple generation mutant; Ausubel etc., the same and above-mentioned discussion of document).Briefly, the preferred method that produces several Nucleotide replacements has utilized oligonucleotide, and described oligonucleotide leap is one or more to be treated mutating alkali yl and contain one or more mutating alkali yls.Oligonucleotide and the hybridization of complementary single-chain nucleic acid cause the synthetic of second chain by oligonucleotide.The preparation double-strandednucleic acid is generally intestinal bacteria, but also can be other prokaryotic organism, yeast or other eukaryote to be converted into host cell.The screening of use standard and carrier cultural method are identified mutant nucleotide sequence and are obtained high yield.

Similarly, can make up the disappearance and/or the insertion of the gene of Codocyte apoptosis pathway protein by in the multiple currently known methods discussed above any.For example, available constraints enzymic digestion gene also reconnects, so that sequence deletion perhaps reconnects to insert or to replace in a large number with other sequence.Available methods known in the art are used other method that produces the variant sequence, and example is seen Sambrook etc., and document is the same; Ausubel etc., document is the same.General drafting by the Restriction Enzyme collection of illustrative plates, sequential analysis or probe hybridization confirm the variant sequence.

Also comprise transgenic plant and vegetable cell in the scope of the invention, it is expressed at proteinic antisense of vegetable cell apoptosis pathway and ribozyme molecule.The expression of antisense rna molecule is by combining with the mRNA of target, stops protein translation and directly blocks the translation of mRNA.Also can use the expression of ribozyme to come the blocking protein translation, described ribozyme is can catalysis RNA specificity cracked enzymatic RNA molecule.The mechanism of action of ribozyme comprises the sequence-specific hybridization of ribozyme molecule and complementary target rna, then carries out the endonuclease enzymatic lysis.Within the scope of the invention, be tup primitive ribozyme molecule through transforming, it can be specifically and catalysis endonuclease cleaving rna sequence effectively.Can produce the RNA molecule by the dna sequence dna of transcribing the coding RNA molecule.

(example is seen WO93/01286, U. S. application serial number 07/723,454 can to synthesize nucleic acid used herein and oligonucleotide by any method known to those skilled in the art; United States Patent (USP) 5,218,088; United States Patent (USP) 5,175,269; United States Patent (USP) 5,109,124).Can use method well-known in the art to identify and be used for the DNA of gene therapy as the ribozyme and the oligonucleotide of the antisense factor.For example, the desired characteristic of described oligonucleotide, length and further feature are well-known.

Antisense nucleotide is with sequence-specific mode and nucleic acid (as mRNA or DNA) bonded oligonucleotide.When combining with the mRNA with complementary sequence, antisense can prevent that (example is seen the United States Patent (USP) 5,168,053 of Altman etc. for the translation of mRNA; The United States Patent (USP) 5,190,931 of Inouye etc.; The United States Patent (USP) 5,135,917 of Burch etc.; The United States Patent (USP) 5,087,617 of Smith and Clusel etc., nucleic acids research, 21:3405-3411,1993, it has described the dumbbell antisense oligonucleotide).The triplex molecule refers to duplex DNA and combines, form collinearity triplex molecule, thereby prevent that (example is seen the United States Patent (USP) 5 of Hogan etc. for the single DNA chain of transcribing, 176,996, it has described the method for preparing synthetic oligonucleotide, and described oligonucleotide can combine with the target site on the duplex DNA).

Useful especially antisense nucleotide and triplex molecule are sense strand or mRNA complementation or the bonded molecules with DNA, what the sense strand of described DNA or mRNA encoded is the protein that participates in the modulation apoptotic pathways, or mediate the protein of any other unnecessary process, thereby the restraining effect to protein translation is catered to the need.

General by connecting the degraded that key is designed to be able to antisense oligonucleotide to resist the endogenous nucleic acid cleavage enzyme below using: thiophosphatephosphorothioate, methylphosphonate, sulfone, sulfuric ester, ketyl, phosphorodithioate, phosphoramidate, (example is seen Agrwal etc., the tetrahedron communication for phosphoric acid ester and other this even key, 28:3539-3542,1987; Miller etc., U.S. chemical institute magazine, 93:6657-6665,1971; Stec etc., tetrahedron communication, 26:2191-2194,1985; Moody etc., nucleic acids research, 12:4769-4782,1989; Uznanski etc., nucleic acids research, 1989; Letsinger etc., tetrahedron, 40:137-143,1984; Eckstein, bioid academic year comments, 54:367-402,1985; Eckstein, bio-science trend, 14:97-100,1989; Stein, oligodeoxynucleotide, the antisense inhibitor of genetic expression, Cohen compiles, Macmillan press, London, p97-117,1989; Jager etc., biological chemistry, 27:7237-7246,1988).

About the apoptotic pathways protein that in plant, produces, can be by the standard method isolated protein, described method such as affinity chromatography, size exclusion chromatography, metal ion chromatography, ion exchange chromatography, HPLC and other known protein matter separation method are (generally referring to Ausubel etc., document is the same, Sambrook etc., document is the same).When using Coomassie blue stain, the protein of separation and purification presents a band on SDS-PAGE.Should understand the present invention includes and produce endogenous vegetable cell apoptosis pathway protein, and reorganization produces non--vegetable cell apoptotic proteins matter, as mammalian cell apoptotic proteins matter, thereby with the carrier of plant as recombinant production.In addition, the present invention also can be used for zoologizeing gene and functional in vegetable cell thereof.Therefore, as hereinafter will be in greater detail, carry the proteinic transgenic plant of the apoptotic apoptotic pathways of inhibition by generation, can be by reducing and recombinant production endogenous or the relevant necrocytosis incident of heterologous polypeptide (as antibody), make the peptide and the polypeptide of any number, comprise that the recombinant production of non--apoptosis related peptides and polypeptide strengthens greatly.

Apoptotic pathways protein can be expressed as six-Histidine (his) fusion rotein, and can be by containing-chromatography of metal, as separating with nickel link coupled pearl.Briefly, make coding His ₆Sequence be connected with Codocyte apoptosis pathway protein DNA sequence.Although His ₆Sequence can be positioned any position of molecule, but preferably is connected to the 3 ' end that is right after terminator codon.Can be by any construction of fusion protein of several different methods.Method is to use and contains His easily ₆The proteinic gene of downstream primer amplification coding apoptotic pathways of codon.

The apoptotic pathways protein of purifying can be used for screening in the test of inhibition compound.These tests can be carried out in external or body, and can utilize any method described herein or known in the art.Also can make crystallization of protein, and it is carried out x-ray analysis measuring its three-dimensional structure, or use it for generation antibody.In addition, as mentioned above, can in plant, express the proteinic heterologous nucleic acids of Codocyte apoptosis pathway, can be used for the vegetable cell of apoptosis inhibitor and toughener shaker test with generation.B. carrier and method for transformation are learned

Can be in multiple host organisms express cell apoptosis pathway protein.Yet as described herein, when placing these protein in the vegetable cell, they can provide beat all advantage, and are particularly all the more so when not identifying similar apoptotic pathways protein.For example, when in vegetable cell, expressing, can use the resistance of apoptotic pathways arrestin increase, comprise resistance fungi and virus infection to biological and abiotic attack.In addition, the ability of modulation vegetable cell death makes it possible to join in plant with expressing Pesticidal compound, for example the toxin (example is seen asks WO97/34926 among the PCT) of Tribactur coding.Therefore, when expressing the pesticide compound of some type or level, can in the plant in normal startup apoptosis cycle, obtain the higher desinsection tolerance of plant.Similarly, by strengthening the resistance of vegetable cell, can obtain higher herbicide tolerant to weedicide inductive apoptosis incident.Shifting genetic material in a lot of vegetation types is routine techniques, and in all vegetation types, the level of transfer can be different.Those skilled in the art should understand, can easily obtain the multiple suitable carrier of expressing in vegetable cell that is applicable to.1. carrier

By vegetable cell is contacted with the carrier that contains heterologous nucleic acid sequence, can produce transgenic plant of the present invention, described nucleotide sequence contains at least one structure gene, the protein of described structural gene coding modulation apoptosis (being apoptosis).Can play a role effectively after making the importing vegetable cell, the desired structure gene should can be operated with effective promotor in vegetable cell and link to each other, to cause required gene transcription.In addition, the polyadenylation sequence or the transcriptional control sequence that can use vegetable cell also to be familiar with.The carrier that preferably carries heterologous nucleic acid sequence also contains one or more selectable marker genes, thereby can be as described herein, easily selects through cell transformed from the cell of culture unconverted.

Those skilled in the art can select appropriate carriers, import heterologous nucleic acid sequence with complete relatively state.Therefore, can use any carrier that can produce the plant that carries the importing dna sequence dna.Correspondingly, can use exposed DNA, let it be to the greatest extent, and transformation efficiency may be lower.Generally instruct the selection of carrier, or do not use carrier by selected method for transformation.

Briefly, Codocyte apoptosis pathway protein DNA sequence is imported the expression vector that is suitable for host plant cell.In certain embodiments, apoptotic pathways protein is apoptotic inhibitor, and in other embodiments, apoptotic pathways protein is apoptotic toughener or initiator.Preferred inhibitors includes but not limited to IAP, Bcl-2, Bcl-x _L, Bcl-W, McL-1, A1, NR13, CED-9, E1B 19K, BHRF1, Bag-1 etc.In other embodiments, the proteinic nucleic acid of Codocyte apoptosis pathway is inserted carrier, and be connected to the nucleic acid molecule of the another kind of polypeptide of coding, to produce fusion rotein.By acquisition apoptotic pathways protein sequence described herein.As discussed above, for each amino acid with multiple codon, sequence can contain another kind of alternative codon.The alternative codon of selected another kind can be the codon of " being suitable for most " host species, and described optimized method is well-known in the art.In addition, generally in primer sequence, mix restriction site, and come the selectional restriction site with reference to the cloning site of carrier.In case of necessity, can in primer sequence, engineering mix translation initiation and terminator codon.

Carrier should contain promoter sequence at least.As mentioned above, " promotor " refers to the nucleotide sequence that contains element, and described element can instruct connection/continuous gene transcription.Promotor should contain the RNA polymerase binding site at least.More typically, in eukaryote, promoter sequence contains the binding site of other transcription factor, speed and arrangement of time that described transcription factor energy controlling gene is expressed.Described site comprises the TATA box, CAAT box, POU box, AP1 binding site etc.Promoter region also can contain enhancer element.When promotor links to each other with gene so that gene can be transcribed the time, this promotor is " can be operatively connected or link to each other " with gene.

Also can comprise other adjusting sequence.This sequence comprises transcription termination signal sequence, intron, secretory signal sequence, replication orgin, selective marker etc.Regulating sequence can operate continuous to transcribe or to translate each other.

Expression vector used herein comprises promotor, described promotor be designed to can be in plant host cell marking protein.Suitable promotor is easy to obtain, and is well-known in the art.Preferred induction type or constitutive promoter.

In other embodiment preferred, carrier also comprises transcription termination sequence." Transcription Termination zone " has signal sequence and/or the polyadenylation signal sequence that stops transcribing by the polysaccharase of discerning selected promotor.

As mentioned above, the used carrier of the present invention generally contains nucleotide sequence, and described nucleotide sequence contains the proteinic structure gene of at least one Codocyte apoptosis pathway, and described structure gene can be operated with promotor and be linked to each other.In order to begin method for transformation of the present invention, at first must make up appropriate carriers, and it is suitably imported vegetable cell.The same with requisite method for transformation, the detailed method that makes up carrier used herein also is the known to the skilled of plant genetic engineering field, yet this paper will discuss this two methods in more detail.Expression vector contains the protokaryon nucleic acid elements of coding bacterium replication orgin (as the intestinal bacteria replication orgin) and reporter gene or selective marker (as antibiotics resistance) usually, to cultivate and to select expression vector in host bacterium; The nucleic acid elements of control transcription initiation is as plant and the used promotor of bacterial growth; The nucleic acid elements of control transcript processing is as Transcription Termination/polyadenylation sequence; With can operate reporter gene or the mark that links to each other with promotor.Useful reporter gene comprises beta-Glucuronidase, beta-galactosidase enzymes, E.C. 2.3.1.28, luciferase etc.Preferred reporter gene is beta-Glucuronidase (GUS) or green fluorescent protein (GFP).Preferred selective marker comprises antibiotic marker, as G418, and Totomycin, kantlex, bialophos (giving) etc. by the bar gene.In certain embodiments, use the synthetic a large amount of constructs of bacterial expression system, digest to downcut required gene with restriction endonuclease subsequently.Then required gene is connected to plant expression vector, and is used for transformed plant cells.Therefore, those skilled in the art can easily understand: can use in bacterium and diverse reporter gene or selective marker used in plant.The preferred amicillin resistance that uses carries out the bacterium selection, and selects transgenic plant cells according to kalamycin resistance.

About the general description of plant expression vector and reporter gene can be referring to Gruber etc., " plant conversion carrier, Zhi Wufenzishengwuxue ﹠amp; Biotechnological means ", volumes such as Glich, p89-119, CRC press, 1993.In addition, GUS expression vector and gus gene box can derive from ClontechLaboratories, Inc., Palo Alto, Calif, and GFP expression vector and GFP box gene can derive from Aurora Biosciences (San Diego, CA).

