CN106442792B - The HPLC detection method of GA content in a kind of Sugarcane Leaves - Google Patents
The HPLC detection method of GA content in a kind of Sugarcane Leaves Download PDFInfo
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Abstract
The invention discloses a kind of HPLC detection methods of GA content in Sugarcane Leaves, comprising the following steps: S1. grinds Sugarcane Leaves to be measured addition liquid nitrogen, methanol extraction, centrifugation, takes supernatant, residue is extracted with methanol, supernatant is taken out after centrifugation, merges supernatant, is evaporated under reduced pressure, petroleum ether extraction decoloration, flowing phased soln, constant volume is added in lower water phase evaporated under reduced pressure, filtering, it is spare as sample solution；S2. using the content of gibberellin in HPLC test sample solution.This method can accurately detect GA content in Sugarcane Leaves, mediate the biochemical mechanism of sugarcane top rot pathogen stress response to provide scientific basis to study it.
Description
Technical field
The present invention relates to sugarcane top rot detection technique field, GA content in especially a kind of Sugarcane Leaves
HPLC detection method.
Background technique
Sugarcane (Saccharum officinarum) it is the most important sugar crop in China, sucrose accounts for China's sugar total yield 92
%.The features such as gradually being shown due to top rot without regional and irregularities, cause the disease increasingly tight to the harm of Sugarcane Industry
Weight.It emerges one after another in all kinds of top rot symptoms of the sugarcane districts such as Brazil, India, Iran, Malaysia and China, especially in high temperature height
Rainy season is easy to break out this disease.According to the test of the Guangxi 2012-2014 sugar cane breed region and the national sugarcane product of 2013-2014
Kind regional testing investigation, tested variety disease incidence reach 100%.This disease can lead to the significant decrease of sugarcane plant height, and yield reduces 5%
~20%, sugar is reduced up to 3%.Sugarcane can generate a series of defense reaction when by top rot Infected with Pathogenic Fungi to protect certainly
Oneself, at the same time, disease fungus also can by change itself toxicity or generate variation, thus influence sugarcane normal physiological biochemistry into
Journey, to infect sugarcane and cause to fall ill.Gibberellin participates in sugarcane physiological metabolism as a kind of important plant endogenous hormones
One outstanding feature of process is that the elongation of sugarcane stem and sugarcane strain is promoted to increase.And sugarcane is the crop using sugarcane stem as harvest product,
Therefore increasing sugarcane plant height degree in production is considerable for improving sugarcane yield.But it there is no foundation so far efficiently, accurately
Sugarcane top rot blade GA content detection method, the trend that gradually aggravates occurs in view of China's sugarcane top rot, therefore
It establishes a set of quickly and effectively sugarcane top rot blade GA content detection method and mediates sugarcane top rot cause of disease to it is studied
The biochemical mechanism of bacterium stress response has very important significance.
Summary of the invention
For the blank of China's sugarcane top rot blade GA content detection method, the purpose of the present invention is to provide one
Plant the HPLC detection method of GA content in simplicity, fast and accurately Sugarcane Leaves.
To achieve the above object, the technical method that the present invention uses is as follows:
The HPLC detection method of GA content in a kind of Sugarcane Leaves, comprising the following steps:
S1. Sugarcane Leaves to be measured are added liquid nitrogen and grind, and are added and are cooled to 2 ~ 5 DEG C of volume fraction in advance for 70 ~ 90% methanol
Aqueous solution, 2 ~ 5 DEG C of 10 ~ 20h of extraction, centrifugation take supernatant, and it is 70 ~ 90% that residue, which adds the pre- volume fraction for being cooled to 2 ~ 5 DEG C,
Methanol aqueous solution extracts 1 ~ 3h, takes out supernatant after centrifugation, repeats residue extraction and centrifugally operated 0 ~ 3 time, merges supernatant, and 30
It is evaporated under reduced pressure at ~ 40 DEG C to methanol is free of, petroleum ether extraction is added and decolourizes 2 ~ 5 times, discards upper organic phase, lower layer's water phase is 30
HPLC detection flowing phased soln used is added in evaporated under reduced pressure at ~ 40 DEG C, and constant volume filters, spare as sample solution；
S2. using the content of gibberellin in HPLC test sample solution.
