CN101942460A - Arabidopsis AtACDO1 gene and expression system and application thereof - Google Patents
Arabidopsis AtACDO1 gene and expression system and application thereof Download PDFInfo
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Abstract
The invention discloses an arabidopsis AtACDO1 gene and an expression system and application thereof. A nucleotide sequence of the arabidopsis AtACDO1 gene is shown as SEQ ID NO: 1. The arabidopsis AtACDO1 gene is silenced by an RNAi method and is transformed into a plant, yellow and green leaf phenotype occurs in the plant, and laminae are lessened and narrowed; by constructing the overexpression plant of the arabidopsis AtACDO1 gene, the degradation of chlorophyll is retarded, and the natural senescence and detached senescence progresses of the laminae can be prolonged obviously by retarding the degradation of the chlorophyll in the laminae, so the photosynthetic period is prolonged and the photosynthetic gross is improved, the good shelf service life of green-leaf vegetables or the postharvest green period of forage crops can be prolonged, and the content of the chlorophyll and protein serving as main nutritional ingredients is increased. In addition, the yellow and green leaf phenotype is controlled by a method of molecular biology, so the arabidopsis AtACDO1 gene can be used as a genetic marker and is used for product identification in agricultural production.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of Arabidopis thaliana AtACDO1 gene and expression system and application.
Background technology
As everyone knows, chlorophyll is one of most important basic substance of carrying out smoothly of photosynthesis of plant.In the plant photosynthesis organ, chlorophyll combines with chlorophyll-binding protein, catches luminous energy, drives photosynthesis and carries out.For human, it is the energy foundation that we depend on for existence that chlorophyll is caught the chemical energy that luminous energy is transformed through photosynthesis.
Chlorophyll is present in all can carry out photosynthetic organism, comprises the blue-green algae (blue bacterium) of green plants, protokaryon and the algae of eucaryon.Chlorophyll has different types: chlorophyll a and b are main types, are present in higher plant and the green alga; Chlorofucsin and d are present in various algae, and normal and chlorophyll a is also deposited; Chlorofucsin is comparatively rare, exists in some chrysophyceae; Bacteriochlorophyll then only exists in some photosynthetic bacterium.
Owing to chlorophyllous synthesizing is successive process from the beginning to the end, the factor (environment and gene) of therefore every energy controlling chlorophyll synthetic enzyme, all the energy controlling chlorophyll synthesizes.The research of chlorophyll degradation is the focus of metabolism research always.To synthetic similar, chlorophyllous degraded also has extremely complicated regulatory pathway, and plant itself influences each other with environmental factor and regulates chlorophyllous degraded (Hortensteiner, Plant Biology 57:55-77,2006).Along with deepening continuously of research, chlorophyllous degraded and regulatory pathway are also distinct day by day.
Chlorophyllous degraded can be divided into commitment and later stage, and it is green that the product of commitment is, and the product in stage in later stage is colourless.Commitment comprises the place to go (going chelatingization) of the modification of tetrapyrrole ring side chain, the hydrolysis of phytyl (taking off phytylization), magnesium ion and the different modifying of different plant tetrapyrrole rings; Later stage then comprises the fracture of tetrapyrrole ring and the oxidation of cleavage product.In leaf senile and ripening of fruits, later stage is particularly important, because this process intermediate product becomes colorless, blade especially fruit normal coloring is had crucial meaning.Because chlorophyllous degraded is the process of a continuous degradation, in most cases the intermediate product of chlorophyll degradation does not accumulate in leaf senile and the ripening of fruits, from this aspect, the yellowish green mutant of control chlorophyll degradation helps the chlorophyll degradation Study on Mechanism very much.
Morphing in some site of Plant Genome can the chlorophyllous metabolism of influence, thereby causes the leaf look of plant to change, and is accompanied by deadly, dwarfing, various features such as sterile.So far, on a lot of plants such as paddy rice, corn, Arabidopis thaliana, barley, rape, all found chlorophyll deficient mutant.Chlorophyll deficient mutant is not only the important materials of photosynthesis, chlorophyll biosynthesizing, chloroplast(id) genetic variation and genetic differentiation and growth etc. of research higher plant, and can be used as mark property and be applied in the heterosis utilization.Therefore, excavate and identify the gene of controlling plant chlorophyll deletion mutantion, the aspect researchs such as the mechanism of action of carrying out location, clone and the mutator gene of chlorophyll deletion mutantion gene all have important significance for theories and using value.
