CN109323911A - A kind of starch iodine absorption spectrum method for the rice silty seed mutant that quick screening starch ingredients change - Google Patents
A kind of starch iodine absorption spectrum method for the rice silty seed mutant that quick screening starch ingredients change Download PDFInfo
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- CN109323911A CN109323911A CN201811530774.XA CN201811530774A CN109323911A CN 109323911 A CN109323911 A CN 109323911A CN 201811530774 A CN201811530774 A CN 201811530774A CN 109323911 A CN109323911 A CN 109323911A
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- 229920002472 Starch Polymers 0.000 title claims abstract description 252
- 239000008107 starch Substances 0.000 title claims abstract description 252
- 235000019698 starch Nutrition 0.000 title claims abstract description 252
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 title claims abstract description 97
- 239000011630 iodine Substances 0.000 title claims abstract description 97
- 229910052740 iodine Inorganic materials 0.000 title claims abstract description 97
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 42
- 235000009566 rice Nutrition 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000004615 ingredient Substances 0.000 title claims abstract description 32
- 230000008859 change Effects 0.000 title claims abstract description 23
- 238000012216 screening Methods 0.000 title claims abstract description 22
- 238000000862 absorption spectrum Methods 0.000 title claims abstract description 19
- 240000007594 Oryza sativa Species 0.000 title 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 96
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 75
- 239000004202 carbamide Substances 0.000 claims abstract description 75
- 241000209094 Oryza Species 0.000 claims abstract description 41
- 239000000284 extract Substances 0.000 claims abstract description 14
- 238000007811 spectroscopic assay Methods 0.000 claims abstract description 14
- 238000010186 staining Methods 0.000 claims abstract description 14
- 238000010183 spectrum analysis Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 238000004043 dyeing Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 67
- 239000000843 powder Substances 0.000 claims description 29
- 238000005119 centrifugation Methods 0.000 claims description 18
- 238000004090 dissolution Methods 0.000 claims description 14
- 239000012535 impurity Substances 0.000 claims description 14
- 238000000746 purification Methods 0.000 claims description 14
- 238000001556 precipitation Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 10
- 230000001376 precipitating effect Effects 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 8
- 238000007873 sieving Methods 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 3
- 230000000996 additive effect Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 2
- 150000003462 sulfoxides Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract 1
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 66
- 239000008367 deionised water Substances 0.000 description 49
- 229910021641 deionized water Inorganic materials 0.000 description 49
- 239000011521 glass Substances 0.000 description 28
- 238000004458 analytical method Methods 0.000 description 25
- 238000010521 absorption reaction Methods 0.000 description 24
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 23
- 229920000856 Amylose Polymers 0.000 description 19
- 239000007788 liquid Substances 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 14
- 238000003756 stirring Methods 0.000 description 14
- 239000000706 filtrate Substances 0.000 description 12
- 230000035772 mutation Effects 0.000 description 10
- 239000013049 sediment Substances 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 230000001086 cytosolic effect Effects 0.000 description 8
- DKNPRRRKHAEUMW-UHFFFAOYSA-N Iodine aqueous Chemical compound [K+].I[I-]I DKNPRRRKHAEUMW-UHFFFAOYSA-N 0.000 description 7
- 241000746966 Zizania Species 0.000 description 7
- 235000002636 Zizania aquatica Nutrition 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011049 filling Methods 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 5
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 229920000945 Amylopectin Polymers 0.000 description 2
- 101100120289 Drosophila melanogaster Flo1 gene Proteins 0.000 description 2
- 101100013145 Drosophila melanogaster Flo2 gene Proteins 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
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- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009990 desizing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
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- 210000002706 plastid Anatomy 0.000 description 1
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- 238000009738 saturating Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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Abstract
The present invention relates to a kind of starch iodine absorption spectrum method of rice silty seed mutant that quickly screening starch ingredients change, the rice silty seed mutant that the method comprising the steps of 1), screening is homozygous；Homozygous rice silty seed is ground up, sieved, extracts starch by step 2)；Starch is obtained starch solution with urea/dmso solution by step 3)；Step 4), starch solution use iodine staining, the starch solution after being dyed, and the starch solution after dyeing is carried out spectroscopic assay, analyzed using conventional spectral analysis method spectrometric result.Simply, conveniently, time-consuming is few, and as a result favorable reproducibility, low in cost for the measuring method, does not need complicated instrument and equipment, and starch that can be good with a small amount of sample extraction effect, and the screening for the rice silty seed mutant of starch ingredients change provides technical support.
