CN106480075A - A kind of gene of regulation and control corn drought stress resistance and its application - Google Patents
A kind of gene of regulation and control corn drought stress resistance and its application Download PDFInfo
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Abstract
The present invention relates to field of plant genetic, specifically, it is related to a kind of gene of regulation and control corn drought stress resistance, the gene is the deletion mutation gene of ZmPP2CA10, and the fragment for being lacked is the continuous base sequence away from ZmPP2CA10 initiation codon ATG upstream 301bp；The continuous base sequence such as SEQ ID NO:Shown in 1；The nucleotide sequence of the promoter of the ZmPP2CA10 after mutation such as SEQ ID NO:Shown in 2.The deletion mutation gene can be used for drought resistance of maize related molecular marker assistant breeding and drought resistance of maize auxiliary identification, easy to operate, and accuracy rate is high.
Description
Technical field
The present invention relates to field of plant genetic, in particular to a kind of regulation and control corn drought stress resistance
Gene and its application.
Background technology
Corn is one of important cereal crops, and in China, the cultivated area of corn has been over paddy rice, leaps to the first
Big crop.Corn (Zea may L.) the different flower of homophyletic, belongs to the annual gramineae plant of cross-pollination.Belong on taxology
Grass family (Graminease) maize race (Maydeae) Zea (Zea L.), originates from Central America, and the initial stage in 16th century passes
Enter to China.Corn is not only important cereal crops, and is also important feed and the raw material of industry, conduct recent years
Energy crop receives very big favor.The change of natural environment has a strong impact on the lifting of corn yield, and arid is impact plant
Grow and its crop yield one of main stress factors, can all cause crop failure to bring huge economic damage every year
Lose.China is the very big country of an arid and semiarid area, and Arid＆semi-arid area accounts for national land area 50% or so.
The heredity of further investigation drought-resistant maize and molecular mechanism, excavation drought resisting new gene, are to improve corn yield further, ensure national grain
Food safety in the urgent need to.
Recent studies indicate that, it is one of important mechanisms of corn reply drought stress to control ABA signal transduction, wherein
PYR/PYL/RCAR albumen, A cluster PP2C phosphatase (PP2CA) and 3 type SnRK2 kinases composition ABA acceptor or other albumen are combined
The early stage conduction of thing mediation ABA signal, but the natural variation of its correlative coding gene and its drought resistance function mechanism are still fresh so far is
People knows.In addition, the drought-resistant maize gene that has identified is based primarily upon transgenosis overexpression or deletion mutant, but also difficult in production
To utilize.
In view of this, the special proposition present invention.
Content of the invention
It is an object of the invention to provide a kind of gene with regulation and control drought-resistant maize function, to solve the above problems.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：
A kind of gene of regulation and control corn drought stress resistance, the gene is the deletion mutation gene of ZmPP2CA10, is lacked
The fragment of mistake is the continuous base sequence away from ZmPP2CA10 initiation codon ATG upstream 301bp；The continuous base sequence is such as
SEQ ID NO:Shown in 1；
The nucleotide sequence of the promoter of the ZmPP2CA10 after mutation such as SEQ ID NO:Shown in 2.
The fragment for being lacked is located at the promoter region of 5 ' UTR promoter region of ZmPP2CA10 upstream from start codon.
By wide coverage, they take part in including arid a lot of members of phosphoprotein phosphatase type 2C (PP2C) family
Reaction is in interior environment stress process.Applicant is from the torrid zone and the jade of 368 kinds in temperate zone used in research before
Mono- various maize population of meter Zu Cheng simultaneously have found a candidate gene ZmPP2CA10 (Genome-Wide Analysis of
ZmDREB Genes and Their Association with Natural Variation in Drought
Tolerance at Seedling Stage of Zea mays L；PLoS Genet, 2013-9-26, Liu S et al).
