CN101665786B - Method for adjusting starch composition of root crops - Google Patents
Method for adjusting starch composition of root crops Download PDFInfo
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- CN101665786B CN101665786B CN2008100425453A CN200810042545A CN101665786B CN 101665786 B CN101665786 B CN 101665786B CN 2008100425453 A CN2008100425453 A CN 2008100425453A CN 200810042545 A CN200810042545 A CN 200810042545A CN 101665786 B CN101665786 B CN 101665786B
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Abstract
The invention relates to a method and a substance for adjusting starch composition of root crops. The invention discloses polynucleotide constructs which is expressed by specific interference granule bound starch synthase I (GBSSI) and can be processed after being introduced into plant., and a brand-new way for changing the starch quality of the plant by interrupting the expression of GBSSI genes based on micro-molecular RNA interface technology to achieve the aim of adjusting the starch composition in the plant.
Description
Technical field
The invention belongs to biotechnology or phytology field, be specifically related to regulate material and the control method that root crop starch is formed.
Background technology
Starch (starch) is the reserve substance of higher plant, algae and some microorganism cellss; It is the white powder of no color or smell; Extensively be present in each kind of plant; Especially abundant with seed (like rice, wheat, corn) and piece root (like cassava, sweet potato, yam) content, can be used as the raw material of producing starch in the industry, for example can get by extracting in the amyloid materials such as corn, sweet potato, yam, wild acorn nut, the root of kudzu vine.Starch is used for paste producing essence, SANMALT-S, glucose in the industry except that edible, also be used to modulate the starching of printing paste, textiles, the gluing of paper, the compacting of medicinal tablet etc.
Starch is the polysaccharide that is formed by many glucose molecule condensations, and two kinds of straight chain and side chains are arranged.Amylose starch is made up of the glucose molecule that α-1,4 glycosidic link connects, and is linear chain; Pulullan has α-1,6 glycosidic link to connect in bifurcation, and its linear fraction also is that α-1,4 connects.General starch is the mixture of straight chain and pulullan, and amylose starch and pulullan shared ratio in starch is different with the kind of plant, contains in the plant of starch at majority, contains 20～30% amylose starchs, 70～80% pulullan usually.In mammiferous digestive tube, starch is hydrolyzed to glucose through effects such as glycase, maltin, is absorbed and used.
At present, it is outstanding to be with plant piece root (like tapioca root) that the starch processing industry of raw material occupies ratio, but the processing of various products has proposed diversified demand to raw-material starch quality (like the content ratio of amylose starch and amylose starch).Rely on traditional breeding method to change starch quality, length consuming time needs a large amount of manpower and materials, is difficult to satisfy the eager demand of starch processing industry.
Those skilled in the art have attempted regulating the composition and the content of starch in the plant through regulating starch synthetic oligogene, yet have multiplely with the synthetic relevant gene of starch, and how finding a kind of suitable target gene is to be worth the problem furtherd investigate; In addition, how finding suitable method to regulate genes involved also is to be worth further investigation.
Summary of the invention
The material and the control method that the object of the present invention is to provide root crop starch to form.
In first aspect of the present invention, a kind of isolating polynucleotide are provided, described polynucleotide are parts of particle mating type amylosynthease I (GBSSI) gene, its length is 100-500bp; Preferably 150-250bp; Better about 180-210bp.
In another preference, described polynucleotide are:
(a) nucleotide sequence shown in position, (1371 ± 10)～(1570 ± 10) among the SEQ ID NO:1;
(b) with the sequence that (a) limits complementary sequence basically.
In another preference, described polynucleotide are: the sequence shown in position, (1371 ± 5)～(1570 ± 5) among the SEQ ID NO:1.
In another preference, described polynucleotide are: the nucleotide sequence shown among the SEQ ID NO:1 the 1371st～1570.
In second aspect of the present invention, the purposes of described polynucleotide is provided, be used for preparing the construction that reduces the plant amylose content.
In the third aspect of the invention, a kind of construction is provided, said construction contains the structure shown in the formula (I):
Seq
Forward-X-Seq
OppositelyFormula (I),
In the formula (I),
Seq
ForwardBe described polynucleotide, Seq
OppositelyFor with Seq
ForwardBasically complementary polynucleotide;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and said intervening sequence and Seq
ForwardAnd Seq
Instead ToNot complementary.
In another preference, the structure shown in the formula (I) forms the secondary structure shown in the formula (II) after changing vegetable cell over to:
In the formula (II), Seq
Forward, Seq
OppositelyWith the definition of X such as above-mentioned,
‖ is illustrated in Seq
ForwardAnd Seq
OppositelyBetween complementary relationship basically.
