CN114752696B - Chip for detecting scion and evaluating quality of rootstock as well as preparation method and application thereof - Google Patents
Chip for detecting scion and evaluating quality of rootstock as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a chip for detecting scion and evaluating quality of rootstock, a preparation method and application thereof. The method can be used for simply, conveniently and quickly evaluating the performances of the rootstock in heat resistance, drought resistance, leaf spot and rot resistance, iron stress resistance, phosphate stress resistance and the like. The method can be used for evaluating the corresponding characters of various known and unknown stocks by detecting the content of the heterologous mRNA which can be transmitted by the key of the corresponding characteristics of the scion end, quickly and efficiently judging the quality of the stocks, and has important significance for stock breeding and field application. The method has the advantages of simple and quick operation, low operation requirement, strong detection specificity, accurate result and intuitive reaction.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a chip for detecting scion and evaluating the quality of rootstock, a preparation method and application thereof. In particular to a chip for detecting scions and evaluating the quality of apple and pear stocks and a preparation method and application thereof.
Background
Grafting is the most main asexual propagation mode of horticultural crops, and obvious interaction exists between a stock of a grafted plant and a scion, and comprises the regulation and control of the flower formation, the fruit yield and quality, the plant growth and development, the plant resistance and the like of the scion. The regulation and control capability of the rootstock on the scion phenotype is an important index for evaluating the quality of the rootstock, the excellent rootstock can improve the disease resistance, various stress tolerance and the like of plants, and the quality and the yield of horticultural crop products, even the survival condition of the whole plant are determined. The evaluation of the quality of the rootstocks mostly only focuses on the aspect of the rootstocks, and a misregion of forcible recognition that the resistance of the rootstocks is strong and the resistance of the scions is strong exists. The yield and quality of fruit tree production are finally determined by the scion part, so the quality of the rootstock should be evaluated by directly evaluating the scion after grafting. Meanwhile, at present, stocks applied in actual production mostly depend on local original stocks on one hand, and foreign stocks are introduced on the other hand, but the stocks are applied to actual production only after production tests are carried out, growth results of grafted varieties are observed, adaptability to local ecological conditions, resistance to diseases, stress resistance and other properties are observed, and the process is complicated and long.
The rootstock can transport part of rootstock RNA molecules to the scion through grafting, the mRNA molecules can play an important role in the aspects of plant stress resistance, disease and insect resistance and the like, meanwhile, the growth and development of the scion, the fruit quality and the like can be regulated and controlled by the mRNA molecules from the rootstock, and the moving capacity and the moving amount of the RNA molecules from the rootstock to the scion can be used as a mode for evaluating the quality of the rootstock.
At present, the identification method of mRNA molecules still remains in the simple laboratory molecular identification level, including common molecular biology techniques such as polymerase chain reaction PCR, multiplex PCR, real-time fluorescent quantitative PCR, gene chip technique, etc. The technologies have the advantages of strong specificity, large detection amount, relatively short time and the like, and can effectively identify mRNA molecules. But the conditions are also harsh, such as high cost and the need for professional operation in a laboratory environment, etc., under non-laboratory conditions, farmers can only judge the conditions through long-time selection and experience, and cannot be directly, simply and quickly applied to actual production, and cannot be popularized and applied. Therefore, a rapid, inexpensive and highly accurate detection means by the scion is desired.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a chip for detecting and evaluating rootstock so as to simply, conveniently and quickly evaluate the rootstock in the performances of heat resistance, drought resistance, resistance to leaf drop at spots, rot, iron stress, phosphate stress and the like.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
a chip for detecting scion and evaluating the quality of rootstock is characterized in that: the chip comprises a solid phase carrier, and in-vitro reaction reagents are loaded on the solid phase carrier.
Further, the in vitro reaction reagent comprises a mixed buffer solution, a ribonuclease inhibitor, a molecular fingerprint detector, diethyl pyrocarbonate and a color indicating reagent.
The mixed buffer solution can be prepared from E6800
In vitro protein Synthesis kit (NEB Co., ltd.)
Further, the color indicating reagent is chlorophenol red-beta-D-galactopyranose.
Further, the molecular fingerprint detector is an RNA sequence, and comprises a color excitation sequence at the front end of the molecular fingerprint detector and an amplified induced RNA sequence matched with the base of the color excitation sequence, wherein the nucleotide sequence is shown in a sequence with the number of SEQ ID NO.3-4 and SEQ ID NO.38-41 in a sequence table.
It is a further object of the present invention to provide a method for preparing a chip as described above.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
a method for preparing a chip for detecting the quality of scions and evaluating rootstocks is characterized by comprising the following steps:
step one, adding a forward primer and a reverse primer into the plasmid, and carrying out PCR amplification on a PCR premixed solution and water;
the PCR premix may be 2 XTaq MasterMix (Dye);
step two, performing gel electrophoresis on the PCR amplification product obtained in the step one to obtain a DNA recovery product, namely a molecular fingerprint detector;
step three, applying the molecular fingerprint detector obtained in the step two to prepare an in vitro protein reaction reagent;
step four, adding the in-vitro protein reaction reagent obtained in the step three into a solid phase carrier to obtain a chip;
further, the nucleotide sequences of the forward primer and the reverse primer in the step one are the sequences shown in SEQ ID N0.11-14 and SEQ ID N0.31-38 in the sequence table.
Further, the process for preparing the in vitro protein reaction reagent in the third step comprises the following steps: mixing the mixed buffer solution, the RRI, the molecular fingerprint detector, the diethyl pyrocarbonate and the color indicating reagent.