And in this application, by the analysis of the natural variation colony drought-resistant ability to corn, applicants have discovered that one
Deletion mutation gene, the fragment of its disappearance are located on ZmPP2CA10 gene promoter, and the fragment lacked by the gene is a ginseng
With the Conserved Elements of endoplasmic reticulum stress signal path, regulate and control the drought-resistant ability of corn by affecting the expression of ZmPP2CA10.
Application of the deletion mutation gene in drought resistance of maize related molecular marker assistant breeding as above.
ZmPP2CA10 gene is located on No. 6 chromosome of Maize genome, and its gene I/D is GRMZM2G177386；Therefore
The deletion mutation gene also is located at same position, can be applied in assistant breeding as molecular labeling.
Application of the deletion mutation gene as above in drought resistance of maize auxiliary identification.
As ZmPP2CA10 is a gene to drought-resistant maize ability with negative regulation function in corn, and Shen
Ask someone to find, the deletion mutation gene can dramatically increase the drought-resistance ability of corn, this explanation endoplasmic reticulum stress signal path with
Close association is there is between drought stress.And those skilled in the art can be according to the research by ZmPP2CA10 gene and institute
State deletion mutation gene and be applied to drought resistance related molecular marker assistant breeding and drought resistance of maize auxiliary identification field.
Describe for convenience below, below in this application, the deletion mutation unnamed gene is A10p Δ ERSE gene.
A kind of method of auxiliary identification drought resistance of maize, comprises the steps：
Upstream primer and downstream primer are separately designed in the upstream and downstream of continuous base sequence as above；
With genomic DNA, the deletion mutation base of the upstream primer and the downstream primer to corn sample to be measured
Because the negative control DNA of homozygous positive control dna and ZmPP2CA10 genetic homozygous is while carry out electroresis appraisal；Root
According to the molecular weight auxiliary judgment of the amplified production for treating test sample amplified production and positive control and negative control corn sample to be measured
Drought-resistant ability.
Knowable in one embodiment of the application, A10p Δ ERSE gene can significantly increase the drought resisting energy for increasing corn
Power, therefore can carry out auxiliary identification with the presence or absence of the gene to drought resistance of maize.
Above-mentioned auxiliary identifies that the judgment mode of the method for drought resistance of maize is：
If treating, test sample amplified production is consistent with the molecular weight of positive control dna, and less than the molecular weight of negative control DNA,
Then explanation sample to be tested is A10p Δ ERSE genetic homozygous, with stronger drought-resistant ability；
If treating, test sample amplified production is consistent with the molecular weight of negative control DNA, and bigger than the molecular weight of positive control dna,
Then explanation sample to be tested is ZmPP2CA10 genetic homozygous, with weaker drought-resistant ability；
If treating, there are two bands in test sample amplified production, respectively the molecular weight one with negative control DNA and positive control dna
Cause, then explanation sample to be tested is ZmPP2CA10 gene/A10 Δ ERSE genetic heterozygosis, with weaker drought-resistant ability.
Need to make an explanation, those skilled in the art are had the ability according to the content in description of the invention disclosure
Judge the strong and weak impact of A10p Δ ERSE gene and ZmPP2CA10 gene pairs drought-resistant maize ability, therefore the determination methods are not
PCR method is only limitted to, also includes other each regulation and control stages for the two genes, the such as detection of DNA level, mRNA expression
Any detection method of the detection of level and protein expression level etc..
Preferably, the method for auxiliary identification drought resistance of maize as above, the core of the upstream primer and downstream primer
Nucleotide sequence is respectively as SEQ ID NO:3 and SEQ ID NO:Shown in 4.
SEQ ID NO is lacked as A10p Δ ERSE gene is substantially ZmPP2CA10:Genetic fragment shown in 2, therefore
The primer for being adopted can be any primer pair for changing deletion fragment, preferably SEQ ID NO:3 and SEQ ID NO:Drawing shown in 4
Thing pair.
