CN1484705A - Dan encoding a plant lipase transgenic plants and a method for controlling senescence in plants - Google Patents

Abstract

Regulation of expression of senescence in plants is achieved by integration of a gene or gene fragment encoding senescence-induced lipase into the plant genome in antisense orientation. The carnation and Arabidopsis genes encoding senescence-induced lipase are identified and the nucleotide sequences are used to modify senescence in transgenic plants.

Description

The method of the DNA of coded plant lipase, transgenic plant and controlling plant aging

The application is an application Ser. No 09/597,774 part continues, the latter is an application Ser. No 09/250,280 part continuation application, application Ser. No 09/250,280 is the part continuation application of the application Ser. No 09/105,812 of application on June 26th, 1998, and it all is attached to herein by reference.

Invention field

The present invention relates to the coded plant polypeptide and show polynucleotide that old and feeble inductive expresses, the transgenic plant of the described polynucleotide that contain antisense orientation and be used for the method for controlling plant aging.More particularly, the present invention relates to it expresses because old and feeble beginning and derivative vegetable lipase gene and use the aging of described lipase gene controlling plant.

The description of prior art

Aging is the latter stage of biological development in the plant life.It indicates dead, and takes place comprising on the various horizontal biological structure of complete stool, organ, flower and fruit, tissue and individual cells.

It is an old and feeble early stage and basic feature that cytolemma is degenerated.The metabolism of lipid, the particularly metabolism of film fat are one of several biological chemistry performances of cell aging.For example, along with the development of aging, the acyl hydrolase enzymic activity of roseleaf continues to increase, be attended by the film function forfeiture (Borochov etc., Plant Physiol., 1982,69,296-299).It is an old and feeble early stage and distinctive feature that cytolemma is degenerated, cause that permeability increases, ion gradient forfeiture and the crucial membranin reduction (Brown etc. of ionic pump function for example, Plant Physiol.:A Treatise, Vol.X.Academic Press, 1991, the 227-275 pages or leaves).Major part during this membrane structure integrity and functional completeness go down can be owing to the phospholipid metabolism of lipase mediation.Forfeiture (the Thompson that the phosphoric acid lipid is arranged in the fruit in verified petal, blade, cotyledon and the maturation in aging, J.E., Senescence and Aging in Plants, Academic Press, SanDiego, 1988, the 51-83 page or leaf), it seems that this causes the molecular great change of film bilayer, and promote aging, cause cell function impaired.Particularly, the research of carrying out with many old and feeble plant tissues provides evidence (McKersie and Thompson, 1979 for it seems that lipid is separated in the film that is attributable to lipid metabolism thing accumulation in the film double sublayer, Biochim.Biophys.Acta, 508:197-212; Chia etc., 1981, Plant Physiol., 67:415-420).Have more and more evidences to show, in the old and feeble tissue senescence-specific of most lipid metabolism by genetic expression change and finish (Buchanan-Wollaston, V., J.Exp.Bot., 1997,307:181-199).

Old and feeble rising begins to be induced by different inside and outside factors.For example, in many plants, ethene is at for example seed germination, seedling development, fruit maturation and spend in the old and feeble various plant processes and work.In the plant generation of ethene also may be with mechanical trauma, chemical substance, coerce (for example because the temperature and the water yield change and produce) relevant with disease institute inductive wound.Ethene is associated with the adjusting of leaf senile in many plants, and the evidence that obtains with transgenic plant and ethylene reaction type mutant shows that although ethene has effect to aging, but it is not the main instrumentality of this process.For example, in the real ripening process of no transition type plant such as strawberry fruit, in the aging course of some colored for example tawny daylily, and certain plants for example Arabidopsis (Arabidopsis), particularly in monocotyledonous leaf senile process, do not need Ethylene Signal (Smart, C.M., 1994, New Phytology, 126:419-448; Valpuesta etc., 1995, Plant Mol.Biol., 28:575-582).

For example temperature, arid, illumination or nutrition supply are poor and the pathogenic agent invasion and attack to induce old and feeble initial external factor too early to comprise environment-stress.The situation old and feeble with nature (age is correlated with) is the same, the forfeiture that is characterized as cell membrane integrity of environment-stress inductive aging.Specifically, be exposed to environment-stress, bring out the electrolyte leakage (Sharom etc., 1994, Plant Physiol., the 105:305-308 that reflect membrane damage; Wright and Simon, 1973, J.Exp.Botany, 24:400-411; Wright, M., 1974, Planta, 120:63-69; With Eze etc., 1986, Physiologia Plantarum, 68:323-328), the membrane phospholipid level (Wright that descends, M., 1974, Planta is 120:63-69) with lipid phase transformation (Sharom etc., 1994, Plant Physiol., 105:305-308), all these can be owing to the effect of lipase.The plant tissue that is exposed to environment-stress also produces ethene, is commonly referred to coerce ethene (Buchanan-Wollaston, V., 1997, J.Exp.Botany, 48:181-199; Wright, M., 1974, Planta, 120:63-69).As mentioned above, known ethene causes aging in certain plants.The film degeneration that causes permeating also is the essential characteristic of Seed Aging, evidence suggests that this reflects that also the lipid acid in the membrane phospholipid takes off esterification (McKersie, B.D., Senarata, T., Walker, M.A., Kendall, E.J. and Hetherington, P.R. be stated from: Senescence and Aging in Plants, L.D.Nooden and A.C.Leoopold write, Academic Press, 1988. 441-464 pages or leaves).

At present, by inside or the external factors aging that causes of environment-stress initial for example, there not be extensively suitable method for control.Current, as to be used for controlling aging and to increase the shelf life of fresh perishable plant prod (for example fruit, flower and vegetables) technology mainly depends on and reduces the ethene biosynthesizing.For example, United States Patent (USP) 5,824,875 disclose the transgenosis storksbill, and described plant causes that ethylene levels reduces, and increases and show shelf life because one of three 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase genes are expressed with the antisense orientation.Therefore, this technology ethene responsive type plant of being only applicable to be limited in scope.

By reducing the ethene biosynthesizing, cause that maturation takes place more lentamente, the shelf life of some fruit is prolonged.Because the aging of these fruits is induced after maturation, so the effect that the ethene biosynthesizing reduces shelf life is indirect.Being used for postponing the sophisticated another kind of method of fruit is by changing the cell levels at a kind of softening of the cell wall enzyme-polygalacturonase of the early stage synthetic of maturation.This method is similar to the biosynthesizing of control ethene, because it also is only to affect indirectly aging, moreover it is only applicable to the very plant of close limit.

Thereby, need a kind of method that is applicable to the controlling plant aging of various plant.Therefore, exploitation is applicable to that the technology of the adjusting aging of all types plant (no matter whether it is to the ethene sensitivity) is significant.

Summary of the invention

The present invention is based on the aging of coding carnation induces the full length cDNA clone of lipase and the aging of coding Arabidopis thaliana (Arabidopsis thaliana) to induce the discovery and the clone of the full length cDNA clone of lipase.Herein disclosed is described aging and induce the nucleotide sequence and the amino acid sequence corresponding of lipase gene.The nucleotide sequence of successfully inducing lipase gene with the carnation aging detects the corresponding gene or the rna transcription thing that are subjected to similar adjusting in the several plant as the allos probe.

The invention provides a kind of genetically modified plant that is used for controlling old and feeble initial method, described aging or the aging of being correlated with for the age perhaps are the aging of environment-stress inductive.Lipase nucleotide sequence, its fragment or the segmental combination of this class are induced in aging of the present invention, in reverse importing vegetable cell, induce the expression of lipase gene to suppress endogenous aging, induce the level of lipase and change the aging that transforms in the plant thereby reduce endogenous aging.

Use method of the present invention to produce transgenic plant, and monitor its g and D.Select because old and feeblely induce the plant-growth of lipase level due to reducing, bloom, the breakdown of fruit minimizing, Seed Aging reduces and/or the blade yellow reduces the separate part that shows plant that life or shelf life prolong or plant (for example transplant a cutting, spend, nourishing body, fruit, seed or blade), as the desired product with improved characteristics (comprise leaf yellow minimizing, petal come off minimizing, transportation and storage period breakdown of fruit minimizing).Breed these good plants.Equally, option table reveal tolerance to environmental stress is strengthened (for example to low temperature (cold), arid, the susceptibility reduction of infecting etc.) plant as superior prod.

On the one hand, the present invention relates to a kind of aging of encoding induce lipase, with the isolated DNA molecule of SEQ ID NO:1 hybridization or with the functional deriv of the described DNA isolation molecule of SEQ ID NO:1 hybridization.In one embodiment of the invention, described isolated DNA molecule has the sequence of SEQ ID NO:1, promptly with SEQ ID NO:1 100% complementation (sequence identity).In the present invention's another embodiment aspect this, described isolated DNA molecule contains the nucleotide sequence of SEQID NO:4.

The present invention also relates to a kind of aging of encoding induce lipase, with the isolated DNA molecule of SEQ ID NO:18 hybridization or with the functional deriv of the described DNA isolation molecule of SEQ ID NO:18 hybridization.In the embodiment of the present invention aspect this, described isolated DNA molecule has the sequence of SEQ ID NO:18, promptly with SEQ ID NO:18 100% complementation (sequence identity).In the present invention's another embodiment aspect this, described isolated DNA molecule contains the nucleotide sequence of SEQ ID NO:19.

In another embodiment of the present invention, provide a kind of isolating protein or its functional deriv by the above-mentioned dna molecule encode of this paper.A kind of preferred protein has the aminoacid sequence of SEQ IDNO:2, or its functional deriv.

Antisense oligonucleotide or the polynucleotide of coding with at least a portion complementary RNA molecule of the rna transcription thing of above-mentioned dna molecular also are provided in this, and wherein said RNA molecule and the hybridization of described rna transcription thing make endogenous aging induce the expression of lipase to be changed.Described antisense oligonucleotide or polynucleotide can be total lengths, or preferably have about 6 to about 100 Nucleotide.

Described antisense oligonucleotide or polynucleotide and coding aging induce the corresponding section of a chain of dna molecular of lipase complementary basically, the dna molecular that wherein said coding is old and feeble induces lipase and SEQ ID NO:1, SEQ ID NO:18 hybridization or with the two hybridization, perhaps with the corresponding section complementation basically of the coded RNA sequence of the old and feeble dna molecular of inducing lipase of described coding.In one embodiment of the invention, the corresponding section of described antisense oligonucleotide or polynucleotide and SEQ ID NO:1, SEQ ID NO:18 or the two a chain is complementary basically, and is perhaps complementary basically with the corresponding section of the coded rna transcription thing of SEQ ID NO:1.In another embodiment, described antisense oligonucleotide and coding be old and feeble induces the corresponding section complementation basically of about 100-200 Nucleotide of 5 ' the non-encoding part of a chain of dna molecular of lipase or 3 ' end parts, wherein said dna molecular and SEQ ID NO:1, SEQ IDNO:18 or the two hybridization.In another embodiment, the corresponding section of a chain open reading-frame (ORF) of described antisense oligonucleotide or polynucleotide and SEQ ID NO:4 nucleotide sequence or with complementary basically by the corresponding section of the coded rna transcription thing of SEQ ID NO:4.

The invention still further relates to the carrier that is used for transformed plant cells, described carrier comprises:

(a) antisense base sequences, described antisense base sequences (1) and coding be old and feeble induces the corresponding section of a chain of dna molecular of lipase complementary basically, old and feeble dna molecular and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization of inducing lipase of wherein said coding, perhaps the corresponding section complementation basically of the coded RNA sequence of the old and feeble dna molecular of inducing lipase of (2) and described coding; With

(b) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes described antisense base sequences express in its plant transformed cell.

Described adjusting sequence is included in the promotor that function is arranged in the transformed plant cells, described promotor can be induction type or composing type.Described adjusting sequence can randomly comprise a polyadenylation signal.

The plantlet that the present invention also provides the above-mentioned carrier plant transformed of a kind of usefulness cell, produced by this cell or a kind of plant part of ripe plant or this plantlet or plant.

The inventive method also relates to a kind of generation and compares the method that the plant of lipase level reduction is induced in aging with not modified plant, and described method comprises:

(1) transforms plant with above-mentioned carrier;

(2) allow described plant-growth to plantlet stage at least;

(3) aging of lipase activity and/or change and/or aging of environment-stress inductive and/or the aging of ethene inductive that changes are induced in the aging of analyzing the change of described transformed plant or plantlet; With

(4) plant that lipase activity change and/or aging change and/or old and feeble change of environment-stress inductive or ethene inductive aging change are induced in aging is compared in selection and cultivation with non-plant transformed.

Plant of Chan Shenging or filial generation, hybrid, clone or plant part preferably show aging or the old and feeble delay of ethene inductive that lipase expression decreased and old and feeble delay and/or stress-inducing are induced in aging according to the method described above.

The invention still further relates to a kind of method that endogenous aging induces lipase to express in the vegetable cell that suppresses, described method comprises:

(1) make a kind of vector integration in the genome of plant, described carrier comprises:

(A) antisense base sequences, the corresponding section complementation of a chain of the dna molecular of lipase is induced in described nucleotide sequence (i) and the endogenous aging of coding, the dna molecular of lipase and SEQ IDNO:1, SEQ ID NO:18 or the two hybridization are induced in the endogenous aging of wherein said coding, or (ii) with the corresponding section complementation of inducing the coded RNA sequence of lipase gene by described endogenous aging; With

(B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes that described antisense base sequences is expressed; With

(2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described endogenous RNA, cause described aging to induce the expression of lipase gene to be suppressed.

The accompanying drawing summary

Fig. 1 has described to be induced by the aging that derives from carnation cDNA library the deduced amino acid (SEQ ID NO:2) of lipase cDNA clone (SEQ ID NO:1) coding.Consensus motif in the described aminoacid sequence is as follows: single underscore, amidation site; The point underscore, the protein kinase C phosphorylation site; Double underline, N-Semen Myristicae acidylate site; Solid box (box border), the cAMP phosphorylation site; Dash box, casein kinase i I phosphorylation site; Cross hatch frame (cross-hatched box), the consensus sequence of lipase family; And frame of broken lines, the N-glycosylation site.

Fig. 2 has described to induce lipase aminoacid sequence (SEQ ID NO:2) with the total length carnation petal aging of the derivation of the proteic partial sequence of lipase sample comparison.Carlip: the full length sequence (SEQ ID NO:11) of lipase is induced in the aging of carnation petal; Arlip derives from the proteic partial sequence of lipase sample (Gen Bank registration number AL021710) (SEQ ID NO:12) of Arabidopis thaliana; Ipolip derives from the partial sequence (Gen Bank registration number U55867) (SEQ ID NO:13) of the lipase sample sequence of Ipomoea (Ipomea); Arlipi derives from the proteic partial sequence of lipase sample (Gen Bank registration number U93215) (SEQ ID NO:14) of Arabidopis thaliana.Frame has been annotated the same amino acid in three kinds or the four kinds of described sequences.

Fig. 3 shows the western blot analysis of the fusion protein expression products of the carnation lipase cDNA that expresses in the terrible comfortable intestinal bacteria (E.coli).Induce the proteic antibody of lipase to survey described western blotting with anti-described aging.The 1st swimming lane, maltose binding protein; The 2nd swimming lane, the fusion rotein that constitutes by the carnation lipase that merges by means of proteolytic enzyme (factor Xa) restriction enzyme site and maltose binding protein cDNA; The 3rd swimming lane partly is cut into the fusion rotein of free lipase albumen (50.2kDa) and free maltose binding protein with factor Xa.

Fig. 4 is the rna blot analysis of isolating RNA from the carnation petal of different etap.Fig. 4 A is the gel of the ethidium bromide staining of total RNA.Each swimming lane contains 10 μ gRNA.Fig. 4 B is for using ³²The radioautogram of the RNA trace of lipase cDNA detection is induced in the total length carnation aging of P-dCTP mark.

Fig. 5 proves that for the original position of steatolysis Acyl-hydrolase described lytic enzyme is promptly induced the lipase activity of the protein product of lipase cDNA acquisition by overexpression carnation aging in intestinal bacteria.Mal, the Bacillus coli cells that only contains maltose binding protein in basic salt culture medium; MLip contains the Bacillus coli cells of being induced the fusion rotein that lipase constitutes by the carnation aging of merging with maltose binding protein in basic salt culture medium; 40mal/40mLip, only containing maltose binding protein [mal] or containing the Bacillus coli cells of described lipase-maltose binding protein fusion product [mLip] in replenishing the basic salt culture medium of Tween 40; 60mal/60mLip, only containing maltose binding protein [mal] or containing the Bacillus coli cells of described lipase-maltose binding protein fusion product [mLip] in replenishing the basic salt culture medium of Tween 60.

The restriction map of the open reading-frame (ORF) of lipase is induced in the described carnation aging of Fig. 6 A diagram.Numeral refers to the Nucleotide in the open reading-frame (ORF).

Fig. 6 B is the southern blotting technique analysis that digests and induce with the carnation aging carnation genomic dna of lipase cDNA detection with various Restriction Enzymes.

Fig. 7 is the nucleotide sequence (SEQ IDNO:1) that lipase cDNA clone is induced in the carnation aging.The solid line underscore, the non-coding sequence of lipase cDNA is induced in described aging; Non-underscore sequence is an open reading-frame (ORF).

Fig. 8 is the aminoacid sequence (SEQ ID NO:2) that lipase cDNA is induced in the carnation aging.

Fig. 9 A is a rna blot analysis, shows the expression be exposed to carnation lipase described in the II stage petal that 0.5ppm ethene reaches 15 hours.Fig. 9 A is the gel through ethidium bromide staining, demonstrates the carnation RNA (petal: the 1st swimming lane and the 2nd swimming lane that each swimming lane loads constant basis; Blade: the 3rd swimming lane and the 4th swimming lane; +, ethene is handled;-, undressed).Fig. 9 B is the radioautogram of inducing the RNA trace of gel among Fig. 9 A that lipase cDNA surveys with the total length carnation petal aging of mark.

Figure 10 is partial nucleotide sequence (SEQ IDNO:6) and the corresponding deduced amino acid (SEQ ID NO:17) that lipase is induced in the aging of tomato leaf genome.Conservative lipase consensus motif marks with hachure; The sequence that is used for producing the primer of genomic fragment marks with underscore respectively.

Figure 11 is a bar graph, shows the influence of low temperature to membrane permeation.Tomato plant in 8 ° of refrigerations 48 hours, is warmed to room temperature then again.Measurement without the adjoining tree of refrigeration and through the refrigeration plant at transudate during 6 hours and during 24 hours from the seepage (μ Mhos) of leaf disc.

Figure 12 is the rna blot analysis of isolating tomato leaf RNA from be warmed to the plant that envrionment temperature reaches 24 hours in 48 hours then again in 8 ° of refrigerations.Figure 12 A is the gel of the ethidium bromide staining of the total RNA of blade.Figure 12 B uses ³²The radioautogram of the RNA trace of lipase cDNA detection is induced in the total length carnation aging of P-dCTP mark.

