CN101633935B - Method for prolonging shelf life of banana by using gene engineering technology, components and applications thereof - Google Patents
Method for prolonging shelf life of banana by using gene engineering technology, components and applications thereof Download PDFInfo
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Abstract
The invention discloses a method for prolonging the shelf life of banana by using gene engineering technology, components and applications thereof. The method is characterized in that a novel banana gene transfer method is used for transferring a banana ACC oxidase control composite into banana; the banana ACC oxidase control component contains at least one interference RNA, a gene transfer expression vector and a medically acceptable vector agent; and the interference RNA is used for suppressing the expression of mRNA of banana ACC oxidase to further suppress ethylene biosynthesis of banana so as to defer after-ripening of the banana. Therefore, the method can prolong the shelf life of banana.
Description
Technical field
The invention relates to using gene engineering technique to prolong method, constituent and the application thereof of banana fresh-keeping phase, be meant that especially changeing culturing method by gene changes the mRNA expression of growing in banana with inhibitions/reduction banana acc oxidase gene with interferential RNA, and then biosynthetic method, constituent and the application thereof of inhibition/reduction ethene.
Background technology
" after-ripening " means the process of finishing slaking behind the climacteric fruit and vegetable picking voluntarily.In order to transport or to preserve, some climacteric fruits and vegetables needs to pluck in advance.Its objective is,, can prolong fortune, storage phase by the after-ripening of climacteric fruits and vegetables self.Also can take appropriate measures as required (as low temperature, controlled atmosphere, ethylene absorbent, ethylene inhibitor etc.) to suppress/to delay the after-ripening process of climacteric fruits and vegetables, reach the purpose of Long-term Storage.If need listing ahead of time, utilize ethylene agent etc. can promote the fruits and vegetables after-ripening.Some fruit such as banana (Musa spp.) must could better eat through the after-ripening stage.
Banana (Musa spp.) belongs to the monocotyledons of Musaceae (Musaceae) Musa (Musa), and fruit fragrance is good to eat and be of high nutritive value, and is one of important cash crop in the world.Banana (Musa spp.) belongs to climacteric fruit, and after gathering, green ripe banana is by the after-ripening process, produce the climacteric variation, give birth to the hydrolysis etc. of generation, starch and the protopectin-of ethene in comprising, treat that pulp softening, sugariness increase, after fragrance produces, the edibleness beginning can improve.
In the prior art banana is gathered in advance,, can prolong its fortune, storage phase by the after-ripening of banana self.Yet banana often still causes fruit after-ripening, person very because of ethene generates in transportation, and is overdone, ruin.Therefore, reduce the marketable value of banana and influence the popularization of banana.Therefore, by Ethylene Biosynthesis is controlled, so that the method that solves associated problem such as banana after-ripening to be provided.
Ethene (Ethylene) is the hormone that gas form exists, and influences many plant physiology and biochemistry reactions (Burg and Burg, 1962).Ethene is very important to growth and development of plant and adverse circumstance reaction, for example is subjected to the aging and fruit after-ripening of waterflooding, mechanicalness injury, courses of infection, Ye Yuhua when plant, all can produce ethene.The biosynthesizing path of ethene is that MET (methionine) is converted into the sweet MET (S-Adenosyl-methionine of S-gland by the sweet MET synthetic enzyme of S-gland (AdoMet synthetase), AdoMet), again by ACC synthetic enzyme (ACC synthase, ACS) generate 1-amido cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylic acid, ACC), acc oxidase (ACC oxidase, ACO) then oxidation ACC generates ethene (Ethylene) (Yang and Hoffman, 1984).Hence one can see that, and ACO is the last enzyme in ethene biosynthesizing path, therefore, can pass through the biosynthesizing with inhibition/reduction ethene of inhibition ACO gene or protein expression, and then reach the purpose of delayed fruit after-ripening.The present invention is the acc oxidase gene with banana (Musa spp.): Mh-ACO1A (MAO1A, GenBank accession no.AF030411, SEQ ID No:14), Mh-ACO1B (MAO1B, GenBank accession no.AF030410, SEQ ID No:15) and Mh-ACO2 (MAO2, GenBank accession no.U86045, SEQ ID No:16) be target gene, expectation is by suppressing these expression of gene, with the biosynthesizing of inhibition/reduction ethene, reach the sophisticated purpose of delayed fruit.
Past is in order to reduce the method that target gene is expressed, mainly be to grow the antisense strand (antisense strand) of target gene in plant with commentaries on classics, the mRNA sequence complementation (complementary) of the mRNA that is produced and endogenous (endogenous) Shunyi gene, form the structure of double-stranded (duplex), and then degraded or interferencing protein the carrying out of translating, reach the purpose that suppresses endogenous genetic expression; Or make up the mode of the target gene Shunyi chain have great expression (over-expressed) promotor, make the gene great expression and cause common inhibition (co-suppression) phenomenon, and then the expression of suppressor gene.Yet the effect of above-mentioned two kinds of method silences (silience) gene is also bad.Along with gene silencing mechanism is understood gradually, think that double-stranded RNA is only the main factor that causes gene silencing, therefore make up the dna structure that can form double-stranded RNA, and change to grow and enter in the biology, discovery can improve the ability of silencer, wherein if serve as the spacer that forms circle ring (loop) with the intron (intron) of telotism, genetic expression even almost can be by inhibition (Smith et al., 2000) fully.
It is the method that a kind of reduction (knock-down) target gene is expressed that RNA disturbs (RNAi), this method by small-sized list-or two-chain RNA (ssRNA, dsRNA) with silence (silencing) expression of gene.Interferential RNA comprise the small segment RNA interfering (small interfering RNA, siRNA), two strands or strand (ds siRNA or ss siRNA), micro rna (miRNA), small-sized hair clip shape RNA (shRNA) etc.Not bound by theory, RNA disturbs and can produce in vivo, dsRNA will be by class RNaseIII (RNaseIII-like) the enzyme institute identification of a kind of stripping and slicing enzyme by name (Dicer), dsRNA is cut out the small fragment RNA molecule that 3 ' end has 2 Nucleotide outstanding (overhang), be siRNA, its size is about 21 to 23 Nucleotide (Elbashiret al., 2001; Zamore et al., 2000).
SiRNA combines with a kind of protein complex, this protein complex is referred to as RISC, and (RNA-brings out reticent mixture, RNAi silencing complex), RISC has helicase, the siRNA that can untie duplex structure forms single-stranded structure, wherein the antisense of siRNA (antisense) chain (or is cited approvingly and is led chain, guide strand) can combine with RISC with guiding RISC to target mRNA, and with it in conjunction with to carry out the degraded of target mRNA, and then reach expression (Matzke etal., 2001 of reticent target gene; Waterhouse et al., 2001); Wherein this target mRNA is the complementary sequence with the antisense strand of this siRNA promptly.
Smith etc. (2000) grow potato virus (the potato virus Y that contains Shunyi or antisense with commentaries on classics, PVY) Nia-protease (Pro) gene, make the anti-PVY of potato, the ratio that both produce the gene silencing transformed strain is respectively 7% and 4%, but if can form reverse repetition (inverted-repeat) DNA of double-stranded RNA with tool, and the intron (intron) of telotism is configured to the spacer that forms circle ring (loop), then change grow efficient can be up to 96% (22/23).Infer that the intron existence can help RNA to stablize, adjust the RNA orientation, and eukaryote is in the spilicing process, and preRNA meeting of short duration formation duplex (duplex) utilizes this characteristic can help double-stranded RNA to form, improve and suppress effect (Smith et al., 2000).
Generally speaking, change culturing method and be by Agrobacterium carry an external source (exogenous) gene with infecting embryo material (embryogenic material) as embryonic callus (embryogenic callus), embryo's suspended substance (embryogenicsuspension), or somatocyte (somatic cell), grow gene plant to obtain this commentaries on classics.And the gene of banana changes culturing method, the body embryo that is exposed in the banana of Ma Suxuan .1988 in the prior art take place with plant regeneration research in, or in people's such as Dean Engler United States Patent (USP) notification number 6,133,035, a kind of generation changeed pertinent literatures such as method, the patent of growing the gene banana.Yet, knowing this area skill person knows, gene engineering is grown in different plant species or heterogeneic commentaries on classics, gene transmits (deliver) mode, can grow gene structure factors such as (gene structure) because of the genome (genome) or the different commentaries on classics of different plant species, gene is grown in the influence commentaries on classics, gene is sent to the success ratio on the organism, person very more needs requiredly according to different genes or different plant species, changes the mode of growing gene or transmitting gene with improvement.Therefore, desire of the present invention is grown interferential RNA in banana by commentaries on classics, with the biosynthesizing of reduction ethene, and then reaches the purpose that prolongs the fresh preservation phase.
Summary of the invention
Purpose of the present invention promptly is to provide a kind of interferential RNA that utilizes to prolong the constituent of banana fresh-keeping phase, and this constituent comprises interferential RNA, changes plantation technology by gene this interferential RNA commentaries on classics is grown in banana, in order to the biosynthesizing of inhibition/reduction ethene.
An of the present invention purpose is to be to provide a kind of interferential RNA that utilizes to prolong the method for banana fresh-keeping phase, this method by commentaries on classics grow interferential RNA in banana to reduce banana acc oxidase expression of gene, and then the biosynthesizing of inhibition/reduction ethene, to prolong the banana fresh-keeping phase.
Another object of the present invention is to be to provide a kind of banana acc oxidase control combination thing, and this control combination thing comprises interferential RNA, in order to inhibition/reduction banana acc oxidase expression of gene.
Another purpose of the present invention is to be to provide the novelty commentaries on classics of banana to grow genetic method, this commentaries on classics is grown genetic method and is comprised by banana male inflorescence institute inductive callus cell or be material by banana fruit hand substance institute's inductive body protoblast or by the apical meristem institute inductive body protoblast of banana, carry out the gene commentaries on classics and grow, grow the gene banana to obtain changeing.
A further object of the present invention is to be that growing genetic method by the commentaries on classics of novelty grows the commentaries on classics of banana acc oxidase control combination thing in banana, expresses with the mRNA that suppresses the banana acc oxidase, and then the ethene biosynthesizing of inhibition/reduction banana.