Description
Technical field
The present invention relates to a kind of starch iodine of rice silty seed mutant that quickly screening starch ingredients change to absorb light
Spectral method belongs to crop mutant screening technique field.
Background technique
Starch is most important component in paddy endosperm, and an important factor for decision rice eating-quality.Starch is main
It is made of two kinds of glucose polymer components, i.e. the seldom amylose of branch (Amylose) and hyperbranched amylopectin
(Amylopectin).The content and molecular structure of starch each component are the main foundation and important indicator of evaluation starch quality, certainly
Determine the purposes of rice.The biosynthesis of starch is a mistake fine by complexity that a variety of enzymes and regulatory factor participate in jointly
Journey.Currently, although researcher has revealed that out the basic process of Starch synthesis approach by numerous studies, among these according to
It is so unknown there are many link.By synthesizing the research of defect mutant to endosperm starch, it can be found that some new starch
The regulatory factor and its mechanism of action of route of synthesis, to more fully illustrate the biosynthesis pathway of starch.
The mutation of Starch synthesis, metabolism related gene often causes the change of content of starch or structure.It has reported at present
The starch related mutants in road mainly include the types such as white chalk, shrinkage, waxy, silty.Wherein farinaceous albumen mutant refers to embryo
A kind of mutant of the impervious light of milk powder matter is primarily due to amyloplast and proteosome in endosperm and enriches bad, presence each other
Air and the optical characteristics formed.Seed shape is normal, but the color of endosperm is white.Rice silty phenotype may be by more
Kind reason causes, and the starch ingredients of many silty phenotype seeds might not change.Floury gene is rice the most typical
Farinaceous albumen mutant gene.It has been reported that silty mutant have flo1, flo2, flo3, flo4, flo5 and flo6, wherein
Flo1, flo2, flo5 and flo6 are affected to the variation of Starch synthesis and starch ingredients.
Excavating and its studying the influence that starch ingredients change for the relevant silty mutant of more starch facilitates into one
Step supplement improves Starch synthesis and metabolic pathway.The content and molecular structure of starch each component be evaluation starch quality it is main according to
According to and important indicator, determine the purposes of seed.There are many method for measuring starch ingredients, and iodine colorimetry, RNA isolation kit and gel seep
Saturating chromatography is most common starch ingredients analysis method.There are higher costs for RNA isolation kit, complicated for operation, large quantities of can not measure
The disadvantages of determining；The disadvantages of exclusion chromatography is long there are experimental period, depends on high end instrument, experiment condition and high cost requirement；
Iodine colorimetry is also more common, but is mostly used only to analyze the amylose content parameter of starch.Body is studied as mutant is established
The important step of system, the screening technique and process of targeted mutagenesis body are extremely important, therefore need to establish a kind of accurate, fast
Speed, the rice silty seed mutant that screening starch ingredients that completion is had the ability in most of laboratories and that cost is not high change
Method, provide technical support for the creation of mutant and the researchs such as excavation of gene function.
Summary of the invention
The purpose of the present invention is to above-mentioned drawbacks of the existing technology, change in view of existing quick screening starch ingredients
The technological gap of the rice silty seed mutation body method of change proposes a kind of rice silty that quickly screening starch ingredients change
The starch iodine absorption spectrum method of seed mutant.
