CN110317899A - Method for identifying drought resistance of corn based on expression of galacturonase gene - Google Patents
Method for identifying drought resistance of corn based on expression of galacturonase gene Download PDFInfo
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- 240000008042 Zea mays Species 0.000 title claims abstract description 54
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 54
- 230000014509 gene expression Effects 0.000 title claims abstract description 49
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 36
- 235000005822 corn Nutrition 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title abstract description 12
- 230000008641 drought stress Effects 0.000 claims abstract description 22
- 108010059820 Polygalacturonase Proteins 0.000 claims description 24
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 18
- 235000009973 maize Nutrition 0.000 claims description 18
- 210000002966 serum Anatomy 0.000 claims description 12
- 239000002299 complementary DNA Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 6
- 238000003753 real-time PCR Methods 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 229910052902 vermiculite Inorganic materials 0.000 claims description 4
- 235000019354 vermiculite Nutrition 0.000 claims description 4
- 239000010455 vermiculite Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 claims description 3
- 230000000474 nursing effect Effects 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 230000012010 growth Effects 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 2
- 241001057636 Dracaena deremensis Species 0.000 abstract 2
- 230000007613 environmental effect Effects 0.000 description 4
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000010977 jade Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002922 epistatic effect Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Abstract
The invention discloses a method for identifying corn drought resistance based on galacturonase gene expression, which comprises the steps of carrying out drought stress on a corn variety in the growth process of a seedling stage, setting a control group, extracting total RNA of a corn plant, detecting the expression quantity of the galacturonase gene in the corn plant, and obtaining the expression quantity drought resistance index DIE(ii) a Drought resistance index DI using expression levelEAnd (4) evaluating the drought resistance of the corn. 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 drought resistance of maize to identify that field, more particularly to one kind are expressed based on polygalacturonase gene to jade
The method of rice Identification of Drought.
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 galacturonic acid enzyme in plant RNA is measured after drought stress processing, it is final to obtain expression quantity antiG valves
DIE, utilize expression quantity antiG valves DIEEvaluation of drought resistance 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 drought resistance of maize is identified based on polygalacturonase gene expression, comprising the following steps:
Corn variety to be evaluated is selected, carry out drought stress processing in Maize at Seedling Stage, while control group is set；
After drought stress is handled 7 days, the total serum IgE of maize leaf is extracted；
Polygalacturonase gene expression quantity in the total serum IgE of extraction is detected using real-time fluorescence quantitative PCR, is obtained
The expression quantity and control group polygalacturonase gene expression quantity of drought stress polygalacturonase gene；
Calculate corn expression quantity antiG valves DIE, according to following formula:
Expression quantity drought resistance coefficient DCE=drought stress polygalacturonase gene expression quantity ÷ control group galacturonic acid enzyme
Gene expression amount；
DIE=(expression quantity drought resistance coefficient DCE× drought stress polygalacturonase gene expression quantity) all corn varieties of ÷
The average of drought stress expression quantity；
According to corn expression quantity antiG valves DIECorn variety drought-resistant ability is evaluated, wherein DIE>=1.35 be pole
Drought resisting (HR), DIEIt is strong drought resistant (R) for 1.11~1.34；DIEIt is medium drought resisting (MR) for 0.88~1.10；DIEFor 0.64~
0.87 is weak anti-(S)；DIE≤ 0.64 is pole weak drought resistant (HS).
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 first expansion blade is extracted using Trizol reagent.
Preferably, the total serum IgE of extraction is subjected to reverse transcription into cDNA；Then real time fluorescent quantitative is carried out by template of cDNA
PCR detection.
It preferably, include with the following group in the used every 20 μ l of reaction system when the real-time fluorescence quantitative PCR detects
Point: the cDNA template of 1 μ l, the positive and negative primer of 0.5 μ l, 10 μ l SYBR Green mix fluorescent dye, 8 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 polygalacturonase gene expression quantity antiG valves DIEWith corn variety yield
AntiG valves DIPReach positive correlation, the field Direct Identification method qualification result with being widely used at present is consistent；
(3) identification range is wide, can identify that corn hybrid seed can identify corn inbred line again；
(4) determination rates are high, only need can identify more than ten of batch within the time 1 year 20 day from corn seeding to test is completed, and
Only identify a batch within general survey 1 year.
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
In order to make those skilled in the art that the present invention may be better understood, with reference to the accompanying drawings and examples to this hair
Bright technical solution further illustrates.