A kind of interferential RNA that utilizes that can reach the foregoing invention purpose comprises to prolong the constituent of banana fresh-keeping phase:
At least a interferential RNA, gene change grows expression vector and pharmaceutically acceptable supporting agent; Wherein this interferential RNA is to be connected in this gene to change the 3 ' end of growing the expression vector promotor, being implemented in gene with the banana gene order of antisense strand-intron-positive-sense strand in regular turn changes and grows expression vector, and this interferential RNA is in order to suppress in the banana genetic expression with ethene biosynthesizing relevant enzyme;
Wherein this interferential RNA has the sequence as SEQ ID No:1, and the mRNA that can suppress banana acc oxidase-1A (MAO1A) and acc oxidase-1B (MAO1B) in banana simultaneously expresses, and then reduces the biosynthesizing amount of ethene;
Wherein this interferential RNA has the sequence as SEQ ID No:2, can express in order to the mRNA that suppresses banana acc oxidase-2 (MAO2) in banana, and then reduce the biosynthesizing amount of ethene;
The sequence of above-mentioned interferential RNA makes up in the mode of antisense strand-intron-Shunyi chain, wherein this antisense strand, intron or Shunyi chain correspond respectively to the mRNA sequence of banana target gene, have at least 80% sequence complementarity (complementary), or at least 90% sequence identity (Identity).
Wherein this supporting agent can be water or all kinds of suitable damping fluid, can make interferential RNA or its expression vector easy handling, be easy to preserve and comparatively stable difficult degraded.
The present invention further provides a kind of banana acc oxidase control combination thing (control cassette), comprise:
Above-mentioned interferential RNA; And
Gene changes grows expression vector;
Wherein interferential RNA is to be connected in this gene to change the 3 ' end of growing the expression vector promotor, and this promotor is to start the Transcription (transcription) of this interferential RNA in the banana that contains banana acc oxidase control combination thing.
Wherein above-mentioned gene changes grows expression vector, includes but not limited to: pBI101, pBI121, pBIN 19 (ClonTech), pCAMBIA1301, pCAMBIA1305, pGREEN (GenBank Accession No:AJ007829), pGREEN II (GenBank Accession No:EF590266) (www.pGreen.ac.uk), pGreen0029 (John Innes Centre).
The male inflorescence of getting banana places the generation of appropriate culture medium with evoked callus; After treating that callus generates, get callus cell and set up the suspended culture cell that homogenizes with appropriate culture medium; The back Agrobacterium (comprise the gene commentaries on classics and grow expression vector, this carrier can be expressed above-mentioned interferential RNA in plant) that will make the transition changes and grows in callus cell (Agrobacterium mediator method); Behind suitable incubation time, screen these plant to contain suitable microbiotic substratum again, to filter out the plant that successfully commentaries on classics is grown; The plant of screening back survival is replaced in appropriate culture medium inducing with the differentiation of carrying out the body embryo, grow thickly bud and root.
The present invention provides fruit hand substance (fruit finger primordia) or the apical meristem with banana to change the method for growing material as gene in addition, comprises:
Really hand substance or apical meristem place appropriate culture medium to induce the organizer protoblast earlier; The back Agrobacterium (comprise the gene commentaries on classics and grow expression vector, this carrier can be expressed above-mentioned interferential RNA in plant) that will make the transition changes and grows in body protoblast (Agrobacterium mediator method); Behind suitable incubation time, screen these plant to contain suitable microbiotic substratum again, to filter out the plant that successfully commentaries on classics is grown; The plant of screening back survival is replaced in appropriate culture medium inducing with grow thickly bud and root.
The mode that above-mentioned commentaries on classics is grown includes but not limited to: the Agrobacterium mediator method, gene recombined virus infects method, the sub-carrier that jumps changes the method for growing, particle gun changes the method for growing, electroporation, microinjection, the pollen tube method, the liposome media changes the method for growing, the ultrasound media changes the method for growing, the silicon carbide fiber media changes the method (silicon carbide fiber-mediated transformation) of growing, electrophoretic method (electrophoresis), laser microbeam (laser microbeam), macrogol (polyethylene glycol, PEG), calcium phosphate changes the method for growing, DEAE-dextran changes the method etc. of growing.
Term " plant is grown in commentaries on classics " intention is grown effect by commentaries on classics, allogenic gene is changeed grow in the target plant body, and then change the genome composition that plant materials is grown in commentaries on classics, makes allogenic gene can be present in the target commentaries on classics and grows among plant materials and the offspring thereof.
Term " genetic expression " intention mRNA or protein expression.
The method of a kind of prolonging shelf life of banana by using gene engineering technology provided by the present invention, constituent and application thereof when comparing mutually with other prior art, have more following advantage:
1. a kind of RNA interference composition and method thereof that prolongs the banana fresh-keeping phase provided by the present invention can effectively be controlled the ethene biosynthesizing of banana, and generally the after ripening of banana more delays, so more can prolong the banana fresh-keeping phase.
2. a kind of RNA interference composition and method thereof that prolongs the banana fresh-keeping phase provided by the present invention, except can effectively controlling the ethene biosynthesizing of banana, and still can pass through the artificial ripening mode, control its sophisticated time, therefore, can significantly promote the economic worth of banana, the time-histories of storing and transporting.
3. a kind of commentaries on classics culturing method that prolongs the banana fresh-keeping phase provided by the present invention compared to existing commentaries on classics plantation technology, is applicable to that more the gene commentaries on classics of banana is grown, and it is better that the also existing commentaries on classics plantation technology of efficient is grown in commentaries on classics.
Description of drawings
See also the detailed description and the accompanying drawing thereof of following relevant preferred embodiment of the present invention, can further understand technology contents of the present invention and purpose effect thereof; The accompanying drawing of relevant this embodiment is:
Figure 1A-C is the structure flow process of gene silencing universal support pRNAi;
Fig. 2 A-F is for suppressing the structure flow process of banana after-ripening genes involved carrier pBI121-2AnS and pBI121-1AnS;
Fig. 3 changes screening of growing and the regenerative scenario of changeing plant for banana carries out Agrobacterium;
A: through Agrobacterium change grow after in the cell that places screening culture medium; B: the cell mass on substratum; C: body embryonic development situation; D: body embryo enlarged view; E: in the cultivation situation of inducing on the seedling generation substratum; F: regenerated seedling;
Fig. 4 is for changeing the banana plant growth situation of growing reticent banana after-ripening genes involved carrier pBI121-2AnS;
A, C, E, G and I be not for changeing the banana plant control group of growing; B, D, F, H and J, the banana plant of growing for commentaries on classics; K is not for changeing the banana of growing; L is for changeing the banana of growing;
Fig. 5 is the Southern hybridization analysis of banana gene silencing transformed strain genomic dna;
The T-DNA of A:pBI121-2AnS zone makes up and hybridizes used probe; B: with the Mh-ACO2 gene fragment is the Southern results of hybridization of probe.
Fig. 6 grows the Mh-ACO2 genetic expression that is for changeing the northern any of several broadleaf plants commentaries on classics of growing reticent banana after-ripening genes involved carrier;
It is the RT-PCR analytical results of blade that A grows for each commentaries on classics; B grows the RT-PCR that is for different commentaries on classics and expresses quantitative column diagram, and its base of calculation is 100% with the expression amount of WT;
Fig. 7 changes for changeing the northern any of several broadleaf plants grow reticent banana after-ripening genes involved carrier that to grow be the Mh-ACO2 gene expression amount of 2AS-79 Different Organs;
A is that WT and 2AS-79 change that to grow be the RT-PCR analytical results of each organ; B expresses quantitative column diagram for the RT-PCR of each organ, and its base of calculation is 100% with the expression amount of WT;
Fig. 8 changes for changeing the northern any of several broadleaf plants grow reticent banana after-ripening genes involved carrier that to grow be that the siRNA of 2AS-79 different sites expresses situation; The probe of hybridization is the Mh-ACO2 gene fragment, and is control group with synthetic 25nt and 17nt specificity Mh-ACO2 gene fragment; WT-O: do not change the ovary of growing banana; The gynoecium that is is grown in the 79-Pi:2AS-79 commentaries on classics; The stamen that is is grown in the 79-S:2AS-79 commentaries on classics; The petal that is is grown in the 79-Pe:2AS-79 commentaries on classics; The ovary that is is grown in the 79-O:2AS-79 commentaries on classics; The bract that is is grown in the 79-B:2AS-79 commentaries on classics;
Fig. 9 is for changeing the northern any of several broadleaf plants fruit after-ripening pericarp annesl situation of growing reticent banana after-ripening genes involved carrier;
A: the process of pericarp annesl; B: pericarp annesl index map; Standard scale (Bars)=5cm;
Figure 10 changes for respiration rate and the ethene growing amount that changes the northern any of several broadleaf plants fruit after-ripening of growing reticent banana after-ripening genes involved carrier;
A: the respiration rate of fruit after-ripening process changes; B: the ethene growing amount of fruit after-ripening process changes;
Figure 11 is for changeing the pericarp annesl situation after the northern any of several broadleaf plants accelerating ripening of fruit of growing reticent banana after-ripening genes involved carrier is handled;
A: the process of pericarp annesl; B: pericarp annesl index map; Standard scale (Bars)=5cm;
Figure 12 changes for respiration rate and the ethene growing amount that changes after the northern any of several broadleaf plants accelerating ripening of fruit of growing reticent banana after-ripening genes involved carrier is handled;
A: the respiration rate after the accelerating ripening of fruit is handled changes; B: the ethene growing amount after the accelerating ripening of fruit is handled changes.
Embodiment
The present invention is demonstrated with the following examples to illustrating, but the present invention is not limited by following embodiment.Reagent such as nothing that the present invention is used specialize, and are all the commercially available material that is easy to obtain.
1. the structure of gene silencing universal support pRNAi
(1) plasmid material
A.pUC19 plasmid: total length 2.682kb contains intestinal bacteria screening-gene AmpR (GenBank accession no.L09137).
B.pU35STgfp plasmid: have 2X En CaMV 35S promoter, green fluorescent protein (GFP) gene, nopaline synthase (NOS) gene terminator.This original plasmid source is PNAS (1996) 93:5888-5893, behind the 1854bp, inserts and singly cuts in the pUC19 carrier of HindIII under being sure to so that restriction enzyme HindIII is single, promptly becomes pU35STgfp.
(2) gene source
Make up the employed intron of pRNAi carrier (intron), the source is first intron (its sequence such as SEQ ID No:17) of banana acc oxidase gene M AO1A (GenBank accession no.AF030411) and gene M AO1B (GenBank accession no.AF030410), and the sequence of this digenic first intron is identical.
