CN110184384B - Method for identifying drought resistance of corn variety based on real-time quantitative PCR - Google Patents
Method for identifying drought resistance of corn variety based on real-time quantitative PCR Download PDFInfo
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Abstract
The invention discloses a method for identifying drought resistance of a corn variety based on real-time quantitative PCR (polymerase chain reaction), which is used for identifying the drought resistance of the corn variety in the growth process of the seedling stageDrought stress is carried out, a control group is arranged at the same time, total RNA of the corn plants is extracted, DNAJ gene expression quantity is detected, and the expression quantity drought resistance index DI is obtainedE(ii) a Using DIEAnd (4) evaluating the drought resistance of the corn by the expression drought resistance index. The method can realize the rapid and accurate identification of the drought resistance of the corn and provide technical support for screening the drought resistance of the corn.
Description
Technical Field
The invention belongs to the field of drought resistance identification of corn varieties, and particularly relates to a method for identifying the drought resistance of a corn variety based on real-time quantitative PCR.
Background
Corn, the highest-yielding food crop in the world, is severely threatened by drought in terms of productivity. The drought resistance of the corn is limited by the genetic effect and environmental factors of the corn, and any single drought resistance research has certain limitations so far due to different growth and development periods of crops and the influence of biological factors and non-biological factors, so that the drought resistance of the corn is difficult to directly and accurately evaluate. There are many methods for identifying the drought resistance of corn, and the methods mainly comprise a field direct identification method, an artificial environment simulation method, a physiological index method, a molecular biology identification method and the like. The direct field identification method is used for identifying the growth form of crops in different growth and development periods, but the methods have the defects of long time, restriction by environmental factors, large rainfall variation between the years, difficulty in repeating the research data result and the like; the artificial simulation environment method is a method for artificially causing a drought stress environment required by an experiment by regulating and controlling the water content of soil and air in a drought shed, a growth chamber or an artificial climate chamber and evaluating the drought resistance of the water corn by researching the growth and development, the physiological process or the change of yield results of the corn, and the method needs certain equipment, has relatively large energy consumption cost, and creates the drought stress environment which has certain difference from the field production under natural conditions, so that the experiment result has certain difference from the result directly identified in the field; physiological and biochemical indexes for identifying the drought resistance of plants mainly utilize indexes related to leaf moisture, plasma membrane permeability, enzyme activity and the like for identification, but the physical and chemical indexes can be changed due to different growth environments and different growth periods, and errors are easily caused by used reagents and manual operation; the molecular biology identification method is based on modern molecular biology, a saturated molecular genetic map is constructed by using related molecular markers, drought-resistant genes of the corn are located, the molecular markers are used for selecting drought-resistant varieties of the corn, drought resistance belongs to quantitative characters controlled by multiple genes, a plurality of drought-resistant QTLs are discovered in recent years, but the polymorphism frequency of the markers is low, the influence of a single QTL on phenotype difference is small, the epistatic effect is difficult to evaluate and the like, and further research is needed by using molecular marker-assisted selection. Therefore, the establishment of new technologies and the discovery of new markers to solve these problems are urgently required.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting drought resistance of a corn variety, which is characterized by determining the expression quantity of DNAJ in RNA of a corn plant after drought stress treatment and finally obtaining the expression quantity drought resistance index DIEUsing the expression level of drought resistance index DIEAnd (4) evaluating the drought resistance of the corn variety.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for identifying drought resistance of a corn variety based on real-time quantitative PCR comprises the following steps:
selecting a corn variety to be evaluated, sowing the corn variety in a seedling culture tray paved with vermiculite, carrying out drought stress treatment when the corn plant grows to 3-leaf stage, and setting a control group;
extracting total RNA of a first unfolded leaf of a corn plant 7 days after drought stress treatment;
detecting the DNAJ gene expression quantity in the extracted total RNA by adopting real-time quantitative PCR to obtain the expression quantity of drought stress DNAJ and the DNAJ expression quantity of a control group;
calculating drought resistance index DI of corn expression quantityEAccording to the following formula:
expression level drought resistance coefficient DCEDNAJ expression level of control group
DIEExpressed drought resistance coefficient DCEX drought stress DNAJExpression of (d) ÷ average of all maize varieties drought stress expression;
according to DIEEvaluation of drought resistance in maize varieties, wherein DIEGreater than or equal to 1.3 is extremely drought-resistant and DIE1.11-1.19 is strong drought resistance; DIEMedium drought resistance is 0.91-1.10; DIEWeak resistance is 0.1-0.90; DIELess than or equal to 0.70 is extremely weak drought resistance.