Preferably, the method for auxiliary identification drought resistance of maize as above, in its reaction system for expanding, upstream is drawn
The concentration of thing and downstream primer is 8～12 μM, the concentration >=80ng/ μ l of DNA profiling.
It is furthermore preferred that the concentration of upstream primer and downstream primer is 10 μM, the concentration of DNA profiling is 100ng/ μ l.
Preferably, the method for auxiliary identification drought resistance of maize as above, in the response procedures of the amplification, Tm value
For 55～59 DEG C, reaction cycle number of times is 30～40 times；
It is furthermore preferred that response procedures are：
It is furthermore preferred that Tm value is 57 DEG C, reaction cycle number of times is 35 times.
Annealing temperature when generally PCR is expanded is 40 DEG C～60 DEG C.Annealing temperature be by primer Tm value determining,
In Tm value allowed band, select higher annealing temperature greatly reduce the non-specific binding between primer and template, improve PCR
The specificity of reaction.Primer annealing temperature of the present invention is 56 DEG C, with higher specificity.
As the carrying out of reaction, the raw material such as enzyme gradually can be inactivated, dNTPs can gradually use up, there are some non-specific in addition
The amplification of product also accordingly can increase.Thus while with the increase of reaction cycle number, product can increase, but cycle-index is still
Unsuitable excessive, thus the present invention is defined to 38～42 circulations.
Preferably, the method for auxiliary identification drought resistance of maize as above, in the electroresis appraisal, gel used
For 3%～4% Ago-Gel；
It is furthermore preferred that deposition condition is 110～130V of constant voltage, 140～150mA of electric current runs 45～75min
The method of auxiliary identification drought resistance of maize as above is in detection ZmPP2CA10 gene ERSE response element disappearance
Application in mutation.
Description of the drawings
In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, below will be to concrete
Needed for embodiment or description of the prior art, accompanying drawing to be used is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is ZmPP2CA10 corn overexpression transgenic line arid phenotype in the embodiment of the present invention 1；A：Transgenosis material
Material ZmPP2CA10 protein expression situation, CO1 are the conversion background material of transgenosis；B：ZmPP2CA10 corn gene material man
It is 6 and the in vitro dehydration experiment of 12 blade of family；C：To ZmPP2CA10 corn gene material family 6 and family 12 seedling stage material
Do Osmotic treatment 2 weeks rehydrated material growing state；
Fig. 2 is survival rate after ZmPP2CA10 hereditary variation self-mating system Osmotic treatment different from corn in the embodiment of the present invention 2
Data do association analysis；A：At SNP/InDels site in ZmPP2CA10 whole gene group sequence is arid from different self-mating systems
The association analysis of survival rate after reason, it is found that InDel-338 is significantly related to arid；Represent noncoding region,Represent outer
Aobvious son,Represent introne and promoter region；B：InDel-308 and InDel-338 linkage disequilibrium：C:Drought resisting material
With -338 sites to ZmPP2CA10 sequence in arid sensitive material and the sequence alignment in -754 sites；ERSE(ER Stress
Response element):Endoplasmic reticulum stress response element；AREB(ABA Response element):Abscisic acid response element
Part；
Fig. 3 is to detect InDel-338 and ZmPP2CA10 table to Maize at Seedling Stage material Osmotic treatment in the embodiment of the present invention 2
The relation of the amount of reaching；A-C：Drought resisting material and sensitive material ZmPP2CA10 expression (A) under seedling stage normal growing conditions,
Expression increased times (C) after expression (B) and Osmotic treatment after ZmPP2CA10 Osmotic treatment；+ represent ZmPP2CA10's
There is ERSE site in InDel-338 site ,-representative does not have ERSE site in the InDel-338 site of ZmPP2CA10；D-F：
Corn inbred line ZHENG653 (653), CIMBL17 (17), survival rate (D) and two F2 after CIMBL70 (70) Osmotic treatment
Segregating population CIMBL17 X ZHENG653 (17 X 653), deposits after CIMBL70 X ZHENG653 (70 X 653) Osmotic treatment
Motility rate (E, F)；+ /+, -/-, +/- represents homozygosis Sensitive genotype, homozygosis Drought-tolerant gene type, heterozygote genotype respectively；
Fig. 4 is uciferase activity experiment in the embodiment of the present invention 3；The ERSE element of ZmPP2CA10 promoter region is special
The opposite sex has response to endoplasmic reticulum stress signal approach, and (TM is according to mycin：Specific activation endoplasmic reticulum stress signal approach), and not
Participate in abscisic acid signal path (ABA abscisic acid：Specific activation abscisic acid signal path)；EV represents zero load, and A10p is represented
ZmPP2CA10 promoter；
Fig. 5 is the representative electrophoresis result figure in the embodiment of the present invention 4.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.Unreceipted concrete in embodiment
Condition person, the condition that advises according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, are
Can buy, by commercially available, the conventional products for obtaining.