(3) extraction of banana genome DNA
The sample of clip 1g, after liquid nitrogen grinds, adding 15ml extraction buffer (100mM Tris-HCl, pH 8.0; 50mM EDTA; 500mM NaCl), add 1ml 20%SDS again, in 65 ℃ leave standstill 10 minutes after, add 5ml 5M Potassium ethanoate (potassium acetate, KOAc), in leaving standstill 20 minutes on ice.With 25,000xg in 4 ℃ centrifugal 20 minutes, behind nylon net filter, add 10ml Virahol (isopropanol) precipitation after 30 minutes, in 4 ℃ 20, centrifugal 15 minutes of 000xg, remove supernatant liquor after, air-dry throw out.Adding 0.7ml High TE (50mM Tris-HCl, pH 8.0; 10mMEDTA) after the dissolving, add 75 μ l 3M sodium-acetates (sodium acetate, NaOAc) and after 500 μ l Virahols (isopropanol) mix, in 4 ℃ with Eppendorf centrifuge centrifugal 10 minutes.Via air-dry after the 70% and 100% alcohol desalinization of soil by flooding or leaching, standby respectively with 100 μ lTE (pH 8.0) dissolving.
(4) make up flow process
Shown in Figure 1A, at first pUC19 being carried out enzyme with EcoR I and Sal I cuts, remove pUC19 and go up most MCS (Multiple cloning site) zone, carrying out flush end (blunt end) with the Klenow enzyme again handles, through electrophoretic separation, reclaim the 2.6kb fragment, engage (self-ligation) voluntarily, obtain intermediate carrier pUC19m with this fragment.Intermediate carrier pUC19m is carried out enzyme with restriction enzyme HindIII cut, reclaim the fragment of about 2.6kb length; PU35STLgfp is carried out enzyme with restriction enzyme HindIII cut, reclaim the 1.8kb fragment, the two is engaged reaction, the plasmid that obtains is further carried out screening with restriction enzyme and electrophoresis, to obtain intermediate carrier pUC19m-35S.See also shown in Figure 1B, continuous is masterplate with banana genome DNA, by PCR first intron with synthetic banana acc oxidase gene M AO1, as follows in order to the Oligonucleolide primers (oligonucleotide primers) of synthetic this first intron:
Forward primer IMAO-1:(contains the KpnI restriction enzyme and cuts the position)
KpnI
Reverse primer IMAO-2:(contains the BamHI restriction enzyme and cuts the position)
(SEQ?IDNo：4)
BamHI
Synthesize resulting dna fragmentation by IMAO-1 and IMAO-2 primer with PCR, carrying out restriction enzyme with restriction enzyme Kpn I, BamHI cuts, reclaim the fragment (MAO1 intron1) of about 0.12kb, pUC19 (carrying out restriction enzyme with restriction enzyme KpnI, BamHI cuts) after cutting with enzyme, after engaging reaction, obtain the middle interstitial granules pUIN of 2.8kb.Shown in Fig. 1 C, then middle interstitial granules pUC19m-35S is carried out restriction enzyme with limiting enzyme EcoRI and XbaI and cut, reclaim the 3.6kb fragment; In addition middle interstitial granules pUIN being carried out restriction enzyme with limiting enzyme EcoRI, XbaI cuts, first intron of MAO1 gene is downcut, reclaim the 0.13kb fragment, above-mentioned two fragments that obtain (first intron fragment of MAO1 gene, and the pUC19m-35S of enzyme after cutting) are engaged reaction, obtain the reticent universal support pRNAi (Fig. 1 C) of RNA.
2. the structure of the reticent structure of the RNA that reticent banana MAO2 expresses
At first extract total RNA of banana, its method is as follows: the clip vegetable material is ground into powder with liquid nitrogen.Add 20mL65 ℃ and extract damping fluid (Extraction buffer) (2M NaCl, 25mM EDTA, pH 8.0,100mM Tris-HCl, spermidine (spermidine) 0.5g/L, 3% cetyl trimethyl tryptophane bromide (Hexadecyl trimethyl-ammoniumbromide), 3% polyvinylpyrrolidone (polyvinylpyrrolidone-40), 0.4%2-mercaptoethanol (2-mercaptoethanol) after stirring with clarifixator, was handled 10 minutes in 65 ℃.Add equivalent CI (chloroform: primary isoamyl alcohol=49: 1) centrifugal behind the mixing, extract supernatant liquor once again after, add the 8M LiCl of 1/3 times of volume, put 4 ℃ of precipitations and spend the night.Remove supernatant liquors with 4 ℃ of centrifugal backs,, add isopyknic CI concussion and mix the several seconds with 0.5%SDS suspension RNA, with 4 ℃ centrifugal after, get supernatant liquor again, add 100% alcohol of 2 times of volumes, place-20 ℃ of precipitations.Remove supernatant liquor with 4 ℃ of centrifugal backs more afterwards.After adding 70% alcohol of 500 μ l, remove supernatant liquor after centrifugal in 4 ℃.After adding 100% alcohol of 500 μ l, remove supernatant liquor after centrifugal in 4 ℃.Air-dry RNA precipitation.RNA is dissolved in an amount of DEPC water, and quantitatively the back is standby to carry out concentration.
The structure flow process of pRNAi-2AnS plasmid (antisense-sense) sees also shown in Figure 2:
Step 1:
See also shown in Fig. 2 A, at first the total RNA with banana is a masterplate, use One-Step RT-PCR Kit (GeneMark) to react, comprise 0.1 μ g/ μ L template ribonucleic acid in the reaction solution, 50ng/ μ L primer, 1X reaction mixture (Reaction Mix), 1X enhanser (Enhancer), 2% enzyme mixed solution (Enzyme Mix).Temperature of reaction be 50 ℃ 30 minutes, 94 ℃ 2 minutes, afterwards 94 ℃ 30 seconds, 59 ℃ 30 seconds, 72 ℃ were carried out 35 circulations in 1 minute.React again at last 72 ℃ 10 minutes, place 4 ℃ standby.Employed primer is as follows:
Forward primer MAO25L:(contains the BamHI restriction enzyme and cuts the position)
Bam?HI
EcoRI
The nucleotide fragments of the about 0.14bp of PCR composition length (MAO2 fragment), again the MAO2 fragment being carried out enzyme with BamHI and EcoRI cuts and reclaims, engage reaction with the pUC18 (cutting) after enzyme is cut, obtain containing plasmid pUC18-m2p (Fig. 2 A) of MAO2cDNA 139bp with BamHI and EcoRI enzyme.
Step 2:
Plastid pRNAi carries out enzyme with XbaI and cuts, and handles with Klenow enzyme flush end (blunt) again, carries out enzyme with BamHI again and cuts, and reclaims the fragment of 3.8kb; The pUC18-m2p of step 1 gained is carried out enzyme with EcoRI again cut, continuously handle, carry out enzyme with BamHI again and cut, reclaim the fragment of 0.14kb with Klenow enzyme flush end.Dna fragmentation (pUC18-m2p and pRNAi) after aforementioned two enzymes are cut back to close engages reaction, obtains containing the segmental plasmid pRNAi-2xnS of justice (sense) MAO2 Partial cDNA (Fig. 2 B).
Step 3:
Plasmid pRNAi carries out enzyme with KpnI and cuts, and after handling with Klenow enzyme flush end, carries out enzyme with EcoRI again and cuts, and reclaims the fragment of 3.8kb; The pUC18-m2p of step 1 gained is carried out enzyme with BamHI cut, handle with Klenow enzyme flush end again, then carry out enzyme and cut, the fragment of recovery 0.14kb with EcoRI.Dna fragmentation (pUC18-m2p and pRNAi) after aforementioned two enzymes are cut back to close engages reaction, obtain containing antisense (antisense) MAO2 Partial cDNA segmental in the middle of plastid pRNAi-2Asn (Fig. 2 C).
Step 4:
The pRNAi-2Asn of step 3 and the pRNAi-2xnS of step 2 are all cut with XhoI and SacII enzyme, after the fragment that reclaims 150bp and 3.8kb respectively engages reaction, obtain containing the MAO2 Partial cDNA Sequence: antisense (antisense) MAO2 (antisense strand MAO2 fragment sequence such as SEQ ID No:18), the plasmid pRNAi-2AnS of justice (sense) MAO2 (positive-sense strand MAO2 fragment sequence such as SEQ ID No:19) (pRNAi-2AnS has in regular turn (5 ' end is to 3 ' end): the structure sequence of antisense MAO2-first intron-just MAO2, as SEQ ID No:2) (Fig. 2 D).
Step 5:
Plasmid pRNAi-2AnS is cut with the HindIII enzyme, reclaim 1.4kb, engage reaction, obtain the Agrobacterium commentaries on classics and grow with plasmid pBI121-2AnS (Fig. 2 E) with the pBI121 that cuts through the HindIII enzyme (GenBankaccession no.AF485783).
3. the structure of the reticent structure of the RNA that reticent banana MAO1 expresses
The structure flow process of pRNAi-1AnS carrier (antisense-sense) with the construction strategy of MAO2, does not wherein exist together and grows the segmental primer difference of MAO1 for PCR selects, and is as follows:
BamHI
EcoRI
See also the MAO2 construction strategy, to obtain containing the MAO1 Partial cDNA Sequence: antisense MAO1 (antisense strand MAO1 fragment sequence such as SEQ ID No:20), the carrier pBI121-1AnS of justice MAO1 (positive-sense strand MAO1 fragment sequence such as SEQ ID No:21) (has in regular turn (5 ' end to 3 ' end): the structure sequence of antisense MAO1-first intron-just MAO1, as SEQ ID No:1, because the MAO1A of banana and MAO1B tool height keep (conserved) sequence, so MAO1 interferential RNA of the present invention, has sequence through above-mentioned structure gained, as SEQ ID No:1, can suppress the genetic expression of MAO1A and MAO1B simultaneously) (Fig. 2 F).
The foregoing description only is a preferable exemplary illustration, and non-in order to limit building mode of the present invention, other construction strategy that is suitable for also is contained among the present invention.