Codocyte apoptosis pathway nucleic acid sequences to proteins also can comprise secretion signal, is processed and excretory precursor protein matter thereby make the gained polypeptide.Can from vegetable cell or tissue or phloem, reclaim the protein through processing of gained.The secretion signal that is suitable for using is to obtain easily, and is well-known in the art, and example is seen During etc., molecular biology of plants, 15:281-293,1990; Lindsey and Jones, plant cell is selected, p317-335, VCH Weinham, Germany, 1990; Gruber and Crosby, molecular biology of plants and biotechnological means, p89-117, CRC press, Boca Raton, FL, 1993.

Can use Ti-plasmids, root-induce (Ri) plasmid, microparticle bombardment, electroporation and the plant viral vector heterologous nucleic acid sequence that the present invention is used imports vegetable cell, and (comment about described technology and carrier can be referring to for example Weissbach ﹠amp; Weissbach, molecular biology of plants method, Academic press, New York, VHI joint, p421-463,1988; Grierson﹠amp; Corey, molecular biology of plants, the 2nd edition, Blackie, London, 7-9 chapter, 1988 and Horsch etc., science, 227:1229,1985; Shift with plant gene, Potrykus compiles, Springer Verlaa, and 1995, all list this paper in as a reference).

Can genome or synthetic segmental expression vector importing protoplastis or complete tissue or isolated cells will be contained.Preferably expression vector is imported complete tissue.The general method of culturing plants tissue can be referring to Zhi Wufenzishengwuxue ﹠amp; Biotechnological means, volumes such as Glich, p67-88, CRC press, 1993; Philips etc., Yu Mi ﹠amp; The corn improvement, the 3rd edition, volumes such as Sprague, company of U.S. agronomy committee etc., p345-387,1988, hereinafter this method will be described in more detail.2. promotor

In case selected host plant, and determine the method for transgenosis to plant, can be host plant and select composing type, induction type/growth adjustment type, or tissue-specific promoter, make foreign protein mass-energy in the required part of plant (as all cells, some cells, or whole tissue) express.Those skilled in the art can easily understand, and can use any promoters driven structure gene compatible with plant, more specifically are the apoptotic pathways expression of gene.Although can use the endogenous promotor of desired structure gene that gene is carried out transcriptional regulatory, preferred promoter is that external source is regulated sequence.For plant expression vector, suitable viral promotors comprises 35S RNA and 19S RNA promotor (Brisson etc., nature, 310:511,1984 of CaMV; Odell etc., nature, 313:810,1985); The total length transcript promotor of figwort mosaic virus FMV (Gowda etc., the cellular biochemistry magazine, 13D:301,1989 and United States Patent (USP) 5,378,619) and the coat protein promotor (Takamatsu etc., EMBO J6:307,1987) of TMV.Perhaps, also can use plant promoter, as light-inducible promoter (Coruzzi etc., EMBO J 3:1671,1984 of the little subunit of ribulose two-phosphoric acid carboxylase (ssRUBISCO); Bfoglie etc., science, 224:838,1984); Mannopine synthase promotor (Velten etc., EMBO J 3:2723,1984); Nopaline synthase (NOS) and octopine synthase (OCS) promotor (be carried on the root nodule of agrobacterium tumefaciens-induce on the plasmid) or heat-inducible promoter are as soybean hsp17.5-E or hsp17.3-B (Gurley etc., molecular cytobiology, 6:559,1986; Severin etc., molecular biology of plants, 15:827,1990).The relevant comment that is applicable to the multiple known plants promotor of the context of the invention can be referring to the open WO91/19806 of PCT.A. constitutive promoter

The promotor that is used for the context of the invention comprises composing type and induction type natural promoter and the promotor through transforming.Described promotor can derive from plant, virus or other source, include but not limited to the 35S promoter of cauliflower mosaic virus (CaMV), term used herein " CaMV35S " promotor comprises the variant and the analogue of CaMV 35S promoter, for example by being connected with the operator gene zone, at random or controlled mutagenesis and the promotor that obtains, tandem promoter etc.; Seed storage protein gene is (as Zma10Kz or Zmag12, be respectively zein and glutenin gene) or the promotor of " housekeeping gene " (as maize actin gene Zmaact) of in all cells, all expressing (referring to Benfey etc., science, 244:174-181,1989; Elliston, Plant Biotechnology, Kung and Arntzen compile, ButterworthPublishers, Boston, Mass., p.115-139,1989).Therefore, the present invention can utilize known can be in the promotor of the gene of edible plant part high expression level, as the paratin gene promoter of potato, example is seen Wenzler etc., molecular biology of plants, 12:41-45,1989.The CaMV promotor is the common examples of constitutive promoter, ubiquitin promotor (seeing EP patent application 0342926) and chlorella virus dna Methyl transferase promoter (seeing United States Patent (USP) 5,563,328).B. inducible promoter

As mentioned above, inducible promoter also can be used for context of the present invention.Inducible promoter is to react to inductor, directly or indirectly activates the promotor that nucleotide sequence is transcribed.When lacking inductor, sequence can not transcribed.In one embodiment, specificity zygotic induction type promotor exists with the rho factor of the activated transcription form with non-activity, directly or indirectly changes activity form into by inductor then.Inductor can be biological or abiotic attack, chemical reagent for example, as protein, metabolite (sugar, ethanol etc.), growth regulator, weedicide or phenolic compound, or by heat, salt, the physiological stress that the poisonous factor etc. directly cause, or the physiological stress that causes indirectly by the effect of pathogenic agent or disease factor (as virus).Can pass through to use inductor in outside, for example by spraying, water, heating is exposed to light, is exposed to pathogenic agent or similar method, makes the vegetable cell that contains inducible promoter be exposed to inductor.In certain embodiments, inducible promoter can be in induction state in the whole process that seed forms, perhaps be in induction state in the stage of transcribing corresponding to the recombinant nucleic acid molecules nucleotide sequence at least.

In order farthest to play a role, preferred inducible promoter can provide low and express or do not express when lacking inductor; And when having inductor, provide high expression level; The induction scheme that uses can not upset the normal physiologic of plant; And other expression of gene had no impact.The example that can be used for the inducible promoter of the context of the invention comprises by chemokines inductive promotor, as by cupric ion activated yeast metallothionein promoter (Mett etc., Proc.Natl.Acad.Sci.U.S.A.90:4567,1993); Substituted benzsulfamide (as herbicide-safener) activated In2-1 and In2-2 regulate sequence (Hershey etc., molecular biology of plants, 17:679,1991); Separate free N, N-diallyl-2,2-dichloro acetamide (popular name dichloramid) or benzyl-2-chloro-4-(trifluoromethyl)-5-thiazole carboxylic acid salt (popular name flurazole) is induced, and applies for the chemical induction gene promoter sequence of WO90/08830 described corn glutathione-S-transferase 27kD subunit (GSTII) gene as disclosed PCT; GRE by glucocorticoid inducible regulates sequence (Schena etc., Proc.Natl.AcadSci.USA 88:10421,1991); 70kDa heat-inducible promoter (Freeling and the Bennet of D.melanogaster, corn ADN1, heredity is commented academic year, 19:297-323) and by alcohol induced alcoholdehydrogenase promotor (Nagao etc., Miflin compiles, Oxford plant molecular and cytobiology summary, Vol.3, p384-438, Oxford University Press, the Oxford, 1986) or can be caused the Lex A promotor that also can derive from Ligand Pharmaceuticals by chemical treatment.Other inducible promoter comprises by pathogenic agent attacks the inductive promotor, described in PCT publication number WO98/22599 can be by nematode-inductive promotor, can be by promotor (Aoyama and the Chua of glucocorticoid inducible, the plant magazine, 11:605-612,1997), chalcone synthase promotor and defence activated promotor (propl-1) (Strittmatter etc., Bio/Technology 13:1085-1089,1995).The open application EP89/103888.7 that inducible promoter also is described in Ciba-Geigy has wherein identified a large amount of inducible promoters, comprises the PR protein gene, especially tobacco PR protein gene, as PR-1a, PR-1b, PR-1c, PR-1, PR-A, PR-S, cucumber chitinase gene, with acid and alkaline tobacco beta-1,3-glucanase gene.Wound-induced type (WIN) promotor also can be used for context of the present invention, and about discussion can be referring to Lindsey and Jones: plant cell is selected, p317-335, VCH Weinham, Germany, 1990.Other composing type and inducible promoter and enhanser are well known by persons skilled in the art.C. tissue-specific promoter

Tissue-specific promoter also can be used for the present invention.The example of tissue-specific promoter is expression promoter in the branch meristematic tissue (Atanas sova etc., plant magazine, 2:291,1992).Other tissue-specific promoter that can be used for transgenic plant also is well known by persons skilled in the art, and (example is seen Ito etc., molecular biology of plants, 24:863,1994 to comprise cdc2a promotor and cyc07 promotor; Martinez etc., Proc.Natl.Acad.Sci.USA 89:7360,1992; Medford etc., vegetable cell, 3:359,1991; Terada etc., plant magazine, 3:241,1993; Wissenbach etc., plant magazine, 4:411,1993); I class paratin (patatin) promotor (Bevan etc., nucleic acids research, 14:4625-38,1986) at stem tuber; Promotor (Muller etc., Mol.Gen.Genet.224:136-46,1990) with potato tuber ADPGPP gene-correlation; Soybean β-conglycinin (also being known as 7S albumen) promotor, its driving needle is to transcribe (Bray, Planta 172:364-370,1987) of seed; The promotor at seed of corn embryosperm alcohol soluble protein gene (Pedersen etc., cell, 29:1015-26,1982); And pollen specific promoter, as disclosed separation in the United States Patent (USP) 5,086,169 of Mascarenhas from the pollen specific promoter of corn; Disclosed another derives from the pollen specific promoter of corn in the United States Patent (USP) 5,412,085 of Allen etc.; Disclosed anther specific promoter in the United States Patent (USP) 5,477,002 of Tuttle etc.; Disclosed tapetum specific efficient promoter in the United States Patent (USP) 5,470,359 of Huffman etc.; Disclosed separation is from the tapetum specific efficient promoter of Cruciferae among the WO92/11379 of Draper etc.; Plant 8 (1): 55-63, disclosed tobacco pollen specificity promoter in 1995.

Also can in promoter construct, comprise intron sequences, can cause enhanced to be expressed and specificity because comprise intron sequences in the coding region.Therefore, it is comparatively favourable that dna sequence dna to be expressed is linked to each other with promoter sequence, and described promoter sequence contains first intron and the exon sequence of polypeptide, and described polypeptide is a vegetable cell/organize peculiar.In addition, the zone of a promotor can link to each other to obtain chimeric promoters with the zone of different promoters.3. mark

Select or the mark/reporter gene of marking but carrier of the present invention preferably includes also that at least one can work in the host.Identified a plurality of genes that can be used for this purpose (example is seen Fraley, Plant Biotechnology, Kung and Arntzen compile, ButterworthPublishers, Boston, Mass., p.395-407,1989; Weising etc., 22:421-477,1988 are commented in heredity academic year).Selectable marker gene comprises can give the specific phenotype of host or proterties so that transformant can be identified out and any gene that can selective growth.Therefore, the selectable marker gene coding is selected gene product, this product is given the resistance of vegetable cell to chemical reagent or physiological stress, or gives cell differentiable phenotypic characteristic, so that can be chosen by easily selected reagent by recombinant nucleic acid molecules plant transformed cell.The suitable selectable marker gene of host bacterium comprises ampicillin resistance gene (Amp ^r), tetracycline resistance gene (Tc ^r) and kalamycin resistance gene (Kan ^r).The present invention preferably uses ampicillin resistance gene.The suitable mark of eukaryote usually need with host's complementary defective (as at tk ^-Use thymidine kinase (tk) among the host).Yet, also can use drug labelling (as G418 resistance and hygromycin resistance).Suitably the example of selective marker comprises bar gene, adenosine deaminase, Tetrahydrofolate dehydrogenase, Totomycin-B-phosphotransferase, thymidine kinase, xanthine-guanine phosphoribosyl transferase and amino-glucosides 3 '-O-phosphotransferase II (kantlex, Xin Meisu and G418 resistance).Other suitable mark is well known by persons skilled in the art.In addition, can come identified gene to express by the method for carrying out the Northern trace such as the purpose mRNA that plant is produced.4. method for transformation

Basically can transform plant of the present invention (example is seen Enzymology method, Vol.153,1987, Wu and Grossman compile, Academic press lists this paper in as a reference) according in the known several different methods of molecular biology of plants those skilled in the art any.Term used herein " conversion " refers to the genotype that changes host plant by the importing heterologous nucleic acid sequence.