Summary of the invention
The objective of the invention is to provides a kind of Arabidopis thaliana AtACDO1 gene of controlling chlorophyll degraded according to the deficiency in the chlorophyll research that exists in the prior art.
Another purpose of the present invention is to provide the albumen of above-mentioned Arabidopis thaliana AtACDO1 genes encoding.
Another purpose of the present invention is to provide a kind of above-mentioned Arabidopis thaliana AtACDO1 expression carrier that contains.
Another purpose of the present invention is to provide a kind of recombinant bacterial strain that contains above-mentioned expression vector.
Another purpose of the present invention is to provide a kind of transgenic cell line that contains above-mentioned Arabidopis thaliana AtACDO1 gene.
A further object of the invention is to provide the Arabidopis thaliana AtACDO1 application of gene.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
The present invention has cloned the key controlling gene AtACDO1 of Arabidopis thaliana catabolism from Arabidopis thaliana, again by utilizing the RNAi technology and by means of engineered means, creating yellowish green mutant.
The present invention proposes a kind of key gene AtACDO1 that utilizes chlorophyll degradation, create the method for yellowish green mutant.By seeking and the gene of its homology more than 80%, obtain yellowish green mutant, or suppress the AtACDO1 expression of gene, obtain yellowish green leaf plant shape.
Among the present invention, the plant that yellowish green leaf phenotype occurs is a greenery class plant.
As a kind of preferred version, described plant is an Arabidopis thaliana.
Among the present invention, the T-DNA carrier of AtACDO1 gene is the RNAi carrier, by the double-stranded RNA inhibition of gene expression.
Among the present invention, the recombinant vectors of inhibition of gene expression is 35S promoter or model fibroin promotor (Ubiquitin promoter).
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
The primer of amplification AtACDO1 is to also within protection scope of the present invention.
Utilize plant expression vector, control chlorophyll synthetic gene transfered plant cell of the present invention is crossed expression, can obtain photosynthetic efficiency enhanced transgenic cell line and transfer-gen plant.
Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described expression vector also comprises 3 ' end untranslated zone of foreign gene, promptly comprise the polyadenylic acid signal and any other participation mRNA processing or the dna fragmentation of genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene, plant gene 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.
Carry the present invention control the chlorophyll degradation gene plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be monocotyledonss such as paddy rice, corn, wheat, also can be dicotyledonss such as Arabidopis thaliana, tobacco.
Inventor's separating clone first obtains a kind of AtACDO1 gene of regulating the plant chlorophyll degraded, by making up the RNAi carrier, by the agrobacterium mediation converted Arabidopis thaliana, the transfer-gen plant that obtains is carried out phenotype analytical and molecular biology Function Identification, the result shows that the phenotype of yellowish green leaf has appearred in plant, Arabidopis thaliana AtACDO1 strikes turn reducing gene plant (AtACDO1RNAi) in normal culture condition lower blade yellowing, and blade length, width and leaf area diminish, further discover, chlorophyll and carotenoid content significantly are lower than wild-type in the AtACDO1RNAi strain system, are that chlorophyll degradation is accelerated to cause through evaluation.Illustrate that thus the present invention obtains a kind of new gene of controlling chlorophyll degradation, its nucleotide sequence is shown in SEQID NO:1, and called after AtACDO1.The protein of described Arabidopis thaliana AtACDO1 genes encoding, its aminoacid sequence is shown in SEQ ID NO:2, described proteinic aminoacid sequence also can be through 1-10 amino acid whose replacement, disappearance or interpolation, and the protein with control chlorophyll related activity.
A kind of expression vector, this expression vector contain Arabidopis thaliana AtACDO1 gene.
A kind of recombinant bacterial strain, this recombinant bacterial strain contains above-mentioned expression vector.
A kind of transgenic cell line, this transgenic cell line contain Arabidopis thaliana AtACDO1 gene.
