CN105259123B - A kind of brown stain of cut lotus root control method based on molecular regulation - Google Patents
A kind of brown stain of cut lotus root control method based on molecular regulation Download PDFInfo
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Abstract
The brown stain of cut lotus root control method based on molecular regulation that the invention discloses a kind of, the brown stain of cut lotus root control method based on molecular regulation filters out crucial PAL, POD and the PPO for mediating brown stain of cut lotus root, it is further evaluated by effect of the gene expression analysis of early stage to brown stain of cut lotus root control technology, to high volume applications in the quick screening of brown stain of cut lotus root controlling technical method.Individual of the invention is according to different brown stain control technical treatment fresh-cut lotus roots, extract corresponding RNA, design the PAL high with brown stain of cut lotus root degree correlation, POD and PPO gene expression analysis primer, above-mentioned PAL is analyzed, expression quantity of the POD and PPO gene in each sample further evaluates different brown stain control technologies to the effect of fresh-cut lotus root, method is simple, easy to operate.
Description
Technical field
The invention belongs to technical field of bioengineering more particularly to a kind of brown stain of cut lotus root controls based on molecular regulation
Method.
Background technique
Lotus rhizome is most wide China's cultivation, sales volume and the maximum aquatic vegetable of sales range.Fresh-cut lotus root is in storage
Brown stain easily occurs for middle meat, directly affects the organoleptic quality and inherent quality of lotus rhizome, influences product sale and outlet.It grinds the country
The more concern lotus rhizome of the person of studying carefully adopts the technique study of the physiological mechanism and prevent-browning of rear browning mechanism, research shows that the brown stain of lotus rhizome
The mainly participation of enzymatic browning, at present related with the enzymatic browning enzyme of report specifically include that phenylalanine lyase (PAL),
Peroxidase (POD) and polyphenol oxidase (PPO).PAL is one of enzyme important in phenylpropyl alcohol alkanes metabolism approach, catalysis
L-phenylalanine solution ammonia generates trans-cinnamic acid, forms various phenol, lignin, anthocyanidin, alkaloid using a series of transformations
Deng the phenol of formation provides substrate for browning reaction.The researchs such as Xu Chuanjun find PAL base during Phalaenopsis leaves Explant browning
The expression variation of cause is consistent with its brown stain process.The researchs such as Li Zhengguo find Navel orange PAL2 gene expression and pericarp browning
There is Close relation.POD is the ferriporphyrin orgnometallic catalyst for being widely present in plant, and POD is in H2O2There are the case where
Under, phenolic substances and flavonoids oxidation polymerization can be formed to brown material and cause brown stain, the researchs such as Jiang Yihong discovery lily exists
POD activity is positively correlated with brown stain degree in storage, and POD activity change is to cause the principal element of lily brown stain.PPO is
The most important phenolase of fruits and vegetables brown stain.Many plant PPO genes are cloned in succession.Clone obtains 7 polyphenol oxidase bases in tomato
Cause, there are 7 polyphenol oxidase genes in potato.And the Molecular Biology Mechanism about lotus root browning is very few, closes in lotus rhizome
It is reported as only being cloned into 7 cDNA segments and 1 cDNA overall length in PPO.The molecule that PAL, POD and PPO participate in its brown stain is raw
Object mechanism is related to less.
Brown stain of cut lotus root control method is always the hot spot of scientific researcher concern, however conventional brown stain of cut lotus root
Control method evaluation method is all based on conventional physiological and biochemical index analysis, often in the presence of time-consuming consumption manpower and relatively lags behind
Disadvantage, and the brown stain of cut lotus root control method based on molecular regulation, technically simple and usability are extensive, especially suitable for large quantities of
The technical effect of amount is evaluated, while being suitable for other fruit-vegetable quality control technologies and being evaluated.