1 overexpression ZmPP2CA10 of embodiment weakens corn drought tolerance ability
The CDS total length of ZmPP2CA10 gene with corn B73 reference sequences as template design primer, while at primer two ends
Plus partial vector sequences, acquisition fragment is cloned in vitro as template with B73 leaf cDNA, connected by the method for homologous recombination
To on overexpression vector pZZ0153-3HA, and sequence verification obtains correct clone.The overexpression vector plasmid is sent to China
Seed Group Co., Ltd (Wuhan) obtains the transfer-gen plant of the gene.By herbicide screening applicant obtain two pure
The overexpression ZmPP2CA10 corn gene material family 6 and 12 of conjunction.
Transgenic line family 6 and family 12, and the RNA of wild type C01 material is extracted, reverse transcription is done, fixed by fluorescence
Whether amount PCR detection target gene is overexpressed in transcriptional level.Experimental result shows in transgenic line family 6 and family 12
The expression of A10 is higher than wild type C01, illustrates that transgenic line has overexpressed (Figure 1A) really in transcriptional level.
The total protein of ZmPP2CA10 corn gene material family 6 and family 12 and wild type C01 material blade is extracted,
By protein immunoblotting technology, whether overexpressed with target gene A10 in 3HA antibody hybridization detection transgenic line.Real
Test result and show that target gene overexpresses (Figure 1B) in protein level really in transgenic line.
When corn grows to the three leaf wholeheartedly hearts, take family 6 respectively, the 3rd leaf of family 12 and wild type C01, from
Claim leaf weight in different time points in the case of body, finally count the percentage of water loss of each time point blade.Experimental result show turn
With respect to wild type C01 material, dehydration in the identical experiment faster, illustrates that transgenic line is compared and wild type is lost to genetic material
Water is faster (Fig. 1 C).
The double kind of plant in same basin by transgenic line and wild type material, starts when the three leaf wholeheartedly hearts is grown to
Water is irrigated, is not hereafter rewatered and Osmotic treatment is done, Osmotic treatment is after 20 days, to material rehydration and irrigate, observe different materials
Growing state.Experimental result display transgenic line withers, and here degree is significantly stronger than wild type material C 01, and transgenic line phase is described
More more sensitive to arid than in wild type, A10 does negative regulatory factor (Fig. 1 D) in plant is to the response of arid.
Embodiment 2 identifies ZmPP2CA10 and drought-enduring related preference allele
In order to further confirm that the gene mutation details of ZmPP2CA10, applicant from corn difference selfing based material in
Machine selects 100 parts of materials to carry out, to ZmPP2CA10 gene, sequence of resurveying.Genomic DNA with this 100 parts of materials as template, with B73
Reference gene group sequence is defined design primer amplification, surveyed respectively ZmPP2CA10 promoter region and gene regions (including extron and
Introne).