The gene of embodiment 2 bananas changes plantation technology and flow process
1. the commentaries on classics of Agrobacterium mediator method gene is grown
Change the A and the B method of growing, with reference to revising from Ma Suxuan (1988), its step is as follows:
The A method
(1) vegetable material and other material
Banana north any of several broadleaf plants kind (Musa spp.cv.Pei Chiao, AAA group).By male inflorescence institute inductive suspended culture cell, and callus (Callus).Following material is all commercially available getting.
(2) change the media of growing
The Agrobacterium product that use are LBA4404 (Hoekema et al., 1983), pBI121-2AnS or pBI121-1AnS plasmid that conversion (transformation please refer to Molecular Cloning) embodiment 1 is constructed.
(3) the banana gene changes culturing method
Step 1: the inducing of callus
The male inflorescence of getting banana north any of several broadleaf plants kind (Musa spp.cv.Pei Chiao, AAA group) places inducing culture (callus inducing medium is as table 1), with the generation of evoked callus.
The composition of table 1 callus inducing medium
Constituent concentration
MS?salt 1X
VB1-HCl(Thiamine-HCl) 0.1～1mg/L
Nicotinic acid (nicotinic acid) 0.5mg/L
VB6-HCl(pyridoxine-HCl)?0.5mg/L
Padil (Glycine) 2mg/L
Inositol (myo-inositol) 100mg/L
Vitamin H (Biotin) 1mg/L
IAA 1mg/L
NAA 1mg/L
2.4-D 4mg/L
Sucrose (Sucrose) 3～4.5wt%
Agar (Agar) 0.7wt%
Annotate 1: wherein 0.7wt%Agar can be the quartzy agar (Gelrite) of 0.2%～0.3wt%.
Annotate 2: the final pH of substratum is 5.3～5.7.
Step 2: suspension cell is set up
After treating that callus generates, get an amount of callus cell, with suspension culture base (as table 2) with the callus cell suspension culture, with the foundation suspended culture cell that homogenizes.
The composition of table 2 suspension culture base
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5～5mg/L
glycine， 2～5mg/L
myo-inositol 100mg/L
Biotin 1mg/L
Glutaminate (glutamine) 0～100mg/L
Malt extract (malt extract) 0～500mg/L
Proline(Pro) (proline) 0～230mg/L
Picloram (Picloram) 0～1mg/L
2.4-D 1mg/L
Annotate 1: wherein 1mg/L 2.4-D can be the hormone mixture, and this hormone mixture comprises 1mg/L IAA, 1mg/LNAA and 4mg/L 2.4-D.
Annotate 2: the final pH of substratum is 5.3～5.7.
Step 3: cultivate altogether to change and grow
Carry out gene change grow before, Agrobacterium after the transition of the single bacterium colony of inoculation earlier, YEB liquid nutrient medium (the 5g/l extractum carnis (beef extract) that contains an amount of microbiotic (the sharp secondary mycin (Rifamycin) of 50 μ g/ml kalamycins (kanamycin), 20 μ g/ml Streptomycin sulphates (streptomycin) and 100 μ g/ml) in 20ml, 1g/l yeast extract paste (yeast extract), 5g/l peptone (peptone), 5g/l N.F,USP MANNITOL (mannitol), 0.5g/l MgSO
4, pH7.5,12.5g/l agar), concussion was cultivated two days under 28 ℃, 240rpm, treated OD
600To 1.0～1.5; with bacterium liquid through 4; 000rpm (HERMLE Z363 K) is after centrifugal 20 minutes; remove supernatant liquor; grow substratum (as table 3) with common cultivation commentaries on classics again and suspend it again; standby with the suspension bacteria liquid that obtains a conversion back Agrobacterium, this conversion back Agrobacterium includes aforementioned constructed pBI121-2AnS plasmid or pBI121-1AnS plasmid.
Get an amount of callus cell or suspended culture cell, and shake common cultivation after above-mentioned suspension bacteria liquid mixes, be statically placed in 25 ℃ again and cultivated altogether 2-4 days.
Table 3 is cultivated altogether changes the composition of growing substratum
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
Biotin 1mg/L
glutamine 100mg/L
malt?extract 500mg/L
proline 230mg/L
2，4-D 1mg/L
Trimethyl-glycine (betaine) 1mM
Syringylethanone (acetosyringone) 0.1～0.3mM
Annotate 1: wherein MS salt can be SH salt.
Annotate 2: the final of substratum is pH 5.3～5.7.
Step 4: change and grow the back screening
Leave standstill common cultivation after 2-4 days, the cell that commentaries on classics is grown after the common cultivation places solid-state commentaries on classics to grow back screening culture medium (as table 4), changes the screening of growing plant.
Table 4 changes the composition of growing the back screening culture medium
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
Biotin 1mg/L
glutamine 100mg/L
malt?extract 100～500mg/L
proline 230mg/L
2.4-D 1mg/L
Lactose (Lactose) 0～0.1%
Agar 0.7％
Cefotaxime (Cefotaxime) 200mg/L
The G418 proper concn
Annotate 1: wherein 1mg/L 2.4-D can be the hormone mixture, and this hormone mixture comprises 0.05mg/L zeatin (Zeatin), 0.2mg/L 2ip, 0.1mg/L kinetin (kinetin) and 0.2mg/L NAA.
Annotate 2: wherein 0.7%Agar can be 0.2%～0.3%Gelrite.
Annotate 3: wherein the G418 of proper concn can be the Totomycin (hygromycin) of proper concn; This proper concn indication screens for harsh degree (Stringency) from low to high, and preferred embodiments is 50mg/L G418.
Annotate 4: the final pH of substratum is 5.3～5.7.
Step 5: clade embryo (Embryo)
Grow after the back screening culture medium cultivated two months with solid-state commentaries on classics, change again to regeneration culture medium (as shown in table 5) and continue to cultivate, to cell organizer embryo.
The composition of table 5 regeneration culture medium
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
Biotin 1mg/L
malt?extract 0～100mg/L
The hormone mixture
Agar 0.7wt％
The G418 proper concn
Annotate 1: wherein MS salt can be SH salt or B5 salt.
Annotate 2: wherein this hormone mixture can be (1) 0.05mg/L Zeatin, 0.2mg/L 2ip, 0.1mg/L kinetin and 0.2mg/L NAA or (2) 1mg/L BA and 0.1mg/L GA.
Annotate 3: wherein 0.7%Agar can be 0.2%～0.3%Gelrite.
Annotate 4: wherein the G418 of proper concn can be the hygromycin of proper concn; This proper concn indication screens for harsh degree (Stringency) from low to high, and a preferred embodiments is 100mg/L G418.
Annotate 5: the final pH of substratum is 5.3～5.7.
Step 6: induced bundle sprout (induce multiple shoot)
After treating cell organizer embryo, with the body protoblast dislocation induced bundle substratum (as shown in table 6) of sprouting, the inductor embryo germination bud of growing thickly, regeneration becomes seedling.
The sprout composition of substratum of table 6 induced bundle
Constituent concentration
MS?salt
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
Biotin 1mg/L
The hormone mixture
Sucrose 3wt％
Agar 0.7wt％
The G418 proper concn
Annotate 1: wherein MS salt can be 1/2MS salt or B5 salt.
Annotate 2: wherein this hormone mixture can be (1) 1mg/L BA, 0.1mg/L GA or (2) 1mg/L 2iP, 0.1mg/LGA.
Annotate 3: wherein 0.7%Agar can be 0.2%～0.3% Gelrite.
Annotate 4: wherein the G418 of proper concn can be the hygromycin of proper concn; This proper concn indication screens for harsh degree (Stringency) from low to high, and a preferred embodiments is 100mg/L G418.
Annotate 5: the final pH of substratum is 5.3～6.0.
Step 7: induce root
Treat that seedling grows to suitable size, move to again and induce root substratum (as shown in table 7), impel plant to grow up to induce its root of hair.(Fig. 3 A-F)
Table 7 is induced the composition of root substratum
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
IBA 2.5mg/L
BA 2.5mg/L
Sucrose 3wt％
Agar 0.7wt％
The G418 proper concn
Annotate 1: wherein 0.7%Agar can be 0.2%～0.3%Gelrite.
Annotate 2: wherein the G418 of proper concn can be the hygromycin of proper concn; This proper concn indication screens for harsh degree (Stringency) from low to high, and a preferred embodiments is 100mg/L G418.
Annotate 3: the final pH of substratum is 5.3～6.0.
The B method
(1) vegetable material and other material
Banana north any of several broadleaf plants kind (Musa spp.c v.Pei Chiao, AAA group).Following material is all commercially available getting.
(2) change the media of growing
The Agrobacterium product that use are LBA4404 (Hoekema et al., 1983), transform aforementioned constructed pBI121-2AnS plasmid.
(3) the banana gene changes culturing method
Step 1: inductor embryo (Embryo)
With the fruit hand substance of banana or get apical meristem, grow material as commentaries on classics.Really hand substance or apical meristem place inducing culture (as shown in table 8) to induce the organizer protoblast earlier.
Step 2: cultivate altogether to change and grow
Getting body protoblast and aforementioned conversion back agrobacterium liquid (this conversions back Agrobacterium include aforementioned constructed pBI121-2AnS plasmid or pBI121-1AnS plasmid) and inducing culture (as shown in table 8) carries out common cultivation commentaries on classics and grows processing.
Step 3: change and grow the back screening
After cultivating altogether, body is grown in commentaries on classics moved to the inducing culture (as shown in table 8) that contains 50mg/L G418 and change and grow the back screening.
Step 4: cultivate into strain
Body is grown in commentaries on classics after the screening moved to and contain antibiotic inducing culture (as shown in table 8), and grow in commentaries on classics and to handle the about commentaries on classics that can obtain bottle outlet in eight months in back and grow plant.
The composition of table 8 inducing culture
Constituent concentration
MS?salt 1X
Thiamine-HCl 0.1～1mg/L
nicotinic?acid 0.5mg/L
pyridoxine-HCl 0.5mg/L
glycine 2mg/L
myo-inositol 100mg/L
IBA 2.5mg/L
BA 2.5mg/L
Sucrose 3wt％
Agar 0.7wt％
The G418 proper concn
Annotate 1: wherein 0.7%Agar can be 0.2%～0.3%Gelrite.
Annotate 2: wherein the G418 of proper concn can be the hygromycin of proper concn; This proper concn indication screens for harsh degree (Stringency) from low to high, and a preferred embodiments is 100mg/L G418.
Annotate 3: the final pH of substratum is 5.3～6.0.