The RRI may be RNA guard TM
Further, the in vitro reaction reagent preparation conditions in the third step are as follows: incubate at 37 ℃ for 2h.
Still another object of the present invention is to provide an application of the chip for detecting scion to evaluate the quality of rootstock.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
the application of the chip for detecting and evaluating the rootstock is characterized by comprising the following steps:
step one, collecting stock scion phloem RNA;
step two, using the RNA as a template to amplify induced RNA, specifically comprising the following steps:
adding phloem RNA, diethyl pyrocarbonate and RRI into an induced RNA amplification system to react to obtain induced RNA; the induced RNA amplification system comprises five components of buffer solution, NTP mixed solution, dNTP mixed solution, enzyme mixture and primer mixture;
step three, adding the induced RNA obtained in the step two into a chip to perform in-vitro protein reaction;
and step four, observing the reaction discoloration phenomenon, evaluating the corresponding properties of the scion to be tested, and further evaluating the quality of the rootstock.
Further, in the present invention,
the five-component buffer solution in the step two comprises the following components:
tris hydrochloride, potassium chloride, magnesium chloride, dithiothreitol and water;
the components of the enzyme mixture in the second step are as follows:
M-MLV reverse transcriptase, T7 RNA polymerase, AMV reverse transcriptase, ribonuclease and bovine serum albumin;
the components of the primer mixture in the second step are as follows:
primer (R), primer (F), dimethyl sulfoxide and deionized water.
Further, the evaluation process of the discoloration phenomenon described in the fourth step is:
shooting the process of changing a first color of the environmental color of the color indicating reagent to a second color by using an image shooting device under the same shooting condition, and recording the time required for the first color to be converted to the second color, namely the reaction time;
using software to read the change of RGB value in the process of changing the first color to the second color, and calculating the color change rate CCT according to the following formula:
C＝(R+B)/(R+G)；
ΔC＝d[dC/dt]/dt＝d 2 (R+B)/(R+G)/dt 2 ；
CCT＝ΔC×10 5 ：
in the formula, C: color indicating reagent ambient color, R: RGB red number, G: RGB green number, B: RGB blue values, Δ C: change in slope of the curve, t: reaction time, CCT: the rate of change of color.
The method comprises the following steps of evaluating corresponding characters of scions by taking the level of heterologous RNA detected at the scion ends as a basis, further evaluating the quality of stocks, judging the corresponding characters of detection groups by using the rate CCT (Co 1or change rate) of changing first colors into second colors according to specific evaluation indexes based on the environmental colors (C values) of detection group indicators, wherein the visual appearance of the first colors on a solid phase carrier is yellow, and the RGB numerical information of the first colors is as follows: r:200 to 225; g:230 to 240; b:80 to 120 portions; the visual impression of the second color is purple, and the RGB numerical information of the second color is as follows: r:165 to 175; g:30 to 40 percent; b:45 to 55.
In general, there is C A first color ：0.60～0.80；C A second color ：1.00～1.20
The expression quantity of the target gene in the rootstock to be tested is reflected by the color change rate CCT, the higher the CCT (range) value is, the higher the gene expression quantity is, the corresponding character of the scion to be tested is evaluated based on the CCT level, and then the quality of the rootstock is evaluated.
The molecular fingerprint detector-1 performs heat resistance evaluation on the rootstock within the index range of: the CCT is less than 300 and is thermolabile, and the CCT is more than 300 and is more heat-resistant;
the molecular fingerprint detector-2 evaluates the drought resistance of the rootstock to reach the index range of no drought resistance with the CCT less than 260 and drought resistance with the CCT more than 310;
the evaluation index range of the molecular fingerprint detector-3 for the phosphate stress resistance of the rootstock is as follows: the CCT is less than 250 and is not resistant to phosphate stress, and the CCT is more than 300 and is more resistant to phosphate stress;
the molecular fingerprint detector-4 evaluates the iron stress resistance of the rootstock in the index range of: CCT is less than 280, and is not resistant to iron stress, and CCT is more than 310, and is more resistant to iron stress;
the evaluation index range of the molecular fingerprint detector-5 for the rot resistance of the rootstock is as follows: the CCT is less than 330, and the rot is not resistant, and the CCT is more than 330;
the evaluation index range of the molecular fingerprint detector-6 for the anti-alternaria leaf spot of the rootstock is as follows: the resistance to the alternaria leaf spot is not more than 280, and the resistance to the alternaria leaf spot is more than 300;
the chip for detecting and evaluating the rootstock, the preparation method and the application thereof have the advantages that:
the method is simple, convenient and quick to operate (two-step reaction), low in operation requirement (independent of laboratory environment), strong in detection specificity (identification of specific conservative fragments), accurate in result and visual in reaction (visual identification).
By applying the method, the content of the heterologous mRNA which can be transmitted by detecting the key of the corresponding characteristics of the scion end can be detected, the corresponding characteristics of various known and unknown rootstocks can be evaluated, the quality of the rootstocks can be quickly and efficiently judged, and the method has important significance for rootstock breeding and field application.