The ZmPP2CA10 sequence of each material is carried out Multiple Sequence Alignment, will wherein all of SNP/InDel site and pass
Connection population survival rate data (the data source article that others delivers in early stage) does candidate gene association analysis, finds a site
InDel-338 and arid significantly correlated (Fig. 2A).
There is an InDel-308 site in InDel-338 location proximate, InDel-308 position is found by linkage disequilibrium
Point is not chain with InDel-338 site, i.e., InDel-338 site is individually to affect plant drought, rather than and InDel-308
Joint effect drought resisting (Fig. 2 B).
- 338 loci gene types of ZmPP2CA10 in drought resisting material and sensitive material.ERSE(ER Stress Response
element):Endoplasmic reticulum stress response element.AREB(ABA Response element):Abscisic acid response element (Fig. 2 C).
Wherein, CIMBL55, CIMBL70, ZHENG653 etc. represent different corn inbred lines respectively.
In order to the expression for understanding ZmPP2CA10 is that the drought-resistant ability how to corn is impacted, applicant from
18 part and 22 part materials are selected in sensitive material and drought resisting material respectively for Osmotic treatment.Sensitive material is ZmPP2CA10's
InDel-338 loci gene type is for there is ERSE site insertion (+/+).The InDel-338 site deletion of drought resisting material ZmPP2CA10
ERSE element (-/-).These materials are seeded in sandy soil, are normally watered when three leaves are grown into wholeheartedly and samples, and will plant
On thing root, sand uses clear water wash clean, whole plant is exposed in the air and does Osmotic treatment, sample after processing three hours.Take out respectively
Carry the RNA of sample after sample and Osmotic treatment under normal growing conditions, reverse transcription, do fluorescence quantitative PCR detection ZmPP2CA10 and exist
Expression situation of change before and after Osmotic treatment in sensitive material and drought resisting material.
As can be seen from Figure 3：Under normal growing conditions, in sensitive material and drought resisting material, ZmPP2CA10 expression is nothing obvious
Difference (Fig. 3 A)；After Osmotic treatment, expression of the ZmPP2CA10 expression higher than ZmPP2CA10 in drought resisting material in sensitive material
Amount (Fig. 3 B)；After Osmotic treatment, in sensitive material, ZmPP2CA10 expression increased times are significantly higher than in drought resisting material
ZmPP2CA10 expression increases multiple (Fig. 3 C).Above experimental result explanation, ERSE element energy when plant receives drought stress
Strengthen the expression of ZmPP2CA10.
Next, applicant select drought resisting material C IMBL17 and CIMBL70 with miscellaneous to arid sensitive material ZHENG653
Hand over, build F2 segregating population.The two F2 segregating population seeds are multicast in soil, start to do arid place when a leaf is grown to wholeheartedly
Reason, while genotype identification is done in each individual plant sampling, carries out rehydration and processes and count survival rate when plant leaf blade whitens.
Osmotic treatment is done to ZHENG653, CIMBL17, CIMBL70 material, and counts survival rate, find ZHENG653 pair
Arid is sensitive, and CIMBL17, CIMBL70 are relatively drought-enduring, then select these three parents to build F2 segregating population (Fig. 3 D)；CIMBL17
In X ZHENG653 segregating population genotype be InDel-338 ERSE-/-, InDel-338ERSE+/- material survival rate bright
Aobvious higher than InDel-338ERSE+ /+, illustrate that ZmPP2CA10 is an excellent equipotential base in InDel-338 site deletion ERSE
Cause, can strengthen Maize at Seedling Stage drought-resistant ability (Fig. 3 E)；In CIMBL70 X ZHENG653 segregating population, genotype is InDel-338
ERSE-/-, InDel-338 ERSE+/- material survival rate apparently higher than InDel-338ERSE+ /+, illustrate that ZmPP2CA10 exists
InDel-338 site deletion ERSE is a superior allelic, can strengthen Maize at Seedling Stage drought-resistant ability (Fig. 3 F).