The method that expression vector is imported plant tissue comprises physics and chemical process, comprises electroporation, microparticle bombardment, and virus and infectation of bacteria/cultivate altogether with for example agrobacterium tumefaciens.These method for transformation can be used for foreign gene is imported unifacial leaf and dicotyledons, Potrykus, and plant physiology and molecular biology of plants year comment, 42:205-225,1991 and Shimamoto etc., nature, 338:274-276,1989.Cause foreign DNA stable integration to the main method of plant genome DNA to comprise following method: the 1) transgenosis of Agrobacterium-mediation; Horsch etc., science 227:1229,1985; Gruber etc., document is the same; Klee etc., plant physiology academic year comments, 38:467-486,1987; Klee etc., plant nucleus gene molecular biology, 6:2-25,1989; Gatenby, Plant Biotechnology, 93-112,1989; White, Plant Biotechnology, 3-34,1989; With 2) the mediated dna absorption, Paszkowski etc., plant nucleus gene molecular biology, 6:52-68,1989, comprise the method for dna direct being taken in protoplastis, Toriyama etc., Bio/Technology 6:1072-1074,1988; Swash vegetable cell and inductive DNA takes in by brief electrical, Zhang etc., vegetable cell report, 7:379-384,1988 and Fromm etc., nature, 319:791-792,1986.By microparticle bombardment DNA is injected to vegetable cell or tissue, Klein etc., vegetable cell and Recent advances in molecular biology, 56-66,1988; Klein etc., Bio/Technology 6:559-563,1988; McCabe etc., Bio/Technology 6:923-926,1988 and Sanford, plant physiology, 79:206-209,1990, system imports vegetable cell or tissue with DNA by the use micropipette, Hess, international cytology comment, 107:367-395,1987, Neuhaus etc., the applied genetics theory, 75:30-36,1987, Neuhaus and Spangenberg, plant physiology, 79:213-217,1990, or by the direct insulation of the pollen of DNA and sprouting is imported vegetable cell or tissue, DeWet etc. with DNA, EXPERIMENTAL MANIPULATION OF OVULE TISSUES, 197-209,1985, Ohta, Y.Proc.Natl.Acad.Sci.USA 83:715-719,1986; Or 3) use plant virus as genophore, Klee etc., plant physiology academic year comments, 38:467-486,1987; Futterer etc., plant physiology, 79:154-157,1990.

Someone advises using cauliflower mosaic virus (CaMV) (Howell etc., science, 208:1265,1980) and geminivirus infection group (Goodman, the general virology magazine, 54:9,1981) as carrier, but it is reported, kind with Agrobacterium has obtained maximum success (Rorsch etc., science, 227:1229-1231,1985).At present, the method for using in not of the same race based on the conversion system of Agrobacterium a plurality of had been described.Usually, need to use the bacterial isolates of the modified form that carries natural Ti-plasmids,, and can not form root nodule subsequently so that DNA can be transferred to host plant.These methods comprise the border of the DNA insertion Ti-plasmids that will be inserted into Plant Genome, link to each other so that select transformant with selectable marker gene.Described carrier system is described in Hajdukiewicz etc., molecular biology of plants, 25:989-994,1994.This binary system contains first Ti-plasmids and chimeric plasmid, and described Ti-plasmids has virulence region (vir), and this virulence region is that transfer DNA (T-DNA) importing plant is necessary.Chimeric plasmid contains the frontier district at least one wild-type Ti-plasmids T-DNA district, and its flank is in the nucleic acid that shifts.Effectively transformed plant cells (De Framond Biotechnology1:262,1983 of binary Ti-plasmids system have been confirmed; Hoekema etc., nature, 303:179,1983).Other binary system is described in PCT publication number WO99/01563 and WO99/10514.

Bacterium and plant tissue are cultivated together,, selected the plant transformed of regenerating on the substratum then so that foreign DNA is transferred to vegetable cell.As specifically described to the Cruciferae member, the Different Organs of any number and organize the target that all can be used as agriculture bacillus mediated conversion.They comprise thin cellular layer (Charest etc., applied genetics theory, 75:438-444,1988), hypocotyl (DeBlock etc., plant physiology, 91:694-701,1989), blade (Feldman, plant science, 47:63-69,1986), stem (Fry etc., vegetable cell report, 6:321-325,1987), cotyledon (Moloney etc., vegetable cell report, 8:238-242,1989) and class embryo (embryoid) (Neuhaus etc., the applied genetics theory, 75:30-36,1987).Yet those skilled in the art understand: can select different tissues or method for transformation in some crop.

In order to find not have the plant of any position effect, a large amount of produce by any recombinant precursor respectively plant transformed be useful.In certain embodiments, the preferred plant of selecting to contain the heterologous nucleic acids molecule that a copy imports so that reticent effect minimize.

In one embodiment, use the common cultivation of vegetable cell and virus or bacterium that expression vector is imported plant tissue.For example, generally can make under its control that is in promotor, use system based on plant RNA virus by the desired structure gene being inserted the shell promoter region of suitable plant virus.Based on the system description of plant RNA virus in for example United States Patent (USP) 5,500,360; 5,316,931 and 5,589,367 (all listing this paper in as a reference).

As mentioned above, in certain embodiments of the invention, used Agrobacterium-Ti-plasmids system, Watson etc., recombinant DNA concise course, Scientific Beauty compatriots' book series, 164-175,1983.The root nodule of agrobacterium tumefaciens-induce (Ti) plasmid contains the plasmid DNA section that is known as transfering DNA (T-DNA), and this section can be transferred to vegetable cell, is integrated into the plant host genome in cell.The structure of conversion carrier system has two basic steps.At first, the structure plasmid vector that can in intestinal bacteria, duplicate.This plasmid contains the DNA (being antigen protein in the present invention) of the desired protein of encoding; The flank of this DNA is the T-DNA border sequence, and this border sequence can determine that DNA is integrated into the site of Plant Genome.Usually, the gene of the selective marker of also will encoding (as the coding antibiotics resistance, as the gene of kalamycin resistance) inserts between left margin (LB) and right margin (RB) sequence; The expression of this gene in transformed plant cells can provide positive system of selection, thereby identifies the plant or the vegetable cell in the T-DNA zone with integration, Watson etc., recombinant DNA concise course, Scientific Beauty compatriots' book series, 164-175,1983; White, Plant Biotechnology, 3-34,1989.Second step must be transferred to Agrobacterium by intestinal bacteria with plasmid.Via engaging mating system, or can reach this purpose by directly taking in plasmid DNA with Agrobacterium.Subsequently, for T-DNA is transferred to plant, used agrobacterium strains contains virulence (vir) gene, and this gene can be used for T-DNA is transferred to vegetable cell, White, Plant Biotechnology, 3-34,1989; Fraley, Plant Biotechnology, 395-407,1989; Hajdukiewicz etc., molecular biology of plants, 25:989-994,1994.It will be understood by those skilled in the art that: the genetic transformation that can use multiple agrobacterium strains and plasmid construction strategy optimization plant.Will also be understood that agrobacterium tumefaciens is not unique agrobacterium strains that can use.In some applications, other agrobacterium strains may be more suitable as Agrobacterium rhizogenes.The method of inoculation plant tissue has nothing in common with each other, and this depends on species and the Agrobacterium transfer system of plant.Method is blade (leaf disc) method very easily, and available any explant of organizing carries out this method, and described outer planting physical efficiency provides the good source of initial whole plants differentiation.Under certain conditions, it also is desirable adding that child care organizes.Also can carry out other method, for example use agrobacterium tumefaciens vitro conversion regenerated protoplastis, to obtain plant transformed cell, Potrykus, molecular biology of plants, 42:205-225,1991; White, Plant Biotechnology, 3-34,1989.

Relate to and use the method for Agrobacterium to include but not limited to: 1) cultivate altogether with Agrobacterium and through the isolating protoplastis of cultivating; 2) with Agrobacterium-mediated Transformation vegetable cell or tissue; Or 3) use Agrobacterium-mediated Transformation seed, summit or meristematic tissue.In addition, by pressing Bechtold etc., C.R.Acad.Sci.Paris 316:1194, the 1993 described Agrobacterium converted in-situ of using, with press U.S. Patent application 08/667,188 and corresponding PCT publication number WO97/48814 described in method for transformation and the method for transformation that exemplifies of this paper, can realize transgenosis.Briefly, this method is infiltrated based on the vacuum of agrobatcerium cell suspension.

The preferred method that heterologous nucleic acids is imported vegetable cell is to infect described vegetable cell, explant, meristematic tissue or seed with aforesaid through transforming agrobacterium tumefaciens.Under felicity condition known in the art, cultivate through the plant transformed cell forming branch, root, and further develop into plant.In one embodiment, carrier of the present invention contains the Ti-plasmids binary system, wherein heterologous nucleic acid sequence Codocyte apoptosis pathway protein.In certain embodiments, nucleic acid sequence encoding is at least a resists-apoptosis protein matter.Carrier can be chosen wantonly and contain coding second kind or more kinds of apoptotic pathways nucleic acid sequences to proteins.Perhaps, can utilize two or more carriers, wherein each carrier contains heterologous nucleic acid sequence.Can use gene constructed one or more carriers of other apoptotic pathways in a similar fashion.

In certain embodiments, can be by using direct physics or chemical process contact vegetable cell so that heterologous nucleic acid sequence is imported vegetable cell.For example, can be by using micropipette or microparticle bombardment, through micro-injection directly with the nucleic acid physical transfer to vegetable cell.Perhaps, by using polyoxyethylene glycol nucleic acid is transferred to vegetable cell, described polyoxyethylene glycol can form with genetic material can be by the sediment composite of cellular uptake.

Another kind with the method that nucleic acid imports vegetable cell is: carry out the high speed ballistic penetration with the small-particle that contains nucleic acid to be imported, described nucleic acid is included in globule or the particulate matrix, perhaps is positioned at its surface (Klein etc., nature, 327:70,1987; United States Patent (USP) 4,945,050,5,036,006 and 5,100,792).Usually, when using the small-particle bombardment, DNA is adsorbed on the particulate as magnesium sulfate crystals or tungsten particle, and described particulate is accelerated to cell or plant tissue by physics.Although general only the needs imports new nucleotide sequence once, this method generally provides repeatedly and has imported.

Also can heterologous nucleic acids be imported vegetable cell (U.S.A.82:5824 1985, lists this paper in as a reference for Fromm etc., Proc.Natl.Acad.Sci.) by electroporation.In this technology, in the presence of carrier that contains associated nucleic acid sequences or nucleic acid, plant protoplast is carried out electroporation.The electricimpulse of high field intensity can reversibly penetrate film, thereby imports nucleic acid.Plant protoplast through electroporation forms cell walls again, and division also forms plant callus.Use phenotypic markers as described herein can select to contain the transformed plant cells of transforming gene.

In addition, as mentioned above, cauliflower mosaic virus (CaMV) also can be used as heterologous nucleic acids is imported the used carrier of vegetable cell (United States Patent (USP) 4,407,956).CaMV viral DNA genome is inserted parental generation bacterial plasmid, the recombinant DNA molecules that generation can be bred in bacterium.After the clone, clone recombinant plasmid once more, and further modify recombinant plasmid by importing required nucleotide sequence.From the parental generation bacterial plasmid, excise the modified viral part of recombinant plasmid then, and be used to inoculate vegetable cell or plant.

In a plurality of embodiments, the invention provides the method that produces transgenic plant, described transgenic plant have modulated apoptosis, abiotic attack and/or biological attack resistance.Described method comprises makes vegetable cell contact with carrier, described carrier contains heterologous nucleic acid sequence, and described nucleotide sequence contains at least one can be operated with promotor and link to each other the proteinic structure gene of Codocyte apoptosis pathway; Produce plant by described through the plant transformed cell; And option table reveals abiotic or biological attack resistance and/or modulated apoptotic plant.

Term used herein " contact " refers to any method that carrier is imported vegetable cell, comprises aforesaid chemistry, physics or mechanical means.Preferred contact refers to the agrobacterium tumefaciens that transforms via by above-mentioned heterologous nucleic acids, and nucleic acid or carrier are imported vegetable cell (comprising explant, meristematic tissue or seed).

Generally answer the aftergrowth cell to obtain whole plants by method for transformation.The direct product that transforms is called as " transgenosis ".Term used herein " cultivation ", " generation " or " regeneration " refer to by a vegetable cell, one group of vegetable cell, and plant part (comprising seed) or plant tissue (as the protoplastis tissue, callus or tissue part) grow up to the complete plant of a strain.

In the kind of plant and different between planting, but generally at first to prepare the suspension of protoplastis by the method for the complete plant of protoplast regeneration.In some is planted, can induce forming of embryo by protoplastis suspension then, reach the maturation and the germination stage of normal chick embryo.Substratum generally contains growth and regenerate required multiple amino acids and hormone.The example of used hormone comprises plant hormone and cytokinin.Sometimes, adding L-glutamic acid and proline(Pro) are favourable in substratum, concerning especially true such as the species of corn and clover.Effectively substratum is depended in regeneration, genotype and cultivation history.If these variablees are controllable, regeneration also just can be reproduced.

Also can be by plant callus, explant, the complete plant of organ or partial regeneration.Can in organ or plant part regenerated context, transform (referring to Enzymology method, Vol.118 and Klee etc. comment 38:467,1987 plant physiology academic year).Utilize Horsch etc., science, 227:1229, blade-conversion-method of reproduction of 1985 is cultivated blade in selecting substratum, form branch at about 2-4 in week.Downcut the branch that grows from callus, migrate in the suitable selective medium of inducing root.The appropriate selection substratum is known in the art, and is described in Curry and Cassells, culture plant cell method, p31-43, Humana press, Totowa, NJ, 1999; Blackwell etc., IBID 19-30,1999; Franklin and Dixon, culture plant cell, p1-25, IRL press, Oxford, 1994.The plantlet that to take root as early as possible after root occurs migrates in the soil.In case of necessity plantlet is planted again in the flowerpot until reaching ripe.