In another preference, itself does not constitute complementary duplex structure described intervening sequence, and its length is at 50-90b (preferably 60-85bp; That better is 65-75bp).
In another preference, described intervening sequence has the sequence shown in the SEQ ID NO:2.
In fourth aspect of the present invention, a kind of carrier is provided, described carrier contains described construction.
In another preference, described carrier is a binary vector, contains the promotor that links to each other with described construction operability.Preferable described promotor is CaMV35S promotor (constitutive promoter) and/or P54 promotor (cassava vascular-specific expression promotor, adjustable Glutamic acid-rich protein c54 genetic expression, Genbank accession number: AY217353).
In another preference, described carrier is the pCAMBIA1300 carrier.
Aspect the of the present invention the 5th, the purposes of said construction is provided, be used for importing to plant, suppress particle mating type amylosynthease I and express, thereby amylose starch is synthetic in the inhibition plant.
In another preference, described plant is root crop, tuberous plant or spermatophyte;
In a preference, described plant is selected from (but being not limited to): cassava, sweet potato, yam, Chinese yam, taro, the root of kudzu vine.
Aspect the of the present invention the 6th, a kind of method that reduces amylose content in the plant is provided, said method comprises: with described construction or described carrier transfection in plant.
In another preference, said method comprises:
(1) Agrobacterium of carrying described construction or described carrier is provided;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make said construction be transferred to vegetable cell, tissue or organ; With
(3) select vegetable cell, tissue or the organ that changes said construction over to, regeneration plant.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the recombinant vectors pC-35S-GBSSI-RNAi of inventor's structure and the part collection of illustrative plates structure of pC-P54-GBSSI-RNAi.
Fig. 2 has shown that RT-PCR analyzes GBSSI expression of gene in the transgenic cassava.With cassava c15 gene is confidential reference items, and the expression of GBSSI receives inhibition in various degree in the transgenic strain.Wherein, swimming lane M:Marker; Swimming lane WT: the expression of wild-type cassava GBSSI; Swimming lane 1-8: transgenic is the expression of homophyletic chain timbers potato GBSSI not.These strain systems are called TG-1, TG-2, TG-3, TG-4, TG-5, TG-6, TG-7 and TG-8 respectively.
Fig. 3 has shown that wild-type and transgenic cassava TG-7 strain are that the difference that iodine dyes shows that the starch composition of transgenic cassava changes, and amylose content obviously descends.Wherein, amylose starch is met iodine and is presented mazarine, and pulullan presents red-brown.
Fig. 4 has shown that iodine dyes the composition that starch small grain shows starch small grain and obviously changes.Wherein, the left side appears black-and-blue for the iodine of wild-type cassava tissue dyes the result; The right presents reddish-brown for the iodine of GBSSI-RNAi transgenic cassava TG-7 strain system tissue dyes the result.Magnification: 400 times of light microscopics.
Fig. 5 has shown the mensuration wavelength of graphing method selection starch.
Embodiment
The inventor has found a kind of novel method that can regulate root crop starch composition well through extensive studies.Disturb particle mating type amylosynthease I (Granule-Bound Starch Synthase I through in plant, forming microRNA; GBSSI); Can regulate the biosynthesizing level of direct-connected starch or pulullan effectively, and the transgenic plant stabilization characteristics of genetics that obtains.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, described " operability connection " or " operability link to each other " or " being operably connected " interchangeable use refer to functional spatial disposition of two or more nucleic acid region or nucleotide sequence.For example: promoter region is placed in the specific position with respect to the goal gene nucleotide sequence, makes transcribing of nucleotide sequence receive the guiding of this promoter region, thereby promoter region quilt " operability connection " is to this nucleotide sequence.
As used herein, described " plant " is meant the plant that contains the GBSSI gene, normally root crop or plant seed, and described plant includes but not limited to: potato class plant, tuberous plant, kind subclass plant.For example said plant can be selected from: cassava (Manihot esculenta), sweet potato (Ipomoea batatas), yam (Solanum tuberosum), Chinese yam (Dioscorea sp.).More particularly, described plant is cassava (Manihot esculenta).
As used herein, the sequence of " complementation " typically refers to the sequence that converts the sequence of 5 '-3 ' direction into its 3-5 direction and (like 5ATCG3 → GCTA), and then gets its complementary sequence (like GCTA → 5CGAT3)." complementary basically " is meant that the sequence of two sections Nucleotide is enough complementary, can interact with a kind of foreseeable mode, as forming secondary structure (like loop-stem structure).It is complementary that the nucleotide sequence of two usually, " basically complementary " has 70% Nucleotide between mutually at least; Preferably, having 80% Nucleotide at least is complementary; Preferred, having 90% Nucleotide at least is complementary; Further preferred, having 95% Nucleotide at least is complementary; As 96%, 98%, 99% or 100%.