The object of the present invention is achieved like this, a kind of rice silty seed mutant that quick screening starch ingredients change
Starch iodine absorption spectrum method, which comprises the following steps:
The homozygous rice silty seed mutant of step 1), screening；The quality of homozygous rice silty seed is 0.8-
1.2g, containing 40-60 seeds, every seed is taken from self progeny M2, the seed of complete opaque endosperm phenotype,
Seed cross-cutting fault shows as full silty；
Seed in homozygous rice silty seed mutant is ground up, sieved, extracts starch by step 2)；Specifically
The following steps are included:
A, the rice silty seed pestle grind into powder that will have been screened, adds water to continue to be ground into homogenate, homogenate
Successively suspension is obtained by 100 mesh, 400 mesh screens；
B, the suspension collection after sieving is subjected to centrifugal purification in 2mL centrifuge tube, when centrifugal purification, centrifugal force is
3000-4000g, centrifugation time 5-10min；After centrifugation, supernatant is removed, is precipitated；When precipitation surface free from admixture, complete to form sediment
The purifying of powder, enters step e；There is impurity on starch surface after centrifugation, enters step c；
C, continue centrifugal purification after the precipitating after centrifugal purification gently being scraped off precipitation surface yellow impurities with spoon,
When centrifugal purification, centrifugal force 3000-4000g, centrifugation time 5-10min；When centrifuged deposit surface free from admixture, complete to form sediment
The purifying of powder, enters step e；When there is impurity on centrifuged deposit surface, d is entered step；
D, it repeats step c several times, until centrifuged deposit surface free from admixture, completes the purifying of starch, obtain after purification
Starch；
E, the dehydrated alcohol of starch after purification is rinsed 2-3 times, is subsequently placed in 35-40 DEG C of drying device dry 2-3
Its starch after being dried；
F, by the starch ground after step e is dry at starch powder, starch powder is by 100 mesh screens, after sieving
Starch powder is the starch extracted, is then saved starch in drier stand-by；
The starch obtained through step 2) is obtained starch solution with urea/dmso solution by step 3)；With urea/
Dmso solution starch, additive amount are that the starch of every milligram of extraction adds 0.4-0.6mL urea/dimethyl Asia
Sulfone；In urea/dimethyl sulfoxide, the mass percent that urea accounts for dimethyl sulfoxide is 1.0-1.5%；
With urea/dmso solution starch, solution temperature is 95-98 DEG C, dissolution time 50-70min；Dissolution is opened
Stage beginning 5-10min continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5-10s by 1-2min；Later
The 11-60min stage, every 5-10min by urea/dimethyl sulfoxide, starch mixed liquor mix 5-10s after continue to dissolve；
Step 4), the starch solution for obtaining step 3) use iodine staining, the starch solution after being dyed, after dyeing
Starch solution carry out spectroscopic assay, spectrometric result is analyzed using conventional spectral analysis method.
In step 2), spoon is the spoon of 6-8cm.
In step 3), urea/dimethyl sulfoxide, starch mixed liquor are mixed when mixing using turbine mixer.
In step 3), urea/dimethyl sulfoxide dissolves starch in water-bath.
The advanced science of the method for the present invention, in order to solve the above technical problems, the technical scheme is that, screening first is homozygous
Rice silty seed mutant, the homozygous rice silty seed of screening is ground up, sieved, extracts starch, with urea/bis-
Methyl sulfoxide dissolves starch, then iodine staining, spectroscopic assay, spectrum analysis.Wherein, it is prominent to screen homozygous rice silty seed
Seed in variant is selected from and hands in offspring M2, the fully opaque seed of endosperm, the quality of homozygous rice silty seed
For 0.8-1.2g, contain 40-60 seeds.The seed is ground up, sieved, extracts starch, is the rice silty seed that will have been screened
Grain pestle grind into powder adds water to continue to be ground into homogenate, and it is outstanding that homogenate crosses 100 mesh respectively, 400 mesh screens obtain starch
Supernatant liquid, the starch suspension collection after sieving carry out centrifugal purification, centrifugal force 3000-4000g, centrifugation in 2mL centrifuge tube
Time is 5-10min, and desizing surface yellow impurities are gently scraped with spoon, repeats to purify, starch surface is without miscellaneous after centrifugation
Matter is finally washed 2-3 times with dehydrated alcohol, and 35-40 DEG C of dry 2-3d is ground into starch powder, sieves with 100 mesh sieve net, in drier
It saves.Described to use urea/dmso solution starch, additive amount is that the starch of every milligram of extraction adds 0.4-
0.6mL urea/dimethyl sulfoxide, wherein the mass fraction that urea accounts for dimethyl sulfoxide is 1.0-1.5%.
