CN106442792A - HPLC determination method for gibberellin content in sugarcane leaves - Google Patents
HPLC determination method for gibberellin content in sugarcane leaves Download PDFInfo
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Abstract
The invention discloses an HPLC determination method for the gibberellin content in sugarcane leaves. The HPLC detection method comprises the following steps: (S1) adding liquid nitrogen into to-be-detected sugarcane leaves, grinding, carrying out methanol extraction and centrifugation, extracting supernate, extracting residues with methanol, extracting supernate after the centrifugation, combining the types of supernate, carrying out reduced pressure vaporization, carrying out petroleum ether extraction and decoloration, carrying out reduced pressure steaming on a low-layer water phase, adding a flow phase to dissolve, carrying out volume fixation, and filtering to obtain liquid as a sample solution for later use; and (S2) determining the gibberellin content in the sample solution by virtue of an HPLC. By virtue of the HPLC determination method, the gibberellin content in the sugarcane leaves can be determined, and the scientific basis is provided for the research of a biochemical mechanism of the gibberellin in the mediation of the stress response of pathogenic bacteria of the top rot of the sugarcanes.
Description
Technical field
The present invention relates to Caulis Sacchari sinensis top rot detection technique field, GA content in particularly a kind of Sugarcane Leaves
HPLC detection method.
Background technology
Caulis Sacchari sinensis（Saccharum officinarum) it is the most important sugar crop of China, sucrose accounts for the total product 92 of China's sugar
%.Because top rot gradually shows the features such as no regional and irregularities, lead to this disease increasingly tight to the harm of Sugarcane Industry
Weight.Emerge in an endless stream in the sugarcane districts such as Brazil, India, Iran, Malaysia and China all kinds of top rot symptom, especially high in high temperature
Rainy season easily breaks out this disease.According to the test of 2012-2014 Guangxi sugar cane breed region and 2013-2014 country Caulis Sacchari sinensis product
Plant regional testing investigation, tested variety sickness rate reaches 100%.This disease may result in Caulis Sacchari sinensis plant height to significantly reduce, and yield reduces 5%
～20%, sugar reduction reaches 3%.Caulis Sacchari sinensis, when by top rot Infected with Pathogenic Fungi, can produce a series of defense reaction to protect certainly
Oneself, meanwhile, pathogenic fungi also by changing itself toxicity or can produce variation, and thus impact Caulis Sacchari sinensis normal physiological biochemistry enters
Journey, thus infecting Caulis Sacchari sinensis and leading to fall ill.As a kind of important plant endogenous hormones, it participates in Caulis Sacchari sinensis physiological metabolism to gibberellins
One outstanding feature of process is to promote the elongation of sugarcane stem and sugarcane strain to increase.And Caulis Sacchari sinensis are using sugarcane stem as the crop harvesting product,
Therefore increasing sugarcane plant height degree aborning is considerable for improving sugarcane yield.But there is no foundation so far efficiently, accurately
Caulis Sacchari sinensis top rot blade GA content detection method, in view of the trend that the generation of China Caulis Sacchari sinensis top rot gradually increases, therefore
Set up a set of fast and effectively Caulis Sacchari sinensis top rot blade GA content detection method and mediate Caulis Sacchari sinensis top rot cause of disease to studying it
The biochemical mechanism of bacterium stress response has very important significance.
Content of the invention
For the blank of China's Caulis Sacchari sinensis top rot blade GA content detection method, it is an object of the invention to provide one
The HPLC detection method of GA content in kind simplicity, fast and accurately Sugarcane Leaves.
For achieving the above object, the technical method that the present invention adopts is as follows：
In a kind of Sugarcane Leaves, the HPLC detection method of GA content, comprises the following steps：
S1. Sugarcane Leaves to be measured add liquid nitrogen to grind, and add the volume fraction being cooled to 2 ~ 5 DEG C to be in advance that 70 ~ 90% methanol are water-soluble
Liquid, 2 ~ 5 DEG C of extraction 10 ~ 20h, centrifugation, take supernatant, it is 70 ~ 90% methanol that residue adds the pre- volume fraction being cooled to 2 ~ 5 DEG C
Aqueous solution extracts 1 ~ 3h, takes out supernatant after centrifugation, repeats residue extraction and centrifugally operated 0 ~ 3 time, merges supernatant, and 30 ~ 40
It is evaporated under reduced pressure at DEG C to without methanol, adding petroleum ether extraction to decolour 2 ~ 5 times, discards upper organic phase, lower floor's aqueous phase is 30 ~ 40
Evaporated under reduced pressure at DEG C, adds HPLC detection flowing used phased soln, constant volume, filters, standby as sample solution；
S2. adopt the content of gibberellins in HPLC detection sample solution.
