CN110184384A - A method of corn variety drought resistance is identified based on real-time quantitative PCR - Google Patents
A method of corn variety drought resistance is identified based on real-time quantitative PCR Download PDFInfo
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- CN110184384A CN110184384A CN201910634005.2A CN201910634005A CN110184384A CN 110184384 A CN110184384 A CN 110184384A CN 201910634005 A CN201910634005 A CN 201910634005A CN 110184384 A CN110184384 A CN 110184384A
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Abstract
The invention discloses a kind of methods identified based on real-time quantitative PCR corn variety drought resistance, it is to carrying out drought stress in corn variety in seedling stage growth course, control group is set simultaneously, extract the total serum IgE of plant, and wherein DNAJ gene expression amount is detected, obtain expression quantity antiG valves DIE；Utilize DIEExpression quantity antiG valves carry out drought resistance evaluation to corn.The quick and precisely identification to drought resistance of maize can be achieved in the present invention, provides technical support for drought resistance of maize screening.
Description
Technical field
The invention belongs to corn variety Identification of Drought fields, more particularly to a kind of real-time quantitative PCR that is based on is to corn product
The method that kind drought resistance is identified.
Background technique
Corn is as the highest cereal crops of yield in the world, the productivity seriously threat by arid.Drought-resistant maize
Sex expression is restricted jointly by itself hereditary effect and environmental factor, because the growth and development period of crop is different, it is biological because
The drought resisting research of the influence of element and abiotic factor, up to the present any individual event has certain limitation, it is difficult to directly quasi-
The drought resistance of true evaluation corn.The method of drought resistance of maize identification has very much, mainly includes field Direct Identification method, artificial mould
Quasi- Environmental Law, physical signs method, molecular biology identification method etc..Wherein Direct Identification method in field is in different growth and development
Phase identifies plant growth form, but these methods are long there is the time, are restricted by environmental factor, especially drop between year border
Water luffing is larger, and the data result of research is made to be difficult to the disadvantages of repeating；Manual simulation's Environmental Law refer to arid canopy, growth case or
By the water content of regulation soil, air in phjytotron, artificially cause to test required drought stress environment, and by grinding
The method for studying carefully the variation of the growth and development of corn, physiology course or yield result to evaluate water drought resistance of maize, the method need
Certain equipment is wanted, energy consumption cost is relatively large, and field production has one under the conditions of the drought stress environment and nature created
Fixed otherness causes experimental result and the result of field Direct Identification to have different；Physiological and biochemical index identifies that plant is anti-
Drought is mainly to be identified using the relevant index of leaf water, membrane permeability and enzymatic activity etc., still, these physical and chemical indexes
Can because growing environment, growthdevelopmental stage difference due to change, be also easy to bring error because of agents useful for same and manual operation；Molecule
Biological assay refers to based on the basis of modern molecular biology, is lost using the molecule of relevant molecular labeling building saturation
Blit spectrum, and the anti-drought gene of corn is positioned, the selection of Drought Resistant Varieties In Maize is carried out using molecular labeling, drought resisting belongs to
The quantitative character of controlled by multiple genes, many drought resistance QTL of discovered in recent years, but the frequency of polymorphism marked is very low, individually
QTL is small on phenotypic difference influence, and epistatic analysis is difficult to carry out the reason such as assessing, and also needs to carry out using molecular marker assisted selection
A deep step research.Therefore, there is an urgent need to establish new technology and find new marker to solve these problems.
Summary of the invention
It is to utilize in order to solve the above-mentioned technical problems, the present invention provides a kind of corn variety drought resistance detection method
The expression quantity of DNAJ in plant RNA is measured after drought stress processing, it is final to obtain expression quantity antiG valves DIE, utilize table
Up to amount antiG valves DIEDrought-resistant ability evaluation is carried out to corn variety.
In order to achieve the above object, the present invention adopts the following technical scheme that:
A method of corn variety drought resistance is identified based on real-time quantitative PCR, comprising the following steps:
Corn variety to be evaluated is selected, is seeded in the seedlings nursing plate for being covered with vermiculite, grows to 3 in plant
The leaf phase carries out drought stress processing, while control group is arranged；
After drought stress is handled 7 days, the total serum IgE that blade is unfolded in plant first is extracted；
DNAJ gene expression amount in the total serum IgE of extraction is detected using real-time quantitative PCR, obtains drought stress DNAJ
Expression quantity and control group DNAJ expression quantity；
Calculate corn expression quantity antiG valves DIE, according to following formula:
Expression quantity drought resistance coefficient DCE=drought stress DNAJ expression quantity ÷ control group DNAJ expression quantity
DIE=(expression quantity drought resistance coefficient DCE× drought stress DNAJExpression quantity) all corn variety drought stress tables of ÷
Up to the average of amount；
According to DIECorn variety drought-resistant ability is evaluated, wherein DIE>=1.3 be pole drought resisting, DIEIt is 1.11~1.19
For strong drought resistant；DIEIt is medium drought resisting for 0.91~1.10；DIEIt is weak anti-for 0.1~0.90；DIE≤ 0.70 is pole weak drought resistant.
