CN104911184B - Corn abiotic stress response factor ZmERF1 gene promoter sequences and its application - Google Patents

Abstract

ZmERF1 gene promoter sequences of the present invention are in addition to the characteristic feature with plant promoter,Also contain the relevant element of ethylene responses 2,ABA hormone response ABRE elements 1,It is dehydrated correlation MYB1AT elements 4,MYCATERD elements 1,MYCATRD elements 1,Low temperature cold-resistant controlling element 18,CBFHV elements 8,Injury regulation and control WBOXNTERF elements 9,Heat shock CCAATBOX elements 1,Tissue specific expression regulation and control CAATBOX elements 27,Seed development correlation EBOXBNNAPA elements 18,Amylase correlation AMYBOX2 elements 1,CGACGOSAMY elements 1,Sugar correlation WBOXHVISO elements 5,Regulate and control roots development correlation RHERPATEXPA elements 2；The gene promoter has application value to regulation and control corn development and resistance.

Description

Corn abiotic stress response factor ZmERF1 gene promoter sequences and its application

Technical field

The invention belongs to field of plant genetic, and in particular to corn abiotic stress response factor ZmERF1 bases Because of promoter and its application.

Background technology

Corn is one of main cereal crops in China, is played an important role in national food security is ensured.At present I State faces population and is continuously increased dual-pressure with the continuous reduction of cultivated land resource, and improving corn yield and quality can effectively alleviate Food Security.Biotic and abiotic stress, the especially environment-stress such as arid, salt marsh and high temperature, the life of serious threat corn Production, annual world crops caused by abiotic stress production loss 30% more than biotic.Therefore, abiotic stress is to make Into the key constraints of crop yield reduction, current agricultural expanding economy seriously restrict.Met in growing process To arid, damage to plants caused by sudden drop in temperature, heat shock, saline and alkaline when adverse circumstance, plant is in order to preferably grow and maladjustment environment, in long-term evolution A set of distinctive defense mechanism is formd in journey.Therefore, it is anti-to studying to excavate corn anti contravariance related gene and its promoter by clone Inverse molecular genetic mechanism and genetic regulation network have important theory significance and application value.

ERF genes are induced by hormone or environmental factor, are adjusted plant to the responsing reaction of various environment stresses, are improved it Salt tolerance and drought resistance and to damaging to plants caused by sudden drop in temperature and the resistance of osmotic stress.Therefore, the degeneration-resistant relevant ERF gene promoters of corn clone Son, helps to study corn to abiotic stress resistance molecule mechanism, while to improving tolerance of the corn to abiotic stress There is great importance.

The content of the invention

, should it is an object of the invention to provide corn abiotic stress response factor ZmERF1 gene promoters and its application Gene promoter can directly drive foreign gene and express in transgenic plants, such as salt treatment and infiltration under abiotic stress Processing can make the expression of foreign gene rapidly and efficiently, can improve the resistance capacity of plant pair salt stress and osmotic stress.

The present invention provides a kind of new corn abiotic stress response factor ZmERF1 gene promoters, refers to sequence table, Its promoter nucleotide full length sequence is 2217 bp；The application of the gene promoter is provided at the same time, i.e., in salt stress, infiltration Under stress conditions, ZmERF1 gene promoter sequences start target gene is expressed at positions such as root, leaf, inflorescences, is improved Resistance capacity of the plant to salt stress and osmotic stress.

Corn abiotic stress response factor ZmERF1 gene promoters are imported into by the present invention using genetic transfoumation In corn plant genome, it is possible to achieve to the directional operation of target gene, obtain the transfer-gen plant of target gene, this is not only It can realize that target gene is expressed in the case of the adverse circumstances such as salt stress, osmotic stress condition, and it is abiotic inverse for corn The application of border stress provides strong instrument.

Brief description of the drawings

Fig. 1 and Fig. 2 is corn inbred line N04 of the present invention respectivelyZmERF1The expression way figure of gene；

Fig. 2 is the GUS colored graphs of maize leaf of the present invention.

Embodiment

With reference to embodiments and attached drawing is described in further detail the present invention：

Corn inbred line N04 is the applicant's laboratory culture product, selected from pop corn kind BL03, is cultivated in place Zhengzhou City Henan Province Agricultural University Of He'nan of state scientific and educational park.

Cloning vector pMD18-T, DNA marker are purchased from Beijing Quan Shi King Companies；DNA gel QIAquick Gel Extraction Kit, plasmid carry Kit is taken to be purchased from hundred Imtech；E. coli competent reagent preparation box is purchased from Shanghai life work；Primer is synthesized and surveyed Sequence is carried out in Beijing Hua Da company.