Drawings
The invention has the following drawings:
FIG. 1 is a flow chart of the application of a chip for detecting and evaluating rootstock according to the present invention
FIG. 2 is a customizable nucleotide sequence structure of a molecular fingerprint detector and an induced RNA, where N is a customizable interval, and a Complementary region (Complementary region) sequence of the induced RNA is Complementary to the induced RNA, and is a color excitation sequence customizable according to the induced RNA;
FIG. 3 shows that after the molecular fingerprint sensor is activated by the induced RNA, translation of the reporter gene is started in the environment of in vitro protein reaction reagent according to the base complementary pairing principle, and released LacZ protease can crack yellow substrate chlorophenol red-beta-D-galactopyranose to generate purple chlorophenol red product red-beta-D-galactopyranose;
FIG. 4 Whole-gene synthesized induced RNA-1 template plasmid pET-17b (+);
FIG. 5NUPACK analyzes simulated molecular fingerprint detector-1 secondary structure;
FIG. 6 is an agarose gel electrophoresis chart of the amplified rootstock scion induced RNA-1 to be evaluated;
FIG. 7 is a monitoring diagram of in vitro protein reaction of four rootstock scion parts to be evaluated, wherein a reference 1 is non-induced RNA-1, and a reference 2 is a non-molecular detector-1;
FIG. 8 is the C value of the in vitro protein reaction after the induced RNA-1 of the scion of the four rootstocks to be evaluated is added into a molecular fingerprint detector-1;
FIG. 9 shows the relative conductivity and total chlorophyll concentration of four rootstock scions to be evaluated and four rootstocks to be evaluated that are not grafted under heat stress;
FIG. 10 molecular probes synthesized in whole gene-2 template plasmid pET-17b (+);
FIG. 11NUPACK analyzes simulated molecular detector-2 secondary structure;
FIG. 12 is an agarose gel electrophoresis image of a molecular detector-2 for amplifying a rootstock scion part to be evaluated;
FIG. 13 is a monitoring chart of in vitro protein reaction of four rootstock scion parts to be evaluated, wherein a control 1 is non-induced RNA-2, and a control 2 is a non-molecular detector-2;
FIG. 14 is the in vitro protein reaction C value after the induced RNA-2 of the scion of four rootstocks to be evaluated is added into a molecular detector-2;
FIG. 15 shows the relative conductivity and total chlorophyll concentration of four rootstock scions to be evaluated and four rootstocks to be evaluated that are not grafted under heat stress;
FIG. 16 change in soil moisture content (drought resistance) of treatment group and control group under drought stress treatment;
FIG. 17 Assembly of gene evaluation chip;
FIG. 18 is a monitoring chart of in vitro protein reaction of the scion of six pear stocks to be evaluated, wherein a control 1 is non-induced RNA-1, and a control 2 is a non-molecular fingerprint detector-1
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
In this description, each term is compared to the following:
Tris-HCl: tris hydroxymethyl aminomethane hydrochloride
DTT (delay time T): dithiothreitol
M-MLV RT: M-MLV reverse transcriptase
T7 RNA Polymerase: t7 RNA polymerase
RT AMV: AMV reverse transcriptase
RNaseH: ribonuclease H
BSA: bovine serum albumin
RRI: ribonuclease inhibitors
DEPC: pyrocarbonic acid diethyl ester
Chlorophenol red- β -D-galactopyranoside: chlorophenol red-beta-D-galactopyranose
red- β -D-galactopyranoside: red-beta-D-galactopyranose
1. Apple stock evaluation chip and preparation and application thereof
1. Cultivation and sampling of experimental materials
The experimental apple rootstock potted seedling groups used by the invention are annual malus micromalus, malus halliana, M9-T337 and M26, and are transferred and planted in Shanzhuang experimental station of China agricultural university in 2021 month 3. The experimental apple grafted potted seedling groups used by the invention are annual crown of golden crown/malus micromalus, golden crown/malus halliana, golden crown/M9-T337 and golden crown/M26 (scion/rootstock), and are grafted and planted at the Zhuang experimental station of Chinese agriculture university in 3 months in 2021. The total number of the plants is 250, and the plants are uniformly managed.
2. Extraction of sample RNA
Collecting phloem and top leaves with the same height at the scion part of the grafting plant with uniform growth, and collecting fresh leaves of the tissue culture seedling. Sample RNA was extracted using the CTAB method.
3. Amplification System configuration for inducing RNA
Five-component buffer solution: 500mM Tris & HCl;750mM KCL;30mM MgCl 2 ；100mM DTT；pH＝8.3：
Enzyme mixture: M-MLV RT:32U, T7 RNA Polymerase:20U, RT AMV8U, RNaseH 0.2U, BSA 1 mg/ml -1 ；
Primer mixture: 5pmol of primer (R), 5pmol of primer (F), 3.75. Mu.L of DMSO, and addition of H 2 0 to 6 μ L;
example 1: apple rootstock heat resistance evaluation chip and preparation and application thereof (1) chip preparation
A molecular fingerprint detector-1 designed according to the induced RNA-1 is synthesized by adopting a whole gene synthesis method, comprises a color excitation sequence with 36bp complementary to the induced RNA-1, and is shown as SEQ ID N0.1, and the structure of the molecular fingerprint detector-1 predicted by using NUPACK is shown as figure 5.
Taking synthesized pET-17b (+) plasmid as a template, using an amplification primer shown in SEQ ID N0.11-12, adding 2.5 mu L of plasmid as the template, adding 2.5 mu L of forward primer and 2.5 mu L of reverse primer, adding 25 mu L of 2 XTaq MasterMix (Dye) and 17.5 mu L of water for PCR amplification, carrying out gel electrophoresis after obtaining a PCR amplification product, and recovering to obtain a DNA recovery product, namely a molecular fingerprint detector-1.