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 of NanoDrop
(NanoDropTechnologies Inc, Wilmington, DE, USA) detects the concentration and purity for having extracted RNA；
3, polygalacturonase gene is difference expression gene, carries out expression quantity detection, specific method to the gene respectively
It is that the total serum IgE that will be extracted carries out reverse transcription acquisition cDNA, and carries out polygalacturonase gene expression, gene by template of cDNA
The calculating of expression quantity is according to 2-ΔΔCTMethod, the i.e. relative expression quantity of gene=(target gene Ct value-reference gene to be measured to be measured
Ct value)-(control group target gene Ct value-control group reference gene Ct value)., wherein the total volume of reaction system is 20 μ l,
In include the cDNA template of 1 μ l, the positive and negative primer of 0.5 μ l, 10 μ l SYBR Green mix fluorescent dye, 8 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 polygalacturonase gene expression quantity ÷ control group galacturonic acid enzyme
Gene expression amount
DIE=(expression quantity drought resistance coefficient DCEAll corn product of the expression quantity ÷ of) × drought stress polygalacturonase gene
The average of kind drought stress expression quantity, 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>=1.35 be pole drought resisting (HR), DIEFor
1.11~1.34 be strong drought resistant (R)；DIEIt is medium drought resisting (MR) for 0.88~1.10；DIEIt is weak anti-(S) for 0.64~0.87；
DIE≤ 0.64 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 DIP(Yan Weiping, Bian Shaofeng,
Beautiful China waits the evaluation and the northeast screening [J] agricultural sciences of semiarid region drought resisting high yield corn variety, 2017,42 (3): 1-5)
Linear equation is established, coefficient R=0.87 as shown in Figure 1 has reached extremely significant positive correlation (P < 0.01).It can be seen that this hair
The method accuracy rate of bright identification corn variety drought resistance is high, and may be implemented in its drought resistance of Maize at Seedling Stage Rapid identification,
It is harvested without using corn maturation.The corn detection method of the application is directed to Maize at Seedling Stage simultaneously, and determination rates are high, from being seeded into
Identification terminates to take around 20 days, the drought resisting identification of 1 year achievable multiple batch.
The preferred embodiment of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, several deformations can also be made, improves and substitutes, these belong to this hair
Bright protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (5)
1. a kind of express the method identified drought resistance of maize based on polygalacturonase gene, which is characterized in that including following
Step:
Corn variety to be evaluated is selected, carry out drought stress processing in Maize at Seedling Stage, while control group is set；
After drought stress is handled 7 days, the total serum IgE of maize leaf is extracted；
Polygalacturonase gene expression quantity in the total serum IgE of extraction is detected using real-time fluorescence quantitative PCR, obtains arid
Coerce the expression quantity and control group polygalacturonase gene expression quantity of polygalacturonase gene；
Calculate corn expression quantity antiG valves DIE, according to following formula:
Expression quantity drought resistance coefficient DCE=drought stress polygalacturonase gene expression quantity ÷ control group polygalacturonase gene
Expression quantity；
DIE=(expression quantity drought resistance coefficient DCE× drought stress polygalacturonase gene expression quantity) all corn variety arids of ÷
Coerce the average of expression quantity；
According to corn expression quantity antiG valves DIECorn variety drought-resistant ability is evaluated, wherein DIE>=1.35 be pole drought resisting
(HR), DIEIt is strong drought resistant (R) for 1.11~1.34；DIEIt is medium drought resisting (MR) for 0.88~1.10；DIEIt is 0.64~0.87
For weak anti-(S)；DIE≤ 0.64 is pole weak drought resistant (HS).
2. according to claim 1 express the method identified drought resistance of maize, spy based on polygalacturonase gene
Sign is: the drought stress is to control the water content of vermiculite in seedlings nursing plate lower than 20%.
3. according to claim 1 express the method identified drought resistance of maize, spy based on polygalacturonase gene
Sign is, the total serum IgE of plant first expansion blade is extracted using Trizol reagent.
4. according to claim 3 express the method identified drought resistance of maize, spy based on polygalacturonase gene
Sign is, the total serum IgE of extraction is carried out reverse transcription into cDNA；Then real-time fluorescence quantitative PCR detection is carried out by template of cDNA.
5. according to claim 4 express the method identified drought resistance of maize, spy based on polygalacturonase gene
Sign is, includes following components in the used every 20 μ l of reaction system: 1 μ l's when the real-time fluorescence quantitative PCR detects
CDNA template, the positive and negative primer of 0.5 μ l, 10 μ l SYBR Green mix fluorescent dye, 8 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 |
| CN115948591A (en) * | 2022-07-19 | 2023-04-11 | 河南省农业科学院粮食作物研究所 | Haplotype ZmC10.HapDR related to identifying drought tolerance of corn in seedling stage and application thereof |
-
2019
- 2019-08-13 CN CN201910743382.XA patent/CN110317899A/en not_active Withdrawn
Cited By (3)
| 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 |
| CN115948591A (en) * | 2022-07-19 | 2023-04-11 | 河南省农业科学院粮食作物研究所 | Haplotype ZmC10.HapDR related to identifying drought tolerance of corn in seedling stage and application thereof |
| CN115948591B (en) * | 2022-07-19 | 2023-08-15 | 河南省农业科学院粮食作物研究所 | Identification of corn seedling drought tolerance related monomer ZmC10.HapDR and application thereof |
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