Figure 13 is partial nucleotide sequence (SEQ ID NO:15) and the corresponding deduced amino acid (SEQ ID NO:16) of Arabidopsis EST (GenBank registration number N38227), and it is 55.5% identical that described aminoacid sequence and carnation aging induce lipase to have in 64 amino acid districts.Conservative lipase consensus motif marks with hachure.

Figure 14 be the described total length Arabidopsis aging nucleotide sequence of inducing lipase gene (on) (SEQ ID NO:18) and deduced amino acid (descending) (SEQ ID NO:19).

Figure 15 uses ³²That the total length Arabidopsis aging of P-dCTP mark is induced is that lipase is surveyed, from different steps Arabidopsis plant (the 1st swimming lane, 2 age in week plant; The 2nd swimming lane, 3 the week age plant; The 3rd swimming lane, 4 the week age plant; The 4th swimming lane, 5 the week age plant; The 5th swimming lane, 6 age in week plant) the RNA trace of isolating total RNA in the blade.Last figure (15A) is a radioautogram, and figure below (15B) shows the gel of ethidium bromide staining.

Figure 16 uses ³²That the total length Arabidopsis aging of P-dCTP mark is induced is that lipase is surveyed, the RNA trace of isolating total RNA from the 3 all ages Arabidopsis plant leafs of handling with 50 μ M ethrels (a kind of sources of ethylene).Last figure (16A) is a radioautogram, and figure below (16B) shows the gel of ethidium bromide staining.

Figure 17 be 4.6 week the Arabidopsis wild-type plant (left figure) in ages and the total length Arabidopsis aging of antisence direction induce lipase gene, in transfer-gen plant, show the photo of the transfer-gen plant (right figure) of the big or small increase of blade.

Figure 18 be 6.3 week the Arabidopsis wild-type plant (left figure) in ages and lipase gene is induced in the total length Arabidopsis aging of antisence direction, the photo of the transfer-gen plant (right figure) of the big or small increase of performance blade and leaf senile delay in transfer-gen plant.

Figure 19 be 7 week the Arabidopsis wild-type plant (left figure) in ages and the total length Arabidopsis aging of antisence direction induce lipase gene, in transfer-gen plant, show the photo of the transfer-gen plant (right figure) of the big or small increase of blade.

Figure 20 is a width of cloth figure, shows the T that induces lipase gene in the described aging of 3 antisence directions ₁Transgenic arabidopsis belongs to seed production increase in the plant system.Seed production is with the seed body product representation.The SE that shows the n=30 of wild-type plant.

Figure 21 be from 4 age in week Arabidopsis wild-type plant leaf and induce the western blotting of isolating total protein the corresponding rotaring gene plant blade of lipase gene from the described total length aging of antisence direction.(the 1st swimming lane and the 2nd swimming lane load 9 μ g protein, and the 3rd swimming lane and the 4th swimming lane load 18 μ g protein).Described trace uses the antibody of inducing lipase albumen to produce at described Arabidopsis aging to survey.In all transfer-gen plants, the expression decreased of lipase is induced in described aging.

Detailed Description Of The Invention

Be provided for changing old and feeble method and composition of inducing lipase gene to express in the plant cell.Cause old and feeble initial delay and the resistance of environment-stress is improved in old and feeble change of inducing lipase gene to express described in the plant, thereby prolong shelf life and/or the vegetative period of plant.

From the cDNA library that the RNA by the colored old and feeble petal of carnation (Dianthus caryophyllus) prepares, isolate the full length cDNA sequence that coding schedule reveals the carnation lipase gene of old and feeble inductive expression.Use is corresponding to the polynucleotide probes in the institute constituency of described isolating carnation lipase cDNA sequence and total length carnation lipase cDNA, determines the existence of the mRNA of the described lipase gene of coding in tamato fruit in old and feeble carnation blade, the maturation and tender pod Kidney bean blade in the aging and environment-stress (refrigeration) tomato leaf.Use as template, produces polymerase chain reaction (PCR) product with the tomato leaf genomic dna according to the primer of described carnation lipase cDNA design.Described PCR product contains a part open reading-frame (ORF), and coding comprises the partial protein sequence of described conservative lipase consensus motif ITFTGHSLGA (SEQ ID NO:3).Described tomato nucleotide sequence and described carnation aging induce the lipase sequence to have 53.4% sequence identity, have 43.5% identity with Arabidopsis lipase sequence.Described Arabidopsis lipase sequence and described carnation nucleotide sequence have 44.3% identity.

By using the antibody that produces at kytoplasm lipid-protein particulate (a kind of carnation lipase source), the cDNA expression library of the carnation petal preparation of screening from aging is isolated carnation aging of the present invention and is induced lipase gene.Acquisition is induced the positive full length cDNA clone of lipase gene corresponding to described carnation aging and to its order-checking.Described aging induces lipase cDNA clone's nucleotides sequence to be shown among the SEQ ID NO:1.A kind of 447 amino acid whose polypeptide of this cDNA clones coding (SEQ ID NO:2), the molecular weight of the calculating of described polypeptide is 50.2kDa.Described cDNA is cloned in expression in escherichia coli, produces a kind of protein that shows the active expection molecular weight of Acyl-hydrolase, promptly described expressed protein hydrolysis palmitinic acid p-nitrophenyl ester, phosphatide and triacylglycerol.According to expression pattern and the described activity of proteins of described enzyme in developmental carnation, described enzyme is with old and feeble relevant.

Arabidopsis leaf cDNA library in the employing aging, is also isolated Arabidopsis aging of the present invention and is induced lipase gene by PCR as reaction template.Described Arabidopsis aging induces the nucleotide sequence of lipase gene and deduced amino acid to be shown in Figure 14 (SEQID NO:8).According to the expression pattern of this lipase gene in developmental plant, it is with old and feeble relevant.

The RNA trace of the total RNA of carnation petal that surveys with total length carnation cDNA shows, described aging induces the naturally-aged that is expressed in of lipase gene to face and induced (Fig. 4) significantly before initial.Rna blot analysis proves that also aging is induced lipase gene for example to be refrigerated (Figure 12) and ethene (Fig. 4 and Fig. 9) by the environment-stress condition and induced, and known ethene is that response environment is coerced and produced.Described rna blot analysis shows, the carnation aging induces the amount of lipase mRNA to be significantly higher than amount in the carnation petal of juvenile phase I, II and III in aging course (etap IV).In addition, the flower of the Phase that stimulates of ethene also demonstrates aging and induces the expression of lipase gene higher.Equally, be exposed to the plant that low temperature returns to envrionment temperature then, also demonstrated derivative aging and induce lipase gene to express and produce simultaneously freeze injury symptom (for example seepage) (Figure 11 and Figure 12).

The Arabidopsis aging induces the expression of lipase gene to be subjected to similar adjusting.The rna blot analysis that derives from total RNA of different developmental phases Arabidopsis plant leaf shows that described lipase gene is just regulated, simultaneously blade senesce (Figure 15).In addition, induce lipase gene the same with the carnation aging, handle by the plant hormone ethene with the inducer blade aging, the Arabidopsis aging induces lipase gene just to be regulated (Figure 16).

Each kind of plant for example carnation, tender pod Kidney bean, tomato, Arabidopsis and various plant tissue for example blade, fruit and spend in overall gene expression pattern prove that lipase gene of the present invention participates in old and feeble initial in these plants and plant tissue.Therefore, expection is by suppressing basically in plant tissue or change the expression that lipase gene is induced in described aging, can delay senility, rotten and corrupt, thus increase the shelf lives of perishable fruit, flower and vegetables.Preferably by with constitutive promoter CaMV 35S promoter for example, perhaps adopt tissue specificity or old and feeble inducible promoter, in fruit, flower, vegetables, farm crop and forest species, produce the transgenic plant that wherein said lipase cDNA or its oligonucleotide fragment are expressed with the antisense configuration, then can reach this target.

It is a kind of single copy gene that lipase gene is induced in the carnation aging.Induce the various Restriction Enzymes cutting carnation genomic dnas of sequence in the lipase cDNA open reading-frame (ORF) with the aging of nonrecognition carnation, carry out the southern blotting technique analysis.With ³²The full-length cDNA of P-dCTP mark (SEQ IDNO:1) is surveyed the genomic dna through described Restriction Enzyme digestion.Under high stringency hybridization condition, only (hybridization and washing are all carried out under 68 ℃ for a restriction fragment and described cDNA clone hybridization; Lavation buffer solution: 0.2% * SSC, 0.1% SDS).Therefore, to induce lipase gene be a kind of single copy gene (Fig. 6) in the carnation aging.This gene is not the fact of multigene family a member in carnation, and pointing out it forcefully is a kind of single copy gene in other plant.

Induce lipase gene or Arabidopsis aging to induce the understanding of the complete nucleotide sequence of lipase gene for the carnation aging, be enough to make people from various other plant speciess, to separate aging and induce lipase gene.In fact, indicated as this paper, successfully used Oligonucleolide primers based on described carnation cDNA sequence,, produced the tomato leaf aging by the polymerase chain reaction and induced the lipase gene fragment as template with the tomato leaf genomic dna.

Described cloning aging alone or in combination induces lipase gene or its fragment when importing with inverse direction (antisense) under constitutive promoter control, can be used for genetically modified plant and change old and feeblely in modified plant, described promotor is figwort mosaic virus (fig wartmosaic virus) 35S promoter, cauliflower mosaic virus promoter CaMV35S promotor or MAS promotor for example.Can realize similar genetic modification with from inducing lipase gene to enjoy the selected antisense sequences of other plant of enough sequence identity with the carnation aging.A result of described genetic modification is that the amount of the endogenous interpretable aging coding mRNA that induces lipase reduces.Therefore, the aging that produces in described vegetable cell induces the amount of lipase to reduce, thereby has reduced the amount of cell membrane damage and Premeabilisation of cells, for example because the aging of the blade due to aging or the environment-stress, fruit and/or flower and corrupt the minimizing.

For example, be used in dual 35S promoter and regulate the Arabidopsis plant that the total length Arabidopis thaliana aging of expressing antisense orientation down induces the carrier of lipase gene to transform, show and compare with the wild-type plant that the blade size increases and plant-growth is totally bigger, as shown in Figure 17 and Figure 18.These plant also show leaf senile and postpone, as shown in Figure 19.

Induce the effect of lipase gene expression decreased by the caused described aging of described total length lipase gene of antisence direction in the transgenic arabidopsis platymiscium, also show as and transform that seed production increases in the plant.Express seed that described total length aging induces the Arabidopsis plant system of lipase gene to produce and be the wild-type plant up to about 2-3 doubly (Figure 20).

Demonstrate in Figure 21, observed effect to biomass, leaf senile and seed production induces the lipase minimizing to cause owing to old and feeble in these plant in transfer-gen plant.Transgenic plant of the present invention are compared with wild-type plant, show aging and induce the expression of lipase significantly to reduce.

Therefore, method of the present invention and sequence can be used for the delay plant corruption and comprise blade or breakdown of fruit and increase phytomass and seed production, generally, change The Plant Senescence.

Isolating nucleotide sequence of the present invention can be used for separating complementary aging basically and induce the lipase nucleotide sequence from other plant or biology.These sequences can be used for transforming plant again, thereby to utilize the same way as of mode shown in the isolating nucleotide sequence shown in this article, change and transform The Plant Senescence.

By induce lipase, its function fragment or its combination to transform plant and observed genetic modification with aging, can cause in described plant that aging induces the permanent change of lipase level, and can or adopt other breeding scheme to pass to progeny plant by selfing.Plant with described hereditary change produces new department of botany, and wherein said change is stably transmitted on generation generation ground.The present invention provides first and can be used for realizing the suitable dna sequence dna that old and feeble genetic stability is modified in the different plant of wide range.

The evaluation of inducing lipase gene about described aging with separate, generally speaking, use ordinary method well known in the art, carry out the preparation of plasmid DNA, the Restriction Enzyme digestion of DNA, the agarose gel electrophoresis of DNA, proteinic polyacrylamide gel electrophoresis, southern blotting technique analysis, rna blot analysis, DNA connection and bacterium and transform.Referring to for example Sambrook, J. etc., Molecular Cloning:A Laboratory Manual, second edition, Cold Spring HarborPress, Cold Spring Harbor, NY, 1989.Sambrook (referring to above) discloses the technology of nucleic acid hybridization.

When being used for this paper, term " plant " is meant or complete stool, the part of plant, a kind of vegetable cell or one group of vegetable cell.Can be used for the type of plant of the inventive method and unrestricted, for example comprise: ethene responsive type and ethene non-sensitive type plant; Fruit-bearing plant, for example apricot, apple, orange, banana, natsudaidai, pears, tomato, strawberry, avocado etc.; Vegetables, for example Radix Dauci Sativae, pea, lettuce, Caulis et Folium Brassicae capitatae, turnip, potato, sprouting broccoli, asparagus etc.; Flower, for example carnation, rose, chrysanthemum etc.; And any plant that generally speaking can absorb and express dna molecular of the present invention.Described plant can comprise the plant of various ploidy level, comprises monoploid, amphiploid, tetraploid and polyploid plant.

Transgenic plant are defined as in some way by the plant of genetic modification at this paper, include but not limited to have the allos or the homologous aging of mixing and induce ESD NA or modified DNA or allos aging to induce ESD NA or homology aging to induce the plant of some part of ESD NA in genome.Described reformed genetic material can coded protein, comprising one section regulates or control sequence, perhaps can be antisense sequences or comprise antisense sequences, or coding to induce ESD NA or mRNA sequence or its part with respect to the endogenous aging of described plant be the sense-rna of antisense." transgenosis " or " transgenic sequence " is defined as being incorporated into foreign gene or the partial sequence in the transgenic plant.

Term " hybridization " when being used for herein, generally be used to refer to be for those skilled in the art conspicuous, be the nucleic acid hybridization under the suitable stringency according to the characteristic of described probe sequence and target sequence.The condition of hybridization and washing is well-known in the art, according to required severity, by changing the ionic strength of soaking time, temperature and/or solution, reaches the judgement to described condition easily.Referring to for example Sambrook, J. etc., Molecular Cloning:ALaboratory Manual, second edition, Cold Spring Harbor Press, Cold SpringHarbor, New York, 1989.The selection of condition is decided by the relative G-C content of the length of the length of hybridization sequences, especially probe sequence, described nucleic acid and the mispairing amount of permission.When need when the interchain with low complementary degree is partly hybridized, preferably hanging down stringency.During when the complete complementarity of needs or near complete complementarity, preferred high stringency.About typical high stringency, hybridization solution contains 6 * S.S.C., 0.01M EDTA, 1 * Denhardt solution and 0.5%SDS.For the cloning dna fragmentation, hybridization is about 3-4 hour under about 68 ℃, and for the total DNA of eucaryon, hybridization was carried out about 12 hours to about 16 hours.For lower severity, hybridization temperature is brought down below described duplex melting temperature(Tm) (T _M) about 12 ℃.Known T _MBecome with G-C content and duplex length and solution ion strength.

When being used for this paper, term " roughly sequence identity " or " roughly homology " are used for representing that nucleotide sequence or aminoacid sequence show with another nucleotide sequence or aminoacid sequence structure or function roughly is equal to.Has sequence identity roughly or roughly any structure between the sequence of homology or the difference of function will be minimum (de minimis); That is to say that they will not influence described sequence is brought into play indicated effect in required application ability.Difference may be because for example due to the heritable variation that codon is selected in the different plant species.If between two or more sequences, there is the sequence of a great deal of overlapping or similar, even perhaps described sequence is different on length or structure, if but similar physical property is revealed in described different sequence table, then think the textural difference minimum.This class feature comprises: the ability of hybridizing under qualifications for example; Perhaps for example immune cross-reactivity, similar enzymic activity etc. with regard to protein.

In addition, if having at least about 40% between two kinds of nucleotide sequences, more preferably at least about 60%, most preferably from about 90% sequence similarity, two kinds of nucleotide sequences " complementary substantially " then.If two seed amino acid sequences have at least 40%, preferred 70% similarity at the active part of described polypeptide, this two seed amino acids sequence homology roughly then.

When being used for herein, phrase " with " DNA or RNA molecule " the corresponding section hybridization " meaning be hybridization molecule for example oligonucleotide, polynucleotide or any nucleotide sequence (sense orientation or antisense orientation) discern and have roughly onesize in another nucleic acid molecule and have enough sequence similarities with it under conditions suitable, realizing the sequence of hybridization, and with described sequence hybridization.For example, derive from the long antisense molecule of 100 Nucleotide of carnation lipase 3 ' coding region or non-coding region, with discern respectively the Arabidopsis aging induce lipase gene or any other plant senescence induce 3 ' coding region of lipase gene or non-coding region inner nucleotide sequence about 100 Nucleotide part and with its hybridization, as long as between these two kinds of sequences, have about 70% or higher sequence similarity.People will appreciate that, the big young pathbreaker of described " corresponding section " allows certain mispairing in the hybridization, therefore described " corresponding section " can less than or greater than with the molecule of its hybridization, for example big or little 20-30% preferably is no more than about 12-15% greatly or for a short time than the molecule with its hybridization.

" functional deriv " of term nucleic acid (perhaps polynucleotide or oligonucleotide) is used to refer to the old and feeble gene of lipase or fragment, varient, homologue or the analogue of nucleotide sequence of inducing of coding at this paper.Functional deriv can keep at least a portion that the function of lipase coding DNA is induced in described aging, and this makes it to use according to the present invention.Such function can comprise: the coding aging of inducing ESD NA with natural carnation aging or deriving from another kind of plant is induced the roughly homologous DNA hybridization of lipase or is promptly hybridized with antisense orientation with its mRNA transcript hybridization, suppressing the ability of transcribing and/or translating that plant senescence is induced lipase mRNA, or suchlike ability.

" fragment " of gene or dna sequence dna is meant any inferior part (subset) of described molecule, for example short polynucleotide or oligonucleotide." varient " be meant and complete genome or the roughly similar molecule of its fragment, for example has one or morely to be substituted Nucleotide but to keep and the ability of described specific gene hybridization or the coding nucleic acid replacement varient with the mRNA transcript of described n DNA hybridization." homologue " is meant fragment or the varient sequence that belongs to or plant from a kind of different plant." analogue " is meant similar substantially with described complete molecule, varient or its fragment or its effect and described complete molecule, varient or the relevant non-native molecules of its fragment.

So-called " expression of change " or " the modified expression " of lipase gene induced in for example described aging of a kind of gene, is meant reformed in some way process of expression or result that the normal expression of described gene for example takes place in not modified carnation or other plant.As what meant herein, the change of genetic expression is that described aging induces lipase gene to express minimizing fully or part reduces, but also can comprise the change of expressing sequential, perhaps described aging induces the expression of lipase gene to be different from the another kind of state of the normal expression that takes place of most probable in not modified plant or Cultivar.The preferred change is to compare the change that causes described The Plant Senescence to induce lipase output to reduce with the output in the not modified plant.