As used herein; " stem ring " structure is meant a kind of nucleic acid construct thing; It can form a kind of double-stranded region (stem that comprises; Contain polynucleotide of the present invention) secondary structure, described double-stranded region is formed by two zones (being positioned on same the poly-nucleotide chain) of this construction, the both sides of two double-stranded parts of regional apportion; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. the strand zone.Even two zones of this construction are not complete complementary, the double-stranded part of Nucleotide also can keep double-stranded state.For example; Insertion, disappearance, replacement etc. can cause not complementary or this zonule self the formation loop-stem structure of a zonule or the secondary structure of other form, yet these two zones still can be complementary basically; And in foreseeable mode, interact, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, and usually behind the nucleic acid that has obtained a nucleotide sequence with primary structure, those skilled in the art can confirm whether this nucleic acid can form loop-stem structure.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by constituting ", " basically by constituting " and " by constituting "; " mainly by constituting ", " basically by constituting " and " by constituting " belong to the subordinate concept of " containing ", " having " or " comprising ".
The inventor studies to the synthetic oligogene that relates to of multiple starch; Discover that the expression that suppresses GBSSI can regulate the composition of starch in the plant well; Can reduce the content of amylose starch in the starch very effectively, improve the content of pulullan in the starch.The sequence of described GBSSI gene can be substantially the same with the sequence shown in the SEQID NO:1, and among the SEQ ID NO:1, the 75-1901 position is ORFs (ORF) sequence.
And, the inventor on the basis of comparing repeatedly, confirmed a kind of for disturb plant in the effective polynucleotide of GBSSI abnormal expression, it is through being sheared by vegetable cell after importing plant or being processed to form microRNA and disturbing the expression of GBSSI effectively.Present known small molecules conflicting mode has following several kinds: the gene silencing of miRNA regulation and control; The common inhibition (Cosuppression) that justice RNA causes; Sense-rna suppresses, and virus-mediated gene silencing (Virus Induced Gene Silencing, VIGS); Hair fastener type RNA (hairpinRNA, hpRNA) gene silencing of mediation.Just RNA in the plant; The efficiency ratio of the gene silencing of sense-rna mediation is lower, and the genetically modified report of success is rarely found, and microRNA knows still less for the Regulation Mechanism institute of plant; Rest on the fundamental research stage at present, it is very few to utilize miRNA to carry out genetically modified applied research.The inventor is surprised to find that, when being used to disturb GBSSI to express, adopting the hpRNA technology and compares based on the conflicting mode of principles such as miRNA, has significant advantage, and interference effect is very good.
Therefore, the present invention provides a kind of isolating polynucleotide, and described polynucleotide are parts of particle mating type amylosynthease I (GBSSI) encoding sox, and its length is 100-500bp; Preferably 150-250bp; Better about 180-210bp.Described polynucleotide can be used for preparing the construction that reduces amylose content in the plant.Preferably, described polynucleotide have: (a) nucleotide sequence shown in (1371 ± 10)～(1570 ± 10) among the SEQ ID NO:1; Or the sequence that (b) and (a) limits complementary sequence basically; Can suppress the expression of GBSSI in the vegetable cell especially effectively based on the construction microRNA that the back forms in importing to vegetable cell of this polynucleotide design.
Described " nucleotide sequences shown in (1371 ± 10)～(1570 ± 10) " have been specified a series of sequence; I.e. " nucleotide sequence shown in 1371～1570 or its contiguous sequence "; Said sequence can originate in arbitrary base in the 1361st to the 1381st among the SEQID NO:1, ends among the SEQ ID NO:1 the 1560th to the 1580th arbitrary base.
According to polynucleotide sequence provided by the present invention, can be processed into the polynucleotide construction that disturbs GBSSI to express by plant after can designing in being imported into plant, thereby through influence the composition that GBSSI expresses starch in the adjusting plant.Therefore, the invention provides a kind of isolating or artificial constructed polynucleotide construction, described polynucleotide construction can be transcribed and becomes described microRNA at cell inner expression by vegetable cell.As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in the formula (I):
Seq
Forward-X-Seq
OppositelyFormula (I),
In the formula (I), Seq
ForwardBe polynucleotide of the present invention, Seq
OppositelyFor with Seq
ForwardBasically complementary polynucleotide; X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and said intervening sequence and Seq
Just ToAnd Seq
OppositelyNot complementary, and intervening sequence itself does not constitute complementary duplex structure yet.