Further, in the step S2, HPLC chromatogram condition are as follows: mobile phase presses volume by methanol, ultrapure water and acetic acid
It is mixed than 200:300:3, column temperature is 32 ~ 38 DEG C, 0.8 ~ 1.2ml/min of flow velocity, ultraviolet detection wavelength 306nm.More into one
Step, HPLC chromatogram condition are as follows: column temperature is 35 DEG C, flow velocity 1ml/min, 10 μ L of sample volume.
Further, in the step S1, the Sugarcane Leaves to be measured are cut under ice chest.
Further, in the step S1, Sugarcane Leaves and residue extraction to be measured is 80% using volume fraction
Methanol aqueous solution.
Further, in the step S1, the temperature of Sugarcane Leaves and residue extraction to be measured is 4 DEG C.
Further, in the step S1, Sugarcane Leaves to be measured are extracted using methanol aqueous solution, and liquid-solid ratio is 0.2 ~
0.5g/mL。
Further, it in the step S5, after flowing phased soln is added, is filtered using 0.45 μm of miillpore filter.
Further, in the step S1, the methanol aqueous solution that Sugarcane Leaves and residue extraction to be measured uses is methanol-
Ultra-pure water solution.
The present invention still further provides above-described detection method and is judging sugar cane breed to top rot perception and resisting
Application in terms of sex differernce.
The beneficial effects of the present invention are:
1) sample-pretreating method extracts gibberellin using methanol-ultrapure water extractant combination ultrasonic extraction；And lead to
Petroleum ether decoloration is crossed to purify sample；It is compared with other methods, good impurity removing effect, extraction time is short, and sample to be tested is pure
Degree is high.
2) through the invention in HPLC chromatogram conditioned measurement sample solution gibberellin content, gibberellin can with other effectively
Ingredient is preferably separated, and sample retention time is stablized, and has better peak shape, in the reality for having high performance liquid chromatograph
GA content detection work can be carried out by this method by testing room.
3) present invention can detect sugarcane blade GA content changes before and after the processing, and testing result shows: inoculation processing with
There are significant differences for the Sugarcane Leaves GA content of normal growth, and otherness trend increases；The Sugarcane Leaves of normal growth
Middle GA content can be gradually increasing, and be inoculated with GA content in the Sugarcane Leaves of processing and be gradually reduced；The testing result is to grind
Study carefully the biochemical mechanism that it mediates sugarcane top rot pathogen stress response and scientific basis is provided.Meanwhile the popularization and use of this method,
For screening disease-resistant variety and sugarcane breeding for disease resistance is instructed to be of great significance and realistic function.
Detailed description of the invention
Fig. 1 is the standard working curve of gibberellin.
Fig. 2 a is the chromatogram that standard solution detects.
Fig. 2 b is the chromatogram that sample solution detects.
Fig. 3 is the trend analysis figure that different stages of growth sugarcane is inoculated with GA content in sample and sugarcane control sample.
Specific embodiment
Combined with specific embodiments below, further details of elaboration is made to the present invention, but embodiments of the present invention are not
It is confined to the range of embodiment expression.These embodiments are merely to illustrate the present invention, range and is not intended to limit the present invention.This
Outside, after reading the contents of the present invention, those skilled in the art can various modifications may be made to the present invention, these equivalent variations are same
Sample falls within the appended claims limited range of the present invention.
The HPLC detection method of GA content in 1 Sugarcane Leaves of embodiment.