Mixing molecular fingerprint detector-1, in vitro protein reaction mixed buffer solution, and RRI (RNA guard) TM ) The chlorophenol red-beta-D-galactopyranose, DEPC and the like are premixed according to the volume parts, stored at the temperature of-20 ℃, and taken out when in use, and the method specifically comprises the following steps:
the 20 μ L system was formulated as follows: the mixed buffer solution is prepared from E6800
The in-vitro protein synthesis kit provides and ensures that the buffer solution A and the buffer solution B in the kit respectively account for 40 percent and 30 percent in the system; RRI:2 percent; chlorophenol red- β -D-galactopyranose: 4 percent; molecular fingerprint Detector-1: 5 percent; DEPC:12 percent. The above system was incubated at 37 ℃ for 2h.
And (3) taking a 96-well enzyme label plate as a solid phase carrier, sterilizing at high temperature for 20min, storing for later use, and adding 19 mu L of the mixed solution containing the molecular fingerprint detector-1 into the enzyme label plate to obtain the heat resistance evaluation chip.
(2) Chip application
(A) Induction of amplification of RNA
Induced RNA-1 is amplified from the scion phloem RNA of the combination of apple scion to be detected, namely golden crown/malus micromalus, golden crown/malus halliana, golden crown/M9-T337 and golden crown/M26, and the amplification result is shown in figure 5. The solution is prepared as follows:
the volume fraction of 25. Mu.L reaction components was:
ear grafting part RNA: y μ L
DEPC：(9.76-x-y)μL
RRI：(RNA guard TM )：x μL
NTP mixed liquid: each 5mM
dNTP mixture: each 2.5mM
Five-component buffer solution: 5 μ L
Enzyme mixture: 3.24 μ L
Primer mixture: 6 μ L
The reaction conditions are as follows: preheating the five-component buffer solution at 37 ℃ for 5min, adding other reactants, and heating in a water bath kettle at 40.5 ℃ for 2h;
(B) In vitro protein response assay
The in vitro protein reaction is: inducing RNA-1 to react with the in-vitro protein reaction system in the chip. mu.L of the induced RNA-1 was added to the chip for evaluating heat resistance as shown in FIG. 7.
The detection sensitivity of induced RNA-1 with different concentrations is set, and each gene has three repetitions.
The molecular fingerprint detector-1 and the induced RNA-1 are observed to be well combined, and the CCT is increased along with the gradual increase of the concentration of the induced RNA-1.
(C) Data processing analysis, namely evaluating the heat resistance of the rootstock through the CCT value of the scion grafting part
The reaction results are shown in FIG. 7, which was taken with an EOS600D Canon camera with the same settings of the shooting parameters and the same angle, using digital colorimeter software
2001-2020Apple Inc. measuring R, G and B values of reaction environment;
and (3) calculating to obtain a C value, drawing by using Graphpad software, carrying out single-factor linear regression analysis, and calculating to obtain a linear regression slope and a CCT value, wherein the calculation results are shown in tables 1 and 2.
TABLE 1 molecular fingerprint Detector-1 in vitro protein reaction C value
TABLE 2 molecular fingerprint Detector-1 in vitro protein reaction CCT value
The CCT value of the molecular fingerprint detector-1 in vitro protein reaction is obtained by analysis: the golden crown/M26 is more than the golden crown/M9-T337 is more than the golden crown/malus halliana, the M26 and M9-T337 are preliminarily predicted to have better efficiency in improving the heat resistance of the golden crown scion according to the CCT value range, and the CCT value range is slightly higher than that of the malus halliana and the malus halliana;
(D) Stress treatment is carried out, and the evaluation result of the gene chip is verified
Carrying out corresponding heat stress treatment on the Chinese malus micromalus, the malus halliana, the M9-T337 and the M26 stock potted seedlings and the grafted seedlings respectively, observing the phenotype, measuring the leaf conductivity and the chlorophyll content, evaluating the heat stress degree of the scions of the grafted seedlings, and simultaneously evaluating the heat stress degree of the stock potted seedlings as supplement.
Heat stress treatment, culturing at 39.5 ℃ for 24h, measuring the leaf conductivity by using a conductivity meter method, detecting the chlorophyll content by using an ethanol extraction method, wherein the measurement results are shown in fig. 8, evaluating the heat resistance of the grafted seedling scion to be detected and the non-grafted stock to be detected, and judging the heat resistance of the golden crown scion of the grafted seedling to be detected by combining phenotype and physiological indexes: the heat resistance of the golden crown/M9-T337 and golden crown/M26 scions is strongest, the heat resistance of the golden crown/filamental flowering crab scions is stronger, the heat resistance of the golden crown/malus micromalus is weakest, and the heat resistance of the stock to be tested is not grafted: the heat resistance of M9-T337 is the strongest, and M26 and Malus halliana are the second, and Malus micromalus is the weakest.
As can be seen from the analysis of the data in tables 1 and 2, the prediction result of the heat resistance evaluation of the molecular fingerprint detector-1 is the same as the heat resistance analysis result after the stress treatment, and meanwhile, the heat resistance comparison result of the rootstock which is not grafted and is to be tested shows that the rootstock with strong heat resistance can improve the heat resistance of the scion part. The molecular fingerprint detector-1 can accurately judge whether the Malus micromalus, the Malus halliana, the M9-T337 and the M26 are used as stocks in the aspect of improving the heat resistance of the scion grafting part.
Example 2: apple rootstock drought resistance evaluation chip and preparation and application thereof
(1) Chip preparation
A whole-gene synthesis method is adopted to synthesize a molecular fingerprint detector-2 designed according to the induced RNA-2, the color excitation sequence containing 36bp and complementary with the induced RNA-2 is shown as SEQ ID N0.2, and the structure of the molecular fingerprint detector-2 predicted by NUPACK is shown as figure 11.