Structure shown in the formula (I) forms the secondary structure shown in the formula (II) (" stem ring " structure) after changing vegetable cell over to:
‖ is illustrated in Seq
ForwardAnd Seq
OppositelyBetween complementary relationship basically.
Usually, described polynucleotide construction is positioned on the expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described polynucleotide construction.Described expression vector also contains promotor, replication orgin and/or marker gene etc. usually.Method well-known to those having ordinary skill in the art can be used to make up expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, like kalamycin, amicillin resistance.
As optimal way of the present invention, described carrier is a binary vector, contains the double-promoter that links to each other with described construction operability.Preferable described double-promoter is CaMV35S promotor (constitutive promoter) and P54 promotor (vascular-specific expression promotor).Preferable, described carrier is the pCAMBIA1300 carrier.
Comprise the carrier of above-mentioned suitable gene order and suitable promotor or control sequence, can be used to transform suitable host.Described host is normally contained the vegetable cell (preferably cassava cell) of GBSSI gene, or also can be any cell that said expression vector also can pass to said expression vector vegetable cell that is suitable for carrying; Preferably, described host is an Agrobacterium.Can use ordinary method regeneration plant known in the art for plant transformed cell, tissue or organ take place, thereby obtain required genetically modified plant.
The invention still further relates to a kind of method through starch composition in the adjusting plant (promptly reduce the content of amylose starch, improve the content of pulullan) improvement plant, this method comprises: suppress GBSSI expression of gene in (interference) plant.Preferably, import the polynucleotide construction that specificity disturbs GBSSI to express behind the vegetable cell, this construction is imported to vegetable cell, thereby regulate the composition of starch in the plant but be prepared in based on polynucleotide of the present invention.
As optimal way of the present invention, a kind of method for preparing the low plant of amylose content comprises:
(1) Agrobacterium of carrying expression vector is provided, but described expression vector contains the polynucleotide construction that specificity disturbs GBSSI to express behind the described importing vegetable cell;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make said construction change vegetable cell over to;
(3) select vegetable cell, tissue or the organ that changes said construction over to, regeneration plant.
After said vegetable cell, tissue or neomorph became plant, corresponding GBSSI expression of gene was different with wild-type plant in these transgenic plant.
In embodiment of the present invention, change the GBSSI in the cassava and expressed, thereby regulated starch composition in the cassava effectively, obtained the cassava transgenic new variety that amylose starch and amylopection content ratio change.It is the important factor that influences its application that the starch of cassava is formed, and conventional tapioca(flour) amylose content is cultivated starch and formed the range of application that different cassava new variety can be expanded tapioca(flour), especially aspect the novel material exploitation between 22%-32%.The inventor dyes test and explains that amylose content is compared with wild-type really bigger variation has taken place through expressing the expression that microRNA with the tapioca(flour) synthesis related gene has disturbed control amylose starch and pulullan synthetic oligogene GBSSI in the cassava storage root, utilize RT-PCR to compare the variation of changeing GBSSI expression of gene amount in the GBSSI-RNAi cassava and storage root iodine.
Qualitatively or quantitatively determining contents of starch in the plant and starch, to form the method for (being the content or the ratio of amylose starch and pulullan) be that those skilled in the art know.A kind of quilitative method of classics is color analysis methods, based on principle be that starch can form the iodine-starch mixture and have special color reaction with iodine, amylose starch is met iodine and is presented mazarine (or black-and-blue), pulullan presents red-brown.In addition, also can adopt the dual wavelength colourimetry to come to identify quantitatively, this method has higher precision relatively.In addition, on molecular level, identify straight chain and amylopection content and ratio in the starch, and the method for each item index (like viscosity, gelatinization point etc.) of starch in industrial production also is known in the art.
Major advantage of the present invention is:
(1) the present invention is based on the microRNA perturbation technique and disturb the GBSSI expression of gene, regulate the purpose that starch is formed in the plant thereby reach, and the transgenic plant stabilization characteristics of genetics that obtains, for a change the plant amylum quality provides brand-new feasible thinking.
(2) can obtain being different from the new plant of wild-type starch quality at short notice, the defective that overcome traditional breeding technique length consuming time, influenced by accidentalia.