Further, in described step S2, HPLC chromatogram condition is：Mobile phase presses volume by methanol, ultra-pure water and acetic acid
Ratio 200:300:3 are mixed, and column temperature is 32 ~ 38 DEG C, flow velocity 0.8 ~ 1.2ml/min, ultraviolet detection wavelength 306nm.More enter one
Step, HPLC chromatogram condition is：Column temperature is 35 DEG C, flow velocity 1ml/min, sample size 10 μ L.
Further, in described step S1, described Sugarcane Leaves to be measured are cut under ice chest.
Further, in described step S1, to use volume fraction be 80% for Sugarcane Leaves to be measured and residue extraction
Methanol aqueous solution.
Further, in described step S1, the temperature of Sugarcane Leaves to be measured and residue extraction is 4 DEG C.
Further, in described step S1, Sugarcane Leaves to be measured are extracted using methanol aqueous solution, liquid-solid ratio be 0.2 ~
0.5g/mL.
Further, in described step S5, after adding flowing phased soln, filtered using 0.45 μm of microporous filter membrane.
Further, in described step S1, Sugarcane Leaves to be measured and residue extraction adopt methanol aqueous solution be methanol-
Ultra-pure water solution.
The present invention still further provides above-described detection method to judge sugar cane breed perceptual to top rot and anti-
The application of sex differernce aspect.
The invention has the beneficial effects as follows：
1）Sample-pretreating method adopts methanol-ultra-pure water extractant to combine ultrasonic extraction and extracts gibberellins；And pass through stone
Oily ether decolouring purifies to sample；Compared with additive method, good impurity removing effect, extraction time is short, testing sample high purity.
2）By the content of gibberellins in HPLC chromatogram conditioned measurement sample solution of the present invention, gibberellins can be effective with other
Composition preferably carries out separating, and sample retention time is stable, has more preferable peak shape, is possessing the reality of high performance liquid chromatograph
Test room and all GA content detection work can be carried out by this method.
3）The present invention can detect that Caulis Sacchari sinensis before processing rear blade GA content changes, and testing result shows：Inoculation process with
There is significant difference in the Sugarcane Leaves GA content of normal growth, and diversity trend increases；The Sugarcane Leaves of normal growth
Middle GA content can be gradually increasing, and in the Sugarcane Leaves that inoculation is processed, GA content is gradually reduced；This testing result is to grind
The biochemical mechanism studying carefully its mediation Caulis Sacchari sinensis top rot pathogen stress response provides scientific basis.Meanwhile, the promoting the use of of the method,
For screening disease-resistant variety and instruct Caulis Sacchari sinensis breeding for disease resistance significant and realistic function.
Brief description
Fig. 1 is the standard working curve of gibberellins.
The chromatogram that Fig. 2 a obtains for standard solution detection.
The chromatogram that Fig. 2 b obtains for sample solution detection.
Fig. 3 inoculates the trend analysiss figure of GA content in sample and Caulis Sacchari sinensis control sample for different growth phases Caulis Sacchari sinensis.
Specific embodiment
With reference to specific embodiment, further details of elaboration is made to the present invention, but embodiments of the present invention are not
It is confined to the scope that embodiment represents.These embodiments are merely to illustrate the present invention, not for restriction the scope of the present invention.This
Outward, after reading present disclosure, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same
Sample falls within appended claims limited range of the present invention.
The HPLC detection method of GA content in embodiment 1 Sugarcane Leaves.
S1. the Sugarcane Leaves choosing infection Caulis Sacchari sinensis top rot cause of disease, as testing sample, take testing sample 2g to add liquid nitrogen
Grind, add volume fraction 80% methanol aqueous solution that 10mL is cooled to 4 DEG C in advance, fresh-keeping film phonograph seal extracts 16h after 4 DEG C；Then in
It is centrifuged 10min with 8000 r/min under the conditions of 4 DEG C and obtains supernatant, residue 5mL is cooled to 4 DEG C its 80% methanol of volume fraction in advance
Aqueous solution extracts 2h, takes out supernatant after centrifugation 10min, merges supernatant twice and is evaporated under reduced pressure to without methanol in 40 DEG C（About
Surplus 0.3mL aqueous solution）, add 15mL petroleum ether extraction to decolour 3 times, discard upper organic phase, decompression at 40 DEG C of lower floor's aqueous phase is steamed
Dry, add 0.5mLHPLC detection flowing used phased soln, be settled to 2mL, through 0.45 μm of filtering with microporous membrane in internal lining pipe
Sample bottle in, as sample solution.