Producing aspect the plant of hereditary change of the present invention, preferably selecting single plantlet of each strain or individual plant according to required proterties (being generally aging induces lipase to express or the generation minimizing).For example can use specific antibody as herein described, measure the expression that lipase is induced in the quantitative assay aging with routine immunization.In addition, can use biochemical method as herein described, measure old and feeble enzymic activity of inducing lipase.

For the gene or the dna sequence dna that make new insertion are expressed, cause the protein of described gene or dna sequence encoding to produce, perhaps with regard to antisense DNA, transcribe in order to make described antisense DNA, produce antisense rna molecule, suitable regulatory element should be present on the suitable position and with suitable orientation for described gene or dna sequence dna and exist.Described regulatory region can comprise that a promotor, 5 ' untranslated leader and one 3 ' polyadenylation sequence and enhanser and other regulate sequence.

Thereby can induce lipase gene to be used in combination to produce the modulator promoter element that justice or antisense transcript are arranged of described gene with described aging, generally speaking comprise any plant promoter, more particularly comprise: constitutive promoter, for example radix scrophulariae mosaic virus 35 S promoter, dual 35S promoter, cauliflower mosaic virus promoter, CaMV35S promotor or MAS promotor; Or tissue specificity or old and feeble inducible promoter, for example carnation petal GST1 promotor or Arabidopsis SAG12 promotor are (referring to for example J.C.Palaqui etc., Plant Physiol., 112:1447-1456 (1996); Morton etc., Molecular Breeding, 1:123-132 (1995); Fobert etc., Plant Journal, 6:567-577 (1 994); With Gan etc., Plant Physiol., 113:313 (1997), described document is attached to herein by reference).Be used for preferably constitutive promoter of promotor of the present invention.

Can use conventional expression system, for example by measuring the reporter gene product, for example imported protein in blade, flower, fruit or other tissue extract of the transgenic plant of promotor/reporter gene or the level of mRNA, test is from the expression level that can be used for promotor of the present invention.

The invention provides: with coding carnation aging induce lipase gene complementation, with the aging of coding Arabidopsis induce lipase gene complementation or with from gene or gene fragment complementary antisense oligonucleotide and polynucleotide another kind of plant, that under low paramount stringency, induce lipase gene hybridization with carnation or Arabidopsis aging.This class antisense oligonucleotide should be about 6 Nucleotide at least, thereby the minimum specificity of hybridization is provided, and can with the chain of old and feeble DNA that induces lipase of coding or mRNA or its part complementation or with genomic dna in participate in regulating old and feeble flanking sequence complementation of inducing lipase gene to express.Described antisense oligonucleotide can be greatly to 100 Nucleotide, and can extend to and surpass with respect to described antisense oligonucleotide on length be the complete encoding sequence that justice is arranged.Described antisense oligonucleotide can be strand or the DNA of two strands or the chimeric mixture of RNA or DNA and RNA, perhaps is its derivative or modified forms.

The effect of described antisense oligonucleotide can cause old and feeble change of inducing lipase gene to express in the cell, mainly is the inhibition to described genetic expression.Generality about antisense nucleic acid is discussed, referring to: Alberts etc., Molecular Biology of the Cell, second edition, GarlandPublishing, Inc.New York, New York (1989,195-196 page or leaf especially, the document is attached to herein by reference).

Described antisense oligonucleotide can be induced any part complementation of lipase gene with described aging.In one embodiment, for example, described antisense oligonucleotide can a long 6-100 Nucleotide, and can induce 5 ' non-coding sequence of lipase sequence or 3 ' end complementary with described aging.Known is effective inhibitor of the genetic expression of the encoding transcription factor with 5 ' non-coding sequence complementary antisense oligonucleotide mainly.Branch，M.A.，Molec.Cell?Biol.，13：4284-4290(1993)。

Preferred antisense oligonucleotide and the old and feeble corresponding section complementation substantially of inducing the mRNA of lipase of coding.For example, expection will import in the plant with the old and feeble full length cDNA clone of lipase of inducing of antisense orientation coding, cause successfully changing the expression that lipase gene is induced in aging.In addition, import the partial sequence that the lipase gene specific part is induced in the described aging of target, may be effectively same.

The minimum homology that the present invention needs is such homology, described homology is enough to cause enough complementarity, thereby reach described particular target RNA of identification or DNA and inhibition or reduce its translation or its function, and do not influence function and other expression of gene of other RNA or dna molecular.Though antisense oligonucleotide of the present invention comprises at least a portion complementary sequence of inducing the rna transcription thing of lipase gene with described aging, does not need absolute complementarity, although preferred absolute complementary.The ability of hybridization can be depending on the length and the complementary degree of described antisense oligonucleotide.Generally speaking, hybrid nucleic acid is long more, and what it can contain induces the base mispairing of lipase target sequence then many more with described aging, and still can form stable duplex.Those skilled in the art can utilize standard method for example to measure the melting temperature(Tm) of the mixture of hybridization, and determine the mispairing degree of allowing.

Transcribe by the nucleotide sequence that imports from external source, can in cell, produce described antisense rna oligonucleotide.By for example mixed with a kind of carrier effectively be connected with suitable adjustable element (comprising promotor), the coding described aging induce the lipase antisense sequences DNA plasmid or virus transforms or transfection or infection, described antisense molecule can be passed to cell.In described cell, described exogenous DNA array is expressed, and produces the sense-rna that lipase gene is induced in described aging.

Carrier can be a plasmid, and preferably plasmid perhaps can be virus or other carrier that duplicates in vegetable cell or bacterial cell and express coded gene known in the art.Described vector integration makes it to be transcribed and produces required aging and induce the lipase sense-rna in karyomit(e).This class plasmid or virus vector can make up with the recombinant DNA technology method of this area standard.For example, described carrier can be to contain dubbing system that function is arranged and according to the plasmid vector of antisense oligonucleotide of the present invention or polynucleotide in prokaryotic hosts.On the other hand, described carrier can be to contain dubbing system that function is arranged and according to the plasmid of antisense oligonucleotide of the present invention or polynucleotide in Agrobacterium.The plasmid that can duplicate in Agrobacterium is well-known in the art.Referring to Miki etc., Procedures for Introducing Foreign DNAInto Plants, Methods in Plant Molecular Biology and Biotechnology, B.R.Glick and J.E.Thompson write, CRC Press (1993), the 67-83 page or leaf.

In such a way, described carnation lipase gene is cloned in the plasmid vector with antisense orientation.By pUC18 framework construction pCD plasmid, this plasmid contains the 35S promoter that derives from cauliflower mosaic virus (CaMV), after connect a multiple clone site and an octopine synthase terminator sequence, use the described carnation lipase gene of pCD plasmid clone.By the described total length carnation lipase gene with antisense orientation, subclone makes up pCd-lipase (antisense) plasmid in the Hind3 site and EcoR1 site of pCd.Equally, by at first with the pcr amplified fragment of carnation glutathione s-transferase 1 (GST1) promotor (703 to+19bp), subclone makes up pCD Δ 35S-GST1-lipase (antisense) plasmid to the BamH1 and the Sal1 site of pCd carrier.Then total length carnation lipase gene is arrived the Hind3 and the EcoR1 site of described construct with the antisense orientation subclone.By at first with the pcr amplified fragment of carnation glutathione s-transferase 1 (GST1) promotor (703 to+19bp), subclone makes up another kind of plasmid-pGd Δ 35S-GST1-GUS plasmid to the BamH1 and the Sal1 site of pCd carrier.Then with Sal1 and the EcoRI site of β-glucuronidase (GUS) reporter gene subclone to described construct.With Sma1 and the Hind3 site of dual 35S promoter (two copies that contain the polyphone of CaMV 35S promoter) subclone, make up pCd-35S2-lipase (antisense) plasmid by at first to the pCd carrier.Then total length carnation lipase gene is arrived the Hind3 and the EcoR1 site of described construct with the reverse subclone of antisense.

Preferably be about 6-100 Nucleotide and induce the oligonucleotide of the target complement sequence of lipase, for example can prepare, perhaps can synthesize from single oligonucleotide or short oligonucleotide with the recombinant nucleotide technology with aging.Automatic DNA synthesizer DNA is applicable to chemosynthesis oligonucleotide of the present invention and polynucleotide.The method that is used to make up according to recombinant nucleotide molecule of the present invention is disclosed in Sambrook etc., Molecular Cloning:A Laboratory Manual, second edition, ColdSpring Harbor PreSS, Cold Spring Harbor, N.Y., (1989) all are attached to the document herein.Coding induces the oligonucleotide of lipase sequence complementary sense-rna can adopt the well-known method of those skilled in the art to prepare with old and feeble.At Maniatis, T. etc., Molecular mechanisms in the Control of Gene expression, Nierlich etc. write, and Acad.Press among the N.Y. (1976), provides the details about these class methods.

In an alternate embodiment of the present invention, it is to be imported into external source aging in the described vegetable cell by overexpression to induce lipase gene or gene fragment and the common result who suppresses that the inhibition of the expression of lipase is induced in plant endogenous aging.In this embodiment of the present invention, the carrier of lipase is induced in the aging of coding sense orientation, import in the described cell about the described same way as of antisense molecule with this paper.Lipase is induced in described aging, and preferably for example figwort mosaic virus promotor or CaMV35S effectively are connected with constitutive promoter by force.

Transform by adopting any Plant Transformation method known in the art to carry out DNA, can prepare transgenic plant according to the present invention's preparation.Methods for plant transformation comprises: directly plant, tissue or cell are cultivated or direct infection (Miki etc. with agrobacterium tumefaciens (Agrobacterium tumerfaciens) is common, Meth.in Plant Mol.Biol.and Biotechnology, (1993), p.67-88); Directly transfer to gene in the protoplastis or protoplastis picked-up (Paszkowski etc., EMBO J., 12:2717 (1984)); Electroporation (Fromm etc., Nature, 319:719 (1986)); Particle bombardment (Klein etc., BioTechnology, 6:559-563 (1988)); Be expelled in the meristematic tissue of seedling or plant (De LaPena etc., Nature, 325:274-276 (1987)); Be expelled in the protoplastis of cultured cells and tissue (Reich etc., BioTechnology, 4:1101-1104 (1986)).

Generally obtain complete stool with conversion method.By regeneration plants such as protoplastis, callus, tissue part or explants.Derive from aging and induce the plant part of the reformed regeneration plant of expression of lipase, for example blade, flower, fruit, seed etc. are included in the definition of " plant " used herein.The filial generation of regeneration plant, varient and mutant are also included within the definition of " plant ".

The present invention also provide by the carnation of cDNA molecule encoding of the present invention or Arabidopsis aging induce lipase albumen and with the protein that plays cross reaction at described carnation or the proteic antibody of Arabidopsis.This proteinoid has the aminoacid sequence described in the SEQ ID NO:2 shown in Figure 1, with enjoy cross reactivity at the described proteinic antibody of SEQ ID NO:2, have the aminoacid sequence (being shown in Figure 14) described in the SEQ ID NO:19 or enjoy cross reactivity with proteinic antibody described in the anti-SEQ IDNO:19.

Carnation or Arabidopsis aging induce lipase albumen or its functional deriv preferably with recombinant technology, randomly unite with chemical synthesis and produce.In one embodiment of the invention, described aging induces lipase to express as the fusion rotein of inducing lipase to constitute by the described aging of merging with maltose binding protein.Encode clone's the expression of described recombination fusion protein produces a kind of fusion rotein with hydrolysis palmitinic acid p-nitrophenyl ester, phosphatide and triglyceride level (this is a kind of index of lipase activity) of expection molecular weight.Described reorganization is old and feeble induces lipase albumen after inducing the antibody mediated immunity engram analysis of lipase with anti-carnation aging, demonstrates a master tape in western blot analysis.Induce lipase (50.2Kda) by handle the free aging that described fusion rotein discharges with proteolytic enzyme factor Xa, in western blot analysis, also induce lipase antibody response (Fig. 3) with described aging.Described aging is induced the motif search of lipase aminoacid sequence, demonstrate exist one can be for tetradecanoic acid by the covalently bound potential N-Semen Myristicae acidylate site (Fig. 1) of amido linkage (referring to Johnson etc., Ann.Rev.Biochem., 63:869-914 (1994); Towler etc., Ann.Rev.Biochem., 57:67-99 (1988); And R.J.A.Grand, Biochem.J., 258:625-638 (1989)).The protein motif search also shows: described carnation aging induces lipase to contain one section sequence ITFAGHSLGA (SEQ ID NO:4) for conservative lipase consensus motif (table 1).The described conservative lipase consensus sequence that derives from various plant is shown in the following table.

Table 1

Plant species	Conservative lipase sequence
		Carnation	ITFAGHSLGA(SEQ?ID?NO：4)
Tomato	ITFTGHSLGA(SEQ?ID?NO：3)
		Arabidopsis	ITTCGHSLGA(SEQ?ID?NO：9)
Ipomoeanil	ITVTGHSLGS(SEQ?ID?NO：10)

Aging of the present invention induces lipase albumen all to demonstrate in external test and in-site detecting to have lipase activity.About in-vitro measurements, with palmitinic acid p-nitrophenyl ester and soybean phospholipid (40% phosphatidylcholine and 60% other phosphatide) as substrate, with spectrophotometry measure respectively reaction product-p-NP and free fatty acids (Pencreac ' h and Baratti, 1996; Nixon and Chan, 1979; Lin etc., 1983).In addition, adopt improving one's methods of Nixon and (1983) described methods such as Chan (1979) and Lin, by gas-chromatography at the in-vitro measurements lipase activity.In the final volume of 100 μ l, reaction mixture contains 100mM Tris-HCl (pH 8.0), 2.5mM substrate (trilinolein, soybean phospholipid or dilinoleoylphosphatidylcholine) and zymoprotein (100 μ g).The described substrate of emulsification joins it in reaction mixture then in 5% gum arabic., substrate is dissolved in the chloroform for this reason, joins in the gumwater, carried out emulsification in 30 seconds by supersound process.After emulsification, evaporate chloroform with nitrogen gas stream.React on 25 ℃ and reach 2 hours different time at the most.The lipid of abstraction reaction mixture by TLC purifying free fatty acids, also passes through GC quantitatively (McKegney etc., 1995) with its derivatize then.

Described and according to (1983) such as Furukawa by (1996) improved methods such as Tsuboi, in site measurement steatolysis acyl hydrolase enzymic activity.In this a kind of mensuration in back, allow with the old and feeble intestinal bacteria of inducing the full length cDNA clone conversion of lipase of coding, in being supplemented with Tween 40 or Tween 60 (these two kinds of materials all are long chain fatty acid esters) basic salt culture medium, grow as sole carbon source.Therefore, the carbon of using for bacterial growth only just can obtain after fatty acid ester is by the lipase hydrolysis.Find after initial lag period, in Tween 40-basis salt culture medium and Tween 60-basis salt culture medium, to grow with the intestinal bacteria that described aging induces lipase cDNA to transform, and the intestinal bacteria control cultures that is not transformed is not grown, and this discovery has confirmed the lipase activity (Fig. 5) of coded recombinant protein.That is to say that described aging induces lipase to discharge stearic acid (Tween 60) and palmitinic acid (Tween 40), thereby obtain essential carbon for growth usefulness.

It is to keep described aging to induce lipase activity or induce the aging of at least a portion of cross reactivity of the specific antibody immunity of lipase to induce fragment, varient, analogue or the chemical derivative of lipase with aging that lipase proteic " functional deriv " is induced in aging as herein described.Described aging induces the proteic fragment of lipase to be meant any inferior part of described molecule.The variation peptide can be synthetic by direct chemical, for example adopt method well known in the art to carry out chemosynthesis prepares.Aging induces the analogue of lipase to be meant and described whole protein or the similar substantially non-natural protein of its fragment.Aging induces the chemical derivative of lipase to contain extra chemical part, and described chemical part is not the part of peptide G38 or peptide fragment usually.Target amino acid residue by making described peptide with can with organic derivatize agent reaction of selected side chain or terminal residue reaction, modification can be incorporated into described aging and induce in lipase peptide or its fragment.

By cultivating with nucleotide sequence of the present invention (sense orientation) cell transformed, allow described cell synthesize described protein, separate described protein then from substratum or from cell extract, described protein is according to used clone's scheme or be free protein or for fusion rotein, then can produce according to aging of the present invention and induce lipase albumen or peptide.On the other hand, can produce described protein with cell free system.Ranu etc., Meth.Enzymol., 60:459-484, (1979).

Described the present invention now comprehensively, by consulting following examples, can more easily understand the present invention, it is in order to illustrate that described embodiment is provided, and is not to be used for limiting the present invention.

Embodiment 1

In order to separate the vegetable material of described carnation lipase cDNA

Be used in the carnation plant (the white Sim of Dianthus caryophllus L.cv. improvement) that grows in the greenhouse and keep, separate the nucleotide sequence of inducing lipase gene corresponding to described aging.With the colored tissue collecting of old and feeble petal (deriving from the different etap) form in damping fluid, or be stored in-70 ℃ stand-by.

From the carnation petal of before aging begins, collecting just, separate the kytoplasm lipid granule.With carnation petal (25g/150ml damping fluid) in 4 ℃ in homogenate buffer (50mMEpps-0.25M sorbyl alcohol pH 7.4,10mM EDTA, 2mM EGTA, 1mM PMSF, 1mM benzamidine (benzamadine), 10mM amino-n-caproic acid and 4% polyvinylpolypyrrolidone), homogenate is 45 seconds in Omnimizer, homogenate 1 minute again in the Polytron homogenizer.Make homogenate pass through 4 layers of cheese cloth and filter, with filtrate in 4 ℃ with 10, centrifugal 20 minutes of 000g.Supernatant liquor is with 250, and centrifugal 1 hour of 000g is with separating particles body film.Adopting Hudak and Thompson, (1997), Physiol.Plant., the method of 114:705-713 is carried out flotation centrifugal (floatation centrifugation) back by collecting lipid granule, obtains described lipid granule from back microsome supernatant liquor (post-microsomal supematant).Supernatant liquor is made the solution of 10% (w/v) with sucrose, and the described supernatant liquor of 23ml is injected in the 60Ti Beckman centrifuge tube, covers the 1.5ml dissociating buffer, in 4 ℃ with 305, centrifugal 12 hours of 000g.From the dissociating buffer coverture, take out described particle with Pasteur's dropper.With 3ml particle suspension liquid application of sample to aseptic PBS (10mM sodium phosphate buffer pH 7.5 adds 0.85% sodium-chlor) equilibrated Sepharose G-25 post, with aseptic PBS wash-out suspension.Wash-out goes out to contain described particulate void volume, with Centricon-10 filter (can derive from Amicon) it is concentrated into the protein concn of 600 μ g.Use described lipid granule then, in the described particulate rabbit of inoculation 300 μ g, produce antibody.IgG by western blot analysis test blood tires.