Summary of the invention
The purpose of the present invention is to provide a kind of brown stain of cut lotus root technique evaluating method based on molecular regulation, it is intended to solve
The shortcomings that certainly there is time-consuming consumption manpower and relatively lag behind in conventional brown stain of cut lotus root control method evaluation method；Based on molecule tune
The technically simple problem of the brown stain of cut lotus root control method of control.
The invention is realized in this way a kind of brown stain of cut lotus root control method based on molecular regulation, described to be based on dividing
The brown stain of cut lotus root control method of son regulation filters out crucial PAL, POD and the PPO for mediating brown stain of cut lotus root, utilizes the base
Because further being evaluated by effect of the gene expression analysis of early stage to brown stain of cut lotus root control technology, in high volume answering
Quick screening for brown stain of cut lotus root controlling technical method.The technology can be applied to the brown stain control of other fresh-cut fruit and vegetables simultaneously
Technology evaluation and screening processed, high-efficient, the period is short, easy to operate.
Further, the brown stain of cut lotus root control method based on molecular regulation the following steps are included:
Step 1, specifying different temperatures influences brown stain of cut lotus root: bright by brown stain degree and color difference combination organoleptic analysis
True 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C of influences to brown stain of cut lotus root, brown stain most serious and brown stain it is most light be used for subsequent RNA-
seq；
Step 2 clearly mediates related gene PAL, POD and PPO gene of brown stain of cut lotus root, is based on selection not equality of temperature
Degree handles two inconsistent samples of lower browning degree and is sequenced for RNA-seq, analyzes brown stain related gene based on sequencing result
PAL, POD and PPO expression quantity and browning degree correlation, pick out the key gene high with brown stain of cut lotus root correlation, use
In the brown stain of cut lotus root control technology evaluation in later period；
Step 3, after being used for based on PAL, POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies of phase, PAL, the POD high with brown stain of cut lotus root degree correlation are designed
With PPO gene expression analysis primer, different brown stain control technical roles is extracted in fresh-cut lotus root sample RNA, is analyzed above-mentioned
PAL, POD and PPO gene expression further evaluate different brown stain control technologies to the effect of fresh-cut lotus root.
Further, the clear different temperatures influences method to brown stain of cut lotus root are as follows: in conjunction with fresh-cut lotus root in not equality of temperature
Degree stores the lower influence of brown stain degree and color difference analysis sensory evaluation different temperatures to brown stain of cut lotus root, the wherein measurement of brown stain degree
Method is to take sample grinding broken, takes 0.5g to be put into 10ml centrifuge tube, and take 5ml distilled water injection centrifuge tube to be put into ice with liquid-transfering gun
On；Using refiner under 4000r/min revolving speed homogeneous 2min；2min is centrifuged under centrifuge 10000r/min revolving speed；It takes on 4ml
Clear liquid surveys light absorption value in 10ml centrifuge tube, using spectrophotometer under the conditions of wavelength 410nm, and brown stain degree is indicated with A410*10,
Each group is done 3 times in parallel.
Further, related gene PAL, POD and PPO gene for clearly mediating brown stain of cut lotus root uses RNA-
The method of seq participates in related gene PAL, POD and PPO of brown stain of cut lotus root to screen, and it is brown to be based further on RNA-seq analysis
Covert correlation gene PAL, POD and PPO expression quantity and browning degree correlation.
Further, described to be used based on PAL, the POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies of later period, completed using the iQ5 instrument of Bio-Rad.Using 20 μ l
System: wherein 10 μ l 2 × Ssofast EvaGreen Supermix, 1.0 μ l upstream primers (10 μM), 1.0 μ l downstream primers
(10 μM), the diluted cDNA of 2.0 μ l and 6.0 μ l H2O.PCR response procedures are as follows: 94 DEG C, 10min；94 DEG C of 10s of 45 circulations
With 60 DEG C of 30s.
The brown stain of cut lotus root controlling party based on molecular regulation is used another object of the present invention is to provide a kind of
Method realizes the screening of Fresh-cut Apples quality control method.