2. change the screening and growth situation of growing banana
Grow northern any of several broadleaf plants plant and compare with not changeing, the reticent northern any of several broadleaf plants transformed strain of Mh-ACO2 is during the tissue culture, after the field planting, and before about 1.5 meters of plant height, the growth situation is grown the contrast equal no significant difference of strain (Fig. 4 A-H) with changeing.Continue cultivation after about five months, change and grow the contrast strain and grow to plant height more than 3 meters, about 10 of the strong number of sheets, but all only about 2.5 meters of transformed strain plant height, and the strong number of sheets also is about 10 leaves (Fig. 4 I-L) with to contrast strain similar.
Banana changes cell colonization and is regenerated as seedling through antibiotic-screening, after utilizing GUS active mass chemical coloring process to carry out the affirmation of reporter gene, further can carry out the analysis of molecular level, present embodiment utilizes the Southern hybridization analysis, confirms that the dna fragmentation that the desire commentaries on classics is grown is integrated in the banana genome.
Get 20 μ g plant genome DNAs, utilize suitable restriction enzyme to carry out enzyme and cut, carry out electrophoretic analysis with 0.7%agarose gel.The electrophoresis colloid was handled 15 minutes twice with 0.25N HCl, and (pH 7.2,1mMNa for 1.5MNaCl, 0.5M Tris-HCl with the sex change damping fluid again
2EDTA) handled 15 minutes twice, again with neutralization buffer (1.5MNaCl, 0.5M Tris-HCl, pH7.2,1mM Na
2EDTA) handle 15 minutes twice.The intravital DNA of glue is transferred to Hybond N to be changeed on the stain film (Amersham), is UV 120mJ/cm with the condition
2Hybridization instrument (cross-linker) (Spectrolinker XL-1500) DNA be fixed in change on the stain film, place 80 ℃ of vacuum drying ovens one hour with fixed dna.
Change the stain film with prehybridization solution (prehybridization solution): [6X SSPE (20X SSPE:175.3g/L NaCl, 31.2g/L NaH
2PO
42H
2O, 7.4g/L Na
2EDTA, pH 7.4), 0.5%SDS, 5X BFP (100X BFP:2%BSA, 2%Ficoll-40,000,2%PVP-360,000), the salmon essence of 50 μ g/mL sex change (salmon sperm) DNA, 10% sulfuration dextrin (dextrin sulfate)), reacted at least two hours down at 65 ℃, add and contain the hybridization solution (hybridization solution) that radioactive rays are demarcated probe: [6X SSPE (20X SSPE:175.3g/L NaCl, 31.2g/L NaH
2PO
42H
2O, 7.4g/L Na
2EDTA, PH 7.4), 0.5%SDS, 5X BFP (100X BFP:2%BSA, 2% glycan body-40000 (Ficoll-40,000), 2%PVP-360,000), the salmon sperm DNA of 50 μ g/mL sex change, 10% dextrin sulfate], react more than 16 hours down in 65 ℃, (2X SSPE 0.1%SDS), washes under room temperature 15 minutes twice with Wash I solution, again with WashII solution (1X SSPE, 0.1%SDS) under 65 ℃, washed 15 minutes twice,, then utilize X-ray sheet (Kodak XAR film) exposure at last with the probe of the non-specificity hybridization of flush away.
Show that by the analysis of experiments result banana genome is utilized EcoRI and HindIII to carry out specificity and cut the enzyme of position and cut, and estimates to obtain two kinds of dna fragmentation sizes, is respectively 1267bp and 3,040bp, so utilize different probes, demarcate different alien genes.With the Mh-ACO2 gene fragment is that probe (utilizes restriction enzyme XhoI and Sac II to carry out double digestion with plasmid pRNAi-2ANS, obtained 160bp Mh-ACO2 gene fragment is as probe, its sequence such as SEQ ID No:9) carry out result's confirmation of hybridization analysis, contained the Mh-ACO2 gene fragment in the transformed strain genome really.Though except clip size occurring as expected, still occurred 3,040bp fragment signal, yet change in the dna fragmentation of growing not 3, the size of 040bp is so this dna fragmentation of supposition is the interior living Mh-ACO2 gene fragment (Fig. 5 A-B) in the banana genome.
1. (reverse transcription-polymerase chain reaction RT-PCR) observes commentaries on classics and grows the gene transcription restraining effect with the reverse transcription polymerase chain reaction
Be used as template with the total RNA that extracts, use One-Step RT-PCR Kit (GeneMark) to react, comprise 0.1 μ g/ μ L template ribonucleic acid, 50ng/ μ L primer, 1X Reaction Mix, 1X Enhancer, 2%Enzyme Mix in the reaction solution.Temperature of reaction be 50 ℃ 30 minutes, 94 ℃ 2 minutes, afterwards 94 ℃ 30 seconds, 59 ℃ 30 seconds, 72 ℃ were carried out 35 circulations in 1 minute.React again at last 72 ℃ 10 minutes, place 4 ℃ standby.Employed primer is as follows:
The Mh-ACO2 gene:
Forward primer MAO2-5RT:
5’-atggattcctttccggttatcgaca-3’ (SEQ?ID?No：10)
Reverse primer MAO2-3RT:
5’-attccttcatcgccttccta-3’ (SEQ?ID?No：11)
Banana actin gene:
Forward primer BACT5:
5’-tagcggacgtaccacaggtat-3’ (SEQ?ID?No：12)
Reverse primer BACT3:
5’-gtaagcaagcttctccttgat-3’ (SEQ?ID?No：13)
Utilize RT-PCR to detect Mh-ACO2 expression situation between different transformed strains, total RNA tests with the young leaves organization material.The result compares with not changeing the total RNA of banana young leaves that grows as shown in Figure 6, and the Mh-ACO2 expression amount in the transformed strain all has the situation of minimizing, and between different transformed strain, reticent effect has the difference on the degree.Grow control group Mh-ACO2 expression amount and be used as 100% not change, wherein transformed strain system numbers the 2AS-1 transformed strain, Mh-ACO2 genetic expression reduces by 79.3%, 2AS-6 reduces by 96.0%, 2AS-78 falls 86.3%, and 2AS-79 falls 54.4%, and 2AS-80 reduces by 89.2%, 2AS-82 reduces by 96.0%, and 2AS-87 reduces by 37.8% (Fig. 6 A-B).
Observe Mh-ACO2 and grow contrast strain and the reticent transformed strain of Mh-ACO2, the expression situation in tissues such as leaf, stamen, gynoecium, petal, ovary and bract in not changeing.The result grows in the control group not changeing shown in Fig. 7 A-B, the Mh-ACO2 gene, except in blade, express less, on reproductive organ such as stamen, gynoecium, petal, ovary and bract, find that all Mh-ACO2 presents a large amount of expression.Grow the contrast strain and compare with not changeing, transformed strain is on petal, stamen and gynoecium, the Mh-ACO2 gene expression amount has tangible reticent effect, in petal, suppressed 71.0% Mh-ACO2 expression, reticent effect reaches 61.5% in stamen, in gynoecium, reduced by 60.5% Mh-ACO2 expression amount, and the expression in the transformed strain blade, transformed strain is similar, and expression amount is all less, in addition, in ovary and bract, grow the contrast strain and compare with not changeing, Mh-ACO2 does not then have obvious reduction situation, expression amount with contrast strain similar (Fig. 7 A-B).
2. observe to change with small fragment RNA Northern hybridization analysis and grow the gene transcription restraining effect
The geneome RNA of drawing materials adds 10 μ L urea indicator (Urea loading dye) (8M urea (urea), 20mMEDTA-Na
2, 5mM Tris-HCl pH 7.5,0.5% tetrabromophenol sulfonphthaleins (bromphenol blue)) after, in 100 ℃ the heating 10 minutes after, place standby on ice.To contain 15% polyacrylamide (polyacryamide) colloid of 8M urea, (10X TBE is 0.9M Tris with 65 ℃ of 1X TBE of preheating, 0.9M boric acid (boric acid), 20mM EDTA) is used as electrophoresis liquid, voltage carries out electrophoretic separation with 250V, and colloid utilizes electrophoretic blotting groove (Tanan VE-186) that the RNA commentaries on classics is steeped on Hybond N nylon membrane (Amersham), and changeing the stain electrophoresis liquid is 0.5X TBE, voltage is 50V, changes stain one hour.To change the stain film shift out air-dry, with UV 120mJ/cm
2Cross-linker (Spectrolinker XL-1500) carry out crosslink after, again with 80 ℃ of vacuum-dryings 1 hour, fixedly RNA.Nucleic acid probe prepares and the isotropic substance scaling method, described in the Southern hybridization analysis of embodiment 3.
To change the stain film with prehybridization solution (5X SSPE, 50% methane amide (formamide), 0.5%SDS, 5X BFP, reacted at least two hours down at 42 ℃, add and contain hybridization solution (5X SSPE, the 0.5%SDS that radioactive rays are demarcated probe, 5XBFP, the salmon sperm DNA of 200 μ g/mL sex change, candy (dextran sulfate) gathers in 10% sulfuric acid Portugal, in reacting more than 16 hours under the room temperature, with Wash I solution (2X SSPE, 0.1%SDS), under room temperature, washed 15 minutes twice, again with WashII solution (1X SSPE, 0.1%SDS) under 42 ℃, washed 15 minutes twice, utilize X-ray sheet (Kodak XAR film) at last in-80 ℃ of exposures.
Utilize the RNA perturbation technique, reticent target gene can produce the RNA fragment of 21 to 27nt sizes, utilizes northern hybridization analysis to detect this a little small fragment RNAs, to confirm the rnai expression situation.Stamen, gynoecium, petal, ovary and bract tissue with numbering 2AS-79 transformed strain, extract total RNA, utilize RNA denaturing polyacrylamide gel body (denaturedpolyacrylamide gel) to carry out electrophoretic separation again, and utilize the cDNA of Mh-ACO2 to be used as probe and detect less than the RNA below the 100nt.The result as shown in Figure 8, the petal position at the 2AS-79 transformed strain detects the expression of siRNA, clip size is about 25-27nt, the result learns thus, what the RNA interference effect was certain is executed among the transformed strain.But at stamen, gynoecium, ovary and bract tissue, then do not detect obvious siRNA and express, this result points out, the effect of RNAi and the generation of siRNA have the differential expression (Fig. 8) between histoorgan.
