CN102888402A - Cloning and application of corn flooding stress response zmERF2 gene promoter - Google Patents

Abstract

The invention discloses cloning and application of a corn flooding stress response zmERF2 gene promoter. The nucleotide sequence of the gene promoter is shown as SEQ ID NO:1, the promoter contains one ARE, one GARE, one GC motif, two Skn-1motif, one CGTCA-motif and one MBS site cis element; the cis element is related to the plant hormones and anaerobic stress response; and the promoter is a promoter with specific tissues and can be expressed in a root system only, and is also an inducible promoter and can be introduced by flooding to be efficiently expressed. The promoter is connected with an insect-resistant gene, a disease-resistant gene, an adversity-resistant gene and other targeted genes; and under the conditions of flooding and waterlogging, the insect resistance capacity, the disease resistance capacity and the adversity resistance capacity of the root of a transgenic plant can be improved. Meanwhile, the promoter is only expressed in the root, and the problem of food safety on the overground part of an edible transgenic plant is not involved. The zmERF2 promoter has good application prospect in the waterlogging resistance genetic improvement of xerophyte.

Description

Clone and the application thereof of corn flooding stress response zmERF2 gene promoter