Preferably, the drought stress is that the water content of the vermiculite in the seedling raising tray is controlled to be lower than 20%.
Preferably, Trizol reagent is used to extract total RNA from the top leaf of a maize plant.
Preferably, the expression level of the DNAJ gene in the extracted total RNA is detected by adopting real-time fluorescent quantitative PCR.
Further preferably, the extracted total RNA is subjected to reverse transcription into cDNA; then, the cDNA is taken as a template to carry out real-time fluorescence quantitative PCR detection.
More preferably, when the real-time fluorescent quantitative PCR is used for detection, the reaction system comprises the following components in 20 μ l: mu.l of cDNA template, 0.5. mu.l of forward and reverse primers, 10. mu.l of SYBR Green mix fluorescent dye, 7. mu.l of sterile water.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method is simple, can singly identify the drought resistance of the corn through a fluorescent quantitative PCR technology, and does not need to be combined with other characters and indexes;
(2) accurate identification, the invention utilizes DNAJ gene expression quantity drought resistance index DIEThe yield drought resistance index ID of different corn varieties achieves extremely obvious positive correlation and is consistent with the identification result of the field direct identification method widely adopted at present;
(3) the identification range is wide, so that the maize hybrid can be identified, such as the Nongdan 476 and the Zhongxin 978, and the maize inbred line can be identified, such as the 8112, the heddle 31 and the Mo 17;
(4) the identification efficiency is high, the identification can be carried out in the seedling stage of the corn, and the method for detecting the drought resistance of the corn by the fluorescent quantitative PCR established on the basis of the DNAJ gene provides an advanced technical means for identifying the drought resistance of the corn.
Drawings
FIG. 1 shows the expression level drought resistance index DI in example 1 of the present inventionEAnd yield drought resistance index DIPThe correlation graph of (2).
Detailed Description
Aiming at twelve corn varieties of Xian Yu 335 purchased in the market, nong Hua 101, Jixiang No. 1, Zheng Dan 1002, Shaandan 609, Zheng Dan 958, Jidan 50, Shaandan 618, Vitaceae 702, Liaodan 588, Shanghai 605 and Doudan No. 10, the drought resistance detection method comprises the following steps:
1. planting purchased corn seeds in a seedling raising tray paved with vermiculite, carrying out drought stress treatment in the seedling stage, controlling the water content of the vermiculite to be lower than 20%, and setting a control group for normal watering;
2. after 7 days of drought stress treatment, total RNA of the first expanded leaf of the drought stress treated maize plant and total RNA of the first expanded leaf of the maize plant in the normally watered control group were extracted, respectively, total RNA of leaf tissue was isolated using Trizol reagent (Invitrogen, USA) according to the contents of the specification, and concentration and purity of the extracted RNA were measured using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA);
3. DNAJ is differential expression gene, and the expression quantity of said gene is respectively detected, and its concrete method is that the reverse transcription of extracted total RNA is made to obtain cDNA, and the cDNA is used as template to make real-time quantitative PCR, and the calculation of gene expression quantity is according to 2-ΔΔCTAnd (3) the relative expression quantity of the gene is equal to (Ct value of the target gene to be detected-Ct value of the reference gene to be detected) - (Ct value of the target gene of the control group-Ct value of the reference gene of the control group). Wherein the total volume of the reaction system is 20 mul, and the reaction system comprises 2 mul of cDNA template, 0.5 mul of positive primer and negative primer, 10 mul of SYBR Green mix fluorescent dye and 7 mul of sterile water, and is detected;
4. drought resistance finger for calculating corn expression quantityDigital DIEAccording to the following formula:
expression level drought resistance coefficient DCEDrought stress DNAJExpression amount ÷ control group DNAJAmount of expression
DIEExpressed drought resistance coefficient DCE) X drought stress DNAJExpression of the corn variety, average of all the corn varieties drought stress expression, and corn expression drought resistance index DI of each corn varietyEAs shown in table 1;
5. according to DIEEvaluation of drought resistance in maize varieties, wherein DIEGreater than or equal to 1.3 is extremely drought resistant (HR), DIE1.01 to 1.29 is strong drought resistance (R); DIEMedium drought resistance (MR) is 0.91-1.10; DIEWeak resistance (S) is 0.70-0.90; DIELess than or equal to 0.70 is extremely weak drought resistance (HS).