1. banana after-ripening test
The test banana, after the green ripe stage, separately each really referred to clean, (vaseline) smears otch with petrolatum, and be standby behind the natural air drying.Nature after-ripening test system really refers to each to be positioned over 25 ℃, makes its natural after-ripening.The degree of banana after-ripening, general assessment mode commonly used, by observing the degree of pericarp annesl, and foundation fruit colour index (color index) is carried out classification, fruit colour is occurred by green to physiology spot, be divided into into 8 grades: the 1st grade is green (all green) entirely, be with atomic Huang (green-trace of yellow) for green for the 2nd grade, 3rd level is green more than yellow (more green than yellow), the 4th grade is yellow more than green (more yellow than green), the 5th grade is two ends green (green tip), the 6th grade is complete yellow (all yellow), the 7th grade is physiology spot appearance (yellow-flecked with brown), and the 8th grade is that spot enlarges (yellow with largebrown areas).
Shown in Fig. 9 A-B, under natural after-ripening is handled, can find not change the fruit of growing the contrast strain, after about 5 days, promptly begin to have annesl situation slowly, arrive 3rd level at about the 15th day, and after about the 20th day, arrive the 4th grade, arrived the 5th grade at the 28th day, arrive the 6th grade in the 32nd day, arrived the 7th grade on the 35th day, arrived the 8th grade on the 37th day.And the reticent transformed strain banana fruit of Mh-ACO2 look annesl situation, grow the contrast banana and compare with not changeing, after-ripening is expressed and is obviously delayed, the transformed strain pericarp began to have slow annesl after the 6th day, in the time of about the 20th day, reach 3rd level, and between 3rd level and the 4th grade, kept about 20 days, and after the 40th day, just entered the 4th grade, and reached the 5th grade (Fig. 9 A-B) at the 43rd day.
2. the banana respiration rate is measured
Respectively the single fruit of banana is referred to, through behind the weighing, place the sealing of 1L to breathe cylinder, in 25 ℃ leave standstill 1 hour after, draw the gas in the 1mL breathing cylinder, with gas chromatograph [day island proper Tianjin GC-8AIT of company, collocation thermal conductivity detector (Thermal conductivity detector, TCD)], carry out the mensuration of carbonic acid gas, separating tubing string is 1/8 " * stainless-steel tubing pillar of 6ft, fill Porapak Q in the tubing string, 80-100mesh; the tubing string oven temperature is decided to be 40 ℃; the injection port temperature is set at 80 ℃, for carrying a promoting the circulation of qi body, pressure is set in 1kg/cm with hydrogen
2The carbonic acid gas peak length of gained after gas chromatograph detects, calculate the banana respiration rate:
Respiratory rate (ml CO
2/ g/hr)=
[(sample peak height-blank peak height)/standard gas peak height * standard gas concentration (%) * 1/100 * cumulative volume (m1)]/[example weight (g) * time (hr)]
Utilize the carbon dioxide generating amount of GC mensuration nature after-ripening fruit, to calculate after-ripening fruits respiratory rate.The result before the 17th day, does not change and grows the contrast banana and change the respiration rate of growing banana shown in Figure 10 A, all presents the stable expression of low amount, and the two no significant difference after the 18th day, does not change and grows the about 0.03ml CO of contrast respiration of fruits rate
2/ g/hr began to rise, and arrived respiratory climacteric 0.05ml CO until the 27th day
2/ g/hr begins to descend then.And the transformed strain fruit still continues keeping the stably express (Figure 10 A) of low amount to last the 32nd day of measuring the date of test.
3. banana ethene growing amount is measured
Respectively the single fruit of banana is referred to, through behind the weighing, place the sealing of 1L to breathe cylinder, in 25 ℃ leave standstill 1 hour after, draw the gas in the 1mL breathing cylinder, with gas chromatograph [CHROMPACK CP9001, the collocation flame ionic detector (Flame ionization detector, FID)], separating tubing string is 1/8 " * stainless-steel tubing pillar of 6ft; fill activated alumina in the tubing string, 80-100mesh, tubing string oven temperature are decided to be 90 ℃; the injection port temperature is set at 150 ℃; the detector temperature is set 130 ℃, and for carrying a promoting the circulation of qi body, pressure is set in 20kPa with hydrogen.Combustion gases are hydrogen and air.The ethene peak length of gained after gas chromatograph detects, calculate banana ethene growing amount:
Ethene growing amount (μ l C
2H
4/ g/hr)=
[(sample peak height-blank peak height)/standard gas peak height * standard gas concentration (ppm) * cumulative volume (ml)]/[example weight (g) * time (hr)]
Utilize GC to measure the ethene growing amount of nature after-ripening fruit.The result before the 20th day, does not change and grows the contrast banana and change the ethene growing amount of growing banana shown in Figure 10 B, all present the stable expression of extremely low amount, and the expression of the two does not have difference.After the 20th day, change and grow the contrast strain and begin to generate in a large number ethene, arrive ethene at about 27 days and generate peak, about 1.7 μ lC
2H
4/ g/hr then descends afterwards fast.And transformed strain still presents the expression situation of extremely low amount, and only at about about 30 days, 0.2 μ l C has an appointment
2H
4The small amount of ethylene of/g/hr is expressed and is detected (Figure 10 B).
Banana is grown in embodiment 6 commentaries on classics of handling of accelerating the ripening
1. the banana test of accelerating the ripening
The test banana, after the green ripe stage, separately each really referred to clean, (vaseline) smears otch with petrolatum, and be standby behind the natural air drying.This after-ripening test is taked the nature after-ripening respectively and is executed the ethene two portions that accelerate the ripening outward.Nature after-ripening part really refers to each to be positioned over 25 ℃, makes its natural after-ripening.Execute the ethene part of accelerating the ripening outward, under 25 ℃, fruit be positioned among the breathing cylinder of sealing, breathe the ethene that cylinder contains concentration 500ppm, handle 24 hours after, shift out banana, in the exhausting cupboard, remove remaining ethene after, be positioned over 25 ℃ and make its after-ripening.
To be the 0th day before accelerating the ripening, be to be used as benchmark on the 1st day after accelerating the ripening.The result points out that the contrast strain banana through after accelerating the ripening was accelerating the ripening back first day, promptly begins with one day grade to rising, in the 8th grade of arrival in the 8th day.And the transformed strain fruit arrived the 2nd grade on the 3rd day at the 2nd genius beginning annesl, arrived 3rd level on the 4th day, and the annesl situation delays a little afterwards, to the 4th grade of the 6th talent's arrival, with the speed of one day rising one-level, arrived the 8th grade (Figure 11 A-B) until the 10th day afterwards.
2. the banana respiration rate after accelerating the ripening is measured
The carbon dioxide generating amount of fruit after utilizing GC mensuration to accelerate the ripening is to calculate after-ripening fruits respiratory rate.The result shows, accelerating the ripening back the 1st day, changes and grows contrast strain fruits respiratory rate, promptly rises to the 2nd day immediately and arrives peak expression, and keep stable height and breathe counting rate meter and reach, and maintains 0.13ml CO approximately
2/ g/hr began to descend by the 5th day, and respiration rate rises once again after the 6th day.The respiration rate of transformed strain is expressed, and is accelerating the ripening the 1st day, and same respiration rate has rising rapidly, rises to 0.075mlCO
2Behind/the g/hr, just descended immediately, continue to maintain about 0.04ml CO afterwards at the 2nd day
2The low respiration rate of/g/hr was expressed, and just begins to rise rapidly after the 6th day, arrived respiratory climacteric 0.11CO by the 8th day
2/ g/hr then begins to descend afterwards, then begins again after the 10th day rise once again (Figure 12 A).
3. the banana ethene growing amount after accelerating the ripening is measured
Utilize GC to measure to accelerate the ripening handle after, the ethylene production amount of after-ripening fruit.The result shows, changes the contrast strain banana of growing, and is accelerating the ripening processing after the 1st day, and the ethene growing amount promptly rose rapidly, arrived about 3 μ l C at the 2nd day
2H
4Behind/the g/hr, keep expression approximately after the 6th day, beginning was risen once again, arrived ethene growing amount peak 5.2 μ l C to about the 7th day
2H
4/ g/hr descends subsequently rapidly.And the transformed strain banana after accelerating the ripening, is still being kept and is being lower than 0.1 μ l C
2H
4The expression of/g/hr just began to rise after the 3rd day, and arrived ethene growing amount peak 3.1 μ l C at the 5th day
2H
4/ g/hr then slows down decline slowly afterwards, and maintains 2-3 μ l C
2H
4Between/the g/hr (Figure 12 B).Therefore, this commentaries on classics is grown banana and still can be handled by artificial ripening, to control its sophisticated time.
Above-listed detailed description is at the specifying of possible embodiments of the present invention, and this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implements or change, all should be contained in the claim of this case.