Described to use urea/dmso solution starch, temperature is 95-98 DEG C, time 50-70min；It is put into water-bath
Dissolution in pot, starts the 5-10min stage, takes out every 1-2min, mixes 5-10s on turbine mixer at once, then proceed to
Dissolution；The 11-60min stage later takes out every 5-10min, mixes 5-10s on turbine mixer at once.The urine
The starch solution dissolved after sample dissolves well, is stood room temperature cooling, time of repose is by element/dmso solution starch
20-30min.Iodine staining, the concentration of iodine solution are 0.18-0.22%, wherein the mass percent of potassium iodide is 1.8- in iodine solution
2.2%.
Iodine staining, spectroscopic assay, spectrum analysis are that (starch solution uses the starch obtained after iodine staining by sample
Solution), iodine solution and water mixing, react 15-20min, with spectrophotometer scanning 400-900nm locate absorbance, be starch iodine suction
Spectrum is received, each sample OD620, OD620/550 and maximum absorption wave wavelength X max are analyzed.
The spectroscopic assay, spectrophotometer scanning speed are that at a slow speed, sweep time is each sample 2-3min.Spectrum point
Analysis is to choose measured OD620, OD620/550 and maximum absorption wave wavelength X max, and difference OD620 exists compared with parent
0.030 or more perhaps in 5nm, the above are the materials that starch ingredients change in 0.05 or more or λ max by OD620/550.
Mutant verifying, refers to according to Spectroscopic analysis results, it is believed that is the mutant that starch ingredients change, is tried with amylose
Agent box measures its starch ingredients, to verify whether the starch ingredients compared with parent change.
Possessed by of the invention the utility model has the advantages that
(1) simply, conveniently, time-consuming is few for whole operation and analytic process.
(2) low in cost, do not need complicated instrument and equipment.
(3) starch isolation effect is good, and measurement result is more accurate.
(4) sample size needed for is smaller, and as a result reproducibility is preferable.
Detailed description of the invention
Fig. 1 is the stereoscope picture of wild rice Kitaake seed and silty mutant seed and crosscutting in the present invention
Cross-section diagram.
Fig. 2 is the iodine abosrption spectrogram of wild rice Kitaake-1 starch in the present invention.
Fig. 3 is the iodine abosrption spectrogram of wild rice Kitaake-2 starch in the present invention.
Fig. 4 is the iodine abosrption spectrogram of wild rice Kitaake-3 starch in the present invention.
Fig. 5 is the iodine abosrption spectrogram of Rice Powder cytoplasmic mutation body Y17-51 starch in the present invention.
Fig. 6 is the iodine abosrption spectrogram of Rice Powder cytoplasmic mutation body Y17-231 starch in the present invention.
Fig. 7 is the iodine abosrption spectrogram of Rice Powder cytoplasmic mutation body Y17-252 starch in the present invention.
Fig. 8 is the iodine abosrption spectrogram of Rice Powder cytoplasmic mutation body Y17-493 starch in the present invention.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, right combined with specific embodiments below
A specific embodiment of the invention is described in detail.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by the specific embodiments disclosed below.
A kind of starch iodine absorption spectrum method for the rice silty seed mutant that quick screening starch ingredients change, screening
Homozygous rice silty seed mutant, seed is ground up, sieved, extracts starch, with urea/dmso solution starch, iodine
Liquid dyeing, spectroscopic assay, spectrum analysis, analysis include OD620, OD620/550 and maximum absorption wave wavelength X max, mutant sieve
Choosing, mutant verifying, the mutant screened is verified with amylose kit compared with parent, whether starch ingredients change
Become.
Embodiment 1
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) seed is selected: selecting Kitaake seed 50, weighing: 0.93g, number Kitaake-1.
(2) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(3) homogenate crosses 100 mesh, 400 mesh screens respectively, obtains filtrate (suspension)；
(4) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(5) remaining precipitating is suspended again with deionized water, repeated centrifugation, until centrifuged deposit surface free from admixture；
(6) precipitating of last surface free from admixture is washed 3 times with dehydrated alcohol, and 40 DEG C of dry 2d obtain Kitaake-1 starch；
(7) starch is sieved with 100 mesh sieve and is saved in drier after netting.