S1. the Sugarcane Leaves of infection sugarcane top rot cause of disease are chosen as sample to be tested, take sample to be tested 2g that liquid nitrogen is added
It grinds, 80% methanol aqueous solution of volume fraction that 10mL is cooled to 4 DEG C in advance is added, in 4 DEG C of extraction 16h after preservative film sealing；Then in
Supernatant is obtained with 8000 r/min centrifugation 10min under the conditions of 4 DEG C, residue is cooled to its 80% methanol of 4 DEG C of volume fraction with 5mL in advance
Aqueous solution extracts 2h, takes out supernatant after being centrifuged 10min, merge supernatant twice be evaporated under reduced pressure in 40 DEG C to without methanol (about
Surplus 0.3mL aqueous solution), 15mL petroleum ether extraction is added and decolourizes 3 times, discards upper organic phase, depressurizes and steam at 40 DEG C of lower layer's water phase
It does, 0.5mLHPLC detection flowing phased soln used is added, 2mL is settled to, through 0.45 μm of filtering with microporous membrane in internal lining pipe
Sample bottle in, as sample solution.
S2. using salicylic content in HPLC test sample solution.
S2-1. chromatographic condition: ARigol L3000 high performance liquid chromatograph；Chromatographic column: Kromasil C18 reverse-phase chromatography
Column (250mm × 4.6mm, 5 μm)；Mobile phase: 3mL acetic acid is added in 200mL methanol and the mixing of 300mL ultrapure water；Column temperature: 35 DEG C；
The time lose shape as 20min；Flow velocity: 0.8 mL/min；Detection wavelength: 306nm；Sampling volume: 10 μ L.
S2-2. gibberellin standard items 1.25mg is weighed, the dissolution of 10mL water is added, is configured to the standard solution of 125 μ g/mL
Mother liquor；Then it is diluted to the mark that concentration is respectively 25 μ g/mL, 12.5 μ g/mL, 1.25 μ g/mL again with standard solution mother liquor
Quasi- solution, using the chromatographic condition examination criteria solution mother liquor and standard solution of S2-1, correspond to peak area be respectively 51.256,
7.311,4.571,0.272；Then using peak area as ordinate, do standard curve by abscissa of gibberellin concentration, standard is bent
Line is as described in Figure 1, as seen from Figure 1, in institute's detection range (1.25 μ of μ g/mL~125 g/mL), regression equation be y=
0.4175x-1.2383, liquid chromatogram peak area and gibberellin concentration have good linear relationship (R2= 0.9971)。
S2-3. specificity is investigated: taking the standard solution mother liquor and sample solution injection liquid chromatograph of 125 μ g/mL respectively
In, it is detected according to the chromatographic condition of S2-1, the retention time of gibberellin is 7.77 min, standard solution mother liquor testing result
See Fig. 2 a, sample solution testing result is shown in Fig. 2 b.
S2-4. recovery testu: the concentration for adding 1mL into same sample solution respectively is 10 μ g/mL, 20 μ g/
The gibberellin standard solution of mL, 30 μ g/mL, 50 μ g/mL are detected according to 5.1 liquid phase chromatogram condition, calculate the rate of recovery, knot
Fruit is shown in Table 1.
1 gibberellin determination of recovery rates result of table
S2-5. method precision is tested: taking concentration is the standard solution of 1.25 μ g/mL, according to the chromatographic condition of S2-1
Detection, 5 needle of continuous sample introduction observe retention time and peak area, the results are shown in Table 2.
2 method precision experimental result of table
| Number | Retention time (min) | Peak area (μ v 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 is obtained.
It is detected, is repeated three times using the chromatographic condition of S2-1, the content for obtaining the middle gibberellin of sample is 2.456 μ g/
g。
Water in Sugarcane Leaves can satisfy by the sensitivity of this method, detection limit and precision it can be seen from the present embodiment
The measurement of poplar acid content；It is particularly suitable for the measurement of salicylic acid content in the Sugarcane Leaves of infection top rot, passes through above-mentioned sample
Pre-treating method and chromatographic condition, interference of the top rot pathogen to testing result can be excluded.