Sequence table
＜110〉Huang Penglin
＜120〉method of prolonging shelf life of banana by using gene engineering technology, constituent and application thereof
<160>21
<210>1
<211>388
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>1
gtctcctcga?agtccggagg?gttggacggc?cattcgttgt?cgtctcgaag?aacgaacacg 60
tcctcccagt?ccacgttgtc?caagcgttga?ccatcgcctt?ctttcaccag?tgtgtccaac 120
agctgaacgg?gtttggatcc?gcggaggttt?gccatcttca?ccccgctcct?ctcctctgct 180
ttcatggcta?tgctggtgta?aaggtactat?gttcccgatg?ctctgtatcg?tcgctcgcag 240
ctgtcgacgg?atccaaaccc?gttcagctgt?tggacacact?ggtgaaagaa?ggcgatggtc 300
aacgcttgga?caacgtggac?tgggaggacg?tgttcgttct?tcgagacgac?aacgaatggc 360
cgtccaaccc?tccggacttc?gaggagac 388
<210>2
<211>394
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>2
gccttcctat?actggtcatc?aagatcgggg?atctcagaaa?tgttggagac?ggggagatga 60
cgcaggaaaa?aggtgctttc?ccagtcgagg?tggtcgattt?ctgagtcggc?gttttccagt 120
gctttgttgg?cgaactcgga?tccgcggagg?tttgccatct?tcaccccgct?cctctcctct 180
gctttcatgg?ctatgctggt?gtaaaggtac?tatgttcccg?atgctctgta?tcgtcgctcg 240
cagctgtcga?cggatccgag?ttcgccaaca?aagcactgga?aaacgccgac?tcagaaatcg 300
accacctcga?ctgggaaagc?acctttttcc?tgcgtcatct?ccccgtctcc?aacatttctg 360
agatccccga?tcttgatgac?cagtatagga?aggc 394
<210>3
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer
<400>3
ataggtaccc?cgcggaggtt?tgccatactt?c 31
<210>4
<21129
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer
<400>4
ataggatccg?tcgacagctg?cgagcagac 29
<210>5
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer
<400>5
tatggatccg?agttcgccaa?caaag 25
<210>6
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer
<400>6
ccagaattcg?ccttcctata?ctg 23
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer
<400>7
ataggatcca?aacccgttca?g 21
<210>8
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer
<400>8
catgaattcg?tctcctcgaa?gtccg 25
<210>9
<211>160
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>9
tcgagaatta?attcgccttc?ctatactggt?catcaagatc?ggggatctca?gaaatgttgg 60
agacggggag?atgacgcagg?aaaaaggtgc?tttcccagtc?gaggtggtcg?atttctgagt 120
cggcgttttc?cagtgctttg?ttggcgaact?cggatccgcg 160
<210>10
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer
<400>10
atggattcct?ttccggttat?cgaca 25
<210>11
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer
<400>11
attccttcat?cgccttccta 20
<210>12
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer
<400>12
tagcggacgt?accacaggta?t 21
<210>13
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer
<400>13
gtaagcaagc?ttctccttga?t 21
<210>14
<211>1998
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<220>
<221>CDS
<222>(1)..(1998)
<300>
<308>Genbank/AF030411
<309>2008-07-29
<400>14
tatagattat?tagctttaag?gatataataa?tcattttatt?atactaatct?cttacaacaa 60
attaatttca?aaaactattc?aagttaatta?atatgataaa?cctcattaaa?aaaaatcttt 120
tccattaggc?gattaacaaa?aatgacttaa?cacgagttaa?ttaatgtgat?agtcaagttt 180
atgtatgtgc?ttaagctgac?agtcgactgc?tatattatct?aaaccgaact?caagataaaa 240
ttaatttcct?tcacagaatt?tgactgccac?gtttggcggc?tgctgctgtc?tgcgttgacc 300
acgcattttt?tatgcgtctg?gattgtctca?cgaagaacac?acgaaatcta?ctggggatac 360
gtgtgtctgt?tcctggttgc?ttcctcggat?tgggacccct?acaagatggc?agagcgaagc 420
ttcgcccacc?ataaataggg?cccataaccc?tctaattctc?ttcatccatc?cgagcggtaa 480
tacactccca?aagcttgcag?caatactcgc?tcctctctgt?ccattaattt?cttagttgac 540
atggcgattc?cggtcatcga?tttctccaag?ttggatggca?aggaaagggc?cgaaaccatg 600
gcccggattg?ccaatggatg?cgaggaatgg?ggattctttc?aggtttgcca?tacttcaccc 660
cgctcctctc?ctctgctttc?atggctatgc?tggtgtaaag?gtgctatgtt?cccgatgctc 720
tgtatcgtct?gctcgcagct?ggtgaaccat?gggattccgg?tcgagctgct?ggaacgcgtg 780
aagaaggtca?gctccgagtg?ctataagttg?agggaggagc?gcttcaaggg?atccaaaccc 840
gttcagctgt?tggacacact?ggtgaaagaa?ggcgatggtc?aacgcttgga?caacgtggac 900
tgggaggacg?tgttcgttct?tcaagacgac?aacgaatggc?cgtccaaccc?tccggacttc 960
gagtagagtt?cgcatgccgc?tgctgtgctc?gagttttagt?tgctacgata?gccacaaacc 1020
cgatgacgat?gtgatccgat?gattgctctg?cagggagacc?atgaaggagt?acagggaaga 1080
aatcaggaag?ctggcggaga?aaatgatgga?ggtaatggac?gagaatctgg?gcttcgaaaa 1140
gggctgcatc?aagaaagcat?tctctgggga?cggccagcac?ccgcccttct?tcggcaccaa 1200
ggtgagccac?tacccgccgt?gcccgcgcct?ggacctggtg?aagggccttc?gcgcccacac 1260
cgacgccggc?ggcgtcatcc?tcctcttcca?ggacgaccaa?gtcggcggcc?tccagatgct 1320
caaggacggc?cggtggatcg?acgttcagcc?tttggccgac?gccatcgtca?taaacaccgg 1380
agaccagatc?gaggtcctca?gcaacggtcg?ctacaagagc?gcgtggcacc?gggtgctcgc 1440
caccagccac?ggcaaccgcc?gctccatcgc?ttccttctac?aacccctccc?tgaaggcgac 1500
catcgctcca?gccgccggcg?ccgccaccga?ggaagctgcc?cctcctgctc?tgtacccaaa 1560
gtttctgttc?ggggactaca?tggacgtgta?cgcgaagcag?aagtacgagc?ccaaggaacc 1620
gagatttgag?gcagtcagag?ctatttgagg?atggagaagc?tgcgaatcta?ttgttggatt 1680
ataggaggat?ataattcatt?gtactagttt?ggtgtctcat?gcatggatta?cataattgca 1740
aacatgatct?gcatctgaat?aatggctgct?tgttctcagg?acacgcagtc?gtcaatctgt 1800
aagtcggtgt?tcagaataaa?aaccaagtgc?tatgtttcct?cgtcacatct?tctcgagttg 1860
aattcaaagg?atagaaaaaa?gggaaaacat?agttcccctt?ttgcatcaag?catcttacca 1920
cgacagctag?caaccatagt?cggcaaggca?acaacaatct?taaggaaagg?ctgtgacgaa 1980
tgcttctggt?aatacata 1998
<210>15
<211>1865
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<220>
<221>CDS
<222>(1)..(1865)
<300>
<308>Genbank/AF030410
<309>2008-07-29
<400>15
tgaagatgga?ggttttctga?tgtccaaatg?tgctcgtcga?atggttagta?ttttgttttc 60
acaagagaaa?ctaatatcat?tatatggtgt?tctttcgttg?tcactcgctt?aatttaatta 120
tatatctacc?gtgaaacttg?agaatataat?ttcccccatg?gctgttacca?tcagataagc 180
gtgactcact?ttactacgtt?aagtcttccg?atatgccatc?gtctcgtatg?gcatcggtgc 240
tggtggctgt?tcgtgagtta?agagtcccac?gtttggcggc?tgctgctgtc?tgcgttgacc 300
acgcattttt?tatgcgtctg?gattgtctca?cgaagaacac?acgaaatcta?ctggggatac 360
gtgtgtctgt?tcctggttgc?ttcctcggat?tgggacccct?acaagatggc?agagcgaagc 420
ttcgcccacc?ataaataggg?cccataaccc?tctaattctc?ttcatccatc?cgagcggtaa 480
tacactccca?aagcttgcag?caatactcgc?tcctctctgt?ccattaattt?cttagttgac 540
atggcgattc?cggtcatcga?tttctccaag?ttggatggca?aggaaagggc?cgaaaccatg 600
gcccggattg?ccaatggatg?cgaggaatgg?ggattctttc?aggtttgcca?tacttcaccc 660
cgctcctctc?ctctgctttc?atggctatgc?tggtgtaaag?gtgctatgtt?cccgatgctc 720
tgtatcgtct?gctcgcagct?ggtgaaccat?gggattccgg?tcgagctgct?ggaacgcgtg 780
aagaaggtca?gctccgagtg?ctataagttg?agggaggagc?gcttcgaggg?atccaaaccc 840
gttcagctgt?tggacacact?ggtgaaagaa?ggcgatggtc?aacgcttgga?caacgtggac 900
tgggaggacg?tgttcgttct?tcaagacgac?aacgaatggc?cgtccaaccc?tccggacttc 960
gagtagagtt?cgcatgccgc?tgctgtgctc?gagttttagt?tgctacgata?gccacaaacc 1020
cgatgacgat?gtgatccgat?gattgctctg?cagggagacc?atgaaggagt?acagggaaga 1080
aatcaggaag?ctggcggaga?aaatgatgga?ggtaatggac?gagaatctgg?gcttcgaaaa 1140
gggctgcatc?aagaaagcat?tctctgggga?cggccagcac?ccgcccttct?tcggcaccaa 1200
ggtgagccac?tacccgccgt?gcccgcgcct?ggacctggtg?aagggccttc?gcgcccacac 1260
cgacgccggc?ggcgtcatcc?tcctcttcca?ggacgaccaa?gtcggcggcc?tccagatgct 1320
caaggacggc?cggtggatcg?acgttcagcc?tttggccgac?gccatcgtca?taaacaccgg 1380
agaccagatc?gaggtcctca?gcaacggtcg?ctacaagagc?gcgtggcacc?gggtgctcgc 1440
caccagccac?ggcaaccgcc?gctccatcgc?ttccttctac?aacccctccc?tgaaggcgac 1500
catcgctcca?gccgccggcg?ccgccaccga?ggaagctgcc?cctcctgctc?tgtacccaaa 1560
gtttctgttc?ggggactaca?tggacgtgta?cgcgaagcag?aagtacgagc?ccaaggaacc 1620
gagatttgag?gcagtcagag?ctatttgagg?atggagaagc?tgcgaatcta?ttgttggatt 1680
ataggaggat?ataattcatt?gtactagttt?ggtgtctcat?gcatggatta?cataattgca 1740
aacatgatct?gcatctgaat?aatggctgct?tgttctcagg?acacgcagtc?gtcaatctgt 1800
aagtcggtgt?tcagaataaa?aaccaagtgc?tatgtttcct?cgtcacatct?tctcgagttg 1860
aattc 1865
<210>16
<211>3718