Taking synthesized pET-17b (+) plasmid as a template, using an amplification primer shown as SEQ ID N0.13-14, adding 2.5 mu L of plasmid as the template, adding 2.5 mu L of forward primer and 2.5 mu L of reverse primer, adding 25 mu L of 2 xTaq MasterMix (Dye) and 17.5 mu L of water for PCR amplification, carrying out gel electrophoresis after obtaining a PCR amplification product, and recycling to obtain a DNA recycling product, namely a molecular fingerprint detector-2.
Mixing molecular fingerprint detector-2, in vitro protein reaction mixed buffer solution, and RRI (RNA guard) TM ) The chlorophenol red-beta-D-galactopyranose, DEPC and the like are premixed according to the volume parts, stored at the temperature of-20 ℃, and taken out when in use, and the method specifically comprises the following steps:
the 20 μ L system was formulated as follows: the mixed buffer solution is prepared from E6800
The in-vitro protein synthesis kit provides and ensures that the buffer solution A and the buffer solution B in the kit respectively account for 40 percent and 30 percent in the system; RRI:2 percent; chlorophenol red- β -D-galactopyranose: 4 percent; molecular fingerprint Detector-2: 5 percent; diethyl pyrocarbonate: 12 percent. The above system was incubated at 37 ℃ for 2h.
And (3) taking a 96-hole enzyme label plate as a solid phase carrier, sterilizing at high temperature for 2h, storing for later use, and adding 19 mu L of the mixed solution containing the molecular fingerprint detector-2 into the enzyme label plate to obtain the drought resistance evaluation chip.
(2) Chip application
(A) Induction of amplification of RNA
Induced RNA-2 is amplified from the scion phloem RNA of the combination of apple scion to be detected, namely golden crown/malus micromalus, golden crown/malus halliana, golden crown/M9-T337 and golden crown/M26, and the amplification result is shown in figure 11. The solution was configured as follows:
the volume fraction of 25. Mu.L reaction components was:
ear grafting part RNA: y μ L
DEPC：(9.76-x-y)μL
RRI(RNA guard TM )：x μL
NTP mixed liquid: each 5mM
dNTP mixture: each 2.5mM
Five-component buffer solution: 5 μ L
Enzyme mixture: 3.24 μ L
Primer mixture: 6 μ L
Reaction conditions are as follows: preheating five-component buffer solution at 37 ℃ for 5min, adding other reactants, and heating in a water bath kettle at 40.5 ℃ for 2h;
(B) In vitro protein response assay
The in vitro protein reaction is: inducing RNA-2 to react with the in-vitro protein reaction system of the molecular fingerprint detector-2. mu.L of induced RNA-2 was added to the chip for drought resistance evaluation as shown in FIG. 13.
The detection sensitivity of induced RNA-2 with different concentrations is set, and each gene is repeated three times.
The molecular fingerprint detector-2 and the induced RNA-2 are observed to be well combined, and the CCT is increased along with the gradual increase of the concentration of the induced RNA-2.
(C) Data processing analysis, stock drought resistance evaluation through the CCT value of scion
The reaction results are shown in FIG. 13, which were taken with an EOS600D Canon camera with the same settings of the shooting parameters and the same angles, and digital colorimeter software was used
2001-2020Apple Inc. measuring R, G and B values of reaction environment;
and (3) calculating to obtain a C value, drawing by using Graphpad software, carrying out single-factor linear regression analysis, and calculating to obtain a linear regression slope and a CCT value, wherein the calculation results are shown in tables 3 and 4.
TABLE 3 molecular fingerprint Detector-2 in vitro protein reaction c values
| | Control | 1 | |
Malus micromalus (lour.) Merr | Malus halliana (hook.) Roxb | M9-T337 | M26 |
| 0min | 0.73 | 0.70 | 0.70 | 0.71 | 0.71 | 0.70 | |
| 25min | 0.72 | 0.72 | 0.77 | 0.79 | 0.78 | 0.79 | |
| 40min | 0.69 | 0.68 | 0.74 | 0.76 | 0.76 | 0.77 | |
| 55min | 0.71 | 0.70 | 0.76 | 0.77 | 0.78 | 0.78 | |
| 70min | 0.72 | 0.71 | 0.86 | 0.86 | 0.90 | 0.92 | |
| 85min | 0.74 | 0.73 | 0.87 | 0.91 | 0.90 | 0.92 | |
| 100min | 0.72 | 0.70 | 0.92 | 0.97 | 1.00 | 0.99 | |
| 115min | 0.73 | 0.71 | 1.05 | 1.05 | 1.07 | 1.06 | |
| 130min | 0.73 | 0.72 | 1.11 | 1.12 | 1.13 | 1.14 | |
| 145min | 0.70 | 0.68 | 1.09 | 1.12 | 1.13 | 1.13 |
TABLE 4 molecular fingerprint Detector-2 in vitro protein reaction CCT value
| |
|
Xi FuBegonia | Malus halliana (hook.) Roxb | M9-T337 | M26 | |
| CCT | 1.33 | -0.13 | 255.90 | 313.50 | 326.80 | 326.00 |
The CCT value of the molecular fingerprint detector-2 in vitro protein reaction is obtained by analysis: M9-T337 is more than M26 is more than filamental flowering crab, and the drought resistance M9-T337, M26 and the filamental flowering crab are preliminarily predicted to be similar but far higher than that of the filamental flowering crab according to the CCT range.