Arabidopis thaliana AtACDO1 gene of the present invention can be used for chlorophyll degradation, the aging of prolongation plant leaf, the increase chlorophyll content of plant of controlling plant or increase plant protein content, also can be used as genetic marker, and the product that is used for agriculture production is identified.
Compared with prior art, the present invention has following beneficial effect:
Arabidopis thaliana AtACDO1 gene of the present invention is transformed in the plant after by RNAi method silence, yellowish green leaf phenotype appears in plant, blade diminishes and narrows down, by making up Arabidopis thaliana AtACDO1 gene overexpression plant, to slow down chlorophyllous degraded, and in the blade chlorophyllous degraded slow down the naturally-aged and the stripped senescence process that can prolong blade significantly, and then prolong photosynthetic phase and photosynthetic total amount, can prolong the commodity price life-span of green vegetable or adopting the green phase of back of fodder crop, increase its main nutrient composition chlorophyll and Protein content.
Description of drawings
Fig. 1 makes up synoptic diagram for RNAi strikes the off-load body;
Fig. 2 is clone and the evaluation of purpose fragment Arabidopis thaliana AtACDO1;
Fig. 3 is the phenotype analytical of Arabidopis thaliana different genotype plant;
Fig. 4 is a chlorophyll content in the Arabidopis thaliana different genotype plant leaf;
Fig. 5 participates in the evaluation figure of chlorophyll biosynthesizing relation for Arabidopis thaliana AtACDO1; The form of different genotype seedling after 5h and 10h photo-irradiation treatment of dark cultivation in A:4d age.Scale is 1cm.A, C: the chlorophyll content of different genotype seedling.4d secretly grows seedlings behind photo-irradiation treatment 5h (row of going up) or 10h (row down) age, gets about 90 strain seedling respectively and carries out the chlorophyll extraction with 80% acetone.D: the content of protoporphyrin and former chlorophyllide in the different genotype seedling.
Fig. 6 is the expression of chlorophyll metabolism related gene in the different genotype Arabidopis thaliana; A: chlorophyll anabolism gene expression dose is analyzed in each genotype of different growing stages; B: chlorophyll anabolism gene expression dose analysis in each genotype under the different light intensity.Numeral " 10 ", " 40 " and " 120 " represent that respectively light intensity is 10,40 and 120 μ molm-2s-1 among the figure.
Fig. 7 is the activity of chlorphyllase in the different genotype plant under the natural condition.
Embodiment
Below in conjunction with specific embodiment the present invention is done description further, but specific embodiment is not done any qualification to the present invention.
Embodiment 1AtACDO1 gene RNAi expression vector and the structure of crossing expression vector
1. the cultivation of Arabidopis thaliana
The Arabidopis thaliana seed is placed in the 1.5ml centrifuge tube, earlier with 75% alcohol disinfecting, 1～2min, again with 1% clorox, the 3～5min that sterilizes, behind the aseptic water washing 5 times, with the program request of rifle head the sterilization MS solid medium (pH 5.8 for 1.5% sucrose, 0.8% agar) on, lucifuge, behind 4 ℃ of vernalization 2d, forward between illumination cultivation 22 ℃/18 ℃ of temperature (day/night) to, the 16h/8h photoperiod, the incandescent light illumination cultivation.
2. adopt the Trizol method of this area routine to extract Arabidopis thaliana RNA.
3.RT-PCR detect
3.1 reverse transcription reaction (cDNA is synthetic)
Carry out reverse transcription reaction with the MMLV ThermoScript II, reaction system and reaction conditions get final product with reference to the related kit explanation.
3.2 the RT-PCR of goal gene amplification
CDS and enzyme according to mustard AtACDO1 are cut information, choose respectively proper restriction site with the purpose fragment cloning to corresponding carrier, it is as shown in table 1 wherein to use primer:
Table 1 makes up the RNAi expression vector of mustard AtACDO1 and crosses the expression vector primer sequence
PCR reaction system: ddH
2O 15.4 μ L, 10 * Taq Buffer, 2 μ L, 10mM dNTP mix0.5 μ L, PrimerN (10 μ mol/L) 0.4 μ L, PrimerC (10 μ mol/L) 0.4 μ L, dna profiling 1 μ L and Taq enzyme (5U/ μ L) 0.3 μ L.