It is Bio-Red PCR amplification instruments, Eppendorf desk type high speed constant temperature centrifuge, constant incubator, constant-temperature table, low Temperature refrigerator, electrophoresis apparatus, superclean bench, Normal Agarose Gel imaging system, high-pressure sterilizing pot, liquid-transfering gun.

Embodiment 1

With reference to the expression of corn inbred line N04ZmERF1 genes under the conditions of Fig. 1 and Fig. 2 salt stresses, osmotic stress and The clone of ZmERF1 gene promoters：

A. the extraction of corn inbred line N04DNA

Take about 0.5 g young leaflet tablets to be placed in mortar, add grind into powder after liquid nitrogen, powder is quickly charged with 2.0 mL Centrifuge tube in, add 800 μ L mass fractions, 1% SLS extracting solutions, add 800 μ L proportionally be 25：24：1 phenol, The mixed liquor of chloroform and isoamyl alcohol composition, fully mixes, 65 DEG C of incubation 30min, after being cooled to room temperature, 12000 rpm centrifugations 10 Min, Aspirate supernatant are gone in another 1.5 clean mL centrifuge tubes, and the pre- cold isopropanol for adding 0.6 times of supernatant volume mixes Even, 12000 rpm centrifuge 10 min, abandon supernatant, are rinsed with 75% ethanol, of short duration centrifugation after rinsing, stay DNA to be carried out in tube bottom It is dry, dissolved after DNA dryings with aqua sterilisa, it is spare to be placed in -20 DEG C of refrigerations；Wherein 1% SLS extracting solutions of mass fraction are by 100 mmol.L^-1 Tris-HCl、100 mmol.L^-1 NaCl、20 mmol.L^-1EDTA and 10 g.L^-1 SLS is mixed, and is then adjusted The mixed liquor obtained behind PH=8.0；

The clone of b.ZmERF1 gene promoters

According to the promoter sequence of corn inbred line N04 ZmERF1 genes, two pairs of gene-specific primers are devised, this The fragment of two pairs of primer amplifications overlaps, and gene-specific primer sequence I is：F1391：5'GCGGATCCATTTAAGCTTGAAG TTGATGGC3' and R1391：5'TATTACCCCCATGGACATAGCT3'；Gene-specific primer sequence II：F865：5' AAGCTATGTCCATGGGGGTAAT3' and R865 5'GCAGATCTATGTAGTCGAAGATGATTGCGC3'；It is selfed with corn The DNA for being N04 is template, carries out PCR amplification, and amplification system is：5 μ L Hifi Buffer (10 ×), 2 μ L 2.5 The mol.L-1 μ L Taq DNA Polymerase of dNTP, 1 μ L Primer-F (R), 1,1 μ L template DNAs, ddH2O are supplied 50 μL；PCR amplification system program is：95 DEG C of 4 min of pre-degeneration, 95 DEG C of 45 sec of denaturation, 60 DEG C of 45 sec of annealing, 72 DEG C are prolonged 60 sec are stretched, are circulated 34 times altogether；72 DEG C extend 10 min, 10 DEG C of forever, and amplified production is coagulated with 1% agarose of mass fraction Gel electrophoresis detect, and are recycled with QIAquick Gel Extraction Kit, and are connected on PMD18-T carriers, are sequenced, the promoter sequence of sequencing Respectively PMD18-1391 and PMD18-865, particular sequence refer to sequence table artificial sequence 1 and sequence table artificial sequence 2；

C. the structure of conversion carrier

1391 bp promoter sequences being connected to BamHI and NcoI double digestions in carrier T, while with BamHI and NcoI PCAMBIA1301 carriers, are connected, then convert by double digestion pCAMBIA1301 carriers after recycling with 1391 bp promoter sequences Escherichia coli DH5a, picking monoclonal shake bacterium PCR and detect correct monoclonal upgrading grain, digestion identification；Identification correctly carries The pCAMBIA1301 carriers of 1391 bp promoter sequences use NcoI and BglII double digestions again, while with the double enzymes of NcoI and BglII 865 bp promoter sequences being connected in carrier T are cut, 1391 bp promoter sequences will be carried after recycling PCAMBIA1301 carriers are connected with 865 bp promoter sequences, then convert escherichia coli DH5a, and picking monoclonal shakes bacterium PCR Correct monoclonal upgrading grain is detected, digestion identification, obtains 2217 bp of ZmERF1 gene promoters full length sequence；It will build PCAMBIA1301-ZmERF1 promoter-GUS carriers identified with BamHI and BglII double digestions, identify it is correct after utilize PCAMBIA1301-ZmERF1 promoter-GUS carriers are transformed into Agrobacterium competent cell GV3101 by freeze-thaw method, accurate It is standby to infect arabidopsis；