(D) Stress treatment is carried out, and the evaluation result of the gene chip is verified
Respectively carrying out drought stress treatment on the Chinese malus micromalus, the malus halliana, the M9-T337 and the M26 stock potted seedlings and the grafted seedlings, observing the phenotype, measuring the leaf conductivity and the chlorophyll content, and evaluating the stress degree of the stock potted seedlings and the grafted seedlings.
Drought stress treatment, normal watering of a control group, water cut-off treatment of a test group, measurement of soil water content as shown in figure 16, measurement of leaf conductivity by a conductivity meter method, detection of chlorophyll content by an ethanol extraction method, measurement of results as shown in figure 15, evaluation of heat resistance of a grafted seedling scion to be tested and a non-grafted stock to be tested, judgment by combining phenotype and physiological indexes, strongest drought resistance of golden crown/M9-T337 and golden crown/M26 of a grafted seedling golden crown scion to be tested, weakest drought resistance of golden crown/malus halliana. The drought resistance of the rootstocks to be tested which are not grafted presents a similar trend.
The data in the tables 3 and 4 are analyzed, so that the drought resistance evaluation prediction result of the molecular fingerprint detector-2 is the same as the drought resistance analysis result after stress treatment, the drought resistance comparison result of the rootstock to be tested which is not grafted shows that the rootstock with strong drought resistance can improve the drought resistance of the scion grafting part, and the rootstock of malus micromalus, malus halliana, M9-T337 and M26 serving as the rootstock can be accurately judged in the aspect of improving the drought resistance of the scion grafting part.
2. Pear stock evaluation chip and preparation and application thereof
1. Experimental Material culture and sampling
The experimental pear group used in the invention is scion variety Jinshuisusu, stock variety Du pear, ruby pear, autumn pear, quince coffin, chinese pear and birchleaf pear tissue culture seedlings, the culture medium is MS +0.1mg/L NAA +0.8 mg/L6-BA, subculture is carried out for 1 time every 40d, the culture temperature is 24 +/-1 ℃, the relative humidity is 85%, the light intensity is 16 h/8 h and the light intensity is 15001x.
2. Micro-grafting of tissue culture seedlings
According to the publicly reported tissue culture seedling micro-grafting method, birch pear, ruby pear, autumn pear, quince coffin, chinese pear and birch pear tissue culture seedlings are selected as micro-grafting stock parts, the Jinshuisusu is taken as a scion part for micro-grafting, the micro-grafting is carried out after the micro-grafting is carried out for one week in a dark place, the micro-grafting stock parts are taken out and cultured under normal illumination, the healing condition of a grafting opening is observed, and seedlings which are not survived in grafting and black in the lower part of the scion are removed.
3. Extraction of sample RNA
Collecting phloem and top leaf of scion of the grafting tissue culture seedling with uniform growth vigor, storing the sample in a refrigerator at minus 80 ℃, and extracting RNA of the sample by using a CTAB method.
Example 3: heat resistance evaluation of different pear stocks by using molecular fingerprint detector-1
(1) Chip preparation
Heat resistance evaluation chip was prepared as described in example 1
(2) Chip application
(A) Induction of amplification of RNA
And amplifying the induced RNA-1 from the scion phloem RNA of the combination of the tissue culture seedling stock and the scion of the pear to be detected, namely golden shortbread/birch pear, golden shortbread/ruby pear, golden shortbread/quince coffin, golden shortbread/Chinese pear and golden shortbread/birch pear. The solution was configured as follows:
the volume fraction of 25. Mu.L reaction components was:
ear grafting part RNA:5 μ L
DEPC：4.26μL
RRI(RNA guard TM )：0.5μL
NTP mixed liquid: each 5mM
dNTP mixture: each 2.5mM
Five-component buffer solution: 5 μ L
Enzyme mixture: 3.24 μ L
Primer mixture: 6 μ L
Reaction conditions are as follows: preheating the five-component buffer solution at 37 ℃ for 5min, adding other reactants, and heating in a water bath kettle at 40.5 ℃ for 2h;
(B) In vitro protein response assay
The in vitro protein reaction is: inducing RNA-1 to react with a molecular fingerprint detector-1 in-vitro protein reaction system. mu.L of the induced RNA-1 was added to the chip for evaluating heat resistance as shown in FIG. 18.
The detection sensitivity of induced RNA-1 with different concentrations is set, and each gene has three repetitions.
The molecular fingerprint detector-1 and the induced RNA-1 are observed to be well combined, and the CCT is increased along with the gradual increase of the concentration of the induced RNA-1.
(C) Data processing analysis, stock heat resistance prediction is evaluated through the CCT value of the scion part
Shooting with Canon camera E0S600D with the same shooting parameter and unchanged angle, and digital colorimeter software
2001-2020Apple Inc. measuring R, G and B values of reaction environment;
and (3) calculating to obtain a C value, drawing by using Graphpad software, carrying out single-factor linear regression analysis, and calculating to obtain a linear regression slope and a CCT value, wherein the calculation results are shown in tables 5 and 6.
TABLE 5 molecular fingerprint Detector-1 in vitro protein reaction C value
TABLE 6 molecular fingerprint Detector-1 in vitro protein reaction CCT value
The CCT value of the molecular fingerprint detector-1 in vitro protein reaction is obtained by analysis: du pear (390.5) > ruby pear (360.8) > birchleaf pear (366.2) > Chinese pear (352.8) > autumn pear (317.8) > quince (274.2), then the effect of Du pear and ruby pear in improving the heat resistance of the golden crisp cion is predicted to be better, birchleaf pear and Chinese pear in improving the heat resistance of the golden crisp cion have a common effect, and autumn pear and quince in improving the heat resistance of the golden crisp cion are predicted to be poorer.