S2. adopt salicylic content in HPLC detection sample solution.
S2-1. chromatographic condition：ARigol L3000 high performance liquid chromatograph；Chromatographic column：Kromasil C18 reversed phase chromatography
Post（250mm × 4.6mm, 5 μm）；Mobile phase：200mL methanol and the mixing of 300mL ultra-pure water, add 3mL acetic acid；Column temperature：35℃；
Time of losing shape is 20min；Flow velocity：0.8 mL/min；Detection wavelength：306nm；Sampling volume：10μL.
S2-2. weigh gibberellins standard substance 1.25mg, add 10mL water dissolution, be configured to the standard solution of 125 μ g/mL
Mother solution；Then it is diluted to, with standard solution mother solution, the mark that concentration is respectively 25 μ g/mL, 12.5 μ g/mL, 1.25 μ g/mL again
Quasi- solution, using chromatographic condition examination criteria solution mother solution and the standard solution of S2-1, its corresponding peak area respectively 51.256,
7.311、4.571、0.272；Then with peak area as vertical coordinate, standard curve is done for abscissa with gibberellins concentration, standard is bent
As described in Figure 1, as seen from Figure 1, in institute's detection range (1.25 μ g/mL～125 μ g/mL), regression equation is y=to line
0.4175x-1.2383, liquid chromatograph peak area and gibberellins concentration have good linear relationship (R2= 0.9971).
S2-3. specificity is investigated：Take standard solution mother solution and the sample solution injection chromatograph of liquid of 125 μ g/mL respectively
In, the chromatographic condition according to S2-1 is detected, the retention time of gibberellins is 7.77 min, standard solution mother solution testing result
See Fig. 2 a, sample solution testing result is shown in Fig. 2 b.
S2-4. recovery testu：The concentration adding 1mL respectively in same sample solution is 10 μ g/mL, 20 μ g/
ML, 30 μ g/mL, the gibberellins standard solution of 50 μ g/mL, according to 5.1 liquid phase chromatogram condition detection, calculate the response rate, knot
Fruit is shown in Table 1.
Table 1 gibberellins determination of recovery rates result
S2-5. method precision test：Take the standard solution that concentration is 1.25 μ g/mL, according to the chromatographic condition inspection of S2-1
Survey, continuous sample introduction 5 pin observes retention time and peak area, the results are shown in Table 2.
Table 2 method precision experimental result
| Numbering | Retention time（min） | Peak area（μv sec） |
| 1 | 7.768 | 1.358 |
| 2 | 7.752 | 1.345 |
| 3 | 7.643 | 1.324 |
| 4 | 7.718 | 1.296 |
| 5 | 7.735 | 1.320 |
| Standard deviation | 0.049 | 0.024 |
| Relative standard deviation | 0.629 | 1.801 |
The sample solution analysis that S2-6.S1 obtains.
Using the chromatographic condition detection of S2-1, it is repeated three times, the content obtaining the middle gibberellins of sample is 2.456 μ g/
g.
By the present embodiment as can be seen that the sensitivity of this method, detection limit and precision disclosure satisfy that water in Sugarcane Leaves
The mensure of poplar acid content；It is particularly suited for the mensure of salicylic acid content in the Sugarcane Leaves infect top rot, by above-mentioned sample
Pre-treating method and chromatographic condition, the interference to testing result for the top rot pathogen can be excluded.
Embodiment 2 judge sugar cane breed infect top rot after perception and Resistant Difference research.
（1）The preparation of inoculum：Caulis Sacchari sinensis top rot pathogen is inoculated in PDA culture medium and activates 3 days, picking PDA trains
Foster base edge mycelium inoculation shaken cultivation 3 days in the potato glucose water culture medium of sterilizing, centrifugation, the absorbent cotton with sterilizing
Filter, collect obtained thalline, being configured to concentration with sterilized water and thalline is 1 × 106CFU/ml Caulis Sacchari sinensis top rot pathogen
Spore suspension.
（2）The process of inoculation material：Choosing sugar cane breed YT94-128, sugar cane breed GT37 is inoculation material, greenhouse bar
Under part, each kind plants 30 barrels, every barrel of 4 buds, with 0.3% carbendazim seed soaking 20min before sowing, normal management after emerging is long
Inoculated when going out 5～6 complete leaves.