The separation of messenger RNA(mRNA) (mRNA)

Basically according to Chomczynski and Sachi, Anal.Biochem., the described method of 162:156-159 (1987) is separated total RNA from the carnation of Phase I, II, III or IV.In brief, the 15g petal is organized in the liquid nitrogen freezing, homogenate is 30 seconds in the damping fluid that contains 4M guanidine thiocyanate, 25mM Trisodium Citrate pH 7.0,0.5% dodecyl creatine sodium (sarkosyl) and 0.1M beta-mercaptoethanol.The water saturated phenol of 150ml, 30ml chloroform and 15ml 2M NaOAc pH 4.0 are joined in the homogenate sample.With 10, centrifugal 10 minutes of 000g takes out water layer, therefrom is settled out nucleic acid with the 150ml Virahol with sample.Sample is with 5, centrifugal 10 minutes of 000g, and,, contain the isopropanol precipitating of 0.2M NaOAc pH 5.0 then with 30ml with the extracting of 30ml chloroform with 30ml 4M LiCl washing precipitation 1 time.RNA is dissolved in the water of DEPC processing, and is stored in-70 ℃.

With the PolyA that can derive from Promega ⁺Tract mRNA separation system (PolyA ⁺TractmRNA Isolation System), from total RNA, separate PolyA ⁺MRNA.Use PolyA ⁺MRNA can derive from Stratagene (La Jolla, ZAPExpress Calif.) as template so that use ^It is synthetic that the cDNA synthesis system is carried out cDNA.

Carnation petal cDNA library screening

To use from the cDNA library of the isolating mRNA preparation of Phase IV carnation petal, be diluted to about 5 * 10 ⁶PFU/ml carries out immunoscreening with the lipid granule antiserum(antisera) to it.Use ExAssist ^Helper Phage/SOLR bacterial strain system recoveries positive cDNA clone, and with it at pBluescript ^Cyclisation again in the phagemid (Stratagene).Also use ³²The probe of 19 base pairs of P mark (5 '-ACCTACTAGGTTCCGCGTC-3 ') (SEQ ID NO:5) screening stage III carnation petal cDNA library.From phage, downcut positive cDNA clone, with the method in manufacturer's the explanation, with it in pBK-CMV ^(Stratagene) cyclisation again in the phagemid.Full-length cDNA (1.53kb fragment) is inserted in the pBK-CMV carrier.

Arabidopsis leaf cDNA library screening

By the old and feeble leaf cDNA of screening Arabidopsis library, isolate the full length cDNA clone (1338bp) that lipase gene is induced in an aging that derives from Arabidopis thaliana.The probe that is used for screening described library adopts old and feeble blade library to obtain by PCR as template.According to the described PCR primer of the genome sequence that exists among the GenBank (U93215) design.Forward primer has sequence 5 ' ATG TCT AGA GAA GAT ATT GCG CGG CGA 3 ' (SEQID NO:20), and reverse primer has sequence 5 ' GAT GAG CTC GAC GGA GCTGAG AGA GAT G 3 ' (SEQ ID NO:21).PCR product subclone is used for order-checking in Bluescript.The nucleotide sequence and the aminoacid sequence of used PCR product are shown among Figure 14.

The separation of plasmid DNA, dna sequencing

Alkaline lysis (referring to above) with descriptions such as Sambrook comes isolated plasmid dna.With dideoxy sequencing method the total length positive cDNA clone is checked order.Sanger etc., Proc.Natl.Acad.Sci.USA, 74:5463-5467.The compiling open reading-frame (ORF), and with blast search (GenBank, Bethesda, MD) analyze, with BCM Search Launcher:Multiple SequenceAlignments Pattern-Induced Multiple Alignment Method (referring to F.Corpet, Nuc.Acids Res., 16:10881-10890, (1987)), obtain the sequence alignment of derivation aminoacid sequence of the gene of 5 kinds of protein that homology is the highest and described coding.Identify the function motif that exists in the described derivation aminoacid sequence with MultiFinder.

Expression as the lipase of fusion rotein

Contain the phagemid pBK-CMV that lipase is induced in the aging of total length carnation with EcoRI and XbaI digestion, discharge 1.53Kb lipase fragment, described fragment subclone is arrived in the fusion vector pMalc (New England BioLabs) of EcoRI and XbaI digestion.Contain described aging and induce the pMalc carrier called after pMLip of lipase, with this carrier transformed into escherichia coli BL-21 (DE3) cell.

As (referring to above) such as Sambrook and Ausubel etc., Current Protocols inMolecular Biology, Green Pubilishing Associates and Wiley Interscience, New York, (1987), 16.4.1-16.4.3 described in, separation and purifying are by the fusion rotein (syzygy of lipase and maltose binding protein is induced in described aging) of pMLip coding.In brief, to be resuspended in 3ml/g lysis buffer (50mM Tris with the intestinal bacteria BL-21 cell that pMLip transforms, pH 8.0,100mM NaCl and 1mM EDTA) in, described lysis buffer contains every gram cell 8 μ l 50mM PMSF and 80 μ l 20mg/ml N,O-Diacetylmuramidases, hatches 20 minutes in the room temperature vibration.The DNA enzyme I that adds 80 μ l, 5% Septochol and 40 units then vibrates cell until the complete cracking of cell in room temperature.By the centrifugation cell fragment, the broken lysis buffer that is resuspended in 2 times of volumes of cell sheet is added among 8M urea and the 0.1mM PMSF.After 1 hour, add damping fluid (the 50mM KH of 7 times of volumes ₂PO ₄, 1mM EDTA and 50mM NaCl, pH 7.0) with in and suspension.With the pH to pH8.0 of HCl adjusting cell suspending liquid, sedimentation cell fragment then.Allow supernatant liquor to 20mM Tris pH of buffer 8.0,100mMNaCl and 1mM EDTA in 4 ℃ of dialysed overnight.With amylose starch post (can derive from NewEngland BioLab) purifying maltose binding protein-lipase fusion product (Malip).Cut the flow point that contains described fusion rotein with proteolytic enzyme factor Xa (1 μ g/100 μ g fusion rotein), from fusion product, to separate lipase.By SDS PAGE gel electrophoresis and described fusion rotein of western blot analysis and lipase through cutting.Use in contrast by the maltose binding protein of pMalc coding.The results are shown among Fig. 3.

The RNA blot hybridization of carnation RNA

10 μ g are separated at 1% sex change formaldehyde agarose gel from the isolating total RNA that spends of Phase I, II, III, IV, and be fixed on the nylon membrane.The utilization random primer test kit (Boehringer Mannheim) with ³²The described 1.53Kb EcoRI-XbaI of P-dCTP mark lipase fragment is surveyed described filter membrane (7 * 10 with described labeled fragment ⁷Cpm).Filter membrane with 1 * SSC, 0.1%SDS in room temperature washing 1 time, with 0.2 * SSC, 0.1%SDS in 65 ℃ of washings 3 times.With the filter membrane drying, and X-ray film exposure is spent the night in-70 ℃.The results are shown among Fig. 4.

The RNA blot hybridization of Arabidopsis RNA

10 μ g isolating total RNA from the Arabidopsis blade in 2 weeks of growing, 3 weeks, 4 weeks, 5 weeks and 6 weeks is separated at 1% sex change formaldehyde agarose gel, and be fixed on the nylon membrane.The utilization random primer test kit (Boehringer Mannheim) with ³²Lipase gene is induced in the aging of P-dCTP mark total length Arabidopsis, surveys described filter membrane (7 * 10 with this labeled fragment ⁷Cpm).Filter membrane with 1 * SSC, 0.1%SDS in room temperature washing 1 time, with 0.2 * SSC, 0.1%SDS in 65 ℃ of washings 3 times.With the filter membrane drying, and X-ray film exposure is spent the night in-70 ℃.

The separation of genomic dna and southern blotting technique hybridization

The carnation petal of fresh cutting-out is freezing in liquid nitrogen, grind into powder, and with extraction buffer (0.1M Tris, pH 8.2,50mM EDTA, 0.1M NaCl, 2% SDS and 0.1mg/ml Proteinase K) homogenate (2ml/g), with isolation of genomic DNA.Allow the homogenate material in 37 ℃ of incubations 10 minutes, use phenol-chloroform-primary isoamyl alcohol (25: 24: 1) extracting then.With NaOAc and isopropanol precipitating DNA.The DNA precipitation is dissolved among 1 * TE pH 8.0, uses the phenol extracting again, redeposition, and be resuspended among 1 * TE pH 8.0.

Genomic dna is used restriction endonuclease (Bam HI, XbaI, XhoI, EcoRI, HindIII and SalI) digestion respectively, and will be through DNA fractional separation on 1% sepharose of digestion.Separated DNA suction is printed on the nylon membrane, use ³²P-dCTP mark 1.53Kb lipase fragment is hybridized.Hybridization and washing high stringency (68 ℃) (6 * SSC, 2 * Denhardt reagent, 0.1%SDS) and low stringency (in 42 ℃ of hybridization and washing) (6 * SSC, 5 * Denhardt reagent carry out under 0.1%SDS).The results are shown among Fig. 6.As can be observed, lipase cDNA probe only detects a kind of genomic fragment, shows that the carnation lipase gene is a single copy gene.

The enzymatic determination of lipase

Use palmitinic acid p-nitrophenyl ester and soybean phospholipid as the external source substrate, the steatolysis acyl hydrolase enzymic activity of the lipase fusion rotein by the described purifying of spectrophotometric analysis.For with the independent maltose binding protein that compares, as substrate, there is not detectable lipase activity (table 2) with phosphatide.When with the palmitinic acid p-nitrophenyl ester as substrate and when using independent maltose binding protein, can detect the lipase reaction product of a spot of p-NP-prediction, reflect the background level (table 2) of the p-NP in the commercial formulation of palmitinic acid p-nitrophenyl ester.Yet, under the situation of the lipase fusion rotein that has purifying, obviously have to show as the strong lipase activity (table 2) that free fatty acids discharges and p-NP discharges from phosphatide from the palmitinic acid p-nitrophenyl ester.

Table 2

In expression in escherichia coli and the active spectrophotometer measurement result of steatolysis Acyl-hydrolase by fusion rotein amylose starch column chromatography purifying, maltose binding protein and lipase.

Use two kinds of substrate-palmitinic acid p-nitrophenyl ester and soybean phospholipids.

Active represent with the product that generated (from the p-NP of palmitinic acid p-nitrophenyl ester with from the free fatty acids of soybean phospholipid).

Mean value ± the SE that has shown the n=3 replication.

Product

Kinds of protein pNPP free fatty acids

P-NP (nmol/mg albumen/min)

(nmol/mg/min)

Maltose binding protein 0.71 ± 0.02 ND ^*

Lipase fusion rotein 12.01 ± 1.81 46.75 ± 1.24

^*ND can not detect

In other experiment, by the enzymic activity of gas chromatographic analysis lipase fusion rotein, gas-chromatography is a kind of can quantitatively and the evaluation from the technology of the free fatty acids of described substrate release.As substrate, before by gas chromatographic analysis, take off the lipid acid of esterification by the thin-layer chromatography purifying with trilinolein, soybean phospholipid and dilinoleoylphosphatidylcholine.Consistent with spectrophotometry (table 2), do not detect the lipase activity of independent maltose binding protein with soybean phospholipid or dilinoleoylphosphatidylcholine, show that these substrates do not take off the lipid acid (table 3) of esterification basically.Yet when using the lipase fusion rotein as the enzyme source, palmitinic acid, stearic acid and linolic acid take off esterification from the soybean phospholipid extract, and linolic acid takes off esterification (table 3) from dilinoleoylphosphatidylcholine.Opposite with the phosphatide substrate, but in trilinolein, there is the free linoleic acid of detection level, but the level of free linoleic acid significantly raises when the lipase fusion rotein exists, and shows that described lipase also can make lipid acid take off esterification (table 3) from triglyceride level.

Table 3

In expression in escherichia coli and the active GC measuring result of steatolysis Acyl-hydrolase by fusion rotein amylose starch column chromatography purifying, maltose binding protein and lipase

Product (μ g/mg albumen) ¹

Substrate maltose lipase

Conjugated protein fusion rotein

Trilinolein ²Linolic acid (18: 2) 15.9 ± 0.75 33.4 ± 1.58

Soybean phospholipid ³Palmitinic acid (16: 0) ND ⁴4.80

Stearic acid (18: 0) ND 9.68

Linolic acid (18: 2) ND 5.80

Two inferior oleoyl linolic acid (18: 2) ND 20.00

Phosphatidylcholine ³

¹Allow reaction carry out 2 hours, reaction is not continuous linearity during this period.

²Mean value ± the SE that has shown the n=3 replication.

³Once experiment

⁴Can not detect

As Tsuboi etc., Infect.Immunol., 64:2936-2940 (1996); Wang etc., Biotech., 9:741-746 (1995); And G.Sierra, J.Microbiol.and Serol. described in the 23:15-22 (1957), measures the described proteinic lipase activity that obtains by at expression in escherichia coli lipase cDNA in vivo.Intestinal bacteria BL-21 colony inoculation that single bacterium colony of the intestinal bacteria BL-21 cell that will transform with pMal and another transform with pMLip is in basic salt culture medium (pH 7.0), and described substratum contains (g/L): K ₂HPO ₄(4.3), KH ₂PO ₄(3.4), (NH ₄) ₂SO ₄(2.0), MgCl ₂(0.16), MnCl ₂4H ₂O (0.001), FeSO ₄7H ₂O (0.0006), CaCl ₂2H ₂O (0.026) and NaMoO ₄2H ₂O (0.002).Concentration with 1% adds substrate-Tween 40 (polyethenoxy sorbitan monopalmitate) or Tween 60 (polyethenoxy sorbitan monostearate).By measuring the absorbancy of 600nm, the growing state (Fig. 5) of monitoring bacterial cell under 37 ℃ of vibrations.As what in Fig. 5, can see, can in the basic medium that replenishes Tween 40/Tween 60, grow after initial lag period with the Bacillus coli cells that pMLip transforms.Yet, in the substratum that replenishes Tween, do not grow with the Bacillus coli cells that pMal transforms.

Embodiment 2

The carnation aging induces the ethene of lipase gene to induce

The carnation of Phase and carnation are transplanted a cutting and handled 15 hours with 0.5ppm ethene in sealing chamber.As described below, from the Phase petal that ethene is handled and in the blade of treated cutting and from undressed carnation and flower of transplanting a cutting and blade, extract RNA.

The Arabidopsis plant was handled 1 day, 2 days or 3 days with 50 μ M ethrels in sealing chamber.Followingly from the plant leaf that ethrel is handled, extract RNA.

In liquid nitrogen, grind flower or blade (1 flower or 5g blade).The powder that grinds is mixed with 30ml guanidine damping fluid (4M guanidinium isothiocyanate, 2.5mM NaOAc pH 8.5,0.8% beta-mercaptoethanol).Make mixture pass through 4 layers of cheese cloth and filter, and in 4 ℃ with 10, centrifugal 30 minutes of 000g.Then with supernatant liquor with 26,000g carries out cesium chloride density gradient centrifugation and reaches 20 hours.Sedimentary DNA 75% alcohol flushing is resuspended in the 600 μ l DEPC treated waters, uses 0.75ml 95% ethanol and 30 μ l 3M NaOAc in-70 ℃ of precipitated rnas.The carnation RNA of 10 μ g or Arabidopsis RNA be fractional separation on 1.2% sex change formaldehyde agarose gel, transfers on the nylon membrane then.With what cause at random ³²The film that the total length carnation lipase cDNA (SEQ ID NO:1) of P-dCTP mark contains carnation RNA in 4 ℃ of detections spends the night.With what cause at random ³²The film that the total length Arabidopsis lipase cDNA of P-dCTP mark contains Arabidopsis RNA in 42 ℃ of detections spends the night.Then, described film once reaches 15 minutes with the 1 * SSC that contains 0.1% SDS in room temperature washing, washs each 15 minutes with the 0.2 * SSC that contains 0.1% SDS 3 times in 65 ℃ then.Film is spent the night to X-ray film exposure in-70 ℃.

The results are shown in Fig. 9 (carnation) and Figure 16 (Arabidopsis; The 1st swimming lane was handled in 1 day; The 2nd swimming lane was handled in 2 days; The 3rd swimming lane was handled in 3 days).Just as can be seen, ethrel is induced transcribing of carnation lipase and Arabidopsis lipase in flower and/or blade.

Embodiment 3

Produce tomato PCR product with carnation lipase primer

Employing is induced a pair of Oligonucleolide primers of lipase sequences Design according to the carnation aging, and by nested PCR, the partial-length sequence of lipase is induced in the aging that produces the tomato dna group DNA that obtains from tomato leaf.5 ' primer is a kind of 19 aggressiveness, and its sequence is 5 '-CTCTAGACTATGAGTGGGT (SEQ ID NO:7); 3 ' primer is a kind of 18 aggressiveness, and its sequence is CGACTGGCACAACCTCCA-3 ' (SEQ ID NO:8).Adopt tomato dna group DNA, the following polymerase chain reaction of carrying out:

Reactive component:

Genomic dna 100ng

DNTP (each 10mM) 1 μ l

MgCl ₂(5mM)+10x damping fluid 5 μ l

Primer 1 and primer 2 (each 20 μ M, 0.5 μ l

Taq archaeal dna polymerase 1.25U

Reaction volume 50 μ l

Reaction parameter:

94 ℃ 3 minutes

94 ℃/1 minute, 48 ℃/1 minute, 72 ℃/2 minutes, 45 circulations

72 ℃ 15 minutes.

The tomato partial-length sequence that obtains by PCR has nucleotide sequence SEQ ID NO:6 (Figure 10) and derivation aminoacid sequence shown in Figure 10.Described partial-length sequence contains an intron that inserts (Figure 10, lowercase) between two encoding sequences.Described tomato sequence contains conservative lipase consensus sequence-ITFTGHSLGA (SEQ ID NO:3).

Described tomato sequence and carnation aging induce the lipase sequence that 53.4% sequence identity is arranged, and with Arabidopsis lipase 43.5% sequence identity are arranged, and Arabidopsis lipase and described carnation sequence have 44.3% sequence identity.

Embodiment 4

Low temperature is to the influence of cell membrane integrity in the tomato plants

Tomato plant in 7 ℃ to 8 ℃ refrigerations 48 hours, is returned to room temperature then and reaches 24 hours.By measuring the amount (μ Mho) of electrolyte leakage, estimate the influence of low temperature to blade.