(D) Stress treatment is carried out, and the evaluation result of the gene chip is verified
And simultaneously carrying out heat stress treatment on the golden water shortbread/durum pear, the golden water shortbread/ruby pear, the golden water shortbread/autumn pear, the golden water shortbread/quince coffin, the golden water shortbread/Chinese pear and the golden water shortbread/birchleaf pear, observing the wilting degree of plants, evaluating the heat stress degree of the scion grafting part of the micro-grafting tissue culture seedling, and evaluating the heat stress degree of the tissue culture seedling without the grafting stock as supplement.
And (2) heat stress treatment, namely culturing at a high temperature of 38 ℃ for 48h by using an illumination incubator, after 12h treatment, browning the leaves of the golden water shortbread/quince, after 48h treatment, browning and necrotizing the whole plant, browning most of the leaves of the golden water shortbread/autumn pear (more than 60%), presenting poor heat resistance, and wilting and browning most of the leaves and stems of the golden water shortbread/Chinese pear and golden water shortbread/birch pear after 40h, but keeping the plant in a survival state and presenting general heat resistance, wherein when 48h, the golden water shortbread/ruby and the golden water shortbread/birch pear, except for partial leaves, curl and browning (< 50%), the whole plant does not present an obvious heat damage phenotype. The leaves of the Chinese gooseberry and the birchleaf pear which are not grafted do not show obvious heat damage phenotype and show strong heat resistance, the edges of the leaves of the Chinese gooseberry and the birchleaf pear are curled and browned (less than 30 percent) and the edges of the leaves of the birchleaf pear and the birchleaf pear are apprxed to 50 percent), the heat resistance is general, and after the quince tissue culture seedlings are treated for 48 hours, the whole plant is browned and necrotized, and the heat resistance is poor.
As can be seen from the analysis of the data in tables 5 and 6, the prediction result of the heat resistance evaluation of the molecular fingerprint detector-1 is the same as the heat resistance analysis result after the stress treatment, and the accurate judgment can be performed on the aspect of improving the heat resistance of the scion part by using the pyrus betulaefolia, the ruby pears, the autumn pears, the quince, the Chinese pears and the birchleaf pears as the rootstocks.
Those not described in detail in this specification are within the skill of the art.
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gtagatagag tggcacgatc agatattgat gacatctctc ttgagaaggg acttcctgat 60
gaggttgtga agaaaatcaa aagtcttcgc cgcaattatc agcaggattc tgacccaaac 120
ttgccacctg ccga 134
<210> 22
<211> 195
<212> DNA
<213> university of agriculture in China
<400> 22
ttagctttgg tgaaggaagt gaatacagct atgtgaagca caaggttgat ggaattgaca 60
aagataactt tgtctacaag tacagtttga ttgaaggaga tgtcatttct gacaagattg 120
agaagatctc ttatgagact aagttggtgg cttccggcag cggttctgtc atcaagagca 180
ccagccatta ccaca 195
<210> 23
<211> 45
<212> DNA
<213> university of agriculture in China
<400> 23
aattctaata cgactcacta tagggccgtc ctccatttcc aactc 45
<210> 24
<211> 19
<212> DNA
<213> university of China agriculture
<400> 24
ccgcccacat caacctcaa 19
<210> 25
<211> 45
<212> DNA
<213> university of agriculture in China
<400> 25
aattctaata cgactcacta tagggggtag gagcaacagc aggac 45
<210> 26
<211> 17
<212> DNA
<213> university of China agriculture
<400> 26
agcggaacca agggaca 17
<210> 27
<211> 42
<212> DNA
<213> university of China agriculture
<400> 27
aattctaata cgactcacta tagggtcggc aggtggcaag tt 42
<210> 28
<211> 23
<212> DNA
<213> university of China agriculture
<400> 28
gtagatagag tggcacgatc aga 23
<210> 29
<211> 43
<212> DNA
<213> university of agriculture in China
<400> 29
aattctaata cgactcacta tagggtgtgg taatggctgg tgc 43
<210> 30
<211> 21
<212> DNA
<213> university of China agriculture
<400> 30
ttagctttgg tgaaggaagt g 21
<210> 31
<211> 21
<212> DNA
<213> university of agriculture in China
<400> 31
cttgtttgat ggccgtcgtc t 21
<210> 32
<211> 18
<212> DNA
<213> university of agriculture in China
<400> 32
cttttgcgct gccgccag 18
<210> 33
<211> 22
<212> DNA
<213> university of agriculture in China
<400> 33
acctcggtat ctctcccgct cc 22
<210> 34
<211> 18
<212> DNA
<213> university of agriculture in China
<400> 34
cttttgcgct gccgccag 18
<210> 35
<211> 25
<212> DNA
<213> university of agriculture in China
<400> 35
cctgatgagg ttgtgaagaa aatca 25
<210> 36
<211> 18
<212> DNA
<213> university of agriculture in China
<400> 36
cttttgcgct gccgccag 18
<210> 37
<211> 25
<212> DNA
<213> university of agriculture in China
<400> 37
gatctcttat gagactaagt tggtg 25
<210> 38
<211> 19
<212> DNA
<213> university of China agriculture
<400> 38
cttttgcgct gccgccagg 19
<210> 39
<211> 39
<212> DNA
<213> university of agriculture in China
<400> 39
gaacaaacta ccggcagcag acgctcgtct cgtctgggg 39
<210> 40
<211> 39
<212> DNA
<213> university of agriculture in China
<400> 40
tggagccata gagagggcga ggaagaggcc gacccctcc 39
<210> 41
<211> 39
<212> DNA
<213> university of China agriculture
<400> 41
ggactactcc aacacttctt ttagttttca gaagcggcg 39
<210> 42
<211> 39
<212> DNA
<213> university of agriculture in China
<400> 42
ctagagaata ctctgattca accaccgaag gccgtcgcc 39
Claims (6)
1. A chip for detecting scion and evaluating the quality of rootstock is characterized in that: the chip comprises a solid phase carrier, wherein an in vitro reaction reagent is loaded on the solid phase carrier;
the in vitro reaction reagent comprises a mixed buffer solution, a ribonuclease inhibitor, a molecular fingerprint detector, diethyl pyrocarbonate treated water and a color indicating reagent;
the color indicating reagent is chlorophenol red-beta-D-galactopyranose;
the molecular fingerprint detector is an RNA sequence, and the nucleotide sequence of the molecular fingerprint detector is a sequence which is shown in a sequence number of 3-4 and 39-42 in a sequence table.