D. corn inbred line N04 ZmERF1 gene promoter sequences are analyzed

The obtained promoter sequence of ZmERF1 genes will be cloned take advantage of a situation the forecast analysis of element, forecast analysis is shown The promoter has the characteristic feature of plant promoter, contains ethylene responses related elements 2；ABA hormone response ABRE elements 1 It is a；It is dehydrated correlation MYB1AT elements 4, MYCATERD elements 1 and MYCATRD elements 1；Low temperature cold-resistant controlling element 18 A, CBFHV elements 8；Injury regulation and control WBOXNTERF elements 9；Heat shock CCAATBOX elements 1；Tissue specific expression Regulate and control CAATBOX elements 27；Seed development correlation EBOXBNNAPA elements 18；Amylase correlation AMYBOX2 elements 1, CGACGOSAMY elements 1；Sugar correlation WBOXHVISO elements 5；Regulate and control roots development correlation RHERPATEXPA elements 2；

E. the acquisition of ZmERF1 promoters Arabidopsis plant

Picking be grown in YEP (Rif and Kan) containing pCAMBIA1301-ZmERF1 promoter-GUS carriers Monoclonal bacterial strain, is inoculated in the YEP (Rif and Kan) of liquid, 28 DEG C of shake cultures are stayed overnight.Second day according to 1:50 inoculations It is 0.8 ~ 1 to expand culture in new YEP to OD600, and thalline is collected by centrifugation, 5% sucrose and Silweet are added with 1/2 MS L-77, dips in colored method with Agrobacterium and arabidopsis is infected, and the T0 of harvest is sowed in the MS containing 50 mg/L hygromycin for seed On solid screening and culturing medium, can root on screening and culturing medium for resistant plant, determined after PCR is detected containing The positive plant of ZmERF1 promoters；

F. ZmERF1 promoter function analysis

10% PEG and 150 mM NaCl processing will be irrigated containing the Arabidopsis plant of ZmERF1 promoters, it is right respectively The transgenic arabidopsis seedling of normal growth and adverse circumstance processing carries out GUS histochemical stains, refers to attached drawing 3.It turns out that just GUS bluenesss can also occur in the condition Transfer-gen plant of being frequently grown, but blueness is more obvious under adverse circumstance, shows that the promoter is One infiltration and the inducible promoter of salt stress induced expression.

Embodiment 2

ZmERF1 promoters clone is connected on conversion carrier, then a Tnos terminator is being connected downstream, is making Both are built into a conversion carrier；Then the target gene that converts will be needed it is connected to ZmERF1 to be correctly oriented clone to open Between mover and Tnos terminators, the conversion carrier of target gene is built；Followed by particle bombardment or Agrobacterium Target gene is imported into recipient cell by mediated method, is obtained by technical methods such as tissue cultures, pressure screening and plant regenerations Obtain transgenic positive plant；Under the conditions of salt stress, osmotic stress, it is efficient that ZmERF1 promoters can improve downstream target gene Expression.

The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should It is considered as protection scope of the present invention.

Sequence table

<110>Agricultural University Of He'nan

<120>Corn abiotic stress response factor ZmERF1 gene promoter sequences and its application