Preferably, the drought stress is to control the water content of vermiculite in seedlings nursing plate lower than 20%.
Preferably, the total serum IgE of plant top vane is extracted using Trizol reagent.
Preferably, DNAJ gene expression amount in the total serum IgE of extraction is detected using real-time fluorescence quantitative PCR.
It is further preferred that the total serum IgE of extraction is carried out reverse transcription into cDNA；Then it is carried out using cDNA as template glimmering in real time
Fluorescent Quantitative PCR detection.
It is furthermore preferred that include when the real-time fluorescence quantitative PCR is detected, in the used every 20 μ l of reaction system with
Lower component: the cDNA template of 2 μ l, the positive and negative primer of 0.5 μ l, 10 μ l SYBR Green mix fluorescent dye, 7 μ l sterile waters.
Compared with prior art, the invention has the following beneficial effects:
(1) method is simple, can it is single by fluorescent quantitative PCR technique identify corn drought resistance, do not need and its
His character and index combine；
(2) identification is accurate, and the present invention utilizes DNAJ gene expression amount antiG valves DIEFrom different corn variety yield drought resistings
Index ID reaches extremely significant positive correlation, and the field Direct Identification method qualification result with being widely used at present is consistent；
(3) identification range is wide, can identify corn hybrid seed, such as agriculture list 476, many letters 978, and can identify that corn is selfed
System, such as 8112, comprehensive 31 and Mo17；
(4) determination rates are high, can identify in Maize at Seedling Stage, the corn quantitative fluorescent PCR drought resisting established based on DNAJ gene
Property detection method will for identify corn drought resistance advanced technology means are provided.
Detailed description of the invention
Fig. 1 is expression quantity antiG valves DI in the embodiment of the present invention 1EWith yield antiG valves DIPCorrelation curve figure.
Specific embodiment
For first jade 335, agriculture China 101, Jixiang No.1, the Zheng Dan 1002, Shan single 609, Zheng Dan 958, Ji Dan purchased in the market
50, Shan is single 618, big section 702, the Liao Dynasty are single 588, step on sea 605, holds together single No. 10 12 corn varieties carries out drought resistance detections, including
Following steps:
1, the corn seed of buying is planted in the seedlings nursing plate for be covered with vermiculite, is carried out at drought stress in seedling stage
Reason controls the water content of vermiculite lower than 20%, while the control group that setting is normally watered；
2, after drought stress is handled 7 days, the total serum IgE of blade is unfolded in the plant first for extracting drought stress processing respectively
Blade total serum IgE is unfolded in plant first in the control group normally to water, using Trizol reagent (Invitrogen, USA)
The total serum IgE of content separate blade tissue to specifications uses 1000 spectrophotometer (NanoDrop of NanoDrop
Technologies Inc, Wilmington, DE, USA) detect the concentration and purity for having extracted RNA；
3, DNAJ be difference expression gene, respectively to the gene carry out expression quantity detection, specific method be will extract it is total
RNA carries out reverse transcription and obtains cDNA, and carries out real-time quantitative PCR by template of cDNA, and the calculating of gene expression amount is according to 2-ΔΔCTMethod, the i.e. relative expression quantity of gene=(target gene Ct value to be measured-reference gene to be measured Ct value)-(control group purpose base
Because of Ct value-control group reference gene Ct value)., wherein the total volume of reaction system be 20 μ l, including 2 μ l cDNA template,
The SYBR Green mix fluorescent dye of the positive and negative primer of 0.5 μ l, 10 μ l, 7 μ l sterile waters, through detecting；
4, corn expression quantity antiG valves DI is calculatedE, according to following formula:
Expression quantity drought resistance coefficient DCE=drought stress DNAJExpression quantity ÷ control group DNAJExpression quantity
DIE=(expression quantity drought resistance coefficient DCE) × drought stress DNAJAll corn variety drought stress tables of expression quantity ÷
Up to the average of amount, the corn expression quantity antiG valves DI of each corn varietyEAs shown in table 1；
5, according to DIECorn variety drought-resistant ability is evaluated, wherein DIE>=1.3 be pole drought resisting (HR), DIEIt is 1.01
~1.29 be strong drought resistant (R)；DIEIt is medium drought resisting (MR) for 0.91~1.10；DIEIt is weak anti-(S) for 0.70~0.90；DIE≤
0.70 is pole weak drought resistant (HS).