Specifically, downcut the 1g leaf tissue and place the 50ml test tube, get express developed, and add the 40ml deionized water with distilled water.Add a cover to test tube, in room temperature rotation 24 hours.With the interval of 6 hours and 24 hours, specific conductivity (μ Mho) reading record contrast leaf tissue and low temperature injury leaf tissue, the reflection electrolyte leakage.From Figure 11, can obviously find out, in the low temperature injury leaf tissue, the electrolyte leakage of reflection membrane damage take place during warm again.Derive from the rna blot analysis of the RNA of refrigeration tomato leaf

Without the tomato plant blade (contrast) of refrigeration with also returned to room temperature through refrigeration and reach the blade of tomato plant of 0 hour, 6 hours and 24 hours, extract total RNA from 15g.RNA extracts as described in example 3 above method to carry out.The RNA that 10 μ g is derived from every kind of sample separates on 1.2% sex change formaldehyde gel, and is transferred on the nylon membrane.Described film is used ³²The probe of P-dCTP mark (SEQ ID NO:3) is surveyed, then with embodiment 3 described the same terms under wash.The results are shown among Figure 12.

As what can see from radioautogram (Figure 12 B), the expression of tomato lipase gene is induced in refrigeration, and electrolyte leakage increase relevant (Figure 11) in gene induced pattern and the low temperature injury blade.

Embodiment 5

The generation that the transgenic plant of lipase gene are induced in the reverse aging of antisence

Induce the binary vector pKYLX71 of lipase gene to transform Agrobacterium with containing the total length Arabidopsis aging of under dual 35S promoter is regulated, expressing with antisense orientation.By vacuum filtration,, select to derive from the transformed the seed of the T0 plant that is produced with penbritin with these Agrobacterium-mediated Transformation Arabidopsis plant.

Under greenhouse experiment, cultivate T ₁Plant and wild-type Arabidopsis plant.Observe the difference of blade size, plant size of population, seed production and leaf senile between transfer-gen plant and the wild-type plant at the appointed time.Difference is described among Figure 17,18,19 and 20.

Embodiment 6

Old and feeble generation minimizing of inducing lipase in the transgenic plant

Will from 4 age in week Arabidopsis wild-type plant leaf and be transferred on the nylon membrane as isolating gross protein the blade of the corresponding transgenic plant of preparation as described in the embodiment 5, use the antibody that aging induces lipase albumen to produce at Arabidopsis to survey.Western blotting is shown in (the 1st swimming lane and the 2nd swimming lane load 9 μ g protein, and the 3rd swimming lane and the 4th swimming lane load 18 μ g protein) among Figure 21.In old and feeble expression decreased of inducing lipase described in the transfer-gen plant.

Sequence table＜110〉Thompson, John E.

Wang，Tzann-Wei

Hudak，Katalin

Hong; Yuwen＜120〉method＜130〉10799/14＜140〉PCT/US01/19385＜141〉2001-06-19＜150〉09/610 of DNA, genetically modified plants and control plant senescence of coded plant lipase; 104＜151〉2000-07-05＜150〉09/597,774＜151〉2000-06-09＜160〉21＜170〉PatentIn Ver.2.1＜210〉1＜211〉1537＜212〉DNA＜213〉 ( Dianthus caryophyllus )＜400〉1gcacgagcca ttccaaaact ccttacacca ctcaaaacta ttccaacatg gctgcagaag 60cccaaccttt aggcctctca aagcccggcc caacatggcc cgaactcctc gggtccaacg 120cttgggccgg gctactaaac ccgctcaacg atgagctccg tgagctcctc ctacgctgcg 180gggacttctg ccaggtgaca tacgacacct tcataaacga ccagaactcg tcctactgcg 240gcagcagccg ctacgggaag gcggacctac ttcataagac cgccttcccg gggggcgcag 300accggtttga cgtggtggcg tacttgtacg ccactgcgaa ggtcagcgtc ccagaggcgt 360ttctgctgaa gtcgaggtcg agggagaagt gggataggga atcgaattgg attgggtatg 420tcgtggtgtc gaatgacgag acgagtcggg tggcgggacg aagggaggtg tatgtggtgt 480ggagagggac ttgtagggat tatgagtggg ttgatgttct tggtgctcaa cttgagtctg 540ctcatccttt gttacgcact caacaaacta ctcatgttga aaaggtggaa aatgaggaaa 600agaagagcat tcataaatca agttggtacg actgtttcaa tatcaaccta ctaggttccg 660cgtccaaaga caaaggaaaa ggaagcgacg acgacgatga tgacgacccc aaagtgatgc 720aaggttggat gacaatatac acatcggagg atcccaaatc acccttcaca aaactaagtg 780caagaacaca acttcagacc aaactcaaac aactaatgac aaaatacaaa gacgaaaccc 840taagcataac attcgccggt cacagcctag gcgcgacact atcagtcgtg agcgccttcg 900acatagtgga gaatctcacg accgagatcc cagtcacggc cgtggtcttc gggtgcccaa 960aagtaggcaa caaaaaattc caacaactct tcgactcgta cccaaaccta aatgtcctcc 1020atgtaaggaa tgtcatcgac ctgatccctc tgtatcccgt gaaactcatg ggttacgtga 1080acataggaat cgagctggag atcgactcga ggaagtcgac ctttctaaag gactcgaaaa 1140acccgagtga ttggcataat ttgcaagcaa tattgcatgt tgtaagtggt tggcatgggg 1200ttaaggggga gtttaaggtt gtaaataaga gaagtgttgc attggttaat aagtcatgtg 1260attttcttaa ggaagaatgt ttggttcctc cagcttggtg ggttgtgcag aacaaaggga 1320tggttttgaa taaggatggt gagtgggttt tggctcctcc tgaggaagat cctactcctg 1380aatttgattg ataatatttc atcatgtttt atatttttat aaattttact aaatttacat 1440gacaatttat gggactaagt tacttattta tatgtttatt atatttgaaa tgtgttttaa 1500gttacataaa attgcaatta gttttaaaaa aaaaaaa 1537＜210〉2＜211〉447＜212〉PRT＜213〉 ( Dianthus caryophyllus )＜400〉2Met Ala Ala Glu Ala Gln Pro Leu Gly Leu Ser Lys Pro Gly Pro Thr1 5 10 15Trp Pro Glu Leu Leu Gly Ser Asn Ala Trp Ala Gly Leu Leu Asn Pro

20??????????????????25??????????????????30Leu?Asn?Asp?Glu?Leu?Arg?Glu?Leu?Leu?Leu?Arg?Cys?Gly?Asp?Phe?Cys

35??????????????????40??????????????????45Gln?Val?Thr?Tyr?Asp?Thr?Phe?Ile?Asn?Asp?Gln?Asn?Ser?Ser?Tyr?Cys

50??????????????????55??????????????????60Gly?Ser?Ser?Arg?Tyr?Glu?Lys?Ala?Asp?Leu?Leu?His?Lys?Thr?Ala?Phe65??????????????????70??????????????????75??????????????????80Pro?Gly?Gly?Ala?Asp?Arg?Phe?Asp?Val?Val?Ala?Tyr?Leu?Tyr?Ala?Thr

85??????????????????90??????????????????95Ala?Lys?Val?Ser?Val?Pro?Glu?Ala?Phe?Leu?Leu?Lys?Ser?Arg?Ser?Arg

100?????????????????105?????????????????110Glu?Lys?Trp?Asp?Arg?Glu?Ser?Asn?Trp?Ile?Gly?Tyr?Val?Val?Val?Ser

115?????????????????120?????????????????125Asn?Asp?Glu?Thr?Ser?Arg?Val?Ala?Gly?Arg?Arg?Glu?Val?Tyr?Val?Val

130?????????????????135?????????????????140Trp?Arg?Gly?Thr?Cys?Arg?Asp?Tyr?Glu?Trp?Val?Asp?Val?Leu?Gly?Ala145?????????????????150?????????????????155?????????????????160Gln?Leu?Glu?Ser?Ala?His?Pro?Leu?Leu?Arg?Thr?Gln?Gln?Thr?Thr?His

165?????????????????170?????????????????175Val?Glu?Lys?Val?Glu?Asn?Glu?Glu?Lys?Lys?Ser?Ile?His?Lys?Ser?Ser

180?????????????????185?????????????????190Trp?Tyr?Asp?Cys?Phe?Asn?Ile?Asn?Leu?Leu?Gly?Ser?Ala?Ser?Lys?Asp

195?????????????????200?????????????????205Lys?Gly?Lys?Gly?Ser?Asp?Asp?Asp?Asp?Asp?Asp?Asp?Pro?Lys?Val?Met

210?????????????????215?????????????????220Gln?Gly?Trp?Met?Thr?Ile?Tyr?Thr?Ser?Glu?Asp?Pro?Lys?Ser?Pro?Phe225?????????????????230?????????????????235?????????????????240Thr?Lys?Leu?Ser?Ala?Arg?Thr?Gln?Leu?Gln?Thr?Lys?Leu?Lys?Gln?Leu

245?????????????????250?????????????????255Met?Thr?Lys?Tyr?Lys?Asp?Glu?Thr?Leu?Ser?Ile?Thr?Phe?Ala?Gly?His

260?????????????????265?????????????????270Ser?Leu?Gly?Ala?Thr?Leu?Ser?Val?Val?Ser?Ala?Phe?Asp?Ile?Val?Glu

275?????????????????280?????????????????285Asn?Leu?Thr?Thr?Glu?Ile?Pro?Val?Thr?Ala?Val?Val?Phe?Gly?Cys?Pro

290?????????????????295?????????????????300Lys?Val?Gly?Asn?Lys?Lys?Phe?Gln?Gln?Leu?Phe?Asp?Ser?Tyr?Pro?Asn305?????????????????310?????????????????315?????????????????320Leu?Asn?Val?Leu?His?Val?Arg?Asn?Val?Ile?Asp?Leu?Ile?Pro?Leu?Tyr

325?????????????????330?????????????????335Pro?Val?Lys?Leu?Met?Gly?Tyr?Val?Asn?Ile?Gly?Ile?Glu?Leu?Glu?Ile

340?????????????????345?????????????????350Asp?Ser?Arg?Lys?Ser?Thr?Phe?Leu?Lys?Asp?Ser?Lys?Asn?Pro?Ser?Asp

355?????????????????360?????????????????365Trp?His?Asn?Leu?Gln?Ala?Ile?Leu?His?Val?Val?Ser?Gly?Trp?His?Gly

370?????????????????375?????????????????380Val?Lys?Gly?Glu?Phe?Lys?Val?Val?Asn?Lys?Arg?Ser?Val?Ala?Leu?Val385?????????????????390?????????????????395?????????????????400Asn?Lys?Ser?Cys?Asp?Phe?Leu?Lys?Glu?Glu?Cys?Leu?Val?Pro?Pro?Ala

405?????????????????410?????????????????415Trp?Trp?Val?Val?Gln?Asn?Lys?Gly?Met?Val?Leu?Asn?Lys?Asp?Gly?Glu

420?????????????????425?????????????????430Trp?Val?Leu?Ala?Pro?Pro?Glu?Glu?Asp?Pro?Thr?Pro?Glu?Phe?Asp

435 440 445＜210〉3＜211〉10＜212〉PRT＜213〉 （ Lycopersicon esculentum ）＜400〉3Ile Thr Phe Thr Gly His Ser Leu Gly Ala 1 5 10＜210〉4＜211〉10＜212〉PRT＜213〉 （ Dianthus caryophyllus ）＜400〉4Ile Thr Phe Ala Gly His Ser Leu Gly Ala 1 5 10＜210〉5＜211〉19＜212〉DNA＜213〉 （ Dianthus caryophyllus ）＜400〉5acctactagg ttccgcgtc 19＜210〉6＜211〉923＜212〉DNA＜213〉 （ Lycopersicon esculentum ）＜220〉＜221〉CDS＜222〉 （ （ 6..513，845..921 ） ）＜400〉6ctcta gac tat gag tgg gtg gat gtt tta ggt gct cgt cct gat tca 47。

Asp?Tyr?Glu?Trp?Val?Asp?Val?Leu?Gly?Ala?Arg?Pro?Asp?Ser

1???????????????5??????????????????10gct?gac?tct?ctt?ctt?cat?cct?aaa?tct?ctc?caa?aaa?ggc?att?aac?aac???????95Ala?Asp?Ser?Leu?Leu?His?Pro?Lys?Ser?Leu?Gln?Lys?Gly?Ile?Asn?Asn?15??????????????????20??????????????????25??????????????????30aag?aac?gat?gag?gat?gag?gac?gag?gac?gag?gat?gag?atc?aaa?gta?atg???????143Lys?Asn?Asp?Glu?Asp?Glu?Asp?Glu?Asp?Glu?Asp?Glu?Ile?Lys?Val?Met

35??????????????????40??????????????????45gat?ggg?tgg?ctt?aag?atc?tac?gtc?tca?agt?aac?ccg?aag?tcg?tct?ttc???????191Asp?Gly?Trp?Leu?Lys?Ile?Tyr?Val?Ser?Ser?Asn?Pro?Lys?Ser?Ser?Phe

50??????????????????55??????????????????60acg?aga?cta?agt?gca?aga?gaa?caa?ctt?caa?gca?aag?att?gaa?aag?tta???????239Thr?Arg?Leu?Ser?Ala?Arg?Glu?Gln?Leu?Gln?Ala?Lys?Ile?Glu?Lys?Leu

65??????????????????70??????????????????75aga?aat?gag?tat?aaa?gat?gag?aat?ttg?agc?ata?act?ttt?aca?ggg?cat???????287Arg?Asn?Glu?Tyr?Lys?Asp?Glu?Asn?Leu?Ser?Ile?Thr?Phe?Thr?Gly?His

80??????????????????85??????????????????90agt?ctt?ggt?gct?agc?tta?gct?gtt?tta?gct?tca?ttt?gat?gtg?gtt?gaa???????335Ser?Leu?Gly?Ala?Ser?Leu?Ala?Val?Leu?Ala?Ser?Phe?Asp?Val?Val?Glu95??????????????????100?????????????????105?????????????????110aat?ggt?gtg?cca?gtt?gat?att?cca?gta?tct?gca?att?gta?ttt?ggt?agt???????383Asn?Gly?Val?Pro?Val?Asp?Ile?Pro?Val?Ser?Ala?Ile?Val?Phe?Gly?Ser

115?????????????????120?????????????????125cca?caa?gtt?ggg?aat?aag?gca?ttc?aat?gaa?aga?atc?aag?aaa?ttc?tca???????431Pro?Gln?Val?Gly?Asn?Lys?Ala?Phe?Asn?Glu?Arg?Ile?Lys?Lys?Phe?Ser

130?????????????????135?????????????????140aac?ttg?aat?atc?tta?cat?gtt?aag?aac?aag?att?gat?ctc?att?acc?ctt???????479Asn?Leu?Asn?Ile?Leu?His?Val?Lys?Asn?Lys?Ile?Asp?Leu?Ile?Thr?Leu

145?????????????????150?????????????????155tac?cca?agt?gct?ctg?ttt?ggg?tat?gtg?aat?tca?g?gtattgaagg??????????????523Tyr?Pro?Ser?Ala?Leu?Phe?Gly?Tyr?Val?Asn?Ser

160?????????????????165aaaagatcat?tacaattttg?agctagattt?ctcatatcgt?cacactcaac?taacagttat?????583tatatgagaa?agtcactttc?tttgtgaaaa?aattgaatca?acttttggaa?ataatagtag?????643ttgagtgacc?atatgagaaa?tcaacactct?actaacttta?tgctataaga?gaataggtta?????703aggtccatat?gtttatactg?tctgttcaat?tagaatcata?aaagtattac?tagttaaatt?????763tgactacaat?cttatgtaga?catgaataaa?ataaatccta?cataaataag?atttcctaca?????823actttaatga?ttcttcaaca?g??gt?ata?gag?cta?gtc?atc?gat?agc?aga?aag???????873

Gly?Ile?Glu?Leu?Val?Ile?Asp?Ser?Arg?Lys

170 175tct ccg agt tta aag gat tca aaa gac atg ggc gac tgg cac aac ctc 921Ser Pro Ser Leu Lys Asp Ser Lys Asp Met Gly Asp Trp His Asn Leu180 185 190 195ca 923＜210〉7＜211〉19＜212〉DNA＜213〉＜220〉＜223〉：＜400〉7ctctagacta tgagtgggt 19＜210〉8＜211〉18＜212〉DNA＜213〉＜220〉＜223〉：＜400〉8cgactggcac aacctcca 18＜210〉9＜211〉10＜212〉PRT＜213〉 ( Arabidopsis sp. )＜400〉9Ile Thr Thr Cys Gly His Ser Leu Gly Ala 1 5 10＜210〉10＜211〉10＜212〉PRT＜213〉Ipomoea nil＜400〉10Ile Thr Val Thr Gly His Ser Leu Gly Ser 1 5 10＜210〉11＜211〉447＜212〉PRT＜213〉 ( Dianthus caryophyllus )＜400〉11Met Ala Ala Glu Ala Gln Pro Leu Gly Leu Ser Lys Pro Gly Pro Thr 1 5 10 15Trp Pro Glu Leu Leu Gly Ser Asn Ala Trp Ala Gly Leu Leu Asn Pro

20??????????????????25??????????????????30Leu?Asn?Asp?Glu?Leu?Arg?Glu?Leu?Leu?Leu?Arg?Cys?Gly?Asp?Phe?Cys

35??????????????????40??????????????????45Gln?Val?Thr?Tyr?Asp?Thr?Phe?Ile?Asn?Asp?Gln?Asn?Ser?Ser?Tyr?Cys

50??????????????????55??????????????????60Gly?Ser?Ser?Arg?Tyr?Gly?Lys?Ala?Asp?Leu?Leu?His?Lys?Thr?Ala?Phe?65??????????????????70??????????????????75??????????????????80Pro?Gly?Gly?Ala?Asp?Arg?Phe?Asp?Val?Val?Ala?Tyr?Leu?Tyr?Ala?Thr

85??????????????????90??????????????????95Ala?Lys?Val?Ser?Val?Pro?Glu?Ala?Phe?Leu?Leu?Lys?Ser?Arg?Ser?Arg

100?????????????????105?????????????????110Glu?Lys?Trp?Asp?Arg?Glu?Ser?Asn?Trp?Ile?Gly?Tyr?Val?Val?Val?Ser

115?????????????????120?????????????????125Asn?Asp?Glu?Thr?Ser?Arg?Val?Ala?Gly?Arg?Arg?Glu?Val?Tyr?Val?Val

130?????????????????135?????????????????140Trp?Arg?Gly?Thr?Cys?Arg?Asp?Tyr?Glu?Trp?Val?Asp?Val?Leu?Gly?Ala145?????????????????150?????????????????155?????????????????160Gln?Leu?Glu?Ser?Ala?His?Pro?Leu?Leu?Arg?Thr?Gln?Gln?Thr?Thr?His