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 2217

<212> DNA

<213>Corn（Zea mays L.）

<400> 1

atttaagctt gaagttgatg gcatcaatct gccagtctga ttttcagacc tatccaacct 60

gacagcctga ctttaggcat gattatctct aaatttttca caagaacatg gcttgcactc 120

ttaagcaaat ttgttatcca ttgattgctc tacaattttg atgtggtgac ttagggcaaa 180

aaccctatgc tttacaagtt acaaagccct aaagtggagc ctttaacact gaatttcaga 240

cttagtgtag aactcaaatg aggtccattt tgcatttaag tccaaaactt agggtttggc 300

tttcaagtcc acatatttgt gaaccaaagg acttaaaata cttatttggc ttggtttttg 360

cactttagtc caaaagtgga ccaattttgc atataaaccc ctagggtttg gtttcagggt 420

tttccagggt tttaattagg gtttctggta tcccaggggt acaaaagtga ttcaagttta 480

ttcttagggt cattttatga cttttaccct aaagctttta gattttctca acttgggtta 540

agttttaccc ctttaaccac tatttagggt taagtccttt aaccagggtt ctttttgcaa 600

aacaacacaa cttgtttgaa atttttgcct agtgaatgca ctctaggtgt gtcaaacata 660

tgcaatgcca atgctatgat gccatgctca agttttagtt acagtaacac caggggtgtt 720

acaattgctg tggatgacgc tcaaatccat tgggtcgtct aggcttgagt tgatccaatc 780

ctgcgtcgat ccacttcgct agacgttcct ccccaggccc acaaagggat gaccttgcga 840

aatattgggc ccaaagcctt tcgcgaggtc ttctaacctt ccagtggacc tgggggatat 900

ctgtccccca cacattatgc ccgaagggag ataatgcttc gaaggacaaa ggtctttaac 960

acttaagaat tttgttgcgt tgtttcatga ttaagtaata aaaacaagag atcaacacta 1020

tgctcttttc ttaatcgatc attctaaaaa gtacatgatt tttgtcgact ttacacccac 1080

tcaaaattgg tgtaagcaca tgccatacaa gaggttcata gaaacgatta tcatggcctt 1140

aaagaaatat aagattgtct acagcatgaa acgttctaga tgggtggtgg atatttttaa 1200

attggaaaat aaaattcaga ctgatgcttc aattgacata aaatgatatt ttctccgtgc 1260

caccttctca gcaatttaat tttttctatg atttccatta catataacat agttggcaaa 1320

acattattat gaaggattaa caccagctac ctcgttctac aagctatgtc catgggggta 1380

ataacaggcg aatggaattt gttaggggtg agtggagtac gcatgtttca cgttcaagta 1440

tacattaata aaaacattgg tattttgttc acttatgtct ttgtgtgtag gatgtatata 1500

ttcttccaag aaactttatg atcgatctgt taatttatga ggacaattcg tgccgatatg 1560

tcatccctcc aaacatacag catagactta tcaatatcgc taaagaggat taaattagtg 1620

tacggttgtc gacatattcg tctatcatta aaaattctat attgactatt tagaaataat 1680

ttgtatgata ttgataatgt tcgatattat ttcaatctat tttagtcagc gtatttataa 1740

taattatata tttatatcat caatataata ttattgttat gataatattt tatagtacga 1800

attttatata ttatgaaata tcgtttcata tatatgtttt atattaattt ttctaattta 1860

cttaagcatc tgacgctaag tgtcctgtgc ccggatcggc cagagcggtc aaatggttca 1920

atgctttacc tcctctggtt ggtgtccagc ttgggggcag ctgcctttca tttccattct 1980

tccagttgca cccagaccca ggcaggtcat cggcgcccac aactccaagt tgtataaata 2040

ctccgtcgac cgtctcataa cagtcatacc gccgaaccaa atccaacgtc cacagaaaag 2100

aaaacacatt cccgtcgact cccctagttt catcctattc atttctcccc ccaaacactc 2160

cagctactag ttactcgagg aatccatgtg cggcggcgca atcatcttcg actacat 2217

<210> 2

<211> 1391

<212> DNA

<213>Artificial sequence 1

<400> 1

gcggatccat ttaagcttga agttgatggc atcaatctgc cagtctgatt ttcagaccta 60