The ERSE element of 3 luciferase assay of embodiment checking promoter region is the activation of endoplasmic reticulum stress signal
Necessary to ZmPP2CA10, but activation of the ABA path to ZmPP2CA10 is not affected
ZmPP2CA10 Gene A TG upstream 1000bp promoter fragment in clone's CIMBL55 material, this material
The InDel-338 site deletion ERSE element of ZmPP2CA10, this fragment are named as A10p Δ ERSE, the nucleosides of lack part
Acid sequence such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of the promoter of the ZmPP2CA10 after mutation:2 institutes
Show.Point mutation is done to A10p Δ ERSE, InDel-338 site mutation is containing ERSE element, is named as A10pERSE.By this
Two fragments are connected by digestion, are connected on pGL-Basic-LUC carrier, build pGL-Basic-A10p Δ ERSE-LUC,
pGL-Basic-A10pERSE-LUC.Both plasmids are proceeded to overnight incubation in Corn Protoplast respectively, is detected within second day
Reporter gene luciferase enzyme activity.
In incubated overnight cell, tunicamycin (TM) process is done to cell, TM can specific activation endoplasmic reticulum stress signal
Path.Test result indicate that when TM is processed, turning fluorescein enzyme activity in the cell for having pGL-Basic-A10pERSE-LUC plasmid
Property have the cell Enzyme activity of pGL-Basic-A10p Δ ERSE-LUC plasmid apparently higher than turning, illustrate that ERSE element can be responded interior
Matter net stress signal approach, and the transcriptional activity (Fig. 4 A) of ZmPP2CA10 promoter can be strengthened.
In incubated overnight cell, cell is done abscisic acid (ABA) process, ABA can specific activation come off acid signal lead to
Road.Test result indicate that when ABA is processed, turning uciferase activity in the cell for having pGL-Basic-A10pERSE-LUC plasmid
In the uciferase activity no significant difference for turning the cell for having pGL-Basic-A10p Δ ERSE-LUC plasmid, ERSE element is described
ABA signal path can not be responded, can only specificly-response endoplasmic reticulum stress signal path (Fig. 4 B).
Application of the 4 A10p Δ ERSE fragment of embodiment in drought resisting phenotypic evaluation
Embodiment 1～3 has been proven that A10p Δ ERSE fragment can dramatically increase the drought-resistance ability of corn, the present embodiment
In there is provided A10p Δ ERSE fragment application.
60 parts of collection Drought-resistant corn strain, the leaflet tablet of 50 portions of plants of responsive type corn strain, extract plant genome
DNA；110 parts of DNA samples are expanded.
Wherein, Drought-resistant corn strain is selected from：CIMBL55, CIMBL54, CIMBL17, CIMBL22, CIMBL115 and
CIMBL91 is each 10 plants；
Responsive type corn strain is selected from：CIMBL140, CIMBL121, CIMBL119, ZHENG653 X CIMBL17 hybridizes
F1 generation and ZHENG653X CIMBL70F1 are for each 10 plants；
, selected from the genomic DNA (ERSE-/-) of CIMBL70, negative control is selected from the genome of ZHENG653 for positive control
DNA(ERSE+/+).
In terms of 15 μ l reaction systems, the reaction system of the amplification includes：
The sequence of upstream primer used such as SEQ ID NO:Shown in 3, the sequence such as SEQ ID NO of downstream primer used:4 institutes
Show.
PCR amplification program is：
Fig. 5 shows the electrophoretogram of one of glue in experimental result：
Wherein, 7 swimming lanes and 8 swimming lanes are respectively positive control and negative control；
1st, the corresponding sample of 5,10,13,14,17,20 swimming lanes is respectively：CIMBL55、CIMBL54、CIMBL17、
CIMBL22, CIMBL54, CIMBL115 and CIMBL91；
2nd, the corresponding sample of 9,11,24 swimming lanes is all from ZHENG653 X CIMBL70F1 generation；
4th, the corresponding sample of 6,12,16 swimming lanes is all from ZHENG653 X CIMBL17 hybridization F1；
3rd, the corresponding sample of 15,18,19,21,22,23 swimming lanes is respectively：CIMBL140、CIMBL121、CIMBL119、
CIMBL121, CIMBL140, CIMBL121, CIMBL119 sample.