（3）Injection inoculation：Take each 30 plants of the seedling of growing way identical sugar cane breed YT94-128, sugar cane breed GT37, make
With 1ml injector for medical purpose respectively by 100 μ l Caulis Sacchari sinensis top rot pathogen spore suspension to Caulis Sacchari sinensis+1 leaf position, connect as Caulis Sacchari sinensis
Plant sample；Still further take each 30 plants of the seedling of growing way identical sugar cane breed YT94-128, sugar cane breed GT37, aseptic to inject
Water is comparison, as Caulis Sacchari sinensis control sample；Caulis Sacchari sinensis inoculation keeps indoor temperature 28-30 DEG C, humidity 80% after terminating.
（4）Sample is chosen:Identical bits on same strain age+1 leaf position are selected to be equipped with the Sugarcane Leaves of approximate disease, on ice chest
The blade being cut into 1cm × 1cm carries out standby sample.
（5）Detection method and result
S1. take each growth stage（2、4、8、16d）Caulis Sacchari sinensis inoculation sample YT94-128, GT37 and Caulis Sacchari sinensis control sample YT94-
128th, GT37 blade 2g is separately added into liquid nitrogen and grinds, and adds volume fraction 80% methanol aqueous solution of 10mL4 DEG C of pre-cooling, preservative film
Sealing extracts 16h after 4 DEG C；Then it is centrifuged 10min with 8000 r/min under the conditions of 4 DEG C and obtain supernatant, residue 5mL pre-cooling
Extract 2h to its 80% methanol aqueous solution of 4 DEG C of volume fractions, after centrifugation 10min, take out supernatant, merge twice supernatant in 40
DEG C it is evaporated under reduced pressure to without methanol（About remain 0.3mL aqueous solution）, add 15mL petroleum ether extraction to decolour 3 times, discard upper strata organic
Phase, evaporated under reduced pressure at 40 DEG C of lower floor's aqueous phase, add 0.5mLHPLC detection flowing used phased soln, be settled to 2mL, through 0.45 μm
Filtering with microporous membrane in the sample bottle with internal lining pipe, as study sample solution for standby；
S2. adopt the content of gibberellins in HPLC detection sample solution.
S2-1. chromatographic condition：ARigol L3000 high performance liquid chromatograph；Chromatographic column：Kromasil C18 reversed phase chromatography
Post（250mm × 4.6mm, 5 μm）；Mobile phase：200mL methanol and the mixing of 300mL ultra-pure water, add 3mL acetic acid；Column temperature：35℃；
Time of losing shape is 20min；Flow velocity：0.8 mL/min；Detection wavelength：306nm；Sampling volume：10μL.
S2-2 takes different growth phases（2、4、8、16d）Caulis Sacchari sinensis inoculation sample YT94-128, GT37 and Caulis Sacchari sinensis control sample
The study sample solution that product are made, carries out the mensure of GA content, each sample replication 3 times, the results are shown in Table 3.
Table 3 different growth phases Caulis Sacchari sinensis inoculate the content of gibberellins in sample
S2-3 interpretation.
Gibberellins（GA）As a kind of important plant endogenous hormones, it participates in the prominent spy of Caulis Sacchari sinensis physiological metabolism process one
Point is to promote the elongation of sugarcane stem and sugarcane strain to increase.Identified, YT94-128 shows resistance to top rot, and GT37 is to top rot table
Reveal perception.
The Long-term change trend figure of GA content is obtained by the data that above-mentioned table 3 obtains, as shown in figure 3, wherein, A is comparison
The GA content variation tendency of sample GT37, B is the GA content variation tendency of control sample YT94-128, and C is inoculation
The GA content variation tendency of sample GT37, D is the GA content variation tendency of inoculation sample YT94-128, in conjunction with Fig. 3
Shown, the GA content in control sample is gradually increasing with sugarcane production, the GA content change of susceptible variety GT37
Trend is higher than disease-resistant variety YT94-128；In the Sugarcane Leaves that pathogen inoculation is processed, GA content is continuous with sugarcane production
Reduce, the trend that the GA content of susceptible variety GT37 reduces is higher than YT94-128；Wherein, 2 to 4 days after inoculation, distillation
GA content difference between water inoculation and pathogen inoculation process is not notable, but time, inoculation about inoculation one week after
Process with compare between difference reach extremely notable.