tccaacctga cagcctgact ttaggcatga ttatctctaa atttttcaca agaacatggc 120

ttgcactctt aagcaaattt gttatccatt gattgctcta caattttgat gtggtgactt 180

agggcaaaaa ccctatgctt tacaagttac aaagccctaa agtggagcct ttaacactga 240

atttcagact tagtgtagaa ctcaaatgag gtccattttg catttaagtc caaaacttag 300

ggtttggctt tcaagtccac atatttgtga accaaaggac ttaaaatact tatttggctt 360

ggtttttgca ccttagtcca aaagtggacc aattttgcat ataaacccct agggtttggt 420

ttcagggttt tccagggttt taattagggt ttctggtatc ccaggggtac aaaagtgatt 480

caagtttatt cttagggtca ttttatgact tttaccctaa agcttttaga ttttctcaac 540

ttgggttaag ttttacccct ttaaccacta tttagggtta agtcctttaa ccagggttct 600

ttttgcaaaa caacacaact tgtttgaaat ttttgccttg tgaatgcact ctaggtgtgt 660

caaacatatg caatgccaat gctatgatgc catgctcaag ttttagttac agtaacacca 720

ggggtgttac aattgctgtg gatgacgctc aaatccattg ggtcgtctgg gcttgagttg 780

atccaatcct gcgtcgatcc acttcgctag acgttcctcc ccaggcccac aaagggatga 840

ccttgcgaaa tattgggccc aaagcctttc gcgaggtctt ctaaccttcc agtggacctg 900

ggggatatct gtcccccaca cattatgccc gaagggagat aatgcttcga aggacaaagg 960

tctttaacac ttaagaattt tgttgcgttg tttcatgatt aagtaataaa aacaagagat 1020

caacactatg ctcttttctt aatcgatcat tctaaaaagt acatgatttt tgtcgacttt 1080

acacccactc aaaattggtg taagcacatg ccatacaaga ggttcataga aacgattatc 1140

atggccttaa agaaatataa gattgtctac agcatgaaac gttctagatg ggtggtggat 1200

atttttaaat tggaaaataa aattcagact gatgcttcaa ttgacataaa atgatatttt 1260

ctccgtgcca ccttctcagc aatttaattt tttctatgat ttccattaca tataacatag 1320

ttggcaaaac attattatga aggattaaca ccagctacct cgttctacaa gctatgtcca 1380

tgggggtaat a 1391

<210> 3

<211> 865

<212> DNA

<213>Artificial sequence 2

<400> 1

aagctatgtc catgggggta ataacaggcg aatggaattt gttaggggtg agtggagtac 60

gcatgtttca cgttcaagta tacattaata aaaacattgg tattttgttc acttatgtct 120

ttgtgtgtag gatgtatata ttcttccaag aaactttatg atcgatctgt taatttatga 180

ggacaattcg tgccgatatg tcatccctcc aaacatacag catagactta tcaatatcgc 240

taaagaggat taaattagtg tacggttgtc gacatattcg tctatcatta aaaattctat 300

attgactatt tagaaataat ttgtatgata ttgataatgt tcgatattat ttcaatctat 360

tttagtcagc gtatttataa taattatata tttatatcat caatataata ttattgttat 420

gataatattt tatagtacga attttatata ttatgaaata tcgtttcata tatatgtttt 480

atattaattt ttctaattta cttaagcatc tgacgctaag tgtcctgtgc ccggatcggc 540

cagagcggtc aaatggttca atgctttacc tcctctggtt ggtgtccagc ttgggggcag 600

ctgcctttca tttccattct tccagttgca cccagaccca ggcaggtcat cggcgcccac 660

aactccaagt tgtataaata ctccgtcgac cgtctcataa cagtcatacc gccgaaccaa 720

atccaacgtc cacagaaaag aaaacacatt cccgtcgact cccctagttt catcctattc 780

atttctcccc ccaaacactc cagctactag ttactcgagg aatccatgtg cggcggcgca 840

atcatcttcg actacataga tctcg 865

Claims (2)

1. corn abiotic stress response factor ZmERF1 gene promoters, it is characterised in that：Response factor ZmERF1 genes open Mover full length sequence 2217bp：