(3) when being used for foodstuffs industry, the reduction of amylose content improves the mouthfeel of starch food effectively in the starch.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
According to GBSSI gene order (the NCBI accession number of having announced: X74160); Obtain appropriate region (also being 1371-1570 position among the SEQ ID NO:1) among the GBSSI; (SEQ ID NO:2) carries out forward and reverse connection as intervening sequence through an artificial synthetic intron; Be inserted into CaMV35S (conventional constitutive promoter) or P54 (cassava vascular-specific expression promotor; Genbank accession number: AY217353; Adopt the 1080bp sequence (promptly-1～-1080) of upstream from start codon) among the EcoRI and XbaI site of the pCAMBIA1300 binary vector of promoters driven, obtain recombinant vectors pC-35S-GBSSI-RNAi and pC-P54-GBSSI-RNAi (Fig. 1) respectively, be used to transform cassava and prepare transgenic plant.
Used intron sequences is (SEQ ID NO:2):
5-GTACTATAGTATTTTGGTACCTTTTACAATCGGTTTTTTTACCTTTTCTTCTTTATTTATTAAATTTATAG-3。
Preparation and the plant regeneration of embodiment 2, transgenic cassava, sweet potato
The Agrobacterium that will contain two kinds of reorganization binary vectors of above-mentioned pC-35S-GBSSI-RNAi and pC-P54-GBSSI-RNAi transforms cassava fragility callus respectively, utilizes embryo's generation and adventitious organogenesis regeneration to obtain transfer-gen plant.Concrete grammar is following:
1. contain the Agrobacterium monospecific polyclonal of the binary vector of recombinating with sterilization toothpick picking from the flat board, be placed on the resistance YEB substratum, at 28 ℃, shaking culture is spent the night under the 240rpm.
2. get 25 μ l bacterium liquid, be added in the fresh YEB substratum of 50ml, cultivate 12-20 hour to OD
600Be 0.5-1.0.
3.6000rpm, 4 ℃ of centrifugal bacterium liquid 10min are with 50ml liquid MS medium (PH5.3) suspension flushing, once centrifugal again.Bacterium liquid is resuspended in the liquid MS medium that comprises 200 μ M Syringylethanones OD
600Be 1.0.
4. draw the broken filtering suspending nutrient solution of 2ml in 10ml Agrobacterium bacterium liquid, cultivate 45min altogether.Remove unnecessary bacterium liquid, callus is put into blots residual bacterium liquid on the aseptic filter paper, place on the SH solid medium that comprises 100 μ m Syringylethanones, cultivated altogether 3 days for 25 ℃.
5. from filter paper, the callus that transforms is transferred to the 30ml SH substratum.
6. inhale repeatedly and beat cleaning many times, outwell substratum, repeatedly cleaning many times.
7. callus is forwarded in the SH substratum that contains 12.5mg/l Totomycin and 500mg/l Pyocianil to 137rpm, continuous illumination (about 50 μ mol m
-2s
-1) cultivated 3 days.
With the callus subculture in the SH substratum that contains 25mg/l Totomycin and 500mg/l Pyocianil, 108rpm, continuous illumination (about 50 μ mol m
-2s
-1) cultivate, per 3 days subcultures are once.
9.2 after week, kanamycin-resistant callus tissue grows the cadmium yellow same with not adding the contrast of selective agent, brittle embryo callus subculture.
10. the kanamycin-resistant callus tissue that will screen the suspension culture in 2-3 week forwards on the somatic embryo generation solid medium (MSN) that comprises the 10mg/l Totomycin, and 26 ℃, 16 hours illumination cultivation.
11.2-4 after week, resistance embryo callus forms, and kanamycin-resistant callus tissue is transferred to the embryo maturation medium (CMM) that comprises the 12.5mg/l Totomycin go up cultivation, makes it grow cotyledonary embryos.
12. mature embryo is transferred on the stem elongation medium (CEM), and 2-4 can be from the cotyledon shape embryonic development stem bar that makes new advances after week.
13. new stem section is transferred on the MS minimum medium (CBM), after 3 weeks, downcut the generation that the stem section that grows from lateral bud about 1cm is transferred to minimum medium (CBM) the observation root that contains the 8mg/l Totomycin, use not genetically modified plant to do negative control.
14.1 week, the growing state of root was observed in the back, the plant that can take root is used for Molecular Detection.
Adopt as above-mentioned same method, also prepared the sweet potato transgenic plant, and through identifying the plant that has obtained transgenic positive.
Utilize the situation of RT-PCR GBSSI genetic expression from molecular level detection transgenic cassava.TRIZOL (DP421) reagent with TIANGEN company; From wild-type and the stripped seedling leaf of genetically modified cassava, extract total RNA; With oligo (dT) is first strand primer, and M-MLV reversed transcriptive enzyme (TIANGENER104-03) reverse transcription becomes cDNA, with GBSSI; The specific separately primer of c15 gene carries out the double PCR amplification, obtains a result.