It is as shown in table 1 that drought resistance testing result is carried out for above-mentioned 12 corn varieties.
Table 1
Using Microsoft Excel 2013 to expression quantity DIEWith yield antiG valves DIPEstablish linear equation, such as Fig. 1
Shown coefficient R=0.92 has reached extremely related (p < 0.01).It can be seen that identification corn variety drought resistance of the invention
Method accuracy rate is high, and may be implemented to harvest in its drought resistance of Maize at Seedling Stage Rapid identification without using corn maturation.
The corn detection method of the application is directed to Maize at Seedling Stage simultaneously, and determination rates are high, terminates about from identification is seeded into
20 days are needed, the drought resisting identification of 1 year achievable multiple batch.
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention
It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
Claims (6)
1. a kind of method identified based on real-time quantitative PCR corn variety drought resistance, which is characterized in that including following step
It is rapid:
Corn variety to be evaluated is selected, is seeded in the seedlings nursing plate for being covered with vermiculite, grows to tri-leaf period in plant
Drought stress processing is carried out, while control group is set；
After drought stress is handled 7 days, the total serum IgE that blade is unfolded in plant first is extracted；
DNAJ gene expression amount in the total serum IgE of extraction is detected using real-time quantitative PCR, obtains the table of drought stress DNAJ
Up to amount and control group DNAJ expression quantity；
Calculate corn expression quantity antiG valves DIE, according to following formula:
Expression quantity drought resistance coefficient DCE=drought stress DNAJ expression quantity ÷ control group DNAJ expression quantity
DIE=(expression quantity drought resistance coefficient DCEThe expression quantity of × drought stress DNAJ) all corn variety drought stress expression quantity of ÷
Average；
According to corn expression quantity antiG valves DIECorn variety drought-resistant ability is evaluated, wherein DIE>=1.3 be pole drought resisting,
DIEIt is strong drought resistant for 1.11~1.19；DIEIt is medium drought resisting for 0.91~1.10；DIEIt is weak anti-for 0.71~0.90；DIE≤
0.70 is pole weak drought resistant.
2. the method according to claim 1 identified based on real-time quantitative PCR corn variety drought resistance, feature
Be: the drought stress is to control the water content of vermiculite in seedlings nursing plate lower than 20%.
3. the method according to claim 1 identified based on real-time quantitative PCR corn variety drought resistance, feature
It is, the total serum IgE of plant first expansion blade is extracted using Trizol reagent.
4. the method according to claim 1 identified based on real-time quantitative PCR corn variety drought resistance, feature
It is, DNAJ gene expression amount in the total serum IgE of extraction is detected using real-time fluorescence quantitative PCR.
5. the method according to claim 4 identified based on real-time quantitative PCR corn variety drought resistance, feature
It is, the total serum IgE of extraction is subjected to reverse transcription into cDNA；Then real-time fluorescence quantitative PCR detection is carried out by template of cDNA.
6. the method according to claim 5 identified based on real-time quantitative PCR corn variety drought resistance, feature
It is, includes following components in the used every 20 μ l of reaction system: 2 μ l's when the real-time fluorescence quantitative PCR is detected
CDNA template, the positive and negative primer of 0.5 μ l, 10 μ l SYBR Green mix fluorescent dye, 7 μ l sterile waters.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251537A (en) * | 2020-11-27 | 2021-01-22 | 河北农业大学 | Primer group and method for identifying wheat drought resistance |
| CN115948422A (en) * | 2023-01-05 | 2023-04-11 | 河北农业大学 | Plant drought tolerance related gene ZmDnaJ, and amplification primer, recombinant vector and application thereof |
Citations (1)
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| CN106818101A (en) * | 2016-07-29 | 2017-06-13 | 新疆农业科学院粮食作物研究所 | A kind of method for identifying drought resistance of maize |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106818101A (en) * | 2016-07-29 | 2017-06-13 | 新疆农业科学院粮食作物研究所 | A kind of method for identifying drought resistance of maize |
Non-Patent Citations (2)
| Title |
|---|
| DIEFENBACH J等: "The membrane-bound DnaJ protein located at the cytosolic of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species", 《EUR. J. BIOCHEM.》 * |
| 崔润丽等: "谷子DnaJ蛋白基因的克隆", 《华北农学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251537A (en) * | 2020-11-27 | 2021-01-22 | 河北农业大学 | Primer group and method for identifying wheat drought resistance |
| CN115948422A (en) * | 2023-01-05 | 2023-04-11 | 河北农业大学 | Plant drought tolerance related gene ZmDnaJ, and amplification primer, recombinant vector and application thereof |
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