The PCR response procedures: 94 ℃ of sex change 2min, carry out 30 circulations by 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min then, extend 10min down at 72 ℃ at last.
4. make up the RNAi expression vector of mustard AtACDO1 and cross expression vector
In order to study the function of mustard AtACDO1, we buy two mutant SALK_068628.55.00.x and the SALK_130499.44.50.x that T-DNA is inserted in mustard AtACDO1 gene intron different positions back from U.S.'s SALK mutant library.Behind the Kan resistance screening, find SALK_068628.55.00.x, screen to T6 for the time still have resistance to separate, and three-primer detects individual plants DNA and finds to exist really at this gene place insertion, and be homozygote, expression still arranged but RT-PCR detects mustard AtACDO1 gene.For SALK_130499.44.50.x, the seed of purchasing has isozygotied, and the three-primer qualification result shows that also it for isozygotying is, has than high expression level but RT-PCR still detects AtACDO1.This may be because due to the T-DNA that inserts is sheared after transcribing.
Limited in view of the mutant genetic background bought is understood, the present invention by the RNAi technique construction mustard AtACDO1 clpp gene off-load body.Method is that (product reclaims the back with XhoI and HindIII double digestion, reclaims enzyme again and cuts product for ACDO1pskN, ACDO1PsC) amplification positive-sense strand (SEQ ID NO:3) with the primer of band XhoI and HindIII restriction enzyme site; Cut processing PSK-int plasmid vector with same enzyme.The product enzyme is cut back to close fragment cut back to close fragment with the carrier enzyme and be connected, then with carrying primer order-checking, the clone that Screening and Identification is correct by the T4DNA ligase enzyme.With the carrier of this carrier as two time clonings, change antisense strand (SEQ ID NO:4) continuously over to by similar process, obtain the medial expression vector pSKAtCDO1 (Fig. 1) of RNAi.With Xho1 and this expression vector of BamHI double digestion, obtain the sense:loop:antisense structure fragment, and be connected conversion with the pCanG carrier of cutting processing with the same enzyme enzyme, obtain AtACDO1 clpp gene off-load body pCSKAtCDO1, with its transformed into escherichia coli E.coli DH5 α, filter out positive colony, transform Agrobacterium A.tumefaciens EHA105 with the positive colony plasmid, receive and choose mono-clonal on the substratum of mycin and Rifampin containing card, and identify positive colony with PCR.With Agrobacterium inflorescence infusion method transfection Arabidopis thaliana Col-0 wild-type.The seed of receiving screens on the substratum of 50mg/L Kan, and the resistance seedling moves to soil.
After two weeks, the positive plant through screening moves to the soil growth, treats that the ripe seed of collecting promptly gets T0 generation, and process Kan screens and sxemiquantitative RT-PCR analyzes, and we screen two comparatively ideal transgenic line: AtACDO1RNAi#6 and AtACDO1RNAi#25.The AtACDO1 gene expression dose all obviously descends than wild-type in this two strain system, and strain is AtACDO1RNAi#25 decline degree more remarkable (Fig. 2), for subsequent experimental provides comparatively ideal research material.
Simultaneously, we transform the pCanG carrier with the positive-sense strand of amplification, made up 35S::AtACDO1 and crossed expression vector, and conversion wild-type Col-0 has obtained transfer-gen plant.Identify through hygromycin selection and sxemiquantitative RT-PCR, choose wherein transgenic line 35S::AtACDO1# 3 that two AtACDO1 expression levels obviously raise and 35S::AtACDO1#8 (Fig. 2) as alternative research object.
Above-mentioned plant expression vector RNAi intermediate carrier PSK-int, mistake expression vector pCAMBIA1300-211-myc, PCanG, intestinal bacteria E.coli DH5 α and Agrobacterium A.tumefaciens EHA105 are the experiment material that those skilled in the art use always.The conversion of carrier connection in the aforesaid operations, the conversion of Bacillus coli cells and Agrobacterium is the routine operation of this area.