165?????????????????170?????????????????175Val?Glu?Lys?Val?Glu?Asn?Glu?Glu?Lys?Lys?Ser?Ile?His?Lys?Ser?Ser

180?????????????????185?????????????????190Trp?Tyr?Asp?Cys?Phe?Asn?Ile?Asn?Leu?Leu?Gly?Ser?Ala?Ser?Lys?Asp

195?????????????????200?????????????????205Lys?Gly?Lys?Gly?Ser?Asp?Asp?Asp?Asp?Asp?Asp?Asp?Pro?Lys?Val?Met

210?????????????????215?????????????????220Gln?Gly?Trp?Met?Thr?Ile?Tyr?Thr?Ser?Glu?Asp?Pro?Lys?Ser?Pro?Phe225?????????????????230?????????????????235?????????????????240Thr?Lys?Leu?Ser?Ala?Arg?Thr?Gln?Leu?Gln?Thr?Lys?Leu?Lys?Cys?Leu

245?????????????????250?????????????????255Met?Thr?Lys?Tyr?Lys?Asp?Glu?Thr?Leu?Ser?Ile?Thr?Phe?Ala?Gly?His

260?????????????????265?????????????????270Ser?Leu?Gly?Ala?Thr?Leu?Ser?Val?Val?Ser?Ala?Phe?Asp?Ile?Val?Glu

275?????????????????280?????????????????285Asn?Leu?Thr?Thr?Glu?Ile?Pro?Val?Thr?Ala?Val?Val?Phe?Gly?Cys?Pro

290?????????????????295?????????????????300Lys?Val?Gly?Asn?Lys?Lys?Phe?Gln?Gln?Leu?Phe?Asp?Ser?Tyr?Pro?Asn305?????????????????310?????????????????315?????????????????320Leu?Asn?Val?Leu?His?Val?Arg?Asn?Val?Ile?Asp?Leu?Ile?Pro?Leu?Tyr

325?????????????????330?????????????????335Pro?Val?Lys?Leu?Met?Gly?Tyr?Val?Asn?Ile?Gly?Ile?Glu?Leu?Glu?Ile

340?????????????????345?????????????????350Asp?Ser?Arg?Lys?Ser?Thr?Phe?Leu?Lys?Asp?Ser?Lys?Asn?Pro?Ser?Asp

355?????????????????360?????????????????365Trp?His?Asn?Leu?Gln?Ala?Ile?Leu?His?Val?Val?Ser?Gly?Trp?His?Gly

370?????????????????375?????????????????380Val?Lys?Gly?Glu?Phe?Lys?Val?Val?Asn?Lys?Arg?Ser?Val?Ala?Leu?Val385?????????????????390?????????????????395?????????????????400Asn?Lys?Ser?Cys?Asp?Phe?Leu?Lys?Glu?Glu?Cys?Leu?Val?Pro?Pro?Ala

405?????????????????410?????????????????415Trp?Trp?Val?Val?Gln?Asn?Lys?Gly?Met?Val?Leu?Asn?Lys?Asp?Gly?Glu

420?????????????????425?????????????????430Trp?Val?Leu?Ala?Pro?Pro?Glu?Glu?Asp?Pro?Thr?Pro?Glu?Phe?Asp

435 440 445＜210〉12＜211〉418＜212〉PRT＜213〉arabidopsis (Arabidopsis thaliana)＜400〉12Met Lys Arg Lys Lys Lys Glu Glu Glu Glu Glu Lys Leu Ile Val Thr, 15 10 15Arg Glu Phe Ala Lys Arg Trp Arg Asp Leu Ser Gly Gln Asn His Trp

20??????????????????25??????????????????30Lys?Gly?Met?Leu?Gln?Pro?Leu?Asp?Gln?Asp?Leu?Arg?Glu?Tyr?Ile?Ile

35??????????????????40??????????????????45His?Tyr?Gly?Glu?Met?Ala?Gln?Ala?Gly?Tyr?Asp?Thr?Phe?Asn?Ile?Asn

50??????????????????55??????????????????60Thr?Glu?Ser?Gln?Phe?Ala?Gly?Ala?Ser?Ile?Tyr?Ser?Arg?Lys?Asp?Phe?65??????????????????70??????????????????75??????????????????80Phe?Ala?Lys?Val?Gly?Leu?Glu?Ile?Ala?His?Pro?Tyr?Thr?Lys?Tyr?Lys

85??????????????????90??????????????????95Val?Thr?Lys?Phe?Ile?Tyr?Ala?Thr?Ser?Asp?Ile?His?Val?Pro?Glu?Ser

100?????????????????105?????????????????110Phe?Leu?Leu?Phe?Pro?Ile?Ser?Arg?Glu?Gly?Trp?Ser?Lys?Glu?Ser?Asn

115?????????????????120?????????????????125Trp?Met?Gly?Tyr?Val?Ala?Val?Thr?Asp?Asp?Gln?Gly?Thr?Ala?Leu?Leu

130?????????????????135?????????????????140Gly?Arg?Arg?Asp?Ile?Val?Val?Ser?Trp?Arg?Gly?Ser?Val?Gln?Pro?Leu145?????????????????150?????????????????155?????????????????160Glu?Trp?Val?Glu?Asp?Phe?Glu?Phe?Gly?Leu?Val?Asn?Ala?Ile?Lys?Ile

165?????????????????170?????????????????175Phe?Gly?Glu?Arg?Asn?Asp?Gln?Val?Gln?Ile?His?Gln?Gly?Trp?Tyr?Ser

180?????????????????185?????????????????190Ile?Tyr?Met?Ser?Gln?Asp?Glu?Arg?Ser?Pro?Phe?Thr?Lys?Thr?Asn?Ala

195?????????????????200?????????????????205Arg?Asp?Gln?Val?Leu?Arg?Glu?Val?Gly?Arg?Leu?Leu?Glu?Lys?Tyr?Lys

210?????????????????215?????????????????220Asp?Glu?Glu?Val?Ser?Ile?Thr?Ile?Cys?Gly?His?Ser?Leu?Gly?Ala?Ala225?????????????????230?????????????????235?????????????????240Leu?Ala?Thr?Asp?Ser?Ala?Ile?Asp?Ile?Val?Ala?Asn?Gly?Tyr?Asn?Arg

245?????????????????250?????????????????255Pro?Lys?Ser?Arg?Pro?Asp?Lys?Ser?Cys?Pro?Val?Thr?Ala?Phe?Val?Phe

260?????????????????265?????????????????270Ala?Ser?Pro?Arg?Val?Gly?Asp?Ser?Asp?Phe?Arg?Lys?Leu?Phe?Ser?Gly

275?????????????????280?????????????????285Leu?Glu?Asp?Ile?Arg?Val?Leu?Arg?Thr?Arg?Asn?Leu?Phe?Asp?Val?Ile

290?????????????????295?????????????????300Pro?Ile?Tyr?Pro?Pro?Ile?Gly?Tyr?Ser?Glu?Val?Gly?Asp?Glu?Phe?Pro305?????????????????310?????????????????315?????????????????320Ile?Asp?Thr?Arg?Lys?Ser?Pro?Tyr?Met?Lys?Ser?Pro?Gly?Asn?Leu?Ala

325?????????????????330?????????????????335Thr?Phe?His?Cys?Leu?Glu?Gly?Tyr?Leu?His?Gly?Val?Ala?Gly?Thr?Gln

340?????????????????345?????????????????350Gly?Thr?Asn?Lys?Ala?Asp?Leu?Phe?Arg?Leu?Asp?Val?Glu?Arg?Ala?Ile

355?????????????????360?????????????????365Gly?Leu?Val?Asn?Lys?Ser?Val?Asp?Gly?Leu?Lys?Asp?Glu?Cys?Met?Val

370?????????????????375?????????????????380Pro?Gly?Lys?Trp?Arg?Val?Leu?Lys?Asn?Lys?Gly?Ala?Gln?Gln?Asp?Asp385?????????????????390?????????????????395?????????????????400Gly?Ser?Trp?Glu?Leu?Val?Asp?His?Glu?Ile?Asp?Asp?Asn?Glu?Asp?Leu

405 410 415Asp Phe＜210〉13＜211〉401＜212〉PRT＜213〉Ipomoea kinds (Ipomoea sp.)＜400〉13Met Ser Gly Ile Ala Lys Arg Trp Lys Val Leu Ser Gly Ser Asp Asn, 15 10 15Trp Glu Gly Leu Leu Glu Pro Leu Asp Ser Asp Leu Arg Arg Tyr Leu

20??????????????????25??????????????????30Ile?His?Tyr?Gly?Thr?Met?Val?Ser?Pro?Ala?Thr?Asp?Ser?Phe?Ile?Asn

35??????????????????40??????????????????45Glu?Ala?Ala?Ser?Lys?Asn?Val?Gly?Leu?Pro?Arg?Tyr?Ala?Arg?Arg?Asn

50??????????????????55??????????????????60Leu?Leu?Ala?Asn?Cys?Gly?Leu?Val?Lys?Gly?Asn?Pro?Phe?Lys?Tyr?Glu?65??????????????????70??????????????????75??????????????????80Val?Thr?Lys?Tyr?Phe?Tyr?Ala?Pro?Ser?Thr?Ile?Pro?Leu?Pro?Asp?Glu

85??????????????????90??????????????????95Gly?Tyr?Asn?Val?Arg?Ala?Thr?Arg?Ala?Asp?Ala?Val?Leu?Lys?Glu?Ser

100?????????????????105?????????????????110Asn?Trp?Asn?Gly?Tyr?Val?Ala?Val?Ala?Thr?Asp?Glu?Gly?Lys?Val?Ala

115?????????????????120?????????????????125Leu?Gly?Arg?Arg?Asp?Ile?Leu?Ile?Val?Trp?Arg?Gly?Thr?Ile?Arg?Lys

130?????????????????135?????????????????140Ser?Glu?Trp?Asn?Glu?Asn?Leu?Thr?Phe?Trp?Phe?Val?Lys?Ala?Pro?Leu145?????????????????150?????????????????155?????????????????160Phe?Phe?Gly?Gln?Asn?Ser?Asp?Pro?Leu?Val?His?Lys?Gly?Trp?Tyr?Asp

165?????????????????170?????????????????175Met?Tyr?Thr?Thr?Ile?Asn?Gln?Asp?Ser?Gln?Leu?Asn?Glu?Lys?Ser?Ala

180?????????????????185?????????????????190Arg?Asp?Gln?Ile?Arg?Glu?Glu?Val?Ala?Arg?Leu?Val?Glu?Leu?Tyr?Lys

195?????????????????200?????????????????205Asp?Glu?Asp?Ile?Ser?Ile?Thr?Val?Thr?Gly?His?Ser?Leu?Gly?Ser?Ser

210?????????????????215?????????????????220Met?Ala?Thr?Leu?Asn?Ala?Val?Asp?Leu?Ala?Ala?Asn?Pro?Ile?Asn?Asn225?????????????????230?????????????????235?????????????????240Asn?Lys?Asn?Ile?Leu?Val?Thr?Ala?Phe?Leu?Tyr?Ala?Ser?Pro?Lys?Val

245?????????????????250?????????????????255Gly?Asp?Glu?Asn?Phe?Lys?Asn?Val?Ile?Ser?Asn?Gln?Gln?Asn?Leu?Arg

260?????????????????265?????????????????270Ala?Leu?Arg?Ile?Ser?Asp?Val?Asn?Asp?Ile?Val?Thr?Ala?Val?Pro?Pro

275?????????????????280?????????????????285Phe?Gly?Trp?Lys?Glu?Cys?Asp?Asn?Thr?Ala?Ile?Leu?Tyr?Gly?Asp?Val

290?????????????????295?????????????????300Gly?Val?Gly?Leu?Val?Ile?Asp?Ser?Lys?Lys?Ser?His?Tyr?Leu?Lys?Pro305?????????????????310?????????????????315?????????????????320Asp?Phe?Pro?Asn?Leu?Ser?Thr?His?Asp?Leu?Met?Leu?Tyr?Met?His?Ala

325?????????????????330?????????????????335Ile?Asp?Gly?Tyr?Gln?Gly?Ser?Gln?Gly?Gly?Phe?Glu?Arg?Gln?Glu?Asp

340?????????????????345?????????????????350Phe?Asp?Leu?Ala?Lys?Val?Asn?Lys?Tyr?Gly?Asp?Tyr?Leu?Lys?Ala?Glu

355?????????????????360?????????????????365Tyr?Pro?Ile?Pro?Ile?Gly?Trp?Phe?Asn?Ile?Lys?Asp?Lys?Gly?Met?Gln

370 375 380Gln Asp Asp Gly Asn Tyr Ile Leu Asp Asp His Glu Val Asp Lys Thr385,390 395 400Phe＜210〉14＜211〉448＜212〉PRT＜213〉arabidopsis (Arabidopsis thaliana)＜400〉14Met Thr Ala Glu Asp Ile Arg Arg Arg Asp Lys Lys Thr Glu Glu Glu, 15 10 15Arg Arg Leu Arg Asp Thr Trp Arg Lys Ile Gln Gly Glu Asp Asp Trp

20??????????????????25??????????????????30Ala?Gly?Leu?Met?Asp?Pro?Met?Asp?Pro?Ile?Leu?Arg?Ser?Glu?Leu?Ile

35??????????????????40??????????????????45Arg?Tyr?Gly?Glu?Met?Ala?Gln?Ala?Cys?Tyr?Asp?Ala?Phe?Asp?Phe?Asp

50??????????????????55??????????????????60Pro?Ala?Ser?Lys?Tyr?Cys?Gly?Thr?Ser?Arg?Phe?Thr?Arg?Leu?Glu?Phe?65??????????????????70??????????????????75??????????????????80Phe?Asp?Ser?Leu?Gly?Met?Ile?Asp?Ser?Gly?Tyr?Glu?Val?Ala?Arg?Tyr

85??????????????????90??????????????????95Leu?Tyr?Ala?Thr?Ser?Asn?Ile?Asn?Leu?Pro?Asn?Phe?Phe?Ser?Lys?Ser

100?????????????????105?????????????????110Arg?Trp?Ser?Lys?Val?Trp?Ser?Lys?Asn?Ala?Asn?Trp?Met?Gly?Tyr?Val

115?????????????????120?????????????????125Ala?Val?Ser?Asp?Asp?Glu?Thr?Ser?Arg?Asn?Arg?Leu?Gly?Arg?Arg?Asp

130?????????????????135?????????????????140Ile?Ala?Ile?Ala?Trp?Arg?Gly?Thr?Val?Thr?Lys?Leu?Glu?Trp?Ile?Ala145?????????????????150?????????????????155?????????????????160Asp?Leu?Lys?Asp?Tyr?Leu?Lys?Pro?Val?Thr?Glu?Asn?Lys?Ile?Arg?Cys

165?????????????????170?????????????????175Pro?Asp?Pro?Ala?Val?Lys?Val?Glu?Ser?Gly?Phe?Leu?Asp?Leu?Tyr?Thr

180?????????????????185?????????????????190Asp?Lys?Asp?Thr?Thr?Cys?Lys?Phe?Ala?Arg?Phe?Ser?Ala?Arg?Glu?Gln

195?????????????????200?????????????????205Ile?Leu?Thr?Glu?Val?Lys?Arg?Leu?Val?Glu?Glu?His?Gly?Asp?Asp?Asp

210?????????????????215?????????????????220Asp?Ser?Asp?Leu?Ser?Ile?Thr?Val?Thr?Gly?His?Ser?Leu?Gly?Gly?Ala225?????????????????230?????????????????235?????????????????240Leu?Ala?Ile?Leu?Ser?Ala?Tyr?Asp?Ile?Ala?Glu?Met?Arg?Leu?Asn?Arg

245?????????????????250?????????????????255Ser?Lys?Lys?Gly?Lys?Val?Ile?Pro?Val?Thr?Ala?Val?Leu?Thr?Tyr?Gly

260?????????????????265?????????????????270Gly?Pro?Arg?Val?Gly?Asn?Val?Arg?Phe?Arg?Glu?Arg?Met?Glu?Glu?Leu

275?????????????????280?????????????????285Gly?Val?Lys?Val?Met?Arg?Val?Val?Asn?Val?His?Asp?Val?Val?Pro?Lys

290?????????????????295?????????????????300Ser?Pro?Gly?Leu?Phe?Leu?Asn?Glu?Ser?Arg?Pro?His?Ala?Leu?Met?Lys305?????????????????310?????????????????315?????????????????320Ile?Ala?Glu?Gly?Leu?Pro?Trp?Cys?Tyr?Ser?His?Val?Gly?Glu?Glu?Leu

325?????????????????330?????????????????335Ala?Leu?Asp?His?Gln?Asn?Ser?Pro?Phe?Leu?Lys?Pro?Ser?Val?Asp?Val

340?????????????????345?????????????????350Ser?Thr?Ala?His?Asn?Leu?Glu?Ala?Met?Leu?His?Leu?Leu?Asp?Gly?Tyr

355?????????????????360?????????????????365His?Gly?Lys?Gly?Glu?Arg?Phe?Val?Leu?Ser?Ser?Gly?Arg?Asp?His?Ala

370?????????????????375?????????????????380Leu?Val?Asn?Lys?Ala?Ser?Asp?Phe?Leu?Lys?Glu?His?Leu?Gln?Ile?Pro385?????????????????390?????????????????395?????????????????400Pro?Phe?Trp?Arg?Gln?Asp?Ala?Asn?Lys?Gly?Met?Val?Arg?Asn?Ser?Glu

405?????????????????410?????????????????415Gly?Arg?Trp?Ile?Gln?Ala?Glu?Arg?Leu?Arg?Phe?Glu?Asp?His?His?Ser

420?????????????????425?????????????????430Pro?Asp?Ile?His?His?His?Leu?Ser?Gln?Leu?Arg?Leu?Asp?His?Pro?Cys

435 440 445＜210〉15＜211〉1167＜212〉DNA＜213〉Arabidopsis kinds (Arabidopsis sp.)＜220〉＜221〉CDS＜222〉(1) .. (1044)＜400〉15cgg gtc gac cca cgc gtc cgc gaa aac gct tcc gac tac gag gtt gta 48Arg Val Asp Pro Arg Val Arg Glu Asn Ala Ser Asp Tyr Glu Val Val 15 10 15aac ttc ctc tac gcc aca gct cgt gtt tct ctc ccc gaa ggt ttg ctt 96Asn Phe Leu Tyr Ala Thr Ala Arg Val Ser Leu Pro Glu Gly Leu Leu

20??????????????????25??????????????????30ctc?caa?tca?caa?tca?aga?gat?tct?tgg?gac?cgt?gag?tct?aac?tgg?ttt???144Leu?Gln?Ser?Gln?Ser?Arg?Asp?Ser?Trp?Asp?Arg?Glu?Ser?Asn?Trp?Phe