ATTTAAGCTT GAAGTTGATG GCATCAATCT GCCAGTCTGA TTTTCAGACC TATCCAACCT

GACAGCCTGA CTTTAGGCAT GATTATCTCT AAATTTTTCA CAAGAACATG GCTTGCACTC

TTAAGCAAAT TTGTTATCCA TTGATTGCTC TACAATTTTG ATGTGGTGAC TTAGGGCAAA

AACCCTATGC TTTACAAGTT ACAAAGCCCT AAAGTGGAGC CTTTAACACT GAATTTCAGA

CTTAGTGTAG AACTCAAATG AGGTCCATTT TGCATTTAAG TCCAAAACTT AGGGTTTGGC

TTTCAAGTCC ACATATTTGT GAACCAAAGG ACTTAAAATA CTTATTTGGC TTGGTTTTTG

CACTTTAGTC CAAAAGTGGA CCAATTTTGC ATATAAACCC CTAGGGTTTG GTTTCAGGGT

TTTCCAGGGT TTTAATTAGG GTTTCTGGTA TCCCAGGGGT ACAAAAGTGA TTCAAGTTTA

TTCTTAGGGT CATTTTATGA CTTTTACCCT AAAGCTTTTA GATTTTCTCA ACTTGGGTTA

AGTTTTACCC CTTTAACCAC TATTTAGGGT TAAGTCCTTT AACCAGGGTT CTTTTTGCAA

AACAACACAA CTTGTTTGAA ATTTTTGCCT AGTGAATGCA CTCTAGGTGT GTCAAACATA

TGCAATGCCA ATGCTATGAT GCCATGCTCA AGTTTTAGTT ACAGTAACAC CAGGGGTGTT

ACAATTGCTG TGGATGACGC TCAAATCCAT TGGGTCGTCT AGGCTTGAGT TGATCCAATC

CTGCGTCGAT CCACTTCGCT AGACGTTCCT CCCCAGGCCC ACAAAGGGAT GACCTTGCGA

AATATTGGGC CCAAAGCCTT TCGCGAGGTC TTCTAACCTT CCAGTGGACC TGGGGGATAT

CTGTCCCCCA CACATTATGC CCGAAGGGAG ATAATGCTTC GAAGGACAAA GGTCTTTAAC

ACTTAAGAAT TTTGTTGCGT TGTTTCATGA TTAAGTAATA AAAACAAGAG ATCAACACTA

TGCTCTTTTC TTAATCGATC ATTCTAAAAA GTACATGATT TTTGTCGACT TTACACCCAC

TCAAAATTGG TGTAAGCACA TGCCATACAA GAGGTTCATA GAAACGATTA TCATGGCCTT

AAAGAAATAT AAGATTGTCT ACAGCATGAA ACGTTCTAGA TGGGTGGTGG ATATTTTTAA

ATTGGAAAAT AAAATTCAGA CTGATGCTTC AATTGACATA AAATGATATT TTCTCCGTGC

CACCTTCTCA GCAATTTAAT TTTTTCTATG ATTTCCATTA CATATAACAT AGTTGGCAAA

ACATTATTAT GAAGGATTAA CACCAGCTAC CTCGTTCTAC AAGCTATGTC CATGGGGGTA

ATAACAGGCG AATGGAATTT GTTAGGGGTG AGTGGAGTAC GCATGTTTCA CGTTCAAGTA

TACATTAATA AAAACATTGG TATTTTGTTC ACTTATGTCT TTGTGTGTAG GATGTATATA

TTCTTCCAAG AAACTTTATG ATCGATCTGT TAATTTATGA GGACAATTCG TGCCGATATG

TCATCCCTCC AAACATACAG CATAGACTTA TCAATATCGC TAAAGAGGAT TAAATTAGTG

ACGGTTGTCG ACATATTCGT CTATCATTAA AAATTCTATA TTGACTATTT AGAAATAAT

TTGTATGATA TTGATAATGT TCGATATTAT TTCAATCTAT TTTAGTCAGC GTATTTATAA

TAATTATATA TTTATATCAT CAATATAATA TTATTGTTAT GATAATATTT TATAGTACGA

ATTTTATATA TTATGAAATA TCGTTTCATA TATATGTTTT ATATTAATTT TTCTAATTTA

CTTAAGCATC TGACGCTAAG TGTCCTGTGC CCGGATCGGC CAGAGCGGTC AAATGGTTCA

ATGCTTTACC TCCTCTGGTT GGTGTCCAGC TTGGGGGCAG CTGCCTTTCA TTTCCATTCT

TCCAGTTGCA CCCAGACCCA GGCAGGTCAT CGGCGCCCAC AACTCCAAGT TGTATAAATA

CTCCGTCGAC CGTCTCATAA CAGTCATACC GCCGAACCAA ATCCAACGTC CACAGAAAAG

AAAACACATT CCCGTCGACT CCCCTAGTTT CATCCTATTC ATTTCTCCCC CCAAACACTC

CAGCTACTAG TTACTCGAGG AATCCATGTG CGGCGGCGCA ATCATCTTCG ACTACAT；

The Cloning and sequence analysis of corn inbred line N04 ZmERF1 gene promoters is as follows：

A. the extraction of corn inbred line N04 DNA

Take 0.5 g young leaflet tablets to be placed in mortar, add grind into powder after liquid nitrogen, powder is quickly charged with to the centrifugation of 2.0 mL Guan Zhong, adds 800 μ L mass fractions, 1% SLS extracting solutions, and it is proportionally 25 to add 800 μ L：24：1 phenol, chloroform with The mixed liquor of isoamyl alcohol composition, fully mixes, and 65 DEG C of 30 min of incubation, after being cooled to room temperature, 12000 rpm centrifuge 10 min, Aspirate supernatant is gone in another 1.5 clean mL centrifuge tubes, and the pre- cold isopropanol for adding 0.6 times of supernatant volume mixes, 12000 rpm centrifuge 10 min, abandon supernatant, are rinsed with 75% ethanol, of short duration centrifugation, stays DNA to be done in tube bottom after rinsing It is dry, dissolved after DNA dryings with aqua sterilisa, it is spare to be placed in -20 DEG C of refrigerations；Wherein 1% SLS extracting solutions of mass fraction are by 100 mmol.L^-1 Tris-HCl、100 mmol.L^-1 NaCl、20 mmol.L^-1EDTA and 10 g_.L^-1SLS is mixed, and is then adjusted The mixed liquor obtained behind PH=8.0；