The Function Identification of embodiment 2AtACDO1 gene
1. the mensuration of the phenotype analytical of Arabidopis thaliana transfer-gen plant and physical signs
Because AtACDO1 strikes and subtracts plant in the leaf color yellowing of seedling phase, change in order further to understand its phenotype in the later stage of growing, we carry out the phenotype observation with each genotype plant kind in soil, take pictures after 20 days and observe the growth of seedlings development condition and carry out corresponding morphological index measurement.The result is as shown in Figure 3: the AtACDO1 that cultivates in the soil strike subtract strain be the AtACDO1RNAi# 25 plant leaf than the obvious yellowing of wild-type color, yellowish green mutation type surface appears, and this phenotypic characteristic to compare to seedling more obvious during the phase; And AtACDO1 crosses the strain of expression is that leaf color is compared greener with wild-type.
We find simultaneously, and except the leaf color difference, the AtACDO1RNAi# 25 plant leaf diminishes.Indexs such as blade length and width are measured, and statistical study.The result is as shown in table 2, finds that this strain is that the length and width and the leaf area of blade is all little than wild-type, and blade diminish mainly be reduce by blade length due to, but three kinds of genotype seedling leaves unit surface fresh weights and no significant difference.
The development characteristics of table 2 Arabidopis thaliana different genotype plant
Measure the chlorophyll content of Arabidopis thaliana transfer-gen plant with the HPLC method.
The making of chlorophyll and precursor standard specimen thereof is changed slightly with reference to methods such as (Fang et al., 1998).Get spinach blade 10g, add acetone 40ml, extraction chlorophyll spends the night under 4 ℃ of dark.Gained chlorophyll is concentrated to behind the suitable concn chromatography on the thin-layer silicon offset plate with the vacuum concentration instrument, and chromatographic solution is a normal hexane: ether: acetone=6: 3: 2.Separate the Chla and the Chlb that obtain and scrape, with being standard specimen behind 100% acetone extract with blade.
The extraction of enzyme liquid: get about 4g Arabidopis thaliana wild-type blade, acetone grinds, centrifugal remove chlorophyll after, will precipitate air-dryly, add 4ml extracting solution (41mM TRIS then, the 320mM glycine, 670mM glucose, pH 7.8,0.5%SDS (w/v) and 2% water are not allowed PVP), room temperature shaking table (100rpm) is gone up extracting 4h, 4 ℃ of extractings of spending the night then.Extracting good material 10, the centrifugal 30min of 000g, supernatant is crude enzyme liquid.
The preparation of chlorophyllide a: with above-mentioned The addition of C hla and enzyme liquid reaction (100 μ g total protein), reaction system is 2ml, Chla extracting solution 1.2ml; 0.1M Vc 20 μ l; Dezymotize liquid volume required outside, insufficient usefulness 50% acetone is supplied.Reaction system adds 2ml acetone and the abundant mixing of 4ml normal hexane behind reaction 30min under 40 ℃, and 10, the centrifugal 10min of 000g removes upper strata normal hexane phase (the complete Chla of unreacted is in this phase), and lower floor's acetone is chlorophyllide a mutually.In made chlorophyllide a, add a 1N HCl and be phaeophorbide a.
The sample chlorophyll measuring: get an amount of blade, 100% acetone extraction chlorophyll, extracting solution are crossed the organic filter membrane of 0.45 μ m, carry out HPLC then and analyze.
Adopt U.S. AgiLent-1000LC high performance liquid chromatograph system device to measure, working conditions is:
Chromatographic column: AgiLent ODS (C18) reversed-phase column, 5 μ m, 5.0mm * 25cm
Moving phase: adopt gradient elution, 1-20min 80% methyl alcohol: ethyl acetate (0～50%), 20～45min, 80% methyl alcohol: ethyl acetate (50%: 50%) isocratic elution.The detection wavelength is 658nm.
The chlorophyll content result as shown in Figure 4, discovery is than wild-type, AtACDO1 strikes that chla (chlorophyll a), chlb (chlorophyll b) and chla+chlb (total chlorophyll) content obviously descend in the AtACDO1RNAi# 25 blade that subtracts in the strain system, and chla, chlb and all significantly risings of chla+chlb content in the 35S::AtACDO1# 8 strain system.