The result sees Fig. 2, shows that the GBSSI expression of gene receives interference largely in 8 transgenic cassava strain systems.
Equally, utilize RT-PCR can detect the situation of GBSSI genetic expression the transgenic sweet potato from molecular level, the result finds that the GBSSI expression of gene receives very big interference in the transfer-gen plant.
The variation that embodiment 4, tapioca(flour) are formed
One. the iodine staining microscopy
Blade, stem and the storage root of getting wild-type and transgenic cassava TG-7 strain system carry out 1% iodine staining respectively.Concrete dyeing process is following:
Blade: with 75% ethanol decolorization 30 minutes, distilled water flushing once added 1 milliliter of 1% iodine liquid, and dyeing is spent the night, and removes iodine liquid, the unnecessary iodine liquid of distilled water flushing, microscopy.
The stem section, the storage root: material is thinly sliced,, an amount of iodine drop is added on the thin slice according to thin slice size, after the colour developing, microscopy immediately.
The result sees Fig. 3, proves the composition generation noticeable change of starch in the transgenic cassava.
Two. the composition of starch in the dual wavelength colorimetric method for determining cassava
Adopt the composition of starch in the dual wavelength colorimetric method for determining cassava, according to the dual wavelength colorimetric principle, if certain solute all has absorption in the solution under two wavelength, then the absorption difference of two wavelength is directly proportional with solute concentration.Amylose starch and iodine effect produce pure blue, and pulullan and iodine effect generate red-purple or reddish-brown.With the standardized solution of two kinds of starch respectively with Iod R, in same system of coordinates, scan (400-960mm) then or do absorption curve, obtain result shown in Figure 5.Among the figure, 1 is the absorption curve of amylose starch, and 2 is the absorption curve of pulullan.To containing the unknown sample of amylose starch and pulullan, after the iodine colour developing, as long as go into λ 1 in wavelength selected, λ 2, and λ 3, and λ 4, locate to do colorimetric 4 times, utilize amylose starch and pulullan typical curve can obtain two amyloid content in the sample respectively.
Operation steps:
1. select straight chain, pulullan to measure wavelength, reference wavelength
Amylose starch: get 1mg/ml amylose starch reference liquid 1ml, put into the 50ml volumetric flask, adding distil water 30ml transfers to about pH3.5 with 0.1mol/L HCl solution, adds iodine reagent 0.5ml, and with the zero(ppm) water constant volume.Leaving standstill 20min, is blank with zero(ppm) water, carries out the scanning of visible light all wave band or draws the amylose starch absorption curve with common colourimetry with dual beam spectrophotometer.
Pulullan: get 1mg/ml pulullan reference liquid 1ml, put into the 50ml volumetric flask, below the same amylose starch of operation.In same coordinate, obtain pulullan visible light wave range absorption curve.
According to the method that principle is partly introduced, confirm mensuration wavelength, reference wavelength λ 2, λ 1, λ 4 and the λ 3 of amylose starch and pulullan.
2. make dual wavelength amylose starch typical curve
Absorption 1mg/ml amylose starch standardized solution 0.3,0.5,0.7,0.9,1.1,1.3ml put into 6 different 50ml volumetric flasks respectively; Add zero(ppm) water 30ml; Transfer to about pH3.5 with 0.1mol/LHCl solution, add iodine reagent 0.5ml, and use the zero(ppm) water constant volume.Leave standstill 20min; Is blank with zero(ppm) water, under λ 1,2 liang of wavelength of λ, measures A λ 1 respectively with the 1cm cuvette, A λ 2 promptly get △ A directly=A λ 2_A λ 1 directly is an ordinate zou with △ A; Amylose content (mg) is an X-coordinate, preparation dual wavelength amylose starch typical curve.
3. make dual wavelength pulullan typical curve
Absorption 1mg/ml pulullan standardized solution 2.0,2.5,3.0,3.5,4.0,4.5,5.0ml put into 6 different 50ml volumetric flasks respectively.Below operate same amylose starch.With zero(ppm) water is blank, at λ 3, respectively measures its A λ 3 under 4 liang of wavelength of λ with the 1cm cuvette, and A λ 4 promptly gets △ A and props up=A λ 4-A λ 3.Propping up with △ A is ordinate zou, and amylopection content (mg) is an X-coordinate, preparation dual wavelength pulullan typical curve.