In vegetative crop, breed sophisticated transgenic plant by the intercepting cutting or by tissue culture technique, to produce the identical plant of many strains.Select desirable transgenosis, obtain new variety, these new variety of vegetative propagation are for commercial.With in the crop of seminal propagation, the inbreeding plant of can the sophisticated transgenic plant of selfing isozygotying with generation.The inbreeding plant produces the seed of the heterologous nucleic acid sequence that contains new importing.Can cultivate these seeds to generate the plant that can produce selected phenotype (as pathogen resistance).

The present invention also comprises the part that derives from aftergrowth, as flower, and seed, leaf, branch, fruit etc., condition is that these parts contain cell transformed as stated above.Also comprise offspring and the variant and the mutant of aftergrowth in the scope of the invention, condition is the heterologous nucleic acid sequence that these parts contain importing.

In certain embodiments, consider that vegetable cell is contained the recombinant DNA molecules conversion of at least two dna sequence dnas, or transformed by more than one recombinant DNA molecules.Dna sequence dna in these embodiments or recombinant DNA molecules can link to each other by physics in identical carrier, or physics is dispersed in the different carriers.Cell can be transformed by more than one carrier simultaneously, and condition is that each carrier has unique selectable marker gene.Perhaps, can make and after transforming, can carry out regeneration step immediately successively with more than one carrier transformant with first carrier.In addition, can sexual hybridization between each plant that contains different dna sequence dnas or recombinant DNA molecules or plant lines, preferred dna sequence dna or recombinant molecule are connected or are positioned on the phase homologous chromosomes, select to contain the plant of dna sequence dna or recombinant DNA molecules then from the offspring of hybridization.

Use the Northern engram technology, Southern engram technology and/or marker expression, and other technology well known by persons skilled in the art can be monitored the expression of recombinant DNA molecules in transformed plant cells that contains dna sequence dna described herein and promotor.C. produce plant and plant part with required proterties

The present invention relates to wonderful discovery, promptly allos apoptotic pathways albumen mass-energy works in plant.Therefore, this discovery provides a plurality of advantages, comprises plant and plant part (as cut-flower, vegetables etc.) are strengthened the resistance of biological and abiotic attack, and can suppress to wear out.In addition, the present invention also provides the method for identifying functional plants apoptosis protein matter homologue and the method for identification of cell apoptosis pathway proteinic inhibitor and toughener in the context of vegetable cell.1. biological attack/factor resistance

Biological attack is the attack that plant suffers, and it is the direct or indirect consequence that biotic factor is attacked.Biotic factor comprises for example insect, fungi, bacterium, virus, nematode, viroid, mycoplasma etc.Usually, biotic factor is induced the apoptosis of infected vegetable cell.It is believed that the purpose that produces apoptosis is in order to suppress to invade the propagation of pathogenic agent.Yet in one embodiment of the invention, the nucleic acid sequences to proteins that participates in the downward modulation apoptosis in other organism of encoding is passed to vegetable cell, has illustrated the plant that is grown up to by this vegetable cell multiple biotic factor is had resistance.

In certain embodiments, the plant that biological attack is had a resistance provides the remarkable advantage relevant with the lasting health of crop yield and ornamental plant.Topmost biotic factor comprises multiple pathogenic agent, as fungi and virus.One routine pathogenic agent is the fungal pathogens sclerotinite, and it is one of the most assorted phytopathogen of the poorest and recipe of known specificity (example is seen Purdy, plant pathology, 69:875-880,1979).In addition, other multiple important economically pathogenic agent also is known, comprise the fungi gray botrytis, Magnaportyhe gri sea, the kind of phytophthora, the kind of revolving the mould genus of spore, the kind of F.graminearum schw and fusarium, nematode Meloidogynespp (root nematode), virus, as tobacco mosaic virus (TMV) (TMV), tomato spotted wilf virus (TSWV), tobacco corrosion virus (TEV), tobacco necrosis virus (TNV), Wheat streak mosaic virus (WSMV), autochthonal wheat streak mosaic virus (SBWMV), barly yellow dwarf virus (BYDV), kind of bacterium Rhodopseudomonas and the kind of xanthomonas and a lot of other pathogenic agent.Can obtain the example of a plurality of pathogenic agent, those skilled in the art can easily identify these pathogenic agent, and example is seen plant pathology, and the 4th edition, Agrios compiles, Academic press, San Diego, 1997.

In one embodiment, the invention provides the transgenic plant that biological attack had resistance.In this embodiment, by aforesaid method the proteinic heterologous nucleic acid sequence of Codocyte apoptosis pathway is transferred to plant.In certain embodiments, the apoptotic pathways protein of coding is anti--apoptosis protein matter, and promptly this protein can be born the adjusting cell directly or indirectly and enter the apoptosis cycle, or negative process of regulating the apoptosis cycle.Preferred inhibitors includes but not limited to the dominant conditioning agent, as the caspase molecule of avtive spot cysteine mutation, and the CARD structural domain, the Apaf-1 of clipped form etc., and other negative conditioning agent, as IAP, Bcl-2, Bcl-x _L, Bcl-W, McL-1, A1, NR13, ced-9, E1B 19K, BHRF1 etc.Therefore, the inhibitor that exemplifies of this paper comprises Bcl-2 and Ced-9.These apoptosis inhibitors are given the plant that shifted this inhibitor resistance to biological attack.The biological attack resistance that exemplifies is a pathogen resistance, it comprises the resistance to multiple virus and fungi, as tobacco mosaic virus (TMV) (TMV), tobacco necrosis virus (TNV), tobacco corrosion virus (TEV), tomato spotted wilf virus (TSWV), sclerotinite 1980 (Dickman and Mitra, Physiol.Mol.Plant Pathol, 41:255-263,1992); Gray botrytis (ATCC), tobacco tail spore and enclose small cluster shell (Glomerella cingulate).Yet it should be noted: the present invention is not limited to the resistance to these pathogenic agent that exemplify.Therefore, in some aspects, express and use sterilant and with herbicide treatment can inducing plant morbid state, described morbid state causes the apoptosis incident, therefore, expressing anti--apoptosis polypeptide can increase the ability that specified plant is expressed pesticide compound, and/or increases specified plant to the resistance with multiple herbicide treatment, thereby resists abiotic attack.2. abiotic attack/factor resistance

As mentioned above, the present invention also provides the abiotic attack resistance that increases.Cause the abiotic factor of abiotic attack to comprise for example environmental factors, low humidity (arid) for example, high humidity (floods), nutritive deficiency, radiation level, atmospheric pollution (ozone, acid rain, sulfurous gas etc.), temperature (overheated and cold excessively), and soil toxicity, and weedicide infringement, sterilant damages, or other agricultural operation (for example fertilising is excessive, and the pharmaceutical chemicals sprinkling is improper etc.).Therefore, if described abiotic factor constantly strengthens the effect of various plants type (comprising food crop and ornamental plant) viability, the present invention also can be used for producing the resistance enhanced plant to these attacks.

In one embodiment, the description of relevant biological attack the invention provides the transgenic plant that abiotic attack had resistance as mentioned.In this embodiment, the method that describes in detail by this paper is transferred to plant with the proteinic heterologous nucleic acid sequence of Codocyte apoptosis pathway.As for the biological attack resistance, these apoptosis inhibitors are given the plant that shifted this inhibitor resistance to abiotic attack.

Those skilled in the art can easily understand: with reference to content provided herein, use the whole plants that is suitable for the specificity factor action method or blade can easily detect resistance to particular organisms or abiotic attack/factor.3. aging

Known plants is aging be finally cause necrocytosis be subjected to regulate process (go through generally can be referring to Guiamet etc., plant cell physiology, 31:1123-1130,1990).In addition, it also is accompanied by a lot of biological chemistries and structural changes, for example induces L-Cysteine HCL Anhydrous, RNA enzyme etc., and this is consistent with apoptosis.Therefore, by utilizing the transfer of the proteinic heterologous nucleic acids molecule of Codocyte apoptosis pathway, the invention provides modulation plant aged method.Although can be used to increase the life-span of plant prevailingly, also can realize other advantage.For example, suppress the aging prolongation that can cause vegetables and fruit shelf-lives, and life-span of cut-flower and other ornamental plant is prolonged and so beautiful that it excites men.In addition, for the plant that lives, florescence and fruiting period are prolonged.Therefore, the present invention can be widely used in food products market and ornamental plant market.

In addition, can use method of the present invention to identify endogenous plant apoptosis modulator, this modulator conversely can be in suitable vegetable cell optionally overexpression with delaying retrogradation.D. stride the active inhibitor of method 1. apoptosis and the toughener of kind of transitional cell apoptosis pathway gene

The inhibitor and the toughener of research vegetable cell apoptosis can be the data that design transgenic plant and weedicide and sterilant provide usefulness.In addition, in view of beat all and wonderful discovery, the proteinic gene of the apoptotic pathways of the species that the difference of promptly encoding is very big can be brought into play similarly effect in plant, vegetable cell can be used for test, survey the functional of the proteinic gene of Codocyte apoptosis pathway through shifting.For example, can be with mammalian cell apoptosis pathway gene, be transferred to vegetable cell as caspase and Bcl-2 family member, study the inhibition or the enhancement of pair cell apoptosis by several different methods, described method such as analysis of nucleic acids extract are with identification of cell apoptosis mark (as dna ladder), the TUNEL staining analysis, and/or the microscope inspection cell is with the cohesion of identification of cell apoptosis nuclear.

Candidate inhibitor and toughener separable from or derive from a plurality of sources, as the people, rodent, fly (D.melanogaster), nematode (C.elegans), virus (as baculovirus, simplexvirus), bacterium, fungi, plant, parasite, chemical library, peptide or peptide derivant etc.Also can reasonably design inhibitor and toughener according to the protein structure of measuring by X-radiocrystallography (example is seen Mittl etc., journal of biological chemistry, 272:6539-6547,1997).In certain preferred aspects, the specific apoptotic pathways protein (as bcl-2 or its homologue) of inhibitor target.

Needn't stick to specific mechanism, inhibitor can prevent enzymic activity by preventing the processing of apoptotic pathways proenzyme (as caspase), prevents that pathway component (directly or indirectly) from interacting, or plays a role by another kind of mechanism.Inhibitor self can directly or indirectly play a role.In preferred embodiments, the vegetable cell inhibitors of apoptosis can reduce apoptosis, this point can be measured by following method: dna ladder, terminal deoxidation transferring enzyme-mediated dUTP nick end labeling otch end mark (TUNEL) (commercially available test kit) depends on Ca ²⁺Ribozyme level (Mittler etc., vegetable cell, 7:1951-1962,1995), use vital staining (as trypan blue) or similar approach.In other embodiment preferred, inhibitor is a small molecules.In the most preferred embodiment, inhibitor can prevent apoptosis.The inhibitor of preferred energy penetration cell.

In addition, the toughener that needs apoptosis activity or apoptotic pathways protein expression in some cases.At that time, increase apoptosis and can alleviate the pathogenic agent infringement, all the more so for the pathogenic agent (as virus or fungal pathogens) that needs viable cell to play a role.Therefore, it is favourable expressing this toughener under pathogen-inducible.Toughener can increase the speed or the effectiveness of apoptosis process, and this point can be illustrated by following mechanism: the dna ladder level increases, and the TUNEL positive cell depends on Ca ²⁺Ribozyme raise (Mittler etc., vegetable cell, 7:1951-1962,1995), proenzyme (caspase or its homologue) processing is transcribed or is translated increase, or works by other mechanism.Know that a lot of guidances mentioned above also are applicable to the design toughener with those skilled in the art know that.

The shaker test of inhibitor and toughener is with the type of inhibitor or toughener, different and different with affected living features.Test can be carried out in external or body.Usually, in vitro tests is designed to estimate apoptosis phenotype incident, dna ladder, and TUNEL dyeing depends on Ca ²⁺Ribozyme level (Mittler etc., vegetable cell, 7:1951-1962,1995), protein processing or enzymic activity, and in vivo test is designed to these things are evaluated as reactivity to biological and abiotic attack.In any test, the statistics for suitable contrast increases significantly or reduces the expression resistance, strengthens or inhibition.2. plant identification apoptosis protein matter and genes involved

Can carry out an external test with combining of mammalian proteins matter (or other known apoptotic pathways protein) by checking vegetable cell protein, with the apoptotic pathways protein in the research plant.Briefly, can screen the expression product and the multiple mammalian cell apoptosis pathway component bonded ability of the expression library of plant cDNA.Similarly, can screen plant milk extract.For example, can be with the Apaf-1 proteopexy in solid surface (on post), and contact with the expression product in plant cDNA library.Apaf-1 combines with Mammals, and subsequently by the pH gradient, the protein of wash-outs such as salt gradient is plant caspase-9 homologue.Therefore, any apoptotic pathways protein can be fixed in a similar fashion, and is used to survey the plant cDNA expression library.Those skilled in the art should understand that the apoptosis protein matter that derives from different plant species can keep this wonderful discovery of functionally active to show: any known method that can survey protein-protein interaction all can be used for identifying the plant protein homologue of other known apoptosis protein matter in plant.It will be understood by those skilled in the art that and to use multiple mark and detection method.Described method comprises enzyme linked immunosorbent assay, analytical ultracentrifugation and use BiaCore 3000 ^TM(BiaCore, Uppsula, Sweden).