Relation between 3AtCDO1 gene and chlorophyll are synthetic
Seed with AtACDO1RNAi#25, wild-type and 35S::AtACDO1#8 strain system is laid on the MS plate respectively, under dark fully condition, grew 4 days after the vernalization, sprout seedling this moment and show as the yellow reaction, no chlorophyll is synthetic in the blade, then flat board is moved to (120 μ mol.m-2.s-1) under the light, and during respectively at illumination 5h and 10h, measure the seedling chlorophyll content.Protoporphyrin and former chlorophyllide extract from 20d each genotype seedling leaves in age, and measure in wavelength 628nm and 503nm place with spectrophotometry.The result as shown in Figure 5, along with the growth of light application time, each genotype seedling cotyledon changes green (Fig. 5 A), shows as yellow.Chlorophyll content increases along with the growth of light application time also (Fig. 5 B, C), but significance analysis but finds no matter be that chla, chlb and chlorophyll content all do not have significant difference in each genotype at illumination 5h (Fig. 5 B) or illumination 10h (Fig. 5 C).Measure in 20 days plant leafs the content of ProtoIV and Pchlide simultaneously and find that Pchlide does not all have significant difference in AtACDO1RNAi plant, wild-type and 35S::AtACDO1#8 plant yet.Because the direct precursor of chlorophyll synthetic is Pchlide, and therefore Pchlide content indifference in three kinds of genotype can infer that the AtACDO1 gene also has neither part nor lot in chlorophyllous synthetic.
Each genotype chlorophyll metabolism Expression of Related Genes under 4 different growing stages and the light intensity
In order to identify whether the AtACDO1 gene influences the chlorophyll degradation expression of gene, we have detected in different growing stage (10,20 and 30d) or the following three kinds of genotype plant of different light intensity (10,40 and 120 μ mol.m-2.s-1) condition the degraded genes involved and chlorophyll is synthetic and the Expression of Related Genes level, the results are shown in Figure 6.Fig. 6 B shows that in different growing stages, the expression level of chlorophyll synthetic gene HEMC, CHLH, CHLD and PORA and chlorophyll degradation gene C AO is all along with the increase of growth time is in rising trend; And synthetic gene NYC1 and DVR on a declining curve (Fig. 6 A, B).But in same vegetative period, these expression of gene level and no significant differences in the different genotype plant.With regard to synthetic gene UPM1, high level expression when growth 20 days and 30 days in the growth wild-type; Closely expressed the highlyest in the AtACDO1RNAi plant in the time of 20 days, express the highlyest when 10 days and 20 days and cross the expression plant, during still with regard to 20d, its expression does not have difference among three genotype.Turn to the FeChI1 of protoheme route of synthesis for catalysis ProtoIV, three genotype all are the expression that a higher level was arranged in 20 days, but in crossing the expression plant, almost detect less than expression in 10 days and 30 days.Concerning chlorphyllase gene C LH1 and CLH2, each growth phase does not have considerable change in three kinds of genotype.Because this gene is first gene of catalysis chlorophyll degradation process, so as if RT-PCR result points out, and we AtACDO1 is more to work on protein level.
When the chlorophyll metabolism related gene was expressed under analyzing different light intensity, we did not detect the expression of CLH2 and ACD1 and ACD2 under these condition confidential reference items.Wherein DVR is along with the increase of light intensity, and expressing in each genotype increases; CAO then in contrast.And no matter other genes are to synthesize or degrade all not have evident difference (Fig. 6 C) between each genotype under different light intensity.