The results of drought resistance tests on the twelve maize varieties are shown in table 1.
TABLE 1
Expression level DI using Microsoft Excel 2013EAnd yield drought resistance index DIPA linear equation is established, the correlation coefficient R is 0.92 as shown in FIG. 1, and polar correlation (p) is achieved<0.01). Therefore, the method for identifying the drought resistance of the corn variety has high accuracy, can realize the rapid identification of the drought resistance of the corn at the seedling stage, and does not need to use the mature harvest of the corn.
Meanwhile, the corn detection method is high in identification efficiency aiming at the seedling stage of the corn, 20 days are needed from sowing to identification, and multiple batches of drought resistance identification can be completed in 1 year.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (6)
1. A method for identifying drought resistance of a corn variety based on real-time quantitative PCR is characterized by comprising the following steps:
selecting a corn variety to be evaluated, sowing the corn variety in a seedling culture tray paved with vermiculite, carrying out drought stress treatment when a corn plant grows to a three-leaf period, and setting a control group;
extracting total RNA of a first unfolded leaf of a corn plant 7 days after drought stress treatment;
detecting the DNAJ gene expression quantity in the extracted total RNA by adopting real-time quantitative PCR to obtain the expression quantity of drought stress DNAJ and the DNAJ expression quantity of a control group;
calculating drought resistance index DI of corn expression quantityEAccording to the following formula:
expression level drought resistance coefficient DCEDNAJ expression level of control group
DIEExpressed drought resistance coefficient DCEExpression of x drought stress DNAJ) ÷ average of all maize varieties drought stress expression;
drought resistance index DI according to corn expression quantityEEvaluation of drought resistance in maize varieties, wherein DIEGreater than or equal to 1.3 is extremely drought-resistant and DIE1.11-1.19 is strong drought resistance; DIEMedium drought resistance is 0.91-1.10; DIEWeak resistance is 0.71-0.90; DIELess than or equal to 0.70 is extremely weak drought resistance.
2. The method for identifying drought resistance of a maize variety based on real-time quantitative PCR as claimed in claim 1, wherein: the drought stress is to control the water content of vermiculite in the seedling culture plate to be lower than 20 percent.
3. The method for identifying drought resistance of a maize variety based on real-time quantitative PCR as claimed in claim 1, wherein Trizol reagent is used to extract total RNA from the first expanded leaf of a maize plant.
4. The method for identifying the drought resistance of the corn variety based on the real-time quantitative PCR as claimed in claim 1, wherein the DNAJ gene expression amount in the extracted total RNA is detected by using real-time fluorescent quantitative PCR.
5. The method for identifying drought resistance of a maize variety based on real-time quantitative PCR of claim 4, wherein the extracted total RNA is reverse transcribed into cDNA; then, the cDNA is taken as a template to carry out real-time fluorescence quantitative PCR detection.
6. The method for identifying the drought resistance of the corn variety based on the real-time quantitative PCR as claimed in claim 5, wherein the reaction system adopted in the real-time quantitative PCR detection comprises the following components in 20 μ l: mu.l of cDNA template, 0.5. mu.l of forward and reverse primers, 10. mu.l of SYBR Green mix fluorescent dye, 7. mu.l of sterile water.
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