Therefore, in the proteinic method of plant identification apoptotic pathways, made up fusion rotein, it contains known allos apoptotic pathways protein or its fragment and mark peptide sequence (as glutathione-S-transferase), described mark peptide binding antibody or other molecule (as gsh).Carrier transform bacteria with encoding fusion protein.Purified fusion protein.Briefly, induce pGEX-4T-3 (Pharmacia, Uppsala by IPTG, Sweden) GST in (glutathione-S-transferase) Bcl-2 fusion rotein, and use that gsh-(example is seen Kaelin etc. to this fusion rotein of pearl purifying, cell, 64:521,1991).Carry the cell of expression of plants library construction body with metabolism method mark.With cell extract and the gsh that is full of GST-Bcl-2-Sepharose pearl insulation.Perhaps, immunoprecipitation fusion rotein etc.Wash unconjugated protein, the protein of elution of bound.By the further fractional separation bonded of example gel electrophoresis protein.Then bonded protein is used to produce antibody, amino acid sequence analysis and other in vitro tests.Can be by the clone of any separation coding conjugated protein in the multiple standards method, described method comprises the immunoscreening expression library, probe hybridization (its middle probe is based on partial amino-acid series) and other currently known methods.

In case between expression of plants library or plant milk extract and known apoptotic pathways component, detect protein-protein interaction, can from the expression of plants library, identify protein by order-checking, and identify suitable cDNA.

In one embodiment, provide detection vegetable cell apoptosis pathway method of protein, described method comprises with the expression cDNA library transfection or the transformant that derive from plant; Detect expressed protein of cell and the interaction between the allos apoptotic pathways protein.In another embodiment, provide and detected the vegetable cell apoptosis pathway method of protein of inferring, described method comprises: make plant milk extract or plant protein from expression library contact with known allos apoptotic pathways protein, and detection allos apoptotic pathways protein and derive from interaction between the protein of plant.In multiple other alternative method, can derive from the gene of plant and in vegetable cell, express the functional equivalent that the gene that derives from animal detects plant or animal gene by in zooblast, expressing, perhaps screen its apoptosis activity.For example, in carrier, make up the cDNA library in self-organization or cell source, have report albumen or peptide-labeled fusion rotein with generation.Report albumen or mark can be can be by any protein convenient and that measure sensitively.For example, can use beta-galactosidase enzymes and FLAG peptide.In addition, can use a plurality of marks to detect by sandwich test.Usually, carrier can use the expression of strong promoter with driving cDNA-fusion gene, and can use the replication orgin that is suitable for host cell.Then the plant gene library is transformed or transfection to animal host's cell.Perhaps, by using any method as herein described or other known method to transform the various plants host cell, take this to detect the apoptosis functional equivalent of animal gene, (the Nagata etc. of described plant host cell such as tobacco-(BY-2), international cytology comment, 132:1-30,1992); Corn (Hi-II) (corn handbook, p663-671, volumes such as Freeling, Springer, New York, 1994); Arabidopis thaliana (landsberg erecta) (Fuerst etc., plant physiology, 112:1023-1028,1996).

Perhaps, can pass through other method, as yeast two-heterological system identifies other vegetable cell apoptosis pathway protein.Briefly, in two-heterological system, the proteinic fusions of constructed dna-binding domains-apoptotic pathways (as the GAL4-Bcl-2 fusion rotein), conversion or transfection are to the cell that contains the GAL4 binding site, and described site links to each other with selectable marker gene.Structure merges with GAL4 activation structure territory, derives from the cDNA library of vegetable cell and tissue, and carries out cotransformation or cotransfection.When coding of the cDNA in the fusions of cDNA-GAL4 activation structure territory and Bcl-2 interacting proteins, selective marker is expressed.Cultivate the cell that contains cDNA then, the separation construct is also identified.

Also can carry out other test, whether on function, be equal to known with the protein of measuring by polynucleotide encoding, by the apoptotic pathways protein of another polynucleotide encoding, before perhaps further measuring plant gene or cDNA and whether having-or anti--apoptosis activity.In one embodiment, carry out above-mentioned test by required gene of heterogenous expression in any of several systems, described system comprises that for example mammlian system (comprises the people, the cell of tissue culture or clone), C.elegans nematode system, the nematode gene ced9 that wherein in apoptosis, works, (therefore ced4 or ced3 have sudden change, can seek functional rehabilitation), with fruit bat D.melanogaster, especially be subjected to the fruit bat of the eye-specificity promoter control in genetic engineering modified fruit bat with pigsney is, why having pigsney is because due to the excessive apoptosis of fruit bat retina, and excessively apoptosis is caused by allos overexpression or the apoptotic gene of disappearance control fly's eye eyeball.

When utilizing the Mammals pilot system, expression is under the control of promotor (as CMV) by rotaring redyeing gene or cDNA.Observe the cell of expressing gene, seek the apoptotic evidence of autonomous induction, perhaps make of the stimulation of described cellular exposure, measure the influence of genetic expression pair cell apoptosis kinetics or degree then in the energy cell death inducing.The stimulation of energy cell death inducing is well known by persons skilled in the art, it comprises withdraws somatomedin, ionizing rays, use ligand stimulation, described part can in conjunction with the death receptor of TNF receptor family (as Fas, TNF α, TRAIL), with (pro)-apoptosis gene before the heterogenous expression (as Bax, Bad).Use multiple detection method well known by persons skilled in the art; can measure through the apoptosis of transfectional cell pair cell apoptotic stimulus due to reacting; described detection method comprises induces caspase enzymic activity (ability of the stain remover extract cracking caspase substrate ethanoyl-Asp-Glu-Val-Asp-amino methyl tonka bean camphor by measuring cell is measured activity); dna degradation (measuring) by agarose gel electrophoresis; nuclear condenses (dye by Hoechst or DAPI and measure); by plastosome be released into kytoplasm cytochrome c (by for example cell grade Separation Research or by carry out with the cytochrome c monoclonal antibody immunocytochemistry locate measure); and mitochondrial function (by tetrazolium salts, the determination by reduction of MTT or MTS).Therefore, if the apoptotic effect of required gene pairs (increases or reduces the kinetics or the degree of apoptosis phenotype, or cell death inducing itself) being similar to known on matter and/or amount (as Bcl-2, Bax), can with required genetic testing be and this known functional equivalent; To plant base thereby speech, if certain expression of gene has influence on the kinetics or the degree of apoptosis phenotype, or can cell death inducing itself, then can be with this genetic testing preceding-or resist-apoptosis gene.

About the C.elegans system, if genes encoding is anti--apoptosis protein matter, some or all of cells (for example losing the ced9 rescue in the nematode of ced9 function) are saved in the genetic expression meeting in the function mutation worm (worm) (wherein a lot of or most of worm cells suffer apoptosis) of ced9 disappearance.As fruit gene is preceding-apoptosis gene, the necrocytosis that the genetic expression meeting recovery in the function mutation nematode system (wherein be in developmental hermaphroditic 128 cells and exempt from apoptosis) of ced3 or ced4 disappearance is lost.

About the D.melanogaster system, if certain gene can return to normal size with the eyes of fly, can think that this genes encoding resists-apoptosis protein matter, if this expression of gene causes the fruit bat eyes further to diminish, can think that then this gene is preceding-apoptosis gene.

Generally carry out in vivo test in or stable conversion or cells transfected instantaneous by expression vector, described expression vector contains as described herein, the proteinic gene of Codocyte apoptosis pathway.In the context of vegetable cell, in vivo test generally comprises with biology or abiotic factor attack cells, and plant or plant part are observed the morphology of inoculation site, seek apoptotic sign.Subsequently, dna fragmentationization and TUNEL positive cell number are analyzed with respect to the variation of control sample, further identified these inoculation site.In addition, in the context of zooblast, the caspase processing in the time of can using these cells to measure existence or shortage candidate compound, substrate conversion, or apoptosis.When detecting apoptosis, can use the various kinds of cell analysis, comprise that for example dyeing and microscope inspection are with the fragmentation of checking nucleic acid and the porosity of cell.In addition, can carry out in vivo test, detect when having candidate compound, through transforming or the cells transfected apoptosis pathway protein or the ability of the known substrate of activated protein cleavage thus, described substrate is by cotransfection, cotransformation, or place cell culture medium, can detect and measure substrate conversion by this test.

Detection side's science of law of apoptotic pathways enzyme is the method that is usually used in the enzyme analysis reaction, and it comprises for example SDS-PAGE, spectroscopy, and HPLC analyzes, radioautograph, chemical beam split, color reaction and immunochemistry (as trace, precipitation etc.).In addition, also can use the detection method of multiple making plant apoptosis (being apoptosis).Example is seen Mittler etc., vegetable cell, 7:29-42,1995; Mittler etc., molecular biology of plants, 34:209-221,1997; The TUNEL test kit of Oncor and Boehringer-Mannheim.

When needs are analyzed caspase when active, for example, when the vegetable cell apoptosis pathway gene of inferring is expressed, or when otherwise needing, preferably use chromogenic substrate in zooblast.The transformation energy of these substrates is measured apoptotic pathways directly or indirectly, the enzymic activity of especially one or more caspase molecules.In this, such as through the caspase of mark molecule, lamin, the multiple substrate of PARP and caspase substrate analogue is well known by persons skilled in the art.These substrates also can be purchased from company, as Oncogene ResearchProducts, Cambridge, MA.Can be comprised ZEVD-amc (carbobenzoxy-Glu-Val-Asp-amino methyl tonka bean camphor) by the example of the substrate analogue of fluorescent mark mark, YVAD-amc (ethanoyl-Tyr-Val-Ala-Asp-amino methyl tonka bean camphor) and DEVD-amc (ethanoyl-Asp-Glu-Val-Asp-amino methyl tonka bean camphor).

In addition, in other detects embodiment, can in construct, use eukaryotic promoter, with induction type or constitutive expression preceding-or anti--apoptosis protein matter be passed to cell and detect.For example, can conversion or transfectional cell so that their overexpressions anti--apoptosis polypeptide Bcl-2, thereby the cell that provides membrane product to have higher level Bcl-2 makes to detect to overcome the potentiators of apoptosis that Bcl-2 suppresses.Therefore, available second carrier transformant, this carrier contains the derivable cDNA that derives from the expression of plants library.In this regard, available apoptosis stimulation process cell is so that cell is inducing cDNA can keep the apoptosis balance before transcribing.

The identification of cell inhibitors of apoptosis mentioned above and the method for toughener provide another kind of mensuration apoptosis active mode, because cell is processed, make it can carry out the preparation of apoptosis.By this method, cell synthesizes and/or activates required all of apoptosis must component.Needed only is to cause cell to surmount its holding point (holdingpoint) to enter apoptotic stimulation.Therefore, stimulate under the existence of (as anti--fas antibody, Staurosporine etc.) at apoptosis, toughener can cause cell to enter apoptosis, and inhibitor can postpone or suppress this process.

Prevent that the holding point that cell enters apoptosis from can be the overexpression of apoptotic pathways protein (cell survival (anti--apoptosis) polypeptide), or with known apoptosis inhibitor processing cell.Anti--apoptosis polypeptide is characterised in that: when when suffering through inducing to express in the apoptotic cell or activating these polypeptide, they show the apoptotic ability that prevents.For example, when shortage work anti--during the apoptosis polypeptide, the cell of being handled by potentiators of apoptosis (as preceding-apoptosis factor) can start or quicken apoptosis.Yet, when having the apoptosis polypeptide, handle with preceding-apoptosis factor/toughener and can start the apoptosis approach, but cell can survive, because the one or more incidents in the described approach are suppressed.According to anti--point that the apoptosis polypeptide works, can suppress the apoptosis approach in the early stage or relative late period that the chain of events that finally causes necrocytosis is carried out.Anti--apoptosis polypeptide and coding nucleic acid thereof are well-known in the art, and it comprises for example associated protein Bcl-2 of Bcl-2 family, Bcl-X _L, Mcl-1, E1B-19K, IAP, Ced-9, Bcl-w etc., and the dominant form of caspase.These forms comprise that for example the avtive spot halfcystine has the caspase that inactivation suddenlys change.

Use recombination method for example well known by persons skilled in the art to resist-the apoptosis polypeptide by overexpression.The ordinary method of carrying out this recombinant expression method is described in for example Sambrook etc., molecular cloning: laboratory manual, cold spring harbor laboratory, New York (1992), GreenePublishing Associates and Wiley-Interscience, New York (1995).Can use these methods stable or transient expression anti--the apoptosis polypeptide, expression levels should be enough to prevent cell death inducing.Coding is anti--and the nucleic acid molecule of apoptosis polypeptide can be by the homologous nucleic acid coding that for example derives from same species or cell type, and perhaps nucleic acid molecule can be by the heterologous nucleic acids coding that derives from different plant species or cell type.The source of coding nucleic acid is unimportant, and the apoptosis inhibition is active to be resisted-the apoptosis polypeptide as long as its energy coding schedule reveals.

Be enough to prevent cell death inducing anti--apoptosis expression of polypeptides level is well known by persons skilled in the art, but also this expression level of conventional determining of those skilled in the art.The expression vector and the system that can provide recombinant polypeptide to express are known, and can be purchased.Selection can provide the carrier of expression of enough levels or the routine techniques that system is those skilled in the art in particular host cell.Perhaps, anti-by expressing-the apoptosis polypeptide, measure with preceding-apoptosis factor then and handle; Or whether cell survives after inducing the cDNA molecule that derives from plant, gets final product the expression level that conventional determining is enough to prevent cell death inducing.