The brown stain of cut lotus root controlling party based on molecular regulation is used another object of the present invention is to provide a kind of
Method realizes the screening of fresh-cut yams quality control method.
The brown stain of cut lotus root controlling party based on molecular regulation is used another object of the present invention is to provide a kind of
Method realizes the evaluation of fresh-cut yams quality control technology and screening.
The brown stain of cut lotus root controlling party based on molecular regulation is used another object of the present invention is to provide a kind of
Method realizes the evaluation of fresh-cut long bean quality control technology and screening
Brown stain of cut lotus root control method provided by the invention based on molecular regulation is technically simple and usability is extensive, special
Not Shi Yongyu the evaluation of large batch of technical effect, while being suitable for other fruit-vegetable quality control technologies and evaluating and screen.
Detailed description of the invention
Fig. 1 is the brown stain of cut lotus root control method flow chart provided in an embodiment of the present invention based on molecular regulation.
Fig. 2 is that brown stain of cut lotus root degree changes schematic diagram under different temperatures provided in an embodiment of the present invention.
Fig. 3 is fresh-cut lotus root chromatic aberration schematic diagram under different temperatures provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention is based on RNA-Seq technology, crucial PAL, POD and the PPO for mediating brown stain of cut lotus root are filtered out, further
The effect of brown stain of cut lotus root control technology is evaluated by the gene expression analysis of early stage.This method is suitable for it simultaneously
He evaluates fruit-vegetable quality control technology.
With reference to the accompanying drawing and specific embodiment is further described application principle of the invention.
As shown in Figure 1, the brown stain of cut lotus root control method based on molecular regulation of the embodiment of the present invention includes following step
It is rapid:
S101: clear different temperatures influences brown stain of cut lotus root: clear by brown stain degree and color difference combination organoleptic analysis
Influence of the different temperatures (4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C) to brown stain of cut lotus root, brown stain most serious and brown stain it is most light be used for after
The RNA-seq in face；
S102: clearly mediating related gene PAL, POD and PPO gene of brown stain of cut lotus root, based on selection different temperatures
It handles two inconsistent samples of lower browning degree to be sequenced for RNA-seq, brown stain related gene is analyzed based on sequencing result
PAL, POD and PPO expression quantity and browning degree correlation, pick out the key gene high with brown stain of cut lotus root correlation, use
In the brown stain of cut lotus root control technology evaluation in later period；
S103: the later period is used for based on PAL, the POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies, design PAL, the POD high with brown stain of cut lotus root degree correlation and
PPO gene expression analysis primer extracts different brown stain control technical roles in fresh-cut lotus root sample RNA, analyze above-mentioned PAL,
POD and PPO gene expression further evaluates different brown stain control technologies to the effect of fresh-cut lotus root.
The clear different temperatures of step influences method to brown stain of cut lotus root are as follows: stores in conjunction with fresh-cut lotus root in different temperatures
Hide the lower influence of brown stain degree and color difference analysis sensory evaluation different temperatures to brown stain of cut lotus root.The wherein measuring method of brown stain degree
It is broken to take sample to be ground, take 0.5g to be put into 10ml centrifuge tube, and take 5ml distilled water injection centrifuge tube to be put into ice with liquid-transfering gun
On.Using refiner under 4000r/min revolving speed homogeneous 2min.2min is centrifuged under centrifuge 10000r/min revolving speed.It takes on 4ml
Clear liquid surveys light absorption value in 10ml centrifuge tube, using spectrophotometer under the conditions of wavelength 410nm, and brown stain degree is indicated with A410*10,
Each group is done 3 times in parallel.
The method that related gene PAL, POD and PPO gene for clearly mediating brown stain of cut lotus root uses RNA-seq
Come screen participate in brown stain of cut lotus root related gene PAL, POD and PPO, be based further on RNA-seq analysis brown stain dependency basis
Because of PAL, POD and PPO expression quantity and browning degree correlation.