35??????????????????40??????????????????45ggc?tac?att?gct?gtc?acg?tct?gat?gaa?cgg?tct?aag?gct?tta?gga?cgc???192Gly?Tyr?Ile?Ala?Val?Thr?Ser?Asp?Glu?Arg?Ser?Lys?Ala?Leu?Gly?Arg

50??????????????????55??????????????????60cgt?gag?atc?tat?ata?gct?ttg?aga?gga?acg?agc?agg?aac?tat?gag?tgg???240Arg?Glu?Ile?Tyr?Ile?Ala?Leu?Arg?Gly?Thr?Ser?Arg?Asn?Tyr?Glu?Trp?65??????????????????70??????????????????75??????????????????80gtc?aat?gtt?ttg?ggt?gct?agg?cca?act?tca?gct?gac?ccc?ttg?ctg?cac???288Val?Asn?Val?Leu?Gly?Ala?Arg?Pro?Thr?Ser?Ala?Asp?Pro?Leu?Leu?His

85??????????????????90??????????????????95gga?ccc?gag?cag?gat?ggt?tct?ggt?ggt?gta?gtt?gaa?ggt?acg?act?ttt???336Gly?Pro?Glu?Gln?Asp?Gly?Ser?Gly?Gly?Val?Val?Glu?Gly?Thr?Thr?Phe

100?????????????????105?????????????????110gat?agt?gac?agt?gaa?gat?gaa?gaa?ggg?tgt?aag?gtg?atg?ctc?ggg?tgg???384Asp?Ser?Asp?Ser?Glu?Asp?Glu?Glu?Gly?Cys?Lys?Val?Met?Leu?Gly?Trp

115?????????????????120?????????????????125ctc?aca?atc?tat?act?tct?aat?cac?ccc?gaa?tcg?aaa?ttc?act?aag?ctg???432Leu?Thr?Ile?Tyr?Thr?Ser?Asn?His?Pro?Glu?Ser?Lys?Phe?Thr?Lys?Leu

130?????????????????135?????????????????140agt?cta?cgg?tca?cag?ttg?tta?gcc?aag?atc?aag?gag?ctt?ctg?ttg?aag???480Ser?Leu?Arg?Ser?Gln?Leu?Leu?Ala?Lys?Ile?Lys?Glu?Leu?Leu?Leu?Lys145?????????????????150?????????????????155?????????????????160tat?aag?gac?gag?aaa?ccg?agc?att?gtg?ttg?act?gga?cat?agc?ttg?gga???528Tyr?Lys?Asp?Glu?Lys?Pro?Ser?Ile?Val?Leu?Thr?Gly?His?Ser?Leu?Gly

165?????????????????170?????????????????175cct?aca?gag?gct?gtt?ctg?gcc?gcc?tat?gat?ata?gct?gag?aac?ggt?tcc???576Pro?Thr?Glu?Ala?Val?Leu?Ala?Ala?Tyr?Asp?Ile?Ala?Glu?Asn?Gly?Ser

180?????????????????185?????????????????190agt?gat?gat?gtt?ccg?gtc?act?gct?ata?gtc?ttt?ggt?tgt?cca?cag?gta???624Ser?Asp?Asp?Val?Pro?Val?Thr?Ala?Ile?Val?Phe?Gly?Cys?Pro?Gln?Val

195?????????????????200?????????????????205gga?aac?aag?gag?ttc?aga?gac?gaa?gta?atg?agt?cac?aag?aac?tta?aag???672Gly?Asn?Lys?Glu?Phe?Arg?Asp?Glu?Val?Met?Ser?His?Lys?Asn?Leu?Lys

210?????????????????215?????????????????220atc?ctc?cat?gta?agg?aac?acg?att?gat?ctc?tta?act?cga?tac?cca?ggg???720Ile?Leu?His?Val?Arg?Asn?Thr?Ile?Asp?Leu?Leu?Thr?Arg?Tyr?Pro?Gly225?????????????????230?????????????????235?????????????????240gga?ctt?tta?ggg?tat?gtg?gac?ata?gga?ata?aac?ttt?gtg?atc?gat?aca???768Gly?Leu?Leu?Gly?Tyr?Val?Asp?Ile?Gly?Ile?Asn?Phe?Val?Ile?Asp?Thr

245?????????????????250?????????????????255aag?aag?tca?ccg?ttc?cta?agc?gat?tca?agg?aat?cca?ggg?gat?tgg?cat???816Lys?Lys?Ser?Pro?Phe?Leu?Ser?Asp?Ser?Arg?Asn?Pro?Gly?Asp?Trp?His

260?????????????????265?????????????????270aat?ctt?cag?gcg?atg?tta?cat?gtt?gta?gct?gga?tgg?aat?ggg?aag?aaa???864Asn?Leu?Gln?Ala?Met?Leu?His?Val?Val?Ala?Gly?Trp?Asn?Gly?Lys?Lys

275?????????????????280?????????????????285gga?gag?ttt?aaa?ctg?atg?gtt?aag?aga?agt?att?gca?tta?gtg?aac?aag???912Gly?Glu?Phe?Lys?Leu?Met?Val?Lys?Arg?Ser?Ile?Ala?Leu?Val?Asn?Lys

290?????????????????295?????????????????300tca?tgc?gag?ttc?ttg?aaa?gct?gag?tgt?ttg?gtg?cca?gga?tct?tgg?tgg???960Ser?Cys?Glu?Phe?Leu?Lys?Ala?Glu?Cys?Leu?Val?Pro?Gly?Ser?Trp?Trp305?????????????????310?????????????????315??????????????????320gta?gag?aag?aac?aaa?gga?ctg?atc?aag?aac?gaa?gat?ggt?gaa?tgg?gtt???1008Val?Glu?Lys?Asn?Lys?Gly?Leu?Ile?Lys?Asn?Glu?Asp?Gly?Glu?Trp?Val

325?????????????????330?????????????????335ctt?gct?ccc?gtt?gaa?gaa?gaa?cct?gta?cct?gaa?ttc?taaattgtat????????1054Leu?Ala?Pro?Val?Glu?Glu?Glu?Pro?Val?Pro?Glu?Phe

340 345ttctgtattt ttctctaagg tcatgataaa tcaacaataa gcagttcaac tatgtgatga 1114aaagacccaa gttattatat tgatatgagt ttatgagata aaaaaaaaaa aaa 1167＜210〉16＜211〉348＜212〉PRT＜213〉Arabidopsis kinds (Arabidopsis sp.)＜400〉16Arg Val Asp Pro Arg Val Arg Glu Asn Ala Ser Asp Tyr Glu Val Val, 15 10 15Asn Phe Leu Tyr Ala Thr Ala Arg Val Ser Leu Pro Glu Gly Leu Leu

20??????????????????25??????????????????30Leu?Gln?Ser?Gln?Ser?Arg?Asp?Ser?Trp?Asp?Arg?Glu?Ser?Asn?Trp?Phe

35??????????????????40??????????????????45Gly?Tyr?Ile?Ala?Val?Thr?Ser?Asp?Glu?Arg?Ser?Lys?Ala?Leu?Gly?Arg

50??????????????????55??????????????????60Arg?Glu?Ile?Tyr?Ile?Ala?Leu?Arg?Gly?Thr?Ser?Arg?Asn?Tyr?Glu?Trp?65??????????????????70??????????????????75??????????????????80Val?Asn?Val?Leu?Gly?Ala?Arg?Pro?Thr?Ser?Ala?Asp?Pro?Leu?Leu?His

85??????????????????90??????????????????95Gly?Pro?Glu?Gln?Asp?Gly?Ser?Gly?Gly?Val?Val?Glu?Gly?Thr?Thr?Phe

100?????????????????105?????????????????110Asp?Ser?Asp?Ser?Glu?Asp?Glu?Glu?Gly?Cys?Lys?Val?Met?Leu?Gly?Trp

115?????????????????120?????????????????125Leu?Thr?Ile?Tyr?Thr?Ser?Asn?His?Pro?Glu?Ser?Lys?Phe?Thr?Lys?Leu

130?????????????????135?????????????????140Ser?Leu?Arg?Ser?Gln?Leu?Leu?Ala?Lys?Ile?Lys?Glu?Leu?Leu?Leu?Lys145?????????????????150?????????????????155?????????????????160Tyr?Lys?Asp?Glu?Lys?Pro?Ser?Ile?Val?Leu?Thr?Gly?His?Ser?Leu?Gly

165?????????????????170?????????????????175Pro?Thr?Glu?Ala?Val?Leu?Ala?Ala?Tyr?Asp?Ile?Ala?Glu?Asn?Gly?Ser

180?????????????????185?????????????????190Ser?Asp?Asp?Val?Pro?Val?Thr?Ala?Ile?Val?Phe?Gly?Cys?Pro?Gln?Val

195?????????????????200?????????????????205Gly?Asn?Lys?Glu?Phe?Arg?Asp?Glu?Val?Met?Ser?His?Lys?Asn?Leu?Lys

210?????????????????215?????????????????220Ile?Leu?His?Val?Arg?Asn?Thr?Ile?Asp?Leu?Leu?Thr?Arg?Tyr?Pro?Gly225?????????????????230?????????????????235?????????????????240Gly?Leu?Leu?Gly?Tyr?Val?Asp?Ile?Gly?Ile?Asn?Phe?Val?Ile?Asp?Thr

245?????????????????250?????????????????255Lys?Lys?Ser?Pro?Phe?Leu?Ser?Asp?Ser?Arg?Asn?Pro?Gly?Asp?Trp?His

260?????????????????265?????????????????270Asn?Leu?Gln?Ala?Met?Leu?His?Val?Val?Ala?Gly?Trp?Asn?Gly?Lys?Lys

275?????????????????280?????????????????285Gly?Glu?Phe?Lys?Leu?Met?Val?Lys?Arg?Ser?Ile?Ala?Leu?Val?Asn?Lys

290?????????????????295?????????????????300Ser?Cys?Glu?Phe?Leu?Lys?Ala?Glu?Cys?Leu?Val?Pro?Gly?Ser?Trp?Trp305?????????????????310?????????????????315?????????????????320Val?Glu?Lys?Asn?Lys?Gly?Leu?Ile?Lys?Asn?Glu?Asp?Gly?Glu?Trp?Val

325?????????????????330?????????????????335Leu?Ala?Pro?Val?Glu?Glu?Glu?Pro?Val?Pro?Glu?Phe

340 345＜210〉17＜211〉195＜212〉PRT＜213〉tomato (Lycopersicon esculentum)＜400〉17Asp Tyr Glu Trp Val Asp Val Leu Gly Ala Arg Pro Asp Ser Ala Asp, 15 10 15Ser Leu Leu His Pro Lys Ser Leu Gln Lys Gly Ile Asn Asn Lys Asn

20??????????????????25??????????????????30Asp?Glu?Asp?Glu?Asp?Glu?Asp?Glu?Asp?Glu?Ile?Lys?Val?Met?Asp?Gly

35??????????????????40??????????????????45Trp?Leu?Lys?Ile?Tyr?Val?Ser?Ser?Asn?Pro?Lys?Ser?Ser?Phe?Thr?Arg

50??????????????????55??????????????????60Leu?Ser?Ala?Arg?Glu?Gln?Leu?Gln?Ala?Lys?Ile?Glu?Lys?Leu?Arg?Asn?65??????????????????70??????????????????75??????????????????80Glu?Tyr?Lys?Asp?Glu?Asn?Leu?Ser?Ile?Thr?Phe?Thr?Gly?His?Ser?Leu

85??????????????????90??????????????????95Gly?Ala?Ser?Leu?Ala?Val?Leu?Ala?Ser?Phe?Asp?Val?Val?Glu?Asn?Gly

100?????????????????105?????????????????110Val?Pro?Val?Asp?Ile?Pro?Val?Ser?Ala?Ile?Val?Phe?Gly?Ser?Pro?Gln

115?????????????????120?????????????????125Val?Gly?Asn?Lys?Ala?Phe?Asn?Glu?Arg?Ile?Lys?Lys?Phe?Ser?Asn?Leu

130?????????????????135?????????????????140Asn?Ile?Leu?His?Val?Lys?Asn?Lys?Ile?Asp?Leu?Ile?Thr?Leu?Tyr?Pro145?????????????????150?????????????????155?????????????????160Ser?Ala?Leu?Phe?Gly?Tyr?Val?Asn?Ser?Gly?Ile?Glu?Leu?Val?Ile?Asp

165?????????????????170?????????????????175Ser?Arg?Lys?Ser?Pro?Ser?Leu?Lys?Asp?Ser?Lys?Asp?Met?Gly?Asp?Trp

180?????????????????185?????????????????190His?Asn?Leu

195＜210〉18＜211〉1344＜212〉DNA＜213〉arabidopsis (Arabidopsis thaliana)＜220〉＜221〉CDS＜222〉(1) .. (1341)＜400〉18atg acg gcg gaa gat att cgc cgg cga gat aaa aaa acc gaa gaa gaa 48Met Thr Ala Glu Asp Ile Arg Arg Arg Asp Lys Lys Thr Glu Glu Glu 15 10 15aga aga cta aga gac acg tgg cgt aag atc caa gga gaa gac gat tgg 96Arg Arg Leu Arg Asp Thr Trp Arg Lys Ile Gln Gly Glu Asp Asp Trp

20??????????????????25??????????????????30gcc?ggg?tta?atg?gat?cca?atg?gat?cca?att?ctt?aga?tcg?gag?cta?atc???144Ala?Gly?Leu?Met?Asp?Pro?Met?Asp?Pro?Ile?Leu?Arg?Ser?Glu?Leu?Ile

35??????????????????40??????????????????45cgt?tac?ggc?gaa?atg?gct?caa?gct?tgt?tac?gac?gct?ttc?gat?ttc?gat???192Arg?Tyr?Gly?Glu?Met?Ala?Gln?Ala?Cys?Tyr?Asp?Ala?Phe?Asp?Phe?Asp

50??????????????????55??????????????????60ccc?gct?tcc?aaa?tac?tgc?ggc?acc?tcc?agg?ttc?acg?cga?ctc?gag?ttc???240Pro?Ala?Ser?Lys?Tyr?Cys?Gly?Thr?Ser?Arg?Phe?Thr?Arg?Leu?Glu?Phe?65??????????????????70??????????????????75??????????????????80ttc?gat?tct?ctc?gga?atg?atc?gat?tcc?ggt?tac?gag?gtg?gcg?cgt?tac???288Phe?Asp?Ser?Leu?Gly?Met?Ile?Asp?Ser?Gly?Tyr?Glu?Val?Ala?Arg?Tyr

85??????????????????90??????????????????95ctc?tac?gcg?acg?tcg?aac?atc?aat?ctc?ccg?aac?ttc?ttc?tcg?aaa?tcg???336Leu?Tyr?Ala?Thr?Ser?Asn?Ile?Asn?Leu?Pro?Asn?Phe?Phe?Ser?Lys?Ser

100?????????????????105?????????????????110cgg?tgg?tct?aaa?gtc?tgg?agc?aaa?aac?gct?aat?tgg?atg?gga?tac?gtc???384Arg?Trp?Ser?Lys?Val?Trp?Ser?Lys?Asn?Ala?Asn?Trp?Met?Gly?Tyr?Val

115?????????????????120?????????????????125gcc?gtt?tca?gac?gac?gaa?acg?tct?cgt?aac?cga?ctc?ggc?cgc?cgt?gat???432Ala?Val?Ser?Asp?Asp?Glu?Thr?Ser?Arg?Asn?Arg?Leu?Gly?Arg?Arg?Asp

130?????????????????135?????????????????140atc?gcg?att?gcg?tgg?aga?gga?acc?gtt?acg?aaa?ctt?gaa?tgg?atc?gcg???480Ile?Ala?Ile?Ala?Trp?Arg?Gly?Thr?Val?Thr?Lys?Leu?Glu?Trp?Ile?Ala145?????????????????150?????????????????155?????????????????160gat?cta?aag?gat?tat?tta?aaa?ccg?gta?acc?gaa?aac?aag?atc?cga?tgc???528Asp?Leu?Lys?Asp?Tyr?Leu?Lys?Pro?Val?Thr?Glu?Asn?Lys?Ile?Arg?Cys

165?????????????????170?????????????????175ccc?gac?ccg?gcc?gtt?aaa?gtc?gaa?tcc?gga?ttc?tta?gat?ctc?tac?act???576Pro?Asp?Pro?Ala?Val?Lys?Val?Glu?Ser?Gly?Phe?Leu?Asp?Leu?Tyr?Thr

180?????????????????185?????????????????190gac?aaa?gac?aca?acc?tgc?aaa?ttc?gcg?aga?ttc?tca?gcg?cgt?gaa?cag???624Asp?Lys?Asp?Thr?Thr?Cys?Lys?Phe?Ala?Arg?Phe?Ser?Ala?Arg?Glu?Gln

195?????????????????200?????????????????205att?tta?acg?gag?gtg?aaa?cgg?tta?gtg?gaa?gaa?cac?ggc?gac?gac?gat???672Ile?Leu?Thr?Glu?Val?Lys?Arg?Leu?Val?Glu?Glu?His?Gly?Asp?Asp?Asp

210?????????????????215?????????????????220gat?tcc?gat?tta?agc?atc?acc?gtg?acg?gga?cac?agt?ctc?ggc?ggc?gcg???720Asp?Ser?Asp?Leu?Ser?Ile?Thr?Val?Thr?Gly?His?Ser?Leu?Gly?Gly?Ala225?????????????????230?????????????????235?????????????????240tta?gcg?ata?tta?agc?gcg?tac?gat?ata?gcg?gag?atg?aga?ttg?aat?cgg???768Leu?Ala?Ile?Leu?Ser?Ala?Tyr?Asp?Ile?Ala?Glu?Met?Arg?Leu?Asn?Arg

245?????????????????250?????????????????255agt?aag?aaa?ggg?aaa?gtg?att?ccg?gtg?acg?gtg?ttg?aca?tac?gga?gga???816Ser?Lys?Lys?Gly?Lys?Val?Ile?Pro?Val?Thr?Val?Leu?Thr?Tyr?Gly?Gly

260?????????????????265?????????????????270ccg?aga?gtt?ggg?aac?gtt?agg?ttt?agg?gag?agg?atg?gag?gaa?ttg?gga???864Pro?Arg?Val?Gly?Asn?Val?Arg?Phe?Arg?Glu?Arg?Met?Glu?Glu?Leu?Gly

275?????????????????280?????????????????285gtg?aaa?gtg?atg?aga?gta?gtg?aat?gtt?cac?gac?gtg?gtt?ccc?aag?tcg???912Val?Lys?Val?Met?Arg?Val?Val?Asn?Val?His?Asp?Val?Val?Pro?Lys?Ser