With urea/dmso solution starch, key step are as follows:
(1) 9.9mg Kitaake-1 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 4.95mL 90% is drawn in screw socket glass with 5mL liquid-transfering gun
In pipe；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass bar stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 2 wild rice Kitaake-1 starch is shown in Table 1.
With reference to the amylose content of amylose kit method analysis Kitaake-1 starch, 1 the results are shown in Table.
Embodiment 2
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) seed is selected: selecting Kitaake seed 50, weighing: 0.96g, number Kitaake-2.
(2) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(3) homogenate crosses 100 mesh, 400 mesh screens respectively, obtains filtrate (suspension)；
(4) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(5) remaining precipitating is suspended again with deionized water, repeated centrifugation, until being centrifuged rear surface free from admixture；
(6) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Kitaake-2 starch；
(7) starch sieves with 100 mesh sieve the interior preservation of drier after net.
With urea/dmso solution starch, key step are as follows:
(1) 10.0mg Kitaake-2 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 5.0mL 90% is drawn in screw socket glass tube with 5mL liquid-transfering gun
It is interior；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 3 wild rice Kitaake-2 starch is shown in Table 1.
With reference to the amylose content of amylose kit method analysis Kitaake-2 starch, 1 the results are shown in Table.
Embodiment 3
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) seed is selected: selecting Kitaake seed 50, weighing: 0.94g, number Kitaake-3.
(2) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(3) homogenate crosses 100 mesh, 400 mesh screens respectively, obtains filtrate (starch suspension)；
(4) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(5) remaining precipitating is suspended again with deionized water, repeated centrifugation, until being centrifuged rear surface free from admixture；
(6) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Kitaake-3 starch；
(7) starch sieves with 100 mesh sieve the interior preservation of drier after net.
With urea/dmso solution starch, key step are as follows:
(1) 10.0mg Kitaake-3 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 5.0mL 90% is drawn in screw socket glass tube with 5mL liquid-transfering gun
It is interior；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 4 wild rice Kitaake-3 starch is shown in Table 1.
With reference to the amylose content of amylose kit method analysis Kitaake-3 starch, 1 the results are shown in Table.
Embodiment 4
Screen homozygous rice silty seed mutant, key step are as follows:
(1) creation of rice mutant: with 300Gy dosage60Co gamma Rays handle conventional japonica rice kind
Kitaake。
(2) obtain homozygous offspring: in 2 generations of selfing, obtain M2 generation homozygous seed.
(3) selection of seed silty phenotype: choosing number is the fully opaque seed of Y17-51 endosperm, such as Fig. 1 silty
Mutant seed.
(4) seed is selected: selecting wherein 55, weighing: 1.05g.
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(2) homogenate crosses 100 mesh, 400 mesh screens respectively, obtains filtrate；
(3) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(4) remaining precipitating is suspended again with deionized water, repeated centrifugation, until centrifuged deposit surface free from admixture；
(5) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Y17-51 starch；
(6) it sieves with 100 mesh sieve and is saved in drier after netting.
With urea/dmso solution starch, key step are as follows:
(1) 9.9mg Y17-51 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 4.95mL 90% is drawn in screw socket glass with 5mL liquid-transfering gun
In pipe；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 5 Rice Powder cytoplasmic mutation body Y17-51 starch is shown in Table 1.
Mutant verifying is the amylose content with reference to amylose kit method analysis Y17-51 starch, as a result
It is shown in Table 1.Data are average value ± SD (n=2), examine otherness by t, * * indicates the straight chain of Y17-51 and parent Kitaake
Content of starch is significant in the level difference of p < 0.01.
Embodiment 5
Screen homozygous rice silty seed mutant, key step are as follows:
(1) creation of rice mutant: with 300Gy dosage60Co gamma Rays handle conventional japonica rice kind
Kitaake。
(2) obtain homozygous offspring: in 2 generations of selfing, obtain M2 generation homozygous seed.
(3) selection of seed silty phenotype: choosing number is the fully opaque seed of Y17-231 endosperm, such as Fig. 1 silty
Mutant seed.
(4) seed is selected: selecting wherein 58, weighing: 1.06g.