B. the clone of ZmERF1 gene promoters

According to the promoter sequence of corn inbred line N04 ZmERF1 genes, two pairs of specific primers, these two pair primer are devised The fragment of amplification overlaps, and specific primer sequence I is：F1391：5'GCGGATCCATTTAAGCTTGAAGTTGATGGC3' and R1391：5'TATTACCCCCATGGACATAGCT3'；Specific primer sequence II：F865：5'AAGCTATGTCCATGGGGGTA AT3' and R865：5'GCAGATCTATGTAGTCGAAGATGATTGCGC3'；Using the DNA of corn inbred line N04 as template, carry out PCR amplification, amplification system are：5 μ L Hifi Buffer (10 ×), 2 μ L, 2.5 mol.L^-1DNTP, 1 μ L Primer-F (R), 1 μ L Taq DNA Polymerase, 1 μ L template DNAs, ddH₂O supplies 50 μ L；PCR system amplification program is：95 4 min of DEG C pre-degeneration, 95 DEG C of 45 sec of denaturation, 60 DEG C of 45 sec of annealing, 72 DEG C of 60 sec of extension, are circulated 34 times altogether；72 DEG C are prolonged Stretch 10min, 10 DEG C of forever；Amplified production is detected with 1% agarose gel electrophoresis of mass fraction, is recycled with QIAquick Gel Extraction Kit, And it is connected on PMD18-T carriers, is sequenced；