4. the mensuration of amylose starch, pulullan and total starch in the sample
Sample was pulverized 60 mesh sieves, used ether defatting, took by weighing about degreasing sample 0.1g (being accurate to 1mg), placed the 50ml volumetric flask.Add 0.5mol/L KOH solution 10ml, in boiling water bath, heat 10min, take out, be settled to 50ml if there is foam to adopt ethanol to eliminate with zero(ppm) water), leave standstill.Draw two parts of 2.5ml of sample liquid (being sample determination liquid and blank solution), all adding distil water 30ml transfers to about pH3.5 with 0.1mol/LHCI solution, adds iodine reagent 0.5ml in the sample, and blank solution does not add iodine reagent, all is settled to 50ml then.Leaving standstill 20min, is contrast with sample blank liquid, uses the 1cm cuvette; Measure λ 2 respectively, λ 1, and λ 4; The absorption value A λ 2 of λ 3, A λ 1, A λ 4; A λ 3. obtain △ A straight=A λ 2-A λ 1 △ A props up=A λ 4-A λ 3. looks into two amyloid dual wavelength typical curves respectively, can calculate amylose starch and amylopection content in the degreasing sample.Sum of the two equals total starch content.
Result treatment, adopt following formula:
In the formula,
X1: look into dual wavelength amylose starch typical curve and get amylose content in the sample liquid (mg);
X2: look into dual wavelength pulullan typical curve and get amylopection content in the sample liquid (mg);
M: sample quality (g);
Total starch (%)=amylose starch (%)+pulullan (%).
Reagent is prepared as follows:
Iodine reagent: take by weighing potassiumiodide 2.0g, be dissolved in a small amount of zero(ppm) water, add iodine 0.2g again, wait to dissolve the back and be settled to 100ml with distilled water diluting.
Amylose starch reference liquid: take by weighing the pure article 0.1000g of amylose starch, be placed in the 100ml volumetric flask, add 0.5mol/L KOH10ml, after in hot water, waiting to dissolve, take out adding distil water and be settled to 100ml, be 1mg/ml amylose starch standardized solution.
The pulullan reference liquid: with the 0.1000g pulullan by be prepared into 1mg/ml pulullan standardized solution with quadrat method.
Through measuring, the content of amylose starch accounts for 1% of starch total content in the transgenic cassava TG-7 strain system, and the content of pulullan accounts for 99% of starch total content.
Use the same method and detect the starch content of transgenic sweet potato, through measuring, the content of amylose starch accounts for 2.5% of starch total content in the transgenic sweet potato strain system, and the content of pulullan accounts for 97.5% of starch total content.
The starch small grain of wild-type and transgenic cassava TG-7 strain system is carried out iodine dye observation, method is following:
1: extract and extract starch in the tapioca root: tissue mashing machine grinds, 100 μ m membrane filtrations, and centrifugal collecting precipitation, 40 ℃ of bakings got final product in two days.
2: get appropriate amount of starch and add the colour developing of capacity 1% iodine liquid, microscopy immediately.
The result is as shown in Figure 4, confirms that the starch small grain amylose content in the transgenic cassava strain system storage root obviously descends.
1371～1570 segmental interference performances of vicinity among embodiment 6, the SEQ ID NO:1
Through conventional PCR method; The inventor has also obtained the nucleic acid fragment corresponding to 1367-1567 bit sequence among the SEQ ID NO:1; Like the method for previous embodiment 1, the intron (sequence such as SEQ ID NO:2) through synthetic carries out forward and reverse connection, this nucleic acid construct thing is cloned into the P54/35S promotor makes it to be connected with the promotor operability in the pCAMBIA1300 binary vector at the back; And recombinant vectors is transferred in the Agrobacterium, transform the callus of cassava plant.The transgenic plant that obtain detect the wherein composition of amylose starch and amylose starch like the method for embodiment 4 after maturation, the result finds that the content of amylose starch accounts for below 2% of starch total content, and the content of pulullan accounts for more than 98% of starch total content.