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<220>
<221>CDS
<222>(1)..(3718)
<300>
<308>Genbank/U86045
<309>2003-10-17
<400>16
aagcttcttt?cacgctactg?tagtgtctct?gttgacaaaa?ggttctttgg?cattaggaag 60
atgtagagct?tgacttctag?tttgggagtt?tgtgccctgg?tacaggaatc?ttctcactgt 120
ggtggtgaac?cagtgagtga?agtcgatgtt?aaatgctacc?gtcgacagtg?tgatcgtgtg 180
tctcaagtgg?gtttaacttg?atgattcgag?ctggcttgct?gcacttggtt?ttcttacagt 240
acgtgtgtgt?ctcacagaca?taaacatgac?tctgttgaca?acagggtgaa?ttggcatcat 300
catcctttac?ctgtccctga?tttgagctgt?agtgctgcgc?tcagccagcg?ttttatcgaa 360
gaagataaac?atgcgtgagc?ctacatgcac?caaagcttgc?cggcaagtca?tgcatagctg 420
caaacatgtg?acaggcaccg?aaccaacaat?tgaagaagat?acgataaaca?tgcgtgagcc 480
tacatgcacc?aaagcttgcc?gacaagtcat?gtttgggtgc?acaatgtgtc?ctcatcttac 540
ttgcatatct?gctgttgcac?aacagcagat?tgcatggagg?tgtgttttcc?ggcaatgcaa 600
tctttgatgt?tggttctctt?ttctctcttc?ttgcattgtt?tatagctctg?tttcttgtgc 660
tcttctttta?cgtagattca?tagcgtagct?taagttgtta?tagattacct?gttttactgg 720
gcaaacttgt?gcaacccagg?aatattccca?tgtgcatctt?cttcctgttt?tcctctgtca 780
aactgttctg?ttcatgatga?ggcagcaccg?aatctaagag?aaatatccta?atgttgattg 840
atttaacctc?ataaaacttg?aagcagaata?tgcttgccgc?tttcatgtga?tcaattgaat 900
tgtttgcttg?cttcacgaga?acaccacatt?ctgaacccat?tgctttcttg?tggccaccaa 960
ccggagaagg?gagtctatat?aactagccga?gcgaggattt?tcccatgacc?tgttcatctc 1020
acgtagagat?ggtgatttgg?ttatagttat?agcgatctat?gatcgaagaa?tgagaaaata 1080
cccagataac?ggagatccat?gcgtcaccag?atggaacctc?ggccgagtgc?ggccgagtga 1140
cactgtttgc?acaccggata?cttcatgttc?acggcaatgg?ccgacatgcc?gaacagccat 1200
cgagcgttga?atgtaaggca?ggaatggccc?atttctcaca?tacgagaggg?atacgagtgg 1260
aaagggcgct?ctaatgagct?gtgaatcgaa?acaatttcta?cctatcgatc?cctgttcttt 1320
tgatatgaag?tatagccaac?aggtcaagag?aagacgagta?cacacgcatc?gccgatgctg 1380
tgacgttact?ttctgaggtt?ggcaatttgt?cactacaatc?caagcggaag?ccatgcacgc 1440
gagcgtcgcc?atggaagaac?tcaacaacat?gatgccttcc?cgggtctcct?caaaggggag 1500
agaccgatgg?aagcagccaa?acttggtccc?cgatcgtgat?gggacgcgag?aggtggaagc 1560
aaggagggtg?gagaaccagg?ccaaaggtgg?tggggctgag?agatggccaa?ctgggtcacc 1620
ttatggaatc?ggctccgtta?cgtcttccac?tgctgttgct?ctcgtcgata?gatccttctc 1680
caactttgct?tcttcactca?tttcgtccct?cgacgtcaag?aacgcctata?aattgcctgg 1740
taatcagcag?cacctagcac?actccagata?gaaagcacaa?gtgcaatcag?ggaagaaaga 1800
gcgtgtcatg?gattcctttc?cggttatcga?catggagaag?cttttgggaa?gggagagagg 1860
agcagccatg?gagatcctcc?gagatgcttg?cgagaaatgg?ggcttctttg?aggtgctgaa 1920
gcatacataa?ctggttttgc?ttctttgaac?tatatatatt?gctaaaaatg?tactatttgc 1980
gcatgcaatc?tgtgtgtaga?ttttaaacca?tggcatctca?cattacctca?tggatgaagt 2040
ggagaaggtg?aacaaagaac?agtacaacaa?atgcagggag?caaaagttca?acgagttcgc 2100
caacaaagca?ctggaaaacg?ccgactcaga?aatcgatcac?ctcgactggg?aaagcacctt 2160
tttcctgcgt?catctccccg?tctccaacat?ttctgagatc?cccgatcttg?atgaccagta 2220
taggttgcac?gatctgatca?tgatgtcatc?ttctagcctt?gtcttttcac?cttgctcatc 2280
gtttcgtttc?ttgggacgat?gactgcgtgc?aggaaggcga?tgaaggaatt?tgctgcagcg 2340
atagagaagc?tggcagagcg?gctgctcgac?ttgctgggtg?agaacctgga?gctggagaag 2400
gggtacctga?agaaagcctt?ctctaatgga?tccaaggggc?caacctttgg?gaccaaggtc 2460
agcagctacc?cgccatgccc?gcgcccggac?ctggtgaagg?gcctgagggc?gcacaccgac 2520
gccggaggca?tcatcttgct?cttccaggac?gaccaggtca?gcggcctgca?gttcctcaag 2580
gacggcgagt?ggctggacgt?gccccccatg?cgccacgcca?tcgtcgtcaa?cctcggcgac 2640
cagctcgagg?tttgggtcct?ctttgctctc?gtttccgctg?cccgtcgtct?gtgatgttga 2700
atgcaacgag?gtctgcaggt?aatcaccaat?ggcaagtaca?agagcgtggt?gcaccgcgtg 2760
gtggctcaga?ctgatggcaa?caggatgtcg?attgcctcct?tctacaaccc?cgggagcgac 2820
gctgtgatct?tcccggcccc?cgctcttgtg?gagaaggaag?cagaggagaa?gaaggaggtc 2880
tatccgaggt?tcgtgttcga?ggattacatg?aagctctacg?tcgggcataa?gttccaggcc 2940
aaggagccaa?gattcgaagc?catgaaagcc?atggaagcag?ttgccaccca?cccaatcgct 3000
acctcttaag?tgacagcccc?caagttagtg?catgtcgctg?tacttcgcgt?taggaagctg 3060
tcgtgtatgt?ctatgcaacc?cgatggaagc?gtggtatgta?cgtgtttgag?ccttttctaa 3120
tgaagcaagt?catattatat?atatatatat?atatatatat?atatataaat?aattactctt 3180
caaaattttc?ttgaattttc?tcttttgtta?atctttttat?cccaatattt?gataggaatc 3240
caaagattaa?gaaaaaagag?tatggtaatt?aattagggat?catatatact?gttttaatca 3300
agaaaatttc?cagatttctg?gattagtggc?caaccttaag?gggattagta?ctaaaccgcc 3360
catctttacc?tatctaaaca?cagcctgccg?gaggagtcga?agaactacaa?aaccctaaac 3420
cttgactact?actcctcttc?cttccagaat?catggattct?tctcctccct?cgcatgccag 3480
atccgatcaa?gattcgctac?cgaccgcctc?cgcagctccg?gaccagacga?tcgcggacgg 3540
gggaacaacg?gtggcggcgg?acgacggagg?agaggagaag?aagggagaag?cgaatggagg 3600
aagcgaagta?gaggaggagt?gcggtttctg?cctcttcatg?aagggcggcg?ggtgcaagga 3660
tgcctttgtc?gcgtgggaga?aatgtatgca?ggaagccgag?aagcgcgacg?aggacatc 3718
<210>17
<211>104
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>17
aggtttgcca?tcttcacccc?gctcctctcc?tctgctttca?tggctatgct?ggtgtaaagg 60
tactatgttc?ccgatgctct?gtatcgtcgc?tcgcagctgt?cgac 104
<210>18
<211>137
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>18
gccttcctat?actggtcatc?aagatcgggg?atctcagaaa?tgttggagac?ggggagatga 60
cgcaggaaaa?aggtgctttc?ccagtcgagg?tggtcgattt?ctgagtcggc?gttttccagt 120
gctttgttgg?cgaactc 137
<210>19
<211>137
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>19
gagttcgcca?acaaagcact?ggaaaacgcc?gactcagaaa?tcgaccacct?cgactgggaa 60
agcacctttt?tcctgcgtca?tctccccgtc?tccaacattt?ctgagatccc?cgatcttgat 120
gaccagtata?ggaaggc 137
<210>20
<211>134
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>20
gtctcctcga?agtccggagg?gttggacggc?cattcgttgt?cgtctcgaag?aacgaacacg 60
tcctcccagt?ccacgttgtc?caagcgttga?ccatcgcctt?ctttcaccag?tgtgtccaac 120
agctgaacgg?gttt 134
<210>21
<211>134
<212>DNA
＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))
<400>21
aaacccgttc?agctgttgga?cacactggtg?aaagaaggcg?atggtcaacg?cttggacaac 60
gtggactggg?aggacgtgtt?cgttcttcga?gacgacaacg?aatggccgtc?caaccctccg 120
gacttcgagg?agac 134
Claims (2)
1. one kind is utilized interferential RNA to prolong the constituent of banana fresh-keeping phase, and it is characterized in that: described constituent comprises:
At least a interferential RNA, gene change grows expression vector and pharmaceutically acceptable supporting agent;
Wherein this interferential RNA is connected in this gene changes the 3 ' end of growing the expression vector promotor, described interferential RNA is formed the sequence shown in SEQ ID No:2, can in banana, express, and then reduce the biosynthesizing amount of ethene in order to the mRNA that suppresses banana acc oxidase-2.
2. banana acc oxidase control combination thing is characterized in that: comprise:
Interferential RNA; And expression vector is grown in the gene commentaries on classics;
Wherein this interferential RNA is connected in this gene changes the 3 ' end of growing the expression vector promotor, and this promotor can start the Transcription of this interferential RNA in the banana that contains banana acc oxidase control combination thing;
Described interferential RNA is formed the sequence shown in SEQ ID No:2, can express in order to the mRNA that suppresses banana acc oxidase-2 in banana, and then reduce the biosynthesizing amount of ethene.
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| Title |
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| 卢柏昌.香蕉转殖反义ACC合成酶及氧化酶基因之研究.《台湾大学园艺学研究所硕士学位论文》.2002,21－24. * |
| 吴静等.香蕉ACC合成酶反义基因转化香蕉的研究.《分子植物育种》.2007,第5卷(第4期),497-501. * |
| 林宜佑.应用RNA干扰技术抑制香蕉ACC氧化酶基因表现之研究.《台湾大学园艺学研究所硕士学位论文中文摘要》.2004,1－4. * |
| 王鸿鹤.ACC氧化酶反义基因转化香蕉(Musa spp.)的研究.《中山大学博士学位论文》.2002,全文. * |
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