It is described that the later period is used for based on PAL, the POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies, completed using the iQ5 instrument of Bio-Rad.Using 20 μ l systems: its
In 10 μ l 2 × Ssofast EvaGreen Supermix, 1.0 μ l upstream primers (10 μM), 1.0 μ l downstream primers (10 μM),
The diluted cDNA of 2.0 μ l and 6.0 μ l H2O.PCR response procedures are as follows: 94 DEG C, 10min；94 DEG C 10s and 60 DEG C of 45 circulations
30s。
Combined with specific embodiments below to being further described in shade of the invention.
Embodiment 1: clear different temperatures influences brown stain of cut lotus root:
Step 1, sample treatment: selection is fresh, removes the peel with mud, size, the consistent lotus root washing of shape, is cut into 5mm or so
Thin slice, take about 300 to be placed in water and impregnate 5min, two panels is put into each pallet and carries out pallet packing.Laboratory treatment mistake
Fresh-cut lotus root piece afterwards is put into rapidly in 4,10,15,20 DEG C of insulating boxs, and experiment is used in next step.The part is handled by 2 hours
Ozone sterilization operating room operated.
The measurement of brown stain degree: it takes sample to be shredded, takes 0.5g to be put into 10ml centrifuge tube, and take 5ml distilled water with liquid-transfering gun
Injection centrifuge tube is put into ice.Using refiner under 4000r/min revolving speed homogeneous 2min.Under centrifuge 10000r/min revolving speed
It is centrifuged 2min.It takes 4ml supernatant in 10ml centrifuge tube, surveys light absorption value, brown stain under the conditions of wavelength 410nm using spectrophotometer
Degree indicates that each group is done 3 times in parallel with A410*10.
Color difference measurement: L, a, b colour space are the systems formulated in 1976, and referred to as uniform colour solid indicates system.Axis L
Represent lightness axis, it is upper it is white under it is black, centre is the gray transitions of different light tones.L is known as lightness index, and L=0 represents black, L=
100 represent white.L value can indicate browning degree, if L value is bigger, represent that color is whiter, and brown stain is lighter.L value representative sample
The brightness on surface.If sample surfaces brightness is bigger, L value is bigger；Conversely, sample surfaces brightness is smaller, L value is with regard to smaller.Color
Difference measurement is carried out with color difference meter.Spectrophotometric color measurement instrument is corrected by blank first, is then measured lotus root piece with aperture plate
Value of chromatism, every 3 repetitions, wherein L value is averaged.
Step 2 clearly mediates related gene PAL, POD and PPO gene of brown stain of cut lotus root:
Two samples that browning degree is inconsistent under treatment of different temperature are selected to be sequenced for RNA-seq, based on sequencing knot
Fruit analyzes brown stain related gene PAL, POD and PPO expression quantity and browning degree correlation, picks out related to brown stain of cut lotus root
The high key gene of property.
Step 3, after being used for based on PAL, POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies of phase: with different brown stain control technical treatment fresh-cut lotus roots, extracting
Corresponding RNA designs PAL, the POD and PPO gene expression analysis primer high with brown stain of cut lotus root degree correlation, analyzes above-mentioned
Expression quantity of PAL, POD and PPO gene expression in each sample further evaluates different brown stain control technologies to fresh-cut lotus root
Effect.
Application effect of the invention is described in detail below with reference to experiment.
Experiment 1: influence of the treatment of different temperature to brown stain of cut lotus root；
(1) experimental method
Sample treatment: selection is fresh, removes the peel with mud, size, the consistent lotus root washing of shape, is cut into the thin slice of 5mm or so,
It takes about 300 to be placed in water and impregnates 5min, two panels is put into each pallet and carries out pallet packing.Fresh-cut after laboratory treatment
Lotus root piece is put into rapidly in 4,10,15,20 DEG C of insulating boxs, and experiment is used in next step.It is killed in the ozone by processing in 2 hours the part
Bacterium operating room is operated.