The promoter sequence of sequencing is：

PMD18-1391：

GCGGATCCAT TTAAGCTTGA AGTTGATGGC ATCAATCTGC CAGTCTGATT TTCAGACCTA

TCCAACCTGA CAGCCTGACT TTAGGCATGA TTATCTCTAA ATTTTTCACA AGAACATGGC

TTGCACTCTT AAGCAAATTT GTTATCCATT GATTGCTCTA CAATTTTGAT GTGGTGACTT

AGGGCAAAAA CCCTATGCTT TACAAGTTAC AAAGCCCTAA AGTGGAGCCT TTAACACTGA

ATTTCAGACT TAGTGTAGAA CTCAAATGAG GTCCATTTTG CATTTAAGTC CAAAACTTAG

GGTTTGGCTT TCAAGTCCAC ATATTTGTGA ACCAAAGGAC TTAAAATACT TATTTGGCTT

GGTTTTTGCA CCTTAGTCCA AAAGTGGACC AATTTTGCAT ATAAACCCCT AGGGTTTGGT

TTCAGGGTTT TCCAGGGTTT TAATTAGGGT TTCTGGTATC CCAGGGGTAC AAAAGTGATT

CAAGTTTATT CTTAGGGTCA TTTTATGACT TTTACCCTAA AGCTTTTAGA TTTTCTCAAC

TTGGGTTAAG TTTTACCCCT TTAACCACTA TTTAGGGTTA AGTCCTTTAA CCAGGGTTCT

TTTTGCAAAA CAACACAACT TGTTTGAAAT TTTTGCCTTG TGAATGCACT CTAGGTGTGT

CAAACATATG CAATGCCAAT GCTATGATGC CATGCTCAAG TTTTAGTTAC AGTAACACCA

GGGGTGTTAC AATTGCTGTG GATGACGCTC AAATCCATTG GGTCGTCTGG GCTTGAGTTG

ATCCAATCCT GCGTCGATCC ACTTCGCTAG ACGTTCCTCC CCAGGCCCAC AAAGGGATGA

CCTTGCGAAA TATTGGGCCC AAAGCCTTTC GCGAGGTCTT CTAACCTTCC AGTGGACCTG

GGGGATATCT GTCCCCCACA CATTATGCCC GAAGGGAGAT AATGCTTCGA AGGACAAAGG

TCTTTAACAC TTAAGAATTT TGTTGCGTTG TTTCATGATT AAGTAATAAA AACAAGAGAT

CAACACTATG CTCTTTTCTT AATCGATCAT TCTAAAAAGT ACATGATTTT TGTCGACTTT

ACACCCACTC AAAATTGGTG TAAGCACATG CCATACAAGA GGTTCATAGA AACGATTATC

ATGGCCTTAA AGAAATATAA GATTGTCTAC AGCATGAAAC GTTCTAGATG GGTGGTGGAT

ATTTTTAAAT TGGAAAATAA AATTCAGACT GATGCTTCAA TTGACATAAA ATGATATTTT

CTCCGTGCCA CCTTCTCAGC AATTTAATTT TTTCTATGAT TTCCATTACA TATAACATAG

TTGGCAAAAC ATTATTATGA AGGATTAACA CCAGCTACCT CGTTCTACAA GCTATGTCCA

TGGGGGTAAT A

PMD18-865：

AAGCTATGTC CATGGGGGTA ATAACAGGCG AATGGAATTT GTTAGGGGTG AGTGGAGTAC

GCATGTTTCA CGTTCAAGTA TACATTAATA AAAACATTGG TATTTTGTTC ACTTATGTCT

TTGTGTGTAG GATGTATATA TTCTTCCAAG AAACTTTATG ATCGATCTGT TAATTTATGA

GGACAATTCG TGCCGATATG TCATCCCTCC AAACATACAG CATAGACTTA TCAATATCGC

TAAAGAGGAT TAAATTAGTG TACGGTTGTC GACATATTCG TCTATCATTA AAAATTCTAT

ATTGACTATT TAGAAATAAT TTGTATGATA TTGATAATGT TCGATATTAT TTCAATCTAT

TTTAGTCAGC GTATTTATAA TAATTATATA TTTATATCAT CAATATAATA TTATTGTTAT

GATAATATTT TATAGTACGA ATTTTATATA TTATGAAATA TCGTTTCATA TATATGTTTT

ATATTAATTT TTCTAATTTA CTTAAGCATC TGACGCTAAG TGTCCTGTGC CCGGATCGGC

CAGAGCGGTC AAATGGTTCA ATGCTTTACC TCCTCTGGTT GGTGTCCAGC TTGGGGGCAG

CTGCCTTTCA TTTCCATTCT TCCAGTTGCA CCCAGACCCA GGCAGGTCAT CGGCGCCCAC

AACTCCAAGT TGTATAAATA CTCCGTCGAC CGTCTCATAA CAGTCATACC GCCGAACCAA

ATCCAACGTC CACAGAAAAG AAAACACATT CCCGTCGACT CCCCTAGTTT CATCCTATTC

ATTTCTCCCC CCAAACACTC CAGCTACTAG TTACTCGAGG AATCCATGTG CGGCGGCGCA

ATCATCTTCG ACTACATAGA TCTCG；

C. the structure of conversion carrier

1391 bp promoter sequences being connected to BamHI and NcoI double digestions in carrier T, while with the double enzymes of BamHI and NcoI Cut pCAMBIA1301 carriers；PCAMBIA1301 carriers are connected with 1391 bp promoter sequences after recycling；Then large intestine is converted Bacillus DH5a, picking monoclonal shake bacterium, and PCR detects correct monoclonal extraction plasmid, digestion identification；Identification correctly carries The pCAMBIA1301 carriers of 1391 bp promoter sequences use NcoI and BglII double digestions again, while with the double enzymes of NcoI and BglII Cut 865 bp promoter sequences being connected in carrier T；1391 bp promoter sequences will be carried after recycling PCAMBIA1301 carriers are connected with 865 bp promoter sequences；Then escherichia coli DH5a is converted, picking monoclonal shakes bacterium PCR Correct monoclonal upgrading grain is detected, digestion identification, obtains ZmERF1 gene promoter full length sequences 2217bp;

D. corn inbred line N04 ZmERF1 gene promoter sequences are analyzed

The forecast analysis of obtained ZmERF1 gene promoter sequences progress cis element will be cloned, the results show promoter is removed Outside characteristic feature with plant promoter, also containing the relevant element of ethylene responses 2 and ABA hormone response ABRE elements 1；It is dehydrated correlation MYB1AT elements 4, MYCATERD elements 1 and MYCATRD elements 1；Low temperature cold-resistant controlling element 18 A, CBFHV elements 8；Injury regulation and control WBOXNTERF elements 9；Heat shock CCAATBOX elements 1；Tissue specific expression Regulate and control CAATBOX elements 27；Seed development correlation EBOXBNNAPA elements 18；Amylase correlation AMYBOX2 elements 1, CGACGOSAMY elements 1；Sugar correlation WBOXHVISO elements 5；Regulate and control roots development correlation RHERPATEXPA elements 2.

2. the application of corn abiotic stress response factor ZmERF1 gene promoters, its feature exist according to claim 1 In：ZmERF1 gene promoter sequences are cloned into expression vector, root, leaf or the inflorescence position of transformed plant carry out the salt side of body Compel, osmotic stress processing, then carry out chemical tissue staining and GUS quantitative fluorescence analysis ZmERF1 gene promoters to adverse circumstance Reaction.