290?????????????????295?????????????????300ccg?gga?ttg?ttt?ttg?aac?gag?agt?aga?cct?cac?gcg?ctg?atg?aag?ata???960Pro?Gly?Leu?Phe?Leu?Asn?Glu?Ser?Arg?Pro?His?Ala?Leu?Met?Lys?Ile305?????????????????310?????????????????315?????????????????320gcg?gag?ggg?ttg?ccg?tgg?tgt?tat?agc?cac?gtg?ggg?gag?gag?ctg?gcg???1008Ala?Glu?Gly?Leu?Pro?Trp?Cys?Tyr?Ser?His?Val?Gly?Glu?Glu?Leu?Ala

325?????????????????330?????????????????335ttg?gat?cat?cag?aac?tcg?ccg?ttt?ctt?aaa?cct?tcc?gtt?gat?gtt?tct???1056Leu?Asp?His?Gln?Asn?Ser?Pro?Phe?Leu?Lys?Pro?Ser?Val?Asp?Val?Ser

340?????????????????345?????????????????350act?gct?cat?aat?ctt?gaa?gct?atg?ctt?cat?tta?ctt?gac?ggg?tat?cat???1104Thr?Ala?His?Asn?Leu?Glu?Ala?Met?Leu?His?Leu?Leu?Asp?Gly?Tyr?His

355?????????????????360?????????????????365gga?aaa?gga?gag?aga?ttt?gtg?ctg?tcg?agt?ggg?aga?gac?cat?gcg?cta???1152Gly?Lys?Gly?Glu?Arg?Phe?Val?Leu?Ser?Ser?Gly?Arg?Asp?His?Ala?Leu

370?????????????????375?????????????????380gtg?aac?aaa?gcg?tcg?gac?ttt?ttg?aaa?gag?cat?tta?caa?att?cca?ccg???1200Val?Asn?Lys?Ala?Ser?Asp?Phe?Leu?Lys?Glu?His?Leu?Gln?Ile?Pro?Pro385?????????????????390?????????????????395?????????????????400ttt?tgg?cgt?caa?gac?gcg?aat?aaa?gga?atg?gtt?cgg?aac?agt?gaa?ggt???1248Phe?Trp?Arg?Gln?Asp?Ala?Asn?Lys?Gly?Met?Val?Arg?Asn?Ser?Glu?Gly

405?????????????????410?????????????????415cgt?tgg?att?caa?gcc?gag?cgt?ctc?cgt?ttt?gag?gat?cat?cat?tct?cct???1296Arg?Trp?Ile?Gln?Ala?Glu?Arg?Leu?Arg?Phe?Glu?Asp?His?His?Ser?Pro

420?????????????????425?????????????????430gat?atc?cac?cac?cat?ctc?tct?cag?ctc?cgt?ctt?gat?cat?cct?tgt?taa???1344Asp?Ile?His?His?His?Leu?Ser?Gln?Leu?Arg?Leu?Asp?His?Pro?Cys

435 440 445＜210〉19＜211〉447＜212〉PRT＜213〉arabidopsis (Arabidopsis thaliana)＜400〉19Met Thr Ala Glu Asp Ile Arg Arg Arg Asp Lys Lys Thr Glu Glu Glu, 15 10 15Arg Arg Leu Arg Asp Thr Trp Arg Lys Ile Gln Gly Glu Asp Asp Trp

20??????????????????25??????????????????30Ala?Gly?Leu?Met?Asp?Pro?Met?Asp?Pro?Ile?Leu?Arg?Ser?Glu?Leu?Ile

35??????????????????40??????????????????45Arg?Tyr?Gly?Glu?Met?Ala?Gln?Ala?Cys?Tyr?Asp?Ala?Phe?Asp?Phe?Asp

50??????????????????55??????????????????60Pro?Ala?Ser?Lys?Tyr?Cys?Gly?Thr?Ser?Arg?Phe?Thr?Arg?Leu?Glu?Phe?65??????????????????70??????????????????75??????????????????80Phe?Asp?Ser?Leu?Gly?Met?Ile?Asp?Ser?Gly?Tyr?Glu?Val?Ala?Arg?Tyr

85??????????????????90??????????????????95Leu?Tyr?Ala?Thr?Ser?Asn?Ile?Asn?Leu?Pro?Asn?Phe?Phe?Ser?Lys?Ser

100?????????????????105?????????????????110Arg?Trp?Ser?Lys?Val?Trp?Ser?Lys?Asn?Ala?Asn?Trp?Met?Gly?Tyr?Val

115?????????????????120?????????????????125Ala?Val?Ser?Asp?Asp?Glu?Thr?Ser?Arg?Asn?Arg?Leu?Gly?Arg?Arg?Asp

130?????????????????135?????????????????140Ile?Ala?Ile?Ala?Trp?Arg?Gly?Thr?Val?Thr?Lys?Leu?Glu?Trp?Ile?Ala145?????????????????150?????????????????155?????????????????160Asp?Leu?Lys?Asp?Tyr?Leu?Lys?Pro?Val?Thr?Glu?Asn?Lys?Ile?Arg?Cys

165?????????????????170?????????????????175Pro?Asp?Pro?Ala?Val?Lys?Val?Glu?Ser?Gly?Phe?Leu?Asp?Leu?Tyr?Thr

180?????????????????185?????????????????190Asp?Lys?Asp?Thr?Thr?Cys?Lys?Phe?Ala?Arg?Phe?Ser?Ala?Arg?Glu?Gln

195?????????????????200?????????????????205Ile?Leu?Thr?Glu?Val?Lys?Arg?Leu?Val?Glu?Glu?His?Gly?Asp?Asp?Asp

210?????????????????215?????????????????220Asp?Ser?Asp?Leu?Ser?Ile?Thr?Val?Thr?Gly?His?Ser?Leu?Gly?Gly?Ala225?????????????????230?????????????????235?????????????????240Leu?Ala?Ile?Leu?Ser?Ala?Tyr?Asp?Ile?Ala?Glu?Met?Arg?Leu?Asn?Arg

245?????????????????250?????????????????255Ser?Lys?Lys?Gly?Lys?Val?Ile?Pro?Val?Thr?Val?Leu?Thr?Tyr?Gly?Gly

260?????????????????265?????????????????270Pro?Arg?Val?Gly?Asn?Val?Arg?Phe?Arg?Glu?Arg?Met?Glu?Glu?Leu?Gly

275?????????????????280?????????????????285Val?Lys?Val?Met?Arg?Val?Val?Asn?Val?His?Asp?Val?Val?Pro?Lys?Ser

290?????????????????295?????????????????300Pro?Gly?Leu?Phe?Leu?Asn?Glu?Ser?Arg?Pro?His?Ala?Leu?Met?Lys?Ile305?????????????????310?????????????????315?????????????????320Ala?Glu?Gly?Leu?Pro?Trp?Cys?Tyr?Ser?His?Val?Gly?Glu?Glu?Leu?Ala

325?????????????????330?????????????????335Leu?Asp?His?Gln?Asn?Ser?Pro?Phe?Leu?Lys?Pro?Ser?Val?Asp?Val?Ser

340?????????????????345?????????????????350Thr?Ala?His?Asn?Leu?Glu?Ala?Met?Leu?His?Leu?Leu?Asp?Gly?Tyr?His

355?????????????????360?????????????????365Gly?Lys?Gly?Glu?Arg?Phe?Val?Leu?Ser?Ser?Gly?Arg?Asp?His?Ala?Leu

370?????????????????375?????????????????380Val?Asn?Lys?Ala?Ser?Asp?Phe?Leu?Lys?Glu?His?Leu?Gln?Ile?Pro?Pro385?????????????????390?????????????????395?????????????????400Phe?Trp?Arg?Gln?Asp?Ala?Asn?Lys?Gly?Met?Val?Arg?Asn?Ser?Glu?Gly

405?????????????????410?????????????????415Arg?Trp?Ile?Gln?Ala?Glu?Arg?Leu?Arg?Phe?Glu?Asp?His?His?Ser?Pro

420?????????????????425?????????????????430Asp?Ile?His?His?His?Leu?Ser?Gln?Leu?Arg?Leu?Asp?His?Pro?Cys

435 440 445＜210〉description 20＜211〉27＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence: primer＜400〉20atgtctagag aagatattgc gcggcga 27＜210〉21＜211〉28＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer＜400〉21gatgagctcg acgaagctga gagagatg 28

Claims (53)

1. The aging of encoding induce the isolated DNA molecule of lipase or itself and SEQ IDNO:1, SEQ ID NO:18 hybridization or with the functional deriv of the two hybridization, wherein said dna molecular under low stringency with SEQ ID NO:1, SEQ ID NO:18 hybridization or with the two hybridization.
2. 2. the DNA isolation molecule of claim 1, wherein said dna molecular has the nucleotide sequence of SEQ IDNO:1.
3. 3. the DNA isolation molecule of claim 1, wherein said DNA isolation molecule contains the nucleotide sequence of SEQ ID NO:4.
4. 4. the DNA isolation molecule of claim 1, wherein said dna molecular has the nucleotide sequence of SEQ IDNO:18.
5. 5. lipase or its functional deriv are induced in an isolating aging, described isolating aging induce lipase by under low stringency with SEQ ID NO:1, SEQ ID NO:18 hybridization or coded with the nucleotide sequence of the two hybridization.
6. 6. lipase is induced in the aging of claim 5, and wherein said lipase has the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:19.
7. 7. carrier that is used for transformed plant cells, described carrier comprises:

   (a) antisense base sequences, described antisense base sequences (1) and coding be old and feeble induces the corresponding section of a chain of dna molecular of lipase complementary substantially, the DNA that wherein said coding is old and feeble induces lipase under low stringency with SEQ ID NO:1, SEQ IDNO:18 or the two hybridization, perhaps a corresponding section of the coded RNA sequence of the old and feeble dna molecular of inducing lipase of (2) and described coding is complementary substantially; With

   (b) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes described antisense base sequences express in its plant transformed cell.
8. 8. the carrier of claim 7, wherein said adjusting sequence comprises a promotor and a transcription termination region.
9. 9. the carrier of claim 7, wherein said adjusting sequence comprises a constitutive promoter.
10. 10. the carrier of claim 7, wherein said adjusting sequence comprises a plant tissue specificity promoter.
11. 11. the carrier of claim 7, wherein said adjusting sequence comprises a plant senescence inducible promoter.
12. 12. the carrier of claim 7, wherein said adjusting sequence comprises a viral promotors.
13. 13. the carrier of claim 7, wherein said adjusting sequence comprises a constitutive promoter.
14. 14. the antisense oligonucleotide of a coding RNA molecule or polynucleotide, described RNA molecule and plant senescence induce the corresponding section of rna transcription thing of lipase gene complementary substantially, wherein said plant gene under low stringency with SEQ ID NO:1, SEQ IDNO:18 or the two hybridization.
15. 15. the antisense oligonucleotide of claim 14 or polynucleotide, wherein said oligonucleotide or polynucleotide comprise about 6 to about 100 Nucleotide.
16. 16. the antisense oligonucleotide of claim 14 or polynucleotide, the coding region of wherein said plant gene have nucleotide sequence SEQ ID NO:1.
17. 17. the antisense oligonucleotide of claim 14 or polynucleotide, the coding region of wherein said plant gene have nucleotide sequence SEQ ID NO:18.
18. 18. the antisense oligonucleotide of claim 14 or polynucleotide, wherein said plant gene are a kind of carnation gene.
19. 19. the antisense oligonucleotide of claim 14 or polynucleotide, wherein said plant gene are a kind of Arabidopsis (Arabidopsis) gene.
20. 20. the antisense oligonucleotide of claim 14 or polynucleotide, wherein said plant gene are a kind of tomato dna.
21. 21. the antisense oligonucleotide of claim 14 or polynucleotide, wherein said plant gene are a kind of tender pod Kidney bean gene.
22. 22. the antisense oligonucleotide of claim 14 or polynucleotide, a corresponding section of wherein said antisense oligonucleotide or polynucleotide and described rna transcription thing 5 ' non-coding region are complementary substantially.
23. 23. a carrier, described carrier comprises:

    (a) dna molecular of lipase is induced in a kind of aging of encoding, wherein said dna molecular under low stringency with SEQ ID NO:1, SEQ ID NO:18 or the two hybridization; With

    (b) regulate sequence, described adjusting sequence effectively is connected with described dna molecular, makes described dna molecular express in its plant transformed cell.
24. 24. bacterial cell that transforms with the carrier of claim 23.
25. 25. carrier plant transformed cell with claim 7.
26. A 26. plant and filial generation thereof that produces from carrier plant transformed cell with claim 7.
27. 27. the plant of a claim 26, plant part or plant filial generation.
28. 28. one kind is suppressed the method that endogenous aging induces lipase to express in the plant, described method comprises:

    (1) make a kind of vector integration in the genome of described plant, described carrier comprises:

    (A) antisense base sequences, described nucleotide sequence (i) and the described endogenous aging of coding induce the corresponding section of a chain of dna molecular of lipase complementary substantially, dna molecular and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization of lipase is induced in the endogenous aging of wherein said coding, or (ii) complementary substantially with the corresponding section of being induced the coded RNA sequence of lipase gene by described endogenous aging; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes that described antisense base sequences is expressed; With

    (2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described RNA sequence, thereby suppress the expression that lipase gene is induced in described aging.
29. 29. the corresponding section of the described DNA of complementary or the corresponding section of described RNA comprise 5 ' non-coding sequence substantially with it for the method for claim 28, wherein said antisense oligonucleotide or polynucleotide.
30. 30. the method for claim 28, wherein said inhibition cause described The Plant Senescence to be changed.
31. 31. the method for claim 28, wherein said inhibition cause described plant that the resistance of environment-stress inductive aging is strengthened.
32. 32. the method for claim 28, wherein said inhibition cause the biomass of described plant to increase.
33. 33. the method for claim 28, wherein said inhibition cause the seed production of described plant to increase.
34. 34. the method for claim 28, wherein said adjusting sequence comprise an activated constitutive promoter in described plant.
35. 35. the method for claim 28, wherein said adjusting sequence comprises a dual 35S promoter.
36. 36. the method for claim 28, wherein said adjusting sequence comprise an activated tissue-specific promoter in described plant.
37. 37. the method for claim 28, wherein said adjusting sequence comprise an activated old and feeble inducible promoter in described plant.
38. 38. the method for claim 28, wherein said plant is selected from: fruit-bearing plant, flowering plant, vegetables, farm crop and forest species.
39. 39. the method for claim 28, wherein said plant are tomato.
40. 40. the method for claim 28, wherein said plant are carnation.
41. 41. one kind is suppressed the method that one or more endogenous agings induce lipase gene to express in the vegetable cell, described method comprises:

    (1) make a kind of vector integration in the genome of at least a cell of described plant, described carrier comprises:

    (A) functional deriv of the isolated DNA molecule of lipase or itself and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization is induced in a kind of encoding exogenous aging, wherein said dna molecular under low stringency with SEQID NO:1, SEQ ID NO:18 or the two hybridization; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described dna molecular, makes and induces lipase to be expressed by the external source aging of described dna molecule encode; With

    (2) cultivate described plant, thereby make described dna molecular overexpression, and described one or more endogenous agings induce lipase gene to be induced lipase to suppress by the external source aging.
42. 42. the method for claim 41, wherein said adjusting sequence comprises a kind of constitutive promoter.
43. 43. one kind changes relevant old and feeble relevant old and feeble method with environment-stress of age in the plant, described method comprises:

    (1) make a kind of vector integration in the genome of described plant, described carrier comprises:

    (A) antisense base sequences, described antisense base sequences (i) and the described endogenous aging of coding induce the corresponding section of a chain of dna molecular of lipase complementary substantially, dna molecular and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization of lipase is induced in the endogenous aging of wherein said coding, or (ii) complementary substantially with at least one part of being induced the coded RNA sequence of lipase gene by described endogenous aging; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes that described antisense base sequences is expressed; With

    (2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described RNA sequence, thereby suppress the expression that lipase gene is induced in described aging.
44. 44. a transgenic plant cells, described cell comprises a kind of carrier of claim 7.
45. 45. a transgenic plant cells, described cell comprises a kind of carrier of claim 23.
46. 46. a plasmid, described plasmid comprise a kind of the have dubbing system of function and a kind of antisense oligonucleotide or polynucleotide of claim 14 in prokaryotic hosts.
47. 47. a plasmid, described plasmid comprise a kind of the have dubbing system of function and a kind of antisense oligonucleotide or polynucleotide of claim 14 in Agrobacterium (Agrobacterium).
48. 48. a kind of plant and filial generation thereof, the cell that wherein said plant origin induces the expression of lipase to be suppressed or to reduce in aging, described cell comprises the carrier of claim 7.
49. 49. a kind of plant and filial generation thereof, wherein said plant derives from the cell that aging induces the expression of lipase to be suppressed or to reduce, and wherein said cell produces by following step:

    (1) make a kind of vector integration in the genome of described cell, described carrier comprises:

    (A) antisense base sequences, described antisense base sequences (i) and the described endogenous aging of coding induce the corresponding section of a chain of dna molecular of lipase complementary substantially, dna molecular and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization of lipase is induced in the endogenous aging of wherein said coding, or (ii) complementary substantially with a corresponding section of being induced the coded RNA sequence of lipase gene by described endogenous aging; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences, makes that described antisense base sequences is expressed; With

    (2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described RNA sequence, thereby suppress the expression that lipase gene is induced in described aging.
50. 50. the plant of claim 49 and filial generation thereof, wherein said plant are tomato.
51. 51. the plant of claim 49 and filial generation thereof, wherein said plant are carnation.
52. 52. a method that suppresses Seed Aging, described method comprises:

    (1) make a kind of vector integration in the genome of plant, described carrier comprises:

    (A) antisense base sequences, a corresponding section of a chain of the endogenous aging dna molecular of inducing lipase of described antisense base sequences (i) and coding is complementary substantially, the described endogenous aging DNA and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization that induces lipase that wherein encode, or (ii) with the corresponding section complementation substantially of the RNA sequence of transcribing from the endogenous aging dna molecular of inducing lipase of encoding; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences; With

    (2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described RNA sequence, thereby suppress described aging expression of inducing lipase gene.
53. 53. a method that increases plant seed output, described method comprises:

    (1) make a kind of vector integration in the genome of described plant, described carrier comprises:

    (A) antisense base sequences, a corresponding section of a chain of the endogenous aging dna molecular of inducing lipase of described antisense base sequences (i) and coding is complementary substantially, the described endogenous aging DNA and SEQ ID NO:1, SEQ ID NO:18 or the two hybridization that induces lipase that wherein encode, or (ii) with the corresponding section complementation substantially of the RNA sequence of transcribing from the endogenous aging dna molecular of inducing lipase gene of encoding; With

    (B) regulate sequence, described adjusting sequence effectively is connected with described antisense base sequences; With

    (2) cultivate described plant, thereby described antisense base sequences is transcribed, and described transcript combines with described RNA sequence, thereby suppress described aging expression of inducing lipase gene.

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