5AtACDO1 is to the active influence of chlorphyllase
By top expression figure encode as can be known the gene A tCLH1 and expression and the no significant difference of AtCLH2 in the different genotype plant of chlorphyllase, thereby but whether its protein level change and regulated chlorophyll content and still belong to the unknown.And chlorphyllase (Chlorophyllase) is first enzyme of catalysis chlorophyll degradation approach, its activity is regulated by many-sided factor, as different genotype, ethene, 6-BA and Plant hormones regulators,gibberellins etc., therefore, we have measured the variation (Fig. 7) of chlorophyll enzymic activity in wild-type, AtACDO1RNAi#25 and the 35S::AtACDO1#8 plant under the normal growth state.As can be seen from Figure 7, in the AtACDO1RNAi#25 plant, the activity of chlorphyllase is significantly higher than the wild-type plant, and activity was significantly higher than the expression plant in the wild-type plant.In conjunction with chlorophyll content (Fig. 3) among this wild-type, AtACDO1RNAi#25 and the 35S::AtACDO1#8 in vegetative period, as seen, chlorophyll content and chlorphyllase activity are negative correlation, illustrate AtACDO1RNAi#25 plant content of chlorophyll reduce be since its body inner chlorophyll enzymic activity higher due to.Show that by above data it is because due to the chlorophyllous degraded quickening, promptly AtACDO1 has participated in the metabolic correlated process of chlorophyll degradation that AtACDO1RNAi#25 presents yellowish green mutation type surface.
Claims (10)
1. Arabidopis thaliana AtACDO1 gene, its nucleotide sequence is shown in SEQ ID NO:1.
2. the protein of the described Arabidopis thaliana AtACDO1 of claim 1 genes encoding, its aminoacid sequence is shown in SEQ ID NO:2.
3. the protein of Arabidopis thaliana AtACDO1 genes encoding according to claim 2 is characterized in that described proteinic aminoacid sequence through 1～10 amino acid whose replacement, disappearance or interpolation, and has control chlorophyll related activity.
4. an expression vector is characterized in that described expression vector contains the described Arabidopis thaliana AtACDO1 of claim 1 gene.
5. a recombinant bacterial strain is characterized in that described recombinant bacterial strain contains the described expression vector of claim 4.
6. a transgenic cell line is characterized in that described transgenic cell line contains the described Arabidopis thaliana AtACDO1 of claim 1 gene.
7. the application of the described Arabidopis thaliana AtACDO1 of claim 1 gene is characterized in that described Arabidopis thaliana AtACDO1 gene is used for the chlorophyll degradation of controlling plant, prolongs the plant leaf aging, increases chlorophyll content of plant or increases plant protein content.
8. according to the application of the described Arabidopis thaliana AtACDO1 of claim 7 gene, it is characterized in that described plant is a greenery class plant.
9. the application of described according to Claim 8 Arabidopis thaliana AtACDO1 gene is characterized in that described plant is an Arabidopis thaliana.
10. the application of the described Arabidopis thaliana AtACDO1 of claim 1 gene is characterized in that described Arabidopis thaliana AtACDO1 gene as genetic marker, and the product that is used for agriculture production is identified.
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| CN109912703A (en) * | 2017-12-13 | 2019-06-21 | 中国科学院遗传与发育生物学研究所 | Application of the protein OsARE1 in regulation plant senescence |
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Non-Patent Citations (5)
| Title |
|---|
| 《GenBank数据库》 20030515 Cheuk, R., et al. GenBank登录号：AAP37783 2-3 , 2 * |
| 《GenBank数据库》 20030515 Cheuk, R., et al. GenBank登录号：BT008424 1，4-10 , 2 * |
| 《Plant Physiology》 20080630 Michal Jasinski, et al. AtOSA1, a Member of the Abc1-Like Family, as a NewFactor in Cadmium and Oxidative Stress Response 719-731 3，7-9 第147卷, 第2期 2 * |
| 《纪念殷宏章先生百年诞辰暨全国光合作用学术研讨会论文摘要汇编》 20080901 杨松光 等 拟南芥ABC1 型蛋白激酶调控高光胁迫响应的分子机制 62 1-10 , 2 * |
| 《纪念殷宏章先生百年诞辰暨全国光合作用学术研讨会论文摘要汇编》 20080901 舒展 等 应用叶绿素荧光成像分析拟南芥花色素苷缺失突变体对光氧化处理的敏感性 63 1-10 , 2 * |
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| CN109912703A (en) * | 2017-12-13 | 2019-06-21 | 中国科学院遗传与发育生物学研究所 | Application of the protein OsARE1 in regulation plant senescence |
| CN109912703B (en) * | 2017-12-13 | 2021-03-16 | 中国科学院遗传与发育生物学研究所 | Application of protein OsARE1 in regulation and control of plant senescence |
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