Except overexpression anti--recombination method of apoptosis polypeptide, also can use the cell that has an instinct for can overexpression anti--apoptosis polypeptide.Have an instinct for can overexpression the cell special case of anti--apoptosis polypeptide be B cell lymphoma, Bcl-2 identifies in B cell lymphoma at first.This leukemic karyomit(e) 14 transpositions cause the Bcl-2 high level expression, so cell can be survived to karyomit(e) 18.Therefore, the leukemia phenotype is caused by the cell survival increase.Other have an instinct for can overexpression the clone of anti--apoptosis polypeptide also can be used for method of the present invention similarly.

Because overexpression is anti--apoptosis blocking-up that the apoptosis polypeptide causes and produced the antagonism influence with preceding-apoptosis factor processing cell pair cell.By this method, cell has kept the apoptosis balance basically.Before-apoptosis factor can be multiple different attack of pair cell, comprises molecule, environment and physical stimulation.As mentioned above, this stimulation is well known by persons skilled in the art, can identify this stimulation by the molecule in the activating cells apoptosis pathway.The example of preceding-apoptosis factor comprises inductor, as removing of somatomedin, and Fas part, anti--Fas antibody, Staurosporine, tumour necrosis factor, ultraviolet ray and gamma-irradiation.Therefore, with preceding-apoptosis factor handle overexpression anti--cell of apoptosis polypeptide can the trigger cell apoptosis, because the positive and negative signal can both provide poising action.An advantage of this initiation is: in case receive the signal that overcomes anti--apoptosis polypeptide blocking-up, the dead component of all cells can obtain to realize apoptosis.So just can the rapid induction apoptosis, thus be used for screening at Bcl-2 or Bcl-X _LOr has the active compound of apoptosis-inducing under the associated molecule existence.Preceding-apoptosis polypeptide that this cell derives from for screening in the cDNA expression library of plant is particularly useful.Similarly, can under the mediation of inducible promoter, express before-the apoptosis polypeptide.Therefore, these cells can contain constitutive expression, derive from the cDNA expression construct of plant.Therefore, can not cause necrocytosis, then can infer to exist to such an extent that hang oneself and express the necrocytosis conditioning agent of cDNA if induce.

Also can identify the anti--apoptosis protein matter that derives from the plant cDNA expression library by following method, described method comprises with plant cDNA expression library transformed host cell, as mammalian cell, cell is contacted, described inductor such as Staurosporine, gamma-irradiation with cell death inducer, anti--Fas, or Fas part etc., and detect and can not enter apoptotic cell, thereby identify the cell and the cDNA construct of expressing anti--apoptosis protein matter.Measure the sequence of expression construct then and illustrate sequence.3. high-throughput

Method as herein described also is applicable to high-throughout form (detection method of for example many-well format wherein can be screened a large amount of samples fast and effectively).For example, the form in 96 holes provides the advantage of actually operating, because plate that is suitable for operating and measuring apparatus can be purchased.This method further automatization with the speed and the effectiveness of further increase method.These features combine with the specificity of method, can not have-cell high-throughput ground screening active candidate inhibitor of apoptosis or toughener.E. medicinal application

In the context of the present invention, can use the inhibitor of apoptosis and toughener to bring into play control action kou with the pair cell death process.Therefore, these inhibitor and toughener can be used for being characterised in that the excessive or insufficient disease of apoptosis level, and can avoid biological and abiotic attack by protective plant.Therefore, the vegetable cell inhibitors of apoptosis has the potential therapeutic value to the people, can treat main neurodegenerative disease: apoplexy, Parkinson's disease, presenile dementia and ALS.Inhibitor also can be used for suppressing the myocardial infarction apoptosis of heart afterwards, the apoptosis of kidney and the apoptosis in the hepatic diseases after the acute ischemic.

The present invention also provides pharmaceutical composition.These compositions can contain above-mentioned any inhibitor, toughener, dna molecular, carrier or host cell, and medicine or physiology acceptable carrier, vehicle or thinner.Usually, the described carrier of used dosage and concentration should be to the acceptor nontoxicity.In general, prepare essential mixing treatment agent of this composition and damping fluid, antioxidant, as xitix, lower molecular weight (being lower than about 10 residues) polypeptide, protein, amino acid, carbohydrate, comprise glucose, sucrose or dextrin, sequestrant, as EDTA, gsh and other stablizer and vehicle.Suitably the example of thinner is the salt solution inclusive NAND specific serum albumin blended salt solution of neutral buffered.

In addition, can prepare the pharmaceutical composition of the present invention by multiple different approaches administration, described route of administration comprises for example intraarticular, in the skull bone, and intracutaneous, in the liver, intramuscular, intraocular, intraperitoneal, in the sheath, intravenously, subcutaneous or even directly inject in the tumour.In addition, pharmaceutical composition of the present invention can be placed container, described container is attended by the wrapping material that described pharmaceutical composition operation instruction is provided.Usually, described explanation comprises the clearly statement of describing reagent concentration, in certain embodiments, also comprises the relative content of rebuilding necessary vehicle composition of pharmaceutical composition or thinner (as water, salt solution or PBS).Pharmaceutical composition can be used for diagnosis or therapeutic purpose.

The mode that can be suitable for the disease of (or prevention) to be treated is used pharmaceutical composition of the present invention.Can determine the amount and the frequency of administration by following factor: patient's healthy state, the type of patient disease and severity.In the process of clinical trial, can the most accurately determine dosage.Can pass through proper technology, comprise the sign that the clinical state of an illness increases the weight of, monitoring such as imaging patient's treatment is renderd a service.

In other embodiments, the apoptotic pathways protein that derives from plant can be passed to cell as the part of gene delivery vector.In a lot of diseases and syndrome, apoptosis very little is the key character that they develop to some extent.The apoptosis that can benefit to increase to a lot of autoimmune diseases and tumor treatment.Increasing apoptotic a kind of method is to provide the target cell with caspase gene in effable mode.Can reach this purpose by transmitting the DNA or the cDNA that can transcribe preceding-apoptosis polypeptide in vivo.More specifically, in order to produce the apoptosis polypeptide in vivo, the nucleotide sequence of Codocyte apoptosis polypeptide can be placed under the control of eukaryotic promoter (as pol III promotor, CMV or SV40 promotor).Control more specifically when transcribing when needs, the apoptosis polypeptide can be placed tissue or cell specificity promotor (as target cell) liver, or inducible promoter, under the control as metallothionein(MT).

With a lot of technology of nucleic acid transfered cell is known.Described method comprises retroviral vector and uses retroviral infection subsequently, the mixture of adenovirus or adeno-associated virus vector and infection subsequently and nucleic acid and flocculation agent (as poly--Methionin).These mixtures or virus vector can be by mixing the specific cell type of part target of carrier.The a lot of parts that are specific to tumour cell and other cell are well-known in the art.

In the context of the present invention, can use variety carrier, comprise for example plasmid, virus, retrotransposon and clay.Representational example comprise adenovirus carrier (referring to WO94/26914, WO93/9191; Yei etc., gene therapy, 1:192-200,1994; Kolls etc., PNAS 91 (1): 215-219,1994; Kass-Eisler etc., PNAS90 (24): 11498-502,1993; Guzman etc., circulation, 88 (6): 2838-48,1993; Guzman etc., circulating research, 73 (6): 1202-1207,1993; Zabner etc., cell, 75 (2): 207-216,1993; Li etc., human gene therapy, 4 (4): 403-409,1993; Caillaud etc., European Journal of Neuroscience, 5 (10): 1287-1291,1993), gland follows 1 type (" AAV-1 ") or gland to follow 2 types (" AAV-2 ") carrier (referring to WO95/13365; Flotte etc., PNAS 90 (22): 10613-10617,1993), hepatitis δ carrier is lived, the δ virus and the herpesvirus vector (example is seen United States Patent (USP) 5,288,641) of attenuation, and disclosed carrier in the United States Patent (USP) 5,166,320.Other representational carrier comprises that (example is seen EP0415731 to retroviral vector; WO90/07936; WO91/02805; WO94/03622; WO93/25698; WO93/25234; United States Patent (USP) 5,219,740; WO93/11230; WO93/10218).

Aspect some, can utilize carrier (vehicles) of the present invention, or the proteinic nucleic acid molecule of Codocyte apoptosis pathway be imported host cell by multiple physics method.The representative example of described method comprises uses calcium phosphate precipitation to transform (Dubensky etc., PNAS81:7529-7533,1984), the target cell that the described nucleic acid molecule direct injection of trace is extremely complete (Acsadi etc., nature, 352:815-818,1991), and electroporation, electroporation can make the cell that is suspended in the conductive solution be subjected to the highfield effect, with instantaneous polarization cytolemma, make nucleic acid molecule enter cell.Other method comprises the nucleic acid molecule (Cotton etc. that use links to each other with the adenovirus of non-activity, PNAS 89:6094,1990), lipofection (Felgner etc., Proc.Natl.Acad.Sci.USA 84:7413-7417,1989), microparticle bombardment (Williams etc., PNAS 88:2726-2730,1991), polycationic compounds (as polylysine), the receptor-specific part, bag carries the liposome of nucleic acid molecule, and spheroplast merges and (passes through this method, the intestinal bacteria that contain nucleic acid molecule are peelled off the okioplast wall, use polyoxyethylene glycol that itself and zooblast are merged), virus transduction (Cline etc., pharmacological agent, 29:69,1985; With Friedmann etc., science, 244:1275,1989) and DNA part (Wu etc., journal of biological chemistry, 264:16985-16987,1989), and the psoralene inactivation of viruses, as Sendai virus or adenovirus.Appropriate carriers also comprises United States Patent (USP) 5,763,416 and WO97/38729, and U.S.'s serial number 08/631,334 described gene activation matrix.

The following example is in order to illustrate rather than limit the present invention.EXAMPLE Example I makes up the binary vector that contains IAP

Transform the interior open reading frame (ORF) of carrier pOPIAPRC (Birmbaum etc., Journal of Virology (J.ofVirology) 68:2521-2528,1994) with in 5 ' the terminal NcoI of importing site by PCR, import the BamHI site at 3 ' end.This operates between the Met (the 1st) of natural protein and Ser (the 2nd) residue and has imported the Ala residue.Used PCR condition is as follows: with 94 ℃ 1 minute; 45 ℃ 1 minute; 72 ℃ were carried out 2 in 2 minutes and take turns circulation, then again with 94 ℃ 1 minute; 55 ℃ 1 minute; 72 ℃ were carried out 35 in 2 minutes and take turns circulation.Primer prc-5:5 '-gttgcagaccatggccagctcccgagcattggc-3 '. primer pre-3:5 '-ttttggatccttttattgttacacttgg-3 '

With NcoI and BamHI digestion PCR product and subclone to expression of plants box pRTL2 (by Carrington etc., Journal of Virology 64:1590-1597,1990 are described as pRTLGUS).The gained carrier is called as pPTN144 (Figure 1A).Use HindIII with the 35S-IAP box subclone of pPTN144 to binary vector pZP212 (Hajdukiewicz etc., molecular biology of plants (Plant Mol.Bio.) 25:989-994,1994).The gained carrier is called as pPTN 148 (Figure 1B).Example II makes up the binary vector that contains Ced-9

The ORF that transforms Ced-9 by PCR imports the XbaI site with in 5 ' the terminal NcoI of importing site at 3 ' end.This operates between the Met (the 1st) of natural protein and Thr (the 2nd) residue and has imported the Ala residue.The PCR condition with transform used identical of pOPIAPRC ORF.Primer ced-5:5 '-gaattccggtttgagccatggcgacacgctgcacggcg-3 ' primer ced-3-2:5 '-ttttttctagaaatacgttacttcaagctg-3 '

With NcoI and XbaI digestion PCR product and subclone to expression of plants box pRTL2 (Carrington etc., Journal of Virology 64:1590-1597,1990).The gained carrier is called as pPTN143 (Fig. 3 A).With the ced-9 expression of plants box of pPTN143 as HindIII fragment subclone to binary vector pZP212 (Hajdukiewicz etc., molecular biology of plants 25:989-994,1994).The gained carrier is called as pPTN 147 (Fig. 3 B).EXAMPLE III makes up the binary vector that contains Bcl-2

Transform the cDNA of bcl-2, in 5 ' the terminal NcoI site that imports of bcl-2 ORF, in 3 ' the terminal XbaI site that imports of ORF.Use the condition identical, carry out the PCR reaction with following primer: primer Bcl2-5:5 '-tttttcctctgggagggccatggcgcacgctgg-3 ' primer Bcl2-3:5 '-ttttttctagatgctcttcgggcgtgg-3 ' with example I

This operates between the Met (the 1st) of natural protein and His (the 2nd) residue and has imported the Ala residue.With Nco I and Xba I digestion PCR product, subclone is to expression of plants box pRTL2.The gained carrier is called as pPTN 157 (Fig. 2 A).Then, use endonuclease HindIII, then connect, with total length bcl-2 expression of plants box subclone to binary vector pZP212.This paper is called pPTN161 (Fig. 2 B) with the gained carrier.EXAMPLE IV makes up the binary vector that contains E1B 19K

The EcoRI/BamHI fragment subclone that carrier pCMV19K (White and Cipriani, molecular cytobiology, 10:120-130,1990) is interior is to pBluescriptKS ⁺To carry out sequential analysis.The gained carrier is called as pPTN145 (Fig. 4 C).According to the sequence data that gets self-template pPTN145, the design primer is to import the NcoI site by PCR at the 5 ' end of ORF, in 3 ' the terminal importing BamHI site of ORF.Extra residue is not added in this operation in protein.Primer pPTN 145-5:5 '-gcttcctgaactccatggaggcttggg-3 ' primer pPTN 145-3:5 '-tttttggatccaacattcattcccgaggg-3 '

Because the NcoI site, inside in the E1 B-19K ORF, with three reconnect NcoI/KpnI and KpnI/BamHI with the fragment subclone of pPTN145 to pRTL2 (NcoI/BamHI).The gained carrier is called as pPTN160 (Fig. 4 A).With the 35S-19k box of pPTN160 as HindIII fragment subclone to binary vector pZP212.The gained carrier is called as pPTN 162 (Fig. 4 B).The EXAMPLE V methods for plant transformation

Agrobacterium strains:In transforming, all tobaccos use agrobacterium tumefaciens bacterial strain C58C1 (Mol.Gen.Genet, 204:383,1986).By three-parental generation mating (Ditta etc., Proc.Natl.Acad.Sci.USA 77:7347-7351,1980) with binary vector pPTN147, pPTN148, pPTN161 and pPTN162 are transferred to C58Cl.Be added with the 100mg/l Streptomycin sulphate, the 100mg/l spectinomycin, the 50mg/l Rifampin selects the Agrobacterium switching fit in the AB basic medium of 50mg/l gentamicin and 1.5 sucrose.