The result that these results are obtained in the research of embodiment 1～3 with applicant is consistent.That is CIMBL55, CIMBL54,
CIMBL17, CIMBL115 and CIMBL91 are ERSE-/- homozygote；ZHENG653 X CIMBL17 first familiar generation and
In ZHENG653 X CIMBL70F1 generation, is ERSE+/- heterozygote；CIMBL140, CIMBL121, CIMBL119 be ERSE+ /+
Homozygote.During actual amplification, drought resisting phenotype only has two samples and does not expand out band, and sensitive phenotype only has a sample and do not have
Band is amplified, the genotype of remaining sample is all consistent with its phenotype and survival results.
Finally it should be noted that：Various embodiments above only in order to technical scheme to be described, rather than a limitation；To the greatest extent
Pipe has been described in detail to the present invention with reference to foregoing embodiments, but it will be understood by those within the art that：Its
Still the technical scheme described in foregoing embodiments can be modified, or to which part or all technical characteristic
Carry out equivalent；And these modifications or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. a kind of regulation and control corn drought stress resistance gene, it is characterised in that the gene for ZmPP2CA10 deletion mutation
Gene, the fragment for being lacked are the continuous base sequence away from ZmPP2CA10 initiation codon ATG upstream 301bp；The continuous alkali
Basic sequence such as SEQ ID NO:Shown in 1；
The nucleotide sequence of the promoter of the ZmPP2CA10 after mutation such as SEQ ID NO:Shown in 2.
2. application of the deletion mutation gene described in claim 1 in drought resistance of maize related molecular marker assistant breeding.
3. application of the deletion mutation gene described in claim 1 in drought resistance of maize auxiliary identification.
4. a kind of method that auxiliary identifies drought resistance of maize, it is characterised in that comprise the steps：
The upstream and downstream of described continuous base sequence separately designs upstream primer and downstream primer in claim 1；
Pure to the genomic DNA of corn sample to be measured, the deletion mutation gene with the upstream primer and the downstream primer
The negative control DNA of the positive control dna of zygote and ZmPP2CA10 genetic homozygous is while carry out electroresis appraisal；According to treating
The drought resisting of the molecular weight auxiliary judgment of the amplified production of test sample amplified production and positive control and negative control corn sample to be measured
Ability.
5. according to claim 4 auxiliary identification drought resistance of maize method, it is characterised in that the upstream primer and under
The nucleotide sequence of trip primer is respectively as SEQ ID NO:3 and SEQ ID NO:Shown in 4.
6. the method that auxiliary according to claim 5 identifies drought resistance of maize, it is characterised in that in the reactant which expands
In system, the concentration of upstream primer and downstream primer is 8～12 μM, the concentration >=80ng/ μ l of DNA profiling.
7. the method that auxiliary according to claim 5 identifies drought resistance of maize, it is characterised in that in the reaction of the amplification
In program, Tm value is 55～59 DEG C, and reaction cycle number of times is 30～40 times.
8. according to claim 5 identification drought resistance of maize method, it is characterised in that in the electroresis appraisal, institute
Gel is 3%～4% Ago-Gel.
9. the method for identification drought resistance of maize according to claim 8, it is characterised in that deposition condition is：
110～130V constant voltage, 140～150mA of electric current；45～75min of electrophoresis time.
10. the method for the auxiliary identification drought resistance of maize described in any one of claim 4～9 is in detection ZmPP2CA10 gene
Application in ERSE response element deletion mutation.
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