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(2) homogenate crosses 100 mesh, 400 mesh screens respectively, obtains filtrate；
(3) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(4) remaining precipitating is suspended again with deionized water, repeated centrifugation, the shallow lake shallow lake surface free from admixture after centrifugation；
(5) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Y17-231 starch；
(6) it sieves with 100 mesh sieve and is saved in drier after netting.
With urea/dmso solution starch, key step are as follows:
(1) 9.9mg Y17-231 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 4.95mL 90% is drawn in screw socket glass with 5mL liquid-transfering gun
In pipe；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 6 Rice Powder cytoplasmic mutation body Y17-231 starch is shown in Table 1.
Mutant verifying is the amylose content with reference to amylose kit method analysis Y17-231 starch, as a result
It is shown in Table 1.Data are average value ± SD (n=2), examine otherness by t, * * * indicates that Y17-231 and parent Kitaake's is straight
Chain content of starch is significant in the level difference of p < 0.001.
Embodiment 6
Screen homozygous rice silty seed mutant, key step are as follows:
(1) creation of rice mutant: with 300Gy dosage60Co gamma Rays handle conventional japonica rice kind
Kitaake。
(2) obtain homozygous offspring: in 2 generations of selfing, obtain M2 generation homozygous seed.
(3) selection of seed silty phenotype: choosing number is the fully opaque seed of Y17-252 endosperm, such as Fig. 1 silty
Mutant seed.
(4) seed is selected: selecting wherein 60, weighing: 0.99g.
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(2) homogenate crosses 100 mesh, 400 mesh screens respectively；
(3) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(4) remaining precipitating is suspended again with deionized water, repeated centrifugation, until being centrifuged rear surface free from admixture；
(5) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Y17-252 starch；
(6) it sieves with 100 mesh sieve and is saved in drier after netting.
With urea/dmso solution starch, key step are as follows:
(1) 10.0mg Y17-252 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 5mL 90% is drawn in screw socket glass tube with 5mL liquid-transfering gun
It is interior；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 7 Rice Powder cytoplasmic mutation body Y17-252 starch is shown in Table 1.
Mutant verifying is the amylose content with reference to amylose kit method analysis Y17-252 starch, as a result
It is shown in Table 1.Data are average value ± SD (n=2), examine otherness by t, * * * indicates that Y17-252 and parent Kitaake's is straight
Chain content of starch is significant in the level difference of p < 0.001.
Embodiment 7
Screen homozygous rice silty seed mutant, key step are as follows:
(1) creation of rice mutant: with 300Gy dosage60Co gamma Rays handle conventional japonica rice kind
Kitaake。
(2) obtain homozygous offspring: in 2 generations of selfing, obtain M2 generation homozygous seed.
(3) selection of seed silty phenotype: choosing number is the fully opaque seed of Y17-493 endosperm, such as Fig. 1 silty
Mutant seed.
(4) seed is selected: selecting wherein 53, weighing: 1.01g.
Seed is ground up, sieved, extracts starch, key step are as follows:
(1) water is added to continue to be ground into homogenate seed grind into powder with pestle；
(2) 100 mesh, 400 mesh screens are crossed in homogenate respectively；
(3) be centrifuged filtrate, be collected in 2mL centrifuge tube, condition: 4000g, 5min gently scrape off precipitation surface with spoon
Yellow non-starch impurity；
(4) remaining precipitating is suspended again with deionized water, repeated centrifugation, until centrifuged deposit surface free from admixture；
(5) it is finally washed 3 times with dehydrated alcohol, 40 DEG C of dry 2d obtain Y17-493 starch；
(6) it sieves with 100 mesh sieve and is saved in drier after netting.
With urea/dmso solution starch, key step are as follows:
(1) 9.8mg Y17-493 starch is weighed in 10mL screw socket glass graduated tube；
(2) UDMSO (the 6M urea liquid containing 10%) of 4.90mL 90% is drawn in screw socket glass with 5mL liquid-transfering gun
In pipe；
The preparation of 90% UDMSO (the 6M urea liquid containing 10%):
A. claim 7.2072g urea (urea) into the small beaker for filling 10mL deionized water；
B.40 half an hour is dissolved in DEG C water-bath, is transferred in graduated cylinder；
C. it is cooled to room temperature, is rinsed beaker 2 times with a small amount of water, be settled to 20mL；
D. it is mended with analysis level DMSO to 200mL.
(3) 10s is mixed on turbine mixer, put it into water-bath, 1h is dissolved in 95 DEG C of water-baths, and dissolution starts 5min rank
Section continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s by 1min；55min later, every 10min
Continue to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5s.