Wild-type tobacco Nicotiana tabacum kind Glurk (genotype NN) or N.tabacurn kind Turkish (genotype nn) are used for the pathogenic agent inoculation, (Glurk and Turkish kind can derive from multiple source with being used to make up transgenic plant, comprise Nebraska university, Lincoln, Collection).

Use is carried out the tobacco conversion through the leaf dip method (Horsch etc., science 227:1229-1231,1985) of changing.Preceding 2 leaves of plant prepared explant by 30 to 40 day age.After cultivating altogether 3 days, explant gone down to posterity to be incubated at is added with 1mg/l BAP, the MS/B5 substratum of 0.1mg/l NAA and 100mg/l kantlex.Add microbiotic Pyocianil and cefotaxime so that Agrobacterium is selected.Every 2 weeks, explant gone down to posterity to be incubated in the fresh culture.Cultivate 6-8 after week, downcut the branch that can tolerate kantlex of differentiation.Branch is placed on the substratum based on MS/B5, and described substratum is added with 0.1mg/l NAA and 50mg/l kantlex.The shaker test of example VI transgenic strain

Breeding kalamycin resistance transgenic plant are added with Kanamycin Sulfate (100mg/l), NAA (plant hormone) 0.1mg/l and BAP (cytokinin) 1mg/l in the described substratum in Murashige-5k00G basis salt culture medium (Sigma).

In order to carry out expression study, collect tobacco leaf, freezing and clay into power in liquid nitrogen.By the described separating plant RNA of example VII A I.With the RNA trace and through radiolabeled pPN147, pPTN148, pPTN 1612 or pPTN 162 hybridization.18S rRNA is used as internal contrast to guarantee to go up the RNA of sample equivalent.Under stringent condition, carry out RNA blot hybridization and film washing (Sambrook etc., document is the same).

Western analyzes:Detect TMV albumen on the SDS PAGE by Coomassie blue stain and/or immunoassay.(grind the plant tissue of infection in the 0.2g tissue/1ml), and in the Laemmli of 5 times of volumes sample-loading buffer (Laemmli, natural 227:680-685,1970), boiled 1-5 minute at distilled water.About 15 μ l samples are splined on the 12%SDS-polyacrylamide gel.By electrophoresis protein transduction is moved to nitrocellulose filter, use colorimetric (Immun-Blot test kit then, BioRad) analyze, TMV polyclonal antibody with dilution in 1: 1000 detects, preparation (can derive from ATCC PVAS-135 to described antibody at virion, Manassass, VA) (Figure 11 A and 11B).

Northern and Southern analyze:Southern analyzes (data not shown) and the Northern trace is represented 4 kinds of genetically modified existence and expression.Inoculation of example VII A pathogenic agent and resistant proof

Estimate the resistance of plant to tobacco mosaic virus (TMV) (TMV).In this experiment, transform the tobacco bred that contains (Glurk) and do not contain (Turlish) TMV resistance N gene.With 4 kinds genetically modified each obtain at least 10 kalamycin resistance Glurk and Turkish strain.Southern analyzes (data not shown) and the Northern trace is represented 4 kinds of genetically modified existence and expression (Fig. 5 illustrates IAP and Ced-9 expresses, and Fig. 8 and 9 has shown that respectively Bcl-2 and Ced-9 express).At copy number, there is not dependency between expression level and the plant reaction.When comparing, there is not the physiological difference that to survey between the transgenic plant with the wild-type contrast.All plants all bloom and tie in a similar fashion and plant.Single copy that evaluation is selected at random inserts the reaction of the tobacco of kalamycin resistance to the pathogenic agent attack.

Detect transgene tobacco to tobacco mosaic virus (TMV) (TMV), tobacco necrosis virus (TNV), tobacco corrosion virus (TEV), tomato spotted wilf virus (TSWV), sclerotinite (Sclerotiniasclerotiorum) 1980 (Dickman and Mitra, Physiol. Mol.PlantPathol, 41:255-263,1992); Gray botrytis (ATCC), tobacco tail spore and the resistance of enclosing small cluster shell (Glomerella cingulata).In 25 ℃ of greenhouses, cultivate transgenosis and contrast tobacco with 16 hours photoperiod.

The virus test:On the tobacco leaf of desorption, sprinkle silicon carbide, with the 50 μ l water frictional inoculations that contain 110 μ gTMV.After 5 minutes, water thoroughly washs leaf, places plate, is maintained at 25 ℃ under constant illumination.Inoculate with TNV and TSWV by similar method, difference is to use and is dissolved in the 10mM sodium phosphate buffer (pH7), inoculates by the water through infecting of 1/25 (w/v) dilution.

In the tobacco strain of resistance (NN) and responsive (nn), the expression of anti--apoptosis gene is all influential to TMV.The N gene is given the allergy that TMV is infected, and takes this to prevent that virus from diffusing to whole body.Inoculate back 4 to 5 days with TMV, downright bad infringement occurs, and passing in time, area is increasing.In fact the development of local lesion can be divided into two stages.Originally, little (1-2mm) immersion type infringement occurs, after 2 to 3 days, become uniform necrosis.Then, necrosis area continues to enlarge, and is accompanied by chlorisis, rather than the immersion type edge.Virus moves near the infringement that is limited in growing and healing, and causes the death of whole leaf.TMV induces invisible symptom not containing on the inoculation tobacco leaf of N gene, but still reproducible and diffuse to whole blade.TMV is inoculated in 4 different transgenosis Glurk (NN) strain (using 3 different strains at least) and 2 Turkish (nn) strain.The tobacco of unconverted and only carrier-containing tobacco are used as contrast.

In addition, use the test of desorption leaf in the great majority experiment, because they are favourable in experiment, the result shows that when comparing with the complete plant in the greenhouse reaction 100% that pathogenic agent is attacked is relevant.

The TMV inoculation is to containing and not containing Bcl-2, and the Glurk strain of CED9 and IAP gene can both produce local lesion.In all these researchs, the transgenic plant that contain adenovirus E 1 B-19k in any tobacco bred do not show the resistance to this pathogenic agent, therefore it are not done further evaluation.Yet secondary being grown in of infringement expressed in other genetically modified strain by restriction (Fig. 7,10A and 10B) widely.Therefore, as if Bcl-2, CED-9 and IAP can be by preventing that the resistance that is provided by the N gene is provided to damage expansion.TMV also may be come by initial infringement diffusion, and can not induce further necrosis.In order to determine this point, Western engram analysis (Figure 11 A) is carried out in local lesion and organizing on every side independently.Although the virus in the infringement is very abundant, beyond the infringement border, do not detect virus, this proof Bcl-2 and ced-9 have strengthened N gene resistance really.On the other hand, Bcl-2 and ced-9 are to the not influence (Figure 11 B) of accumulation of TMV in the Turkish strain.

A lot of plant hosts-virus combination also can produce downright bad local lesion, but be not limited to specific host gene type.In this combination, plant is called as the local lesion host of virus.Tobacco is the local lesion host of multiple virus, comprises tobacco necrosis virus (TNV) and tomato spotted wilf virus (TSWV).Different with the N gene with TMV is that the hereditary basis of this general necrotic reaction is unknown, but may be different between a virus and another virus, because the morphology of local lesion is very different.Therefore, measure transgenosis at the influence of the reaction of these not identical plant viruses also highly significant to the host.Surprisingly, after with TNV or TSWV inoculation, the leaf of expressing the tobacco strain of Bcl-2 or ced-9 does not have visible symptom (Figure 12).After with each inoculation in these viruses, necrosis is also limited on the plant of expressing IAP widely.Therefore, the local lesion host that to contain any genetically modified tobacco no longer be these two kinds of viruses.Think as expected like that, identical phenotype appears in NN and nn genetic background, this is because the N gene is that TMV-is specific.

In addition, detect tobacco corrosion virus (TEV), illustrated with the similar N gene of TSWV autonomous by the method the same with above-mentioned other virus.Therefore, with the expression Bcl-2 of TEV inoculation and the cell of Ced-9 visible symptom (data not shown) does not appear.

Make selected tobacco plant self-pollination (promptly with the hybridization of homologous genes type).Importantly, in progeny plants, do not have kalamycin resistance, those plants that lack transgene expression all are susceptible at present.

The kalamycin resistance offspring of express transgenic (sees Figure 13 (Ced-9 exemplifies Bcl-2 and IAP is similar), 14A (Bcl-2 expresses and the TMV inoculation), 14B (Ced-9 expresses and the TMV inoculation), 14C (Ced-9 expresses and the TSWV inoculation)) in fact all have resistance, and identical by the phenotype after the virus attack with observed phenotype in parental generation.

Mycologic test:In order to carry out the test of desorption leaf, the 5mm agar block that contains the mycelia point of active growth by placement, inoculate at least two leaves of each strain of two single transgenic plant of kind 10 strains, described mycelia point derives from the sclerotinite that grows on the Ma Lingzhu agar glucose or the bacterium colony in 3 day age of gray botrytis.

Leaf is placed on the moistening aseptic filter paper of glass dish, be incubated 3 to 7 days with high humidity with 25 ℃.In order to inoculate whole plants (only for sclerotinite), in YPSS liquid nutrient medium (Tuite, 1968), thecaspore (is about 10 ⁴/ ml) be incubated 2 hours, and be sprayed on the tobacco leaf, get off until liquid flow.To place 25 ℃ through the plant of inoculation, relative humidity is 100%, in the dark fog chamber.Each strain is used 5 strain plants at least.All experiments repeat 3 times at least.

The test of making of sclerotinite shows: genetically modified tobacco has very high tolerance, in most of the cases has resistance completely, and this point is different with hypersusceptible wild-type tobacco.Different with multiple other fungi that can only transmit in water or damping fluid, this fungi need inoculate with nutrition source.As shown in figure 15, can be with the fungi of abundant nutrition source inoculation along the surface growth of tobacco leaf, but can not occur infecting or host's residence.Fungi finally stops growing, and supposition may be because exhausted nutrition source, and importantly, even after prolonging soaking time, fungi still can not be resided and infection plant's tissue.In contrast, leaf texture (Figure 15) can be resided and macerate to the wild-type fungi fully through after the identical time.

Estimate the reaction (Figure 16) that autophilous tobacco plant described above is attacked sclerotinite.Do not have kalamycin resistance, the progeny plants that lacks transgene expression all is a susceptible.The selected plant with kalamycin resistance and transgene expression generally all has resistance, and and parental generation, the phenotype of elementary transformant is identical.

When being inoculated in any the transgene tobacco that contains in 3 kinds of transgenosiss, also be that the gray botrytis of the downright bad nutritious fungus pathogenic agent of wide host range demonstrates similar reaction.In these two fungi examples, whole plants is seeded on the phenotypic response and identical (Figure 17 A-Bcl-2 expresses, and 17B-Ced-9 expresses) of arriving with desorption leaf experimental observation.

In addition, the 3 kinds of transgenosis Ced-9 that encode, the plant of Bcl-2 and IAP is to tobacco tail spore (Figure 19) and enclose small cluster shell and also have resistance, and its resistance mode is similar to the resistance (data not shown) to sclerotinite.

At last, transform plant with the carrier that is positioned at encoding wild type bcl-xL on the aforesaid identical parental generation carrier and bcl-xL non-functional mutant form G1 38A.Be exposed to sclerotinite then.Example VII A I separation and trace are through the RNA of transformation of tobacco strain

By make the RNA sample with through radiolabeled probe hybridization, analyze regeneration kantlex-resistant strain through the transformation of tobacco plant, described probe contains the proteinic required gene (Gordon-Kamm etc. of Codocyte apoptosis pathway, vegetable cell (The Plant Cell), 2,603-618,1990).