Through conventional PCR method; The inventor has also obtained the nucleic acid fragment corresponding to 1375-1573 bit sequence among the SEQ ID NO:1; Like the method for previous embodiment 1, the intron (sequence such as SEQ ID NO:2) through synthetic carries out forward and reverse connection, this nucleic acid construct thing is cloned into the P54/35S promotor makes it to be connected with the promotor operability in the pCAMBIA1300 binary vector at the back; And recombinant vectors is transferred in the Agrobacterium, transform the callus of cassava plant.The transgenic plant that obtain detect the wherein composition of amylose starch and amylose starch like the method for embodiment 4 after maturation, the result finds that the content of amylose starch accounts for below 2% of starch total content.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Shanghai Inst. of Life Science, CAS
< 120>regulate the method that root crop starch is formed
<130>083966
<160>2
<170>PatentIn?version3.3
<210>1
<211>2168
<212>DNA
< 213>cassava (Manihot esculenta Crantz)
<400>1
<210>2
<211>71
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>oligonucleotide
<400>2
Claims (11)
1. isolating polynucleotide is characterized in that, described polynucleotide are:
(a) nucleotide sequence shown in position, (1371 ± 10)～(1570 ± 10) among the SEQ ID NO:1; Or
(b) with the sequence complementary sequence that (a) limits.
2. polynucleotide as claimed in claim 1 is characterized in that, described polynucleotide are: the sequence shown in position, (1371 ± 5)～(1570 ± 5) among the SEQ ID NO:1.
3. the purposes of claim 1 or 2 described polynucleotide is characterized in that, is used for preparing the construction that reduces the plant amylose content.
4. a construction is characterized in that, said construction contains the structure shown in the formula (I):
Seq
Forward-X-Seq
OppositelyFormula (I),
In the formula (I),
Seq
ForwardBe claim 1 or 2 described polynucleotide, Seq
OppositelyFor with Seq
ForwardBasically complementary polynucleotide;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and said intervening sequence and Seq
ForwardAnd Seq
Instead ToNot complementary, itself does not constitute complementary duplex structure described intervening sequence.
5. construction as claimed in claim 4 is characterized in that, the structure shown in the formula (I) forms the secondary structure shown in the formula (II) after changing vegetable cell over to:
In the formula (II), Seq
Forward, Seq
OppositelyWith the definition of X such as above-mentioned,
|| be illustrated in Seq
ForwardAnd Seq
OppositelyBetween complementary relationship basically.
6. construction as claimed in claim 4 is characterized in that, described intervening sequence length is at 50-90bp.
7. construction as claimed in claim 4, the sequence of described intervening sequence is shown in SEQ ID NO:2.
8. a carrier is characterized in that, described carrier contains the arbitrary described construction of claim 4-7.
9. the purposes of the arbitrary said construction of claim 4-7 is characterized in that, is used for importing to plant, suppress particle mating type amylosynthease I and express, thereby amylose starch is synthetic in the inhibition plant.
10. a method that reduces amylose content in the plant is characterized in that, said method comprises: with the arbitrary described construction of claim 4-7 or the described carrier transfection of claim 8 in plant.
11. method as claimed in claim 10 is characterized in that, said method comprises:
(1) Agrobacterium of carrying arbitrary described construction of claim 4-7 or the described carrier of claim 8 is provided;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make said construction be transferred to vegetable cell, tissue or organ; With
(3) select vegetable cell, tissue or the organ that changes said construction over to, regeneration plant.
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| CN102206639B (en) * | 2010-03-30 | 2013-11-20 | 中国科学院上海生命科学研究院 | Gene adjusting starch anabolism of plant seeds and application thereof |
| CN105400748B (en) * | 2013-03-05 | 2019-03-12 | 中国科学院上海生命科学研究院 | Saltant type OsGBSS1 enzyme and preparation method thereof and purposes |
| FI20165270A (en) * | 2016-03-31 | 2017-10-01 | Kemira Oyj | Method and system for determining starch in a sample |
| CN113151321A (en) * | 2021-05-25 | 2021-07-23 | 云南中烟工业有限责任公司 | Tobacco particle combined starch synthase gene and application thereof |
Citations (1)
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|---|---|---|---|---|
| CN1314946A (en) * | 1998-07-28 | 2001-09-26 | 纳幕尔杜邦公司 | Modification of starch biosynthetic enzyme gene expression to produce starches in chain crops |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314946A (en) * | 1998-07-28 | 2001-09-26 | 纳幕尔杜邦公司 | Modification of starch biosynthetic enzyme gene expression to produce starches in chain crops |
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| Title |
|---|
| Rui LJ,et al.RNA silencing of Waxy gene results in low levels of amylose in the seeds of transgenic wheat (Triticum aestivum L.).《遗传学报》.2005,第32卷(第8期),第846-854页. * |
| 乔光明等.玉米颗粒结合型淀粉合成酶(GBSS)基因cDNA的克隆及植物表达载体的构建.《农业生物技术学报》.2007,第15卷(第4期),第673-676页. * |
| 郭华等.中国春小麦GBSS与淀粉颗粒结合特性的研究.《作物学报》.2007,第33卷(第2期),第322-326页. * |
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