Iodine staining, spectroscopic assay, spectrum analysis, key step are as follows:
(1) starch solution taken out cools down 20min at room temperature；
(2) prepare 50mL volumetric flask, add 40mL deionized water, add 1mL starch solution and 1mL iodine solution (containing 0.2% iodine
With 2% potassium iodide):
The preparation of iodine solution (containing 0.2% iodine and 2% potassium iodide):
A. claim 0.2g iodine and 2g potassium iodide into 25mL small beaker；
B. plus 2mL deionized water dissolving iodine and potassium iodide, with glass bar stirring and dissolving 5min；
C. plus 10mL deionized water continues to dissolve, with glass stirring and dissolving 5min；
D. it is transferred to 100mL volumetric flask, is settled to 100mL with deionized water, is kept in dark place spare.
(3) it is settled to 50mL with deionized water at once, be mixed by inversion, be placed in reaction 20min in darkroom；
(4) it is formed sediment with 6300 spectrophotometer of Ultrospec (Amersham Bioscience, Cambridge, UK) analysis
The iodine absorption spectrum of powder, Setting pattern: Scanning, speed: Slow, scanning range: 400-900nm, scan unit: 1nm.
(5) analyze OD620, OD620/550 and maximum absorption wave wavelength X max, 620nm place absorbance reflection starch and
The affinity of iodine；OD620/550 reflects the ratio of long-chain and short chain in starch；Maximum absorption wavelength λ max reflection starch is averaged
Chain length.The starch ingredients parameter of the iodine absorption spectroanalysis of Fig. 8 Rice Powder cytoplasmic mutation body Y17-493 starch is shown in Table 1.
Mutant verifying is the amylose content with reference to amylose kit method analysis Y17-493 starch, as a result
It is shown in Table 1.Data are average value ± SD (n=2), examine otherness by t, * indicates the straight chain of Y17-493 and parent Kitaake
Content of starch is significant in the level difference of p < 0.05.
The extraction of starch can produce bigger effect the analysis of starch ingredients, and the difference of extracting method extracts starch
Purity has difference and is affected if extraction purity of starch difference is big for iodine absorption spectrum and parameter.UDMSO is molten simultaneously
Solution starch process ensures to dissolve thoroughly, and blob of viscose is not present, and blob of viscose mainly causes since starch dissolution is not thorough, it is necessary to survey again
It is fixed.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable
Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair
In bright scope of the claims.
1 starch iodine absorption spectrum parameter of table
Claims (4)
1. a kind of starch iodine absorption spectrum method for the rice silty seed mutant that quickly screening starch ingredients change, feature
It is, comprising the following steps:
The homozygous rice silty seed mutant of step 1), screening；The quality of homozygous rice silty seed is 0.8-1.2g, is contained
There are 40-60 seeds, every seed is taken from self progeny M2, the seed of complete opaque endosperm phenotype, and seed is horizontal
Section shows as full silty；
Seed in homozygous rice silty seed mutant is ground up, sieved, extracts starch by step 2)；It specifically includes
Following steps:
A, the rice silty seed pestle grind into powder that will have been screened, adds water to continue to be ground into homogenate, homogenate is successively
Suspension is obtained by 100 mesh, 400 mesh screens；
B, the suspension collection after sieving is subjected to centrifugal purification in 2mL centrifuge tube, when centrifugal purification, centrifugal force 3000-
4000g, centrifugation time 5-10min；After centrifugation, supernatant is removed, is precipitated；When precipitation surface free from admixture, starch is completed
Purifying, enters step e；When precipitation surface has impurity, c is entered step；
C, continue centrifugal purification after the precipitating after centrifugation gently being scraped off precipitation surface yellow impurities with spoon, centrifugal purification
When, centrifugal force 3000-4000g, centrifugation time 5-10min；When centrifuged deposit surface free from admixture, the pure of starch is completed