The measurement of brown stain degree: it takes sample to be shredded, takes 0.5g to be put into 10ml centrifuge tube, and take 5ml distilled water with liquid-transfering gun
Injection centrifuge tube is put into ice.Using refiner under 4000r/min revolving speed homogeneous 2min.Under centrifuge 10000r/min revolving speed
It is centrifuged 2min.It takes 4ml supernatant in 10ml centrifuge tube, surveys light absorption value, brown stain under the conditions of wavelength 410nm using spectrophotometer
Degree indicates that each group is done 3 times in parallel with A410*10.
Color difference measurement: L, a, b colour space are the systems formulated in 1976, and referred to as uniform colour solid indicates system.Axis L
Represent lightness axis, it is upper it is white under it is black, centre is the gray transitions of different light tones.L is known as lightness index, and L=0 represents black, L=
100 represent white.L value can indicate browning degree, if L value is bigger, represent that color is whiter, and brown stain is lighter.L value representative sample
The brightness on surface.If sample surfaces brightness is bigger, L value is bigger；Conversely, sample surfaces brightness is smaller, L value is with regard to smaller.Color
Difference measurement is carried out with color difference meter.Spectrophotometric color measurement instrument is corrected by blank first, is then measured lotus root piece with aperture plate
Value of chromatism, every 3 repetitions, wherein L value is averaged.
(2) experimental result
Experimental result show the storage of the fresh-cut lotus root of (4,10,15,20 DEG C) under different temperatures the result shows that, in certain temperature
It spends in range, reserve temperature is higher, and lotus root browning is more serious (attached drawing 2 and attached drawing 3).
Experiment 2: the screening of related gene PAL, POD and PPO gene of brown stain of cut lotus root is mediated:
(1) experimental method
Two samples that browning degree is inconsistent under treatment of different temperature are selected to be sequenced for RNA-seq, based on sequencing knot
Fruit analyzes brown stain related gene PAL, POD and PPO expression quantity and browning degree correlation, picks out related to brown stain of cut lotus root
The high key gene of property.
(2) experimental result
The sample that browning degree is inconsistent under treatment of different temperature is selected to be sequenced for RNA-seq, most of brown stain is related
Gene PAL, POD and PPO expression quantity and browning degree are positively correlated, higher sample P AL, POD and PPO gene expression of browning degree
Higher (table 1).
The RNA-Seq data of 1 fresh-cut lotus root of subordinate list are analyzed
Experiment 3: after being used for based on PAL, POD and PPO gene high with brown stain of cut lotus root degree correlation screened
In the effect assessment of other brown stain of cut lotus root control technologies of phase
(1) experimental method
With different brown stain control technical treatment fresh-cut lotus roots, corresponding RNA, design and brown stain of cut lotus root degree phase are extracted
High PAL, POD and PPO the gene expression analysis primer of closing property, analyzes above-mentioned PAL, POD and PPO gene expression in each sample
Expression quantity further evaluates different brown stain control technologies to the effect of fresh-cut lotus root.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (8)
1. a kind of brown stain of cut lotus root control method based on molecular regulation, which is characterized in that described based on the fresh of molecular regulation
Cut lotus root browning control method filters out crucial PAL, POD and the PPO for mediating brown stain of cut lotus root, further passes through the base of early stage
Because effect of the expression analysis to brown stain of cut lotus root control technology is evaluated；
The brown stain of cut lotus root control method based on molecular regulation the following steps are included:
Step 1, specifying different temperatures influences brown stain of cut lotus root: passing through brown stain degree and color difference combination organoleptic analysis clear 4
DEG C, 10 DEG C, 15 DEG C, 20 DEG C of influences to brown stain of cut lotus root, brown stain most serious and the most light sample of brown stain are for subsequent
RNA-seq analysis；
Step 2 clearly mediates related gene PAL, POD and PPO gene of brown stain of cut lotus root, at selection different temperatures
Two inconsistent samples of lower browning degree are managed to be sequenced for RNA-seq, based on sequencing result analyze brown stain related gene PAL,
POD and PPO expression quantity and browning degree correlation, pick out the key gene high with brown stain of cut lotus root correlation, after being used for
In the brown stain of cut lotus root control technology evaluation of phase；
Step 3, based on PAL, the POD and PPO gene high with brown stain of cut lotus root degree correlation screened for the later period its
In the effect assessment of his brown stain of cut lotus root control technology, PAL, POD and the PPO high with brown stain of cut lotus root degree correlation are designed
Gene expression analysis primer extracts different brown stain control technical roles in fresh-cut lotus root sample RNA, analyzes above-mentioned PAL, POD
With PPO gene expression, different brown stain control technologies is further evaluated to the effect of fresh-cut lotus root.