Press Strommer etc., molecular biology of plants and biotechnological means, p49-65, CRC press, Boca Raton, FL., 1993 described methods are separated the total RNA in the transformation of tobacco leaf.Downcut about 600 to 700mg leaves from plant, remove the intermediary master pulse, leaf is placed on ice.Use the liquid nitrogen grinding leaf, be transferred in the Eppendorf tube.Add 1ml TRIzol, leaf is melted.Sample is placed under the room temperature insulation 5 minutes, add the 0.2ml chloroform then, mix, be incubated 3 minutes more subsequently.Then in 4 ℃,, supernatant liquor is transferred in the isolating test tube with the centrifugal sample of 12,000 * g 15 minutes.Add the 0.5ml Virahol in supernatant liquor, carefully mix gained solution, insulation is 10 minutes under the room temperature.Then in 4 ℃,, remove supernatant liquor with the centrifugal sample of 12,000 * g 10 minutes.With 1ml 75% washing with alcohol throw out, the careful mixing.In 4 ℃, with the centrifugal throw out 5 minutes of 7,500 * g, remove supernatant liquor through washing, vacuum-drying gained throw out 3 to 5 minutes, or at room temperature dry 10 minutes.Add 20 to 50 μ l DEPC H subsequently ₂O or methane amide were in 55 ℃ of insulations 10 minutes.Use sample then, or sample is placed-80 ℃ stand-by.

Use standard method, differentiate the RNA goods with gel electrophoresis, be transferred on the film and hybridize (referring to Sambrook etc., document is the same).At first, in 65 ℃ of rotary ovens with 25ml solution with film prehybridization 1 hour, described solution contains 12.5ml 1M NaHPO ₄PH7.2,0.25g BSA, 50 μ l EDTA (0.5M liquid storage), 1.75g SDS and add distilled water to 25ml (about 11.5ml).By using ³²P-dCTP Klenow mark prepares probe.Then probe is directly added hybridization solution, incubated overnight in 65 ℃ of rotary ovens.Under room temperature, use washing soln (the 2ml 1M NaHPO of 50ml low stringency then ₄PH7.2,0.25g BSA, 100 μ lEDTA (0.5M liquid storage), 2.5g SDS and add distilled water to 50ml) washing film 10 minutes.Use washing soln (the 8ml 1M NaHPO of the high tight degree of 50ml then ₄PH7.2,400 μ lEDTA (0.5M liquid storage), 2g SDS and add distilled water to 200ml), under room temperature with film washing 1 time, 10 minutes, again in 65 ℃ with film washing 2 times, 10 minutes.Use intensifying screen that film was exposed to X-OMAT AR film 5 to 10 hours in-80 ℃ then.

To the invention describes specific embodiment of the present invention although from description above, be understood that this paper in order illustrating, multiple modification of the present invention not to be deviated from the spirit and scope of the present invention.Therefore, the present invention is not limited to these specific embodiments, and scope of the present invention is limited by appended claims.

All reference of mentioning in the application comprise magazine, and patent and patent application are all listed this paper in as a reference in full.

Claims (79)

1. transgenic plant, it contains vegetable cell, and described cell contains at least one coding and has the apoptotic pathways protein of biological function or the heterologous nucleotide sequence of its functional variant.

2. the transgenic plant of claim 1, it further contains by the described nucleotide sequence coded apoptotic pathways protein with biological function.

3. the transgenic plant of claim 1, wherein said nucleotide sequence coded anti--apoptosis protein matter.

4. the transgenic plant of claim 3, wherein anti--apoptosis protein matter is selected from Ced-9, Bcl-2, Bcl-xL, IAP and E1B 19K.

5. the transgenic plant of claim 3, wherein said anti--apoptosis protein matter is Ced-9 or Bcl-2.

6. claim 1 or 3 transgenic plant, wherein said nucleotide sequence comprises tissue-specific promoter.

7. the transgenic plant of claim 6, wherein said tissue-specific promoter is selected from the αDian Fenmei promotor, paratin promotor and glutenin promoter.

8. claim 1 or 3 transgenic plant, wherein said nucleotide sequence comprises inducible promoter.

9. the transgenic plant of claim 8, wherein said inducible promoter is selected from wound-induced type promotor, alcoholdehydrogenase promotor and chalcone synthase promotor.

10. claim 1 or 3 transgenic plant, wherein said nucleotide sequence comprises constitutive promoter.

11. the transgenic plant of claim 10, wherein said constitutive promoter is selected from the VITAMIN B4 Methyl transferase promoter, 35S promoter and ubiquitin promotor.

12. the transgenic plant of claim 1, wherein apoptotic pathways protein is selected from caspase, rev-caspase, Bcl-2 family member, Apaf-1, Bad, Bax, Ced-9 and Ced-4.

13. the transgenic plant of claim 1, wherein said plant is a dicotyledons.

14. the transgenic plant of claim 1, wherein said plant is a monocotyledons.

15. the transgenic plant of claim 1, wherein said plant has resistance to biological attack.

16. the transgenic plant of claim 15, wherein biological attack is caused by insect.

17. the transgenic plant of claim 15, wherein biological attack is caused by pathogenic agent.

18. the transgenic plant of claim 17, wherein said pathogenic agent is selected from fungi, nematode, bacterium and virus.

19. the transgenic plant of claim 17, wherein said pathogenic agent are virus.

20. the transgenic plant of claim 19, wherein said virus are selected from tobacco mosaic virus (TMV) (TMV), tobacco necrosis virus (TNV), tobacco corrosion virus (TEV) and tomato spotted wilf virus (TSWV).

21. the transgenic plant of claim 17, wherein said pathogenic agent is a fungi.

22. the transgenic plant of claim 21, wherein said fungi is a sclerotinite.

23. the transgenic plant of claim 21, wherein said fungi is selected from sclerotinite, gray botrytis, tobacco tail spore and enclose small cluster shell.

24. the transgenic plant of claim 1, wherein said plant has resistance to abiotic attack.

25. the transgenic plant of claim 24, wherein abiotic attack are by being selected from high humidity, low humidity, and salinity, nutritive deficiency, atmospheric pollution, temperature, soil toxicity, the factor of weedicide and sterilant causes.

26. the transgenic plant of claim 1 wherein show the ageing level of reduction to the described plant of small part.

27. vegetable cell, it contains coding and has the apoptotic pathways protein of biological function or the heterologous nucleotide sequence of its functional variant.

28. the vegetable cell of claim 27, it further contains by the described nucleotide sequence coded apoptotic pathways protein with biological function.

29. the vegetable cell of claim 27, wherein said nucleotide sequence coded resisting-apoptosis protein matter.

30. the vegetable cell of claim 29, wherein anti--apoptosis protein matter is selected from Ced-9, Bcl-2, Bcl-xL, IAP and E1B 19K.

31. the vegetable cell of claim 29, wherein said resisting-apoptosis protein matter is Ced-9 or Bcl-2.

32. the vegetable cell of claim 27 or 29, wherein said nucleotide sequence comprises tissue-specific promoter.

33. the vegetable cell of claim 32, wherein said tissue-specific promoter is selected from the αDian Fenmei promotor, paratin promotor and glutenin promoter.

34. the vegetable cell of claim 27 or 29, wherein said nucleotide sequence comprises inducible promoter.

35. the vegetable cell of claim 34, wherein said inducible promoter are selected from wound-induced type promotor, alcoholdehydrogenase promotor and chalcone synthase promotor.

36. the vegetable cell of claim 27 or 29, wherein said nucleotide sequence comprises constitutive promoter.

37. the vegetable cell of claim 36, wherein said constitutive promoter is selected from the VITAMIN B4 Methyl transferase promoter, 35S promoter and ubiquitin promotor.

38. the vegetable cell of claim 27, wherein apoptotic pathways protein is selected from caspase, rey-caspase, Bcl-2 family member, Apaf-1, Bad, Bax, Ced-9 and Ced-4.

39. the vegetable cell of claim 27, wherein said plant is a dicotyledons.

40. the vegetable cell of claim 27, wherein said plant is a monocotyledons.

41. the vegetable cell of claim 27, wherein said plant has resistance to biological attack.

42. the vegetable cell of claim 41, wherein biological attack is caused by insect.

43. the vegetable cell of claim 41, wherein biological attack is caused by pathogenic agent.

44. the vegetable cell of claim 43, wherein said pathogenic agent is selected from fungi, nematode, bacterium and virus.

45. the vegetable cell of claim 43, wherein said pathogenic agent are virus.

46. the vegetable cell of claim 45, wherein said virus are selected from tobacco mosaic virus (TMV) (TMV), tobacco necrosis virus (TNV), tobacco corrosion virus (TEV) and tomato spotted wilf virus (TSWV).

47. the vegetable cell of claim 43, wherein said pathogenic agent is a fungi.

48. the vegetable cell of claim 47, wherein said fungi is selected from sclerotinite, gray botrytis, tobacco tail spore and enclose small cluster shell.

49. the vegetable cell of claim 43, wherein said pathogenic agent are the fungi sclerotinite.

50. the vegetable cell of claim 27, wherein said plant has resistance to abiotic attack.

51. the vegetable cell of claim 50, wherein abiotic attack are by being selected from high humidity, low humidity, and salinity, nutritive deficiency, atmospheric pollution, temperature, soil toxicity, the factor of weedicide and sterilant causes.

52. the vegetable cell of claim 27, wherein said vegetable cell shows the ageing level of reduction.

53. can sprout into the seed of plant, in its genome, has heterologous nucleic acid sequence, the apoptotic pathways protein that this sequence can be encoded and be had biological function.

54. to the transgenic plant that biological attack has resistance, it contains heterologous nucleic acid sequence, this sequence can be encoded and be had the anti--apoptosis protein matter of biological function.

55. the plant of claim 54, wherein said resisting-apoptosis protein matter is selected from Ced-9, Bcl-2, Bcl-xL, IAP and E1B 19K and homologue thereof.

56. the plant of claim 54, wherein biological attack is a pathogenic agent.

57. to the transgenic plant that abiotic attack has resistance, it contains heterologous nucleic acid sequence, this sequence can be encoded and be had the anti--apoptosis protein matter of biological function.

58. the plant of claim 57, wherein said resisting-apoptosis protein matter is selected from Ced-9, Bcl-2, Bcl-xL, IAP and E1B 19K and functional variant thereof.

59. produce biological or abiotic attack had the method for the transgenic plant of resistance, described method comprises: (a) use the carrier transformed plant cells, described carrier contains at least one heterologous nucleic acid sequence, described nucleic acid sequence encoding has the apoptotic pathways protein of biological function, and described nucleotide sequence can be operated with promotor and link to each other; (b) produce plant by described through the plant transformed cell; (c) select conversion plant that biological or abiotic attack are had resistance.

60. the method for claim 59, wherein transgenic plant have resistance to pathogenic agent.

61. the method for claim 59, wherein step of converting is undertaken by physical method.

62. the method for claim 59, wherein step of converting is undertaken by chemical process.

63. the method for claim 59, wherein said vegetable cell is selected from protoplastis, the cell of cell that generation is assigned in and the complete plant of energy regeneration.

64. the method for claim 59, wherein promotor is a constitutive promoter.

65. the method for claim 59, wherein promotor is an inducible promoter.

66. the method for claim 59, wherein promotor is a tissue-specific promoter.

67. the method for claim 59, wherein plant is a dicotyledons.

68. the method for claim 59, wherein plant is a monocotyledons.

69. the plant that produces by the method for claim 59.

70. plant tissue, the plant that its method that gets free claim 59 produces.

71. the method for modulation vegetable cell apoptosis, described method comprises: (a) use the carrier transformed plant cells, described carrier contains that the coded plant cell can not normally produce, apoptotic pathways nucleic acid sequences to proteins with biological function, described nucleotide sequence can be operated with inducible promoter and link to each other; (b) cultivate under the condition of plant through the plant transformed cell being suitable for forming; (c) condition and time culturing plants to be enough to induce described nucleotide sequence to transcribe.

72. identify to have the method for the active plant gene of apoptotic pathways, described method comprises: (a) with plant cDNA library transformed animal cell, wherein each member in cDNA library can operate with promotor and link to each other; (b) transformant is contacted with cell death inducer; (c) detect apoptosis activity in the described cell, and compare, wherein the active increase of apoptosis or reduce the existence of the gene of Codocyte apoptosis pathway protein in the expression plant cDNA library with the activity of control cells system.

73. the method for claim 72, wherein zooblast has been carried out the preparation of necrocytosis.

74. identify the method for the apoptosis gene that works in plant, described method comprises: (a) transform one or more vegetable cells with at least one heterologous nucleic acids molecule, wherein said nucleic acid molecule can be operated with promotor and link to each other; (b) transformant is contacted with cell death inducer; (c) detect apoptosis activity in the described cell, and compare with the activity of control cells system, wherein active increase of apoptosis or reduction are illustrated in the existence of the apoptotic pathways gene that works in the plant.

75. the method for claim 74, wherein the heterologous nucleic acids molecule contains allos cDNA library, and wherein each member in the cDNA library can operate with promotor and link to each other.

76. the method for claim 74, wherein cell death inducer is a biological attack.

77. the method for claim 76, wherein biological attack is a pathogenic agent.

78. the method for claim 74, wherein cell death inducer is non-biological attack.

79. transgenic plant, it contains vegetable cell, and described cell contains at least one heterologous nucleotide sequence, and described sequence encoding has the proteinic functional variant of apoptotic pathways of biological function.

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