Embodiment 2 judges the perception and Resistant Difference research after sugar cane breed infection top rot.
(1) preparation of inoculum: sugarcane top rot pathogen being inoculated in PDA culture medium and is activated 3 days, picking PDA training
Base edge mycelium inoculation is supported shaken cultivation 3 days in the potato glucose water culture medium of sterilizing, centrifugation, with the absorbent cotton of sterilizing
It filters, collect thallus obtained, being configured to concentration with sterile water and thallus is 1 × 106 CFU/ml sugarcane top rot pathogen
Spore suspension.
(2) processing of inoculation material: choosing sugar cane breed YT94-128, sugar cane breed GT37 is inoculation material, greenhouse item
Each kind plants 30 barrels under part, and every barrel of 4 buds, with 0.3% carbendazim seed soaking 20min before sowing, normal management after emergence is long
It is inoculated with when 5~6 complete leaves out.
(3) injection inoculation: the identical sugar cane breed YT94-128 of growing way, each 30 plants of seedling of sugar cane breed GT37 are taken, is made
With 1ml injector for medical purpose respectively by 100 μ l sugarcane top rot pathogen spore suspensions to+1 leaf position of sugarcane, connect as sugarcane
Kind sample；The identical sugar cane breed YT94-128 of growing way, each 30 plants of seedling of sugar cane breed GT37 are still further taken, it is sterile to inject
Water is control, as sugarcane control sample；Sugarcane is kept for 28-30 DEG C of room temperature after being inoculated with, humidity 80%.
(4) sample is chosen: identical bits are equipped with the Sugarcane Leaves of approximate illness on selection+1 leaf position of same strain age, on ice chest
The blade for being cut into 1cm × 1cm carries out standby sample.
(5) detection method and result
S1. the sugarcane of each growth phase (2,4,8,16d) is taken to be inoculated with sample YT94-128, GT37 and sugarcane control sample
YT94-128, GT37 blade 2g are separately added into liquid nitrogen and grind, and 80% methanol aqueous solution of volume fraction of 10mL4 DEG C of pre-cooling is added, and protect
In 4 DEG C of extraction 16h after fresh film sealing；Then supernatant, residue 5mL are obtained with 8000 r/min centrifugation 10min under the conditions of 4 DEG C
It is cooled to its 80% methanol aqueous solution of 4 DEG C of volume fraction extraction 2h in advance, takes out supernatant after being centrifuged 10min, merge supernatant twice
It is evaporated under reduced pressure in 40 DEG C to without methanol (about surplus 0.3mL aqueous solution), 15mL petroleum ether extraction is added and decolourizes 3 times, discards upper layer
Organic phase, evaporated under reduced pressure at 40 DEG C of lower layer's water phase are added that 0.5mLHPLC detection is used to flow phased soln, are settled to 2mL, pass through
0.45 μm of filtering with microporous membrane in internal lining pipe sample bottle in, as study sample solution for standby；
S2. using the content of gibberellin in HPLC test sample solution.
S2-1. chromatographic condition: ARigol L3000 high performance liquid chromatograph；Chromatographic column: Kromasil C18 reverse-phase chromatography
Column (250mm × 4.6mm, 5 μm)；Mobile phase: 3mL acetic acid is added in 200mL methanol and the mixing of 300mL ultrapure water；Column temperature: 35 DEG C；
The time lose shape as 20min；Flow velocity: 0.8 mL/min；Detection wavelength: 306nm；Sampling volume: 10 μ L.
S2-2 takes the sugarcane of different stages of growth (2,4,8,16d) to be inoculated with sample YT94-128, GT37 and sugarcane control sample
Study sample solution made of product, carries out the measurement of GA content, and each sample replication 3 times the results are shown in Table 3.
3 different stages of growth sugarcane of table is inoculated with the content of gibberellin in sample
S2-3 analysis of experimental results.