2. the brown stain of cut lotus root control method based on molecular regulation as described in claim 1, which is characterized in that described clear
Different temperatures on brown stain of cut lotus root influence method are as follows: in conjunction with fresh-cut lotus root different temperatures storage under brown stain degree and color difference analysis
Influence of the sensory evaluation different temperatures to brown stain of cut lotus root, wherein the measuring method of brown stain degree is to take sample grinding broken, is taken
0.5g is put into 10ml centrifuge tube, and takes 5ml distilled water injection centrifuge tube to be put on ice with liquid-transfering gun；Using refiner in 4000r/
Homogeneous 2min under min revolving speed；2min is centrifuged under centrifuge 10000r/min revolving speed；It takes 4ml supernatant in 10ml centrifuge tube, uses
Spectrophotometer surveys light absorption value under the conditions of wavelength 410nm, and brown stain degree is indicated with A410*10, and each group is done 3 times in parallel.
3. the brown stain of cut lotus root control method based on molecular regulation as described in claim 1, which is characterized in that described clear
It mediates related gene PAL, POD and PPO gene of brown stain of cut lotus root and screens participation fresh-cut lotus using the method for RNA-seq
Related gene PAL, POD and PPO of lotus root brown stain are based further on RNA-seq analysis brown stain related gene PAL, POD and PPO table
Up to amount and browning degree correlation.
4. the brown stain of cut lotus root control method based on molecular regulation as described in claim 1, which is characterized in that described to be based on
PAL, the POD and PPO gene high with brown stain of cut lotus root degree correlation screened is used for other brown stain of cut lotus root of later period
In the effect assessment of control technology, using Bio-Rad iQ5 instrument complete, using 20 μ l systems: wherein 10 μ l2 ×
SsofastEvaGreenSupermix, 1.0 μ l upstream primers, 1.0 μ l downstream primers, 2.0 μ l diluted cDNA and 6.0 μ
lH2O, PCR response procedures are as follows: 94 DEG C, 10min；94 DEG C of 10s and 60 DEG C of 30s of 45 circulations；Wherein, upstream primer and downstream
The concentration of primer is 10 μM.
5. a kind of brown stain of cut lotus root control method using as described in claim 1-4 any one based on molecular regulation is fresh
Cut the evaluation of apple quality control technology and screening.
6. a kind of brown stain of cut lotus root control method using as described in claim 1-4 any one based on molecular regulation is fresh
Cut the evaluation of pineapple quality control technology and screening.
7. a kind of brown stain of cut lotus root control method using as described in claim 1-4 any one based on molecular regulation is fresh
Cut the evaluation of Chinese yam quality control technology and screening.
8. a kind of brown stain of cut lotus root control method using as described in claim 1-4 any one based on molecular regulation is fresh
Cut the evaluation of long bean quality control technology and screening.
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| CN101341904A (en) * | 2008-08-22 | 2009-01-14 | 山东营养源食品科技有限公司 | Method for restraining brown stain of cut lotus root |
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