Change, enters step e；When there is impurity on centrifuged deposit surface, d is entered step；
D, it repeats step c several times, until centrifuged deposit surface free from admixture, completes the purifying of starch, obtain shallow lake after purification
Powder；
E, the dehydrated alcohol of starch after purification is rinsed 2-3 times, is subsequently placed in 35-40 DEG C of drying device dry 2-3 days and obtains
Starch after to drying；
F, by the starch ground after step e is dry at starch powder, starch powder is by 100 mesh screens, the starch after sieving
Powder is the starch extracted, is then saved starch in drier stand-by；
The starch obtained through step 2) is obtained starch solution with urea/dmso solution by step 3)；With urea/diformazan
Base sulfoxide dissolves starch, and additive amount is that the starch of every milligram of extraction adds 0.4-0.6mL urea/dimethyl sulfoxide；Urine
In element/dimethyl sulfoxide, the mass percent that urea accounts for dimethyl sulfoxide is 1.0-1.5%；
With urea/dmso solution starch, solution temperature is 95-98 DEG C, dissolution time 50-70min；Dissolution starts 5-
The 10min stage continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5-10s by 1-2min；11- later
The 60min stage continues to dissolve after urea/dimethyl sulfoxide, starch mixed liquor are mixed 5-10s by 5-10min；
Step 4), the starch solution for obtaining step 3) use iodine staining, the starch solution after being dyed, the shallow lake after dyeing
Powder solution carries out spectroscopic assay, is analyzed using conventional spectral analysis method spectrometric result.
2. a kind of starch iodine of rice silty seed mutant that quickly screening starch ingredients change according to claim 1
Absorption spectrum method, which is characterized in that in step 2), spoon is the spoon of 6-8cm.
3. a kind of starch iodine of rice silty seed mutant that quickly screening starch ingredients change according to claim 1
Absorption spectrum method, which is characterized in that in step 3), urea/dimethyl sulfoxide, starch mixed liquor use vortex mixed when mixing
Device mixes.
4. a kind of starch iodine of rice silty seed mutant that quickly screening starch ingredients change according to claim 1
Absorption spectrum method, which is characterized in that in step 3), urea/dimethyl sulfoxide dissolves starch in water-bath.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102179375A (en) * | 2011-03-09 | 2011-09-14 | 中国科学院合肥物质科学研究院 | Nondestructive detecting and screening method based on near-infrared for crop single-grain components |
| CN103592250A (en) * | 2013-11-08 | 2014-02-19 | 浙江省农业科学院 | Method for quickly measuring apparent amylose content of rice with high flux |
| CN108613943A (en) * | 2018-05-09 | 2018-10-02 | 中国科学院合肥物质科学研究院 | A kind of near-infrared single grain crop component detection method based on the transfer of spectrum form |
| CN108680515A (en) * | 2018-08-27 | 2018-10-19 | 中国科学院合肥物质科学研究院 | A kind of simple grain amylose in rice Quantitative Analysis Model structure and its detection method |
-
2018
- 2018-12-14 CN CN201811530774.XA patent/CN109323911A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102179375A (en) * | 2011-03-09 | 2011-09-14 | 中国科学院合肥物质科学研究院 | Nondestructive detecting and screening method based on near-infrared for crop single-grain components |
| CN103592250A (en) * | 2013-11-08 | 2014-02-19 | 浙江省农业科学院 | Method for quickly measuring apparent amylose content of rice with high flux |
| CN108613943A (en) * | 2018-05-09 | 2018-10-02 | 中国科学院合肥物质科学研究院 | A kind of near-infrared single grain crop component detection method based on the transfer of spectrum form |
| CN108680515A (en) * | 2018-08-27 | 2018-10-19 | 中国科学院合肥物质科学研究院 | A kind of simple grain amylose in rice Quantitative Analysis Model structure and its detection method |
Non-Patent Citations (6)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114149999A (en) * | 2021-12-16 | 2022-03-08 | 安徽农业大学 | Corn starch synthesis regulation gene ZmSSP1 and application thereof |
| CN114149999B (en) * | 2021-12-16 | 2023-11-17 | 安徽农业大学 | Corn starch synthesis regulatory gene ZmSSP1 and application thereof |
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