Gibberellin (GA) participates in sugarcane physiological metabolism process one prominent spy as a kind of important plant endogenous hormones
Point is that the elongation of sugarcane stem and sugarcane strain is promoted 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 made by the data that above-mentioned table 3 obtains, as shown in Figure 3, wherein A is control
The GA content variation tendency of sample GT37, B are 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 for being inoculated with sample YT94-128, in conjunction with Fig. 3
Shown, the GA content in control sample is gradually increasing with sugarcane production, the GA content variation of susceptible variety GT37
Trend is higher than disease-resistant variety YT94-128；GA content is continuous with sugarcane production in the Sugarcane Leaves of pathogen inoculation processing
It reduces, 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 processing is not significant, but is inoculated with latter all left and right time, inoculation
Handle with compare between difference reach extremely significant.
This test illustrates, under space management, the rate of susceptible variety GT37 blade synthesis gibberellin is higher than disease-resistant variety
YT94-128；After being inoculated with pathogen, susceptible variety GT37 top rot disease index is constantly aggravated, and blade synthesizes gibberellin
Ability is significantly affected, while sugarcane strain generation gibberellin after by biological (pathogen) and abiotic (needle thorn) stress is secondary
Synthetic reaction, but the former is greater than the latter, is in significant decreasing trend so as to cause GA content in GT37 blade.Disease-resistant variety
YT94-128 is after being inoculated with pathogen, although its disease index is not serious, sugarcane strain blade synthesize gibberellin ability also by
To influence, it may be possible to which it is not to protrude very much that YT94-128 blade itself, which synthesizes gibberellin rate, has arrived after inoculation 12 to 16 days
It shows to change with the similitude of GT37 blade GA content.
Conclusion: can be seen that by test, same sugar cane breed, after infection top rot the 7th to 10 day, the sugarcane of normal growth
GA content difference is smaller between blade and the Sugarcane Leaves for infecting top rot, which shows resistance to top rot
A possibility that it is bigger, conversely, the sugar cane breed is bigger a possibility that showing perception to top rot.This test result is research
Gibberellin resistance Mechanism of Physiological and Biochemical screens disease-resistant variety and sugarcane breeding for disease resistance is instructed to provide important Technical Reference.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (2)
1. the application of GA content HPLC detection method in a kind of Sugarcane Leaves, it is characterised in that:
GA content HPLC detection method is in terms of judging sugar cane breed to top rot perception and Resistant Difference in Sugarcane Leaves
Application；
Detection method includes the following steps by the HPLC:
S1. Sugarcane Leaves to be measured are cut under ice chest, and liquid nitrogen is added and grinds, and it is 80% that the pre- volume fraction for being cooled to 4 DEG C, which is added,
Methanol aqueous solution, liquid-solid ratio are 0.2 ~ 0.5g/mL, 4 DEG C of extraction 16h, are centrifuged, take supernatant, and residue, which adds, pre- is cooled to 4 DEG C
Volume fraction be that 80% methanol aqueous solution extracts 2h, take out supernatant after centrifugation, merge supernatant, be evaporated under reduced pressure at 40 DEG C to
Without methanol, petroleum ether extraction is added and decolourizes 3 times, discard upper organic phase, lower layer's water phase evaporated under reduced pressure at 40 DEG C is added
HPLC detection is used to flow phased soln, and constant volume is spare as sample solution using 0.45 μm of filtering with microporous membrane；
S2. using the content of gibberellin in HPLC test sample solution, HPLC chromatogram condition are as follows: mobile phase is by methanol, ultrapure water
200:300:3 is mixed by volume with acetic acid, and column temperature is 35 DEG C, flow velocity 1ml/min, 10 μ L of sample volume, ultraviolet detection wave
Long 306nm.
2. the application of GA content HPLC detection method in Sugarcane Leaves according to claim 1, it is characterised in that:
In the step S1, the methanol aqueous solution that Sugarcane Leaves and residue extraction to be measured uses is methanol-ultra-pure water solution.
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