This test illustrates, under space management, the speed that susceptible variety GT37 blade synthesizes gibberellins is higher than disease-resistant variety
YT94-128；After inoculation pathogen, susceptible variety GT37 top rot disease index constantly increases, and its blade synthesizes gibberellins
Ability is significantly affected, and sugarcane strain simultaneously is by biology（Pathogen）With abiotic（Acupuncture）Occur gibberellins secondary after stress
Synthetic reaction, but the former is more than the latter, thus leading to GA content in GT37 blade to be in significantly reduce trend.Disease-resistant variety
After inoculation pathogen, although its disease index is not serious, the ability that sugarcane strain blade synthesizes gibberellins is also subject to YT94-128
To impact it may be possible to YT94-128 blade synthesis gibberellins speed itself is not very prominent, arrive 12 to 16 days after inoculation
Show the similarity change with GT37 blade GA content.
Conclusion：Be can be seen that by test, same sugar cane breed, after infection top rot the 7th to 10 day, the Caulis Sacchari sinensis of normal growth
Between the Sugarcane Leaves of blade and infection top rot, GA content difference is less, and this sugar cane breed shows resistance to top rot
Probability bigger, conversely, this sugar cane breed top rot is shown perception probability bigger.This result of the test is research
Gibberellins resistance Mechanism of Physiological and Biochemical, screen disease-resistant variety and instruct Caulis Sacchari sinensis breeding for disease resistance to provide important Technical Reference.
Specific embodiment described herein is only explanation for example to present invention spirit.The affiliated technology of the present invention is led
The technical staff in domain can be made various modifications or supplement or replaced using similar mode to described specific embodiment
Generation, but the spirit without departing from the present invention or surmount scope defined in appended claims.
Claims (10)
1. in a kind of Sugarcane Leaves the HPLC detection method of GA content it is characterised in that comprising the following steps：
S1. Sugarcane Leaves to be measured add liquid nitrogen to grind, and add the volume fraction being cooled to 2 ~ 5 DEG C to be in advance that 70 ~ 90% methanol are water-soluble
Liquid, 2 ~ 5 DEG C of extraction 10 ~ 20h, centrifugation, take supernatant, it is 70 ~ 90% methanol that residue adds the pre- volume fraction being cooled to 2 ~ 5 DEG C
Aqueous solution extracts 1 ~ 3h, takes out supernatant after centrifugation, repeats residue extraction and centrifugally operated 0 ~ 3 time, merges supernatant, and 30 ~ 40
It is evaporated under reduced pressure at DEG C to without methanol, adding petroleum ether extraction to decolour 2 ~ 5 times, discards upper organic phase, lower floor's aqueous phase is 30 ~ 40
Evaporated under reduced pressure at DEG C, adds HPLC detection flowing used phased soln, constant volume, filters, standby as sample solution；
S2. adopt the content of gibberellins in HPLC detection sample solution.
2. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S2, HPLC chromatogram condition is：Mobile phase is by methanol, ultra-pure water and acetic acid by volume 200:300:3 mix
Conjunction is made, and column temperature is 32 ~ 38 DEG C, flow velocity 0.8 ~ 1.2ml/min, ultraviolet detection wavelength 306nm.
3. in Sugarcane Leaves according to claim 2 GA content HPLC detection method it is characterised in that：
In described step S2, HPLC chromatogram condition is：Column temperature is 35 DEG C, flow velocity 1ml/min, sample size 10 μ L.
4. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S1, described Sugarcane Leaves to be measured are cut under ice chest.
5. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S1, Sugarcane Leaves to be measured and residue extraction use the methanol aqueous solution that volume fraction is 80%.
6. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S1, the temperature of Sugarcane Leaves to be measured and residue extraction is 4 DEG C.
7. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S1, Sugarcane Leaves to be measured are extracted using methanol aqueous solution, and liquid-solid ratio is 0.2 ~ 0.5g/mL.
8. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S5, after adding flowing phased soln, filtered using 0.45 μm of microporous filter membrane.
9. in Sugarcane Leaves according to claim 1 GA content HPLC detection method it is characterised in that：
In described step S1, the methanol aqueous solution of Sugarcane Leaves to be measured and residue extraction employing is methanol-ultra-pure water solution.
10. the detection method as described in any one of claim 1 ~ 9 is judging sugar cane breed to top rot perception and Resistant Difference
The application of aspect.
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| CN104897843A (en) * | 2015-06-24 | 2015-09-09 | 南京信息工程大学 | Method for measuring content of endogenous hormones of burgeons of tea tree |
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