2. A method of preparing a chip according to claim 1, comprising the steps of:
step one, adding a forward primer and a reverse primer into the plasmid, and carrying out PCR amplification on the PCR premix and water;
step two, performing gel electrophoresis on the PCR amplification product obtained in the step one to obtain a DNA recovery product, namely a molecular fingerprint detector;
step three, applying the molecular fingerprint detector obtained in the step two to prepare an in vitro protein reaction reagent;
step four, adding the in-vitro protein reaction reagent obtained in the step three into a solid phase carrier to obtain a chip;
the preparation process of the in vitro protein reaction reagent in the step three comprises the following steps: mixing the mixed buffer solution, RRI, a molecular fingerprint detector, diethyl pyrocarbonate and chlorophenol red-beta-D-galactopyranose; the above system was incubated at 37 ℃ for 2h.
3. A method of manufacturing a chip as claimed in claim 2, characterized in that: the nucleotide sequences of the forward primer and the reverse primer in the step one are sequences with the numbers of 11-14 and 31-38 in the sequence table.
4. Use of a chip as claimed in claim 1, characterized in that it comprises the following steps:
step one, collecting stock scion phloem RNA;
step two, using the RNA as a template to amplify induced RNA, specifically comprising the following steps:
adding phloem RNA, diethyl pyrocarbonate and RRI into an induced RNA amplification system to react to obtain induced RNA; the induced RNA amplification system comprises five components of buffer solution, NTP mixed solution, dNTP mixed solution, enzyme mixture and primer mixture;
step three, adding the induced RNA obtained in the step two into a chip to perform in-vitro protein reaction;
observing the reaction discoloration phenomenon, evaluating the corresponding characters of the scions to be tested, and further evaluating the quality of the stocks;
step three the steps of carrying out in vitro protein reaction are: and (3) adding 1 mu L of induced RNA obtained in the step (II) into the chip, and reacting with the in-vitro protein reaction reagent in the chip.
5. The use of the chip of claim 4, wherein:
the five-component buffer solution in the step two comprises the following components:
tris hydrochloride, potassium chloride, magnesium chloride, dithiothreitol and water;
the components of the enzyme mixture in the second step are as follows:
M-MLV reverse transcriptase, T7 RNA polymerase, AMV reverse transcriptase, ribonuclease and bovine serum albumin;
the components of the primer mixture in the second step are as follows:
primer (R), primer (F), dimethyl sulfoxide and deionized water.
6. The use of the chip of claim 5, wherein: the evaluation process of the discoloration described in step four was:
shooting the process of changing a first color of the environmental color of the color indicating reagent to a second color by using an image shooting device under the same shooting condition, and recording the time required for the first color to be converted to the second color, namely the reaction time;
using software to read the change of RGB value in the process of changing the first color to the second color, and calculating the color change rate CCT according to the following formula:
C＝(R+B)/(R+G)；
ΔC＝d[dC/dt]/dt＝d 2 (R+B)/(R+G)/dt 2 ；
CCT＝ΔC×10 5 ；
in the formula, C: color indicating reagent ambient color, R: RGB red number, G: RGB green number, B: RGB blue values, Δ C: change of slope of the curve, t: reaction time, CCT: the rate of change of color.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210159263.1A CN114752696B (en) | 2022-02-21 | 2022-02-21 | Chip for detecting scion and evaluating quality of rootstock as well as preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210159263.1A CN114752696B (en) | 2022-02-21 | 2022-02-21 | Chip for detecting scion and evaluating quality of rootstock as well as preparation method and application thereof |
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| Publication Number | Publication Date |
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| CN114752696A CN114752696A (en) | 2022-07-15 |
| CN114752696B true CN114752696B (en) | 2023-03-24 |
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| CN104603290A (en) * | 2012-06-29 | 2015-05-06 | Htg分子诊断有限公司 | Nuclease protection methods for detection of nucleotide variants |
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| CN108103235B (en) * | 2018-02-11 | 2021-05-18 | 山东省果树研究所 | SNP molecular marker and primer for identifying cold resistance of apple rootstock and application of SNP molecular marker and primer |
| CN113430197B (en) * | 2021-05-17 | 2022-07-29 | 西北农林科技大学 | Movable miRNA for promoting root development and drought resistance of rootstock and identification method |
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| CN104603290A (en) * | 2012-06-29 | 2015-05-06 | Htg分子诊断有限公司 | Nuclease protection methods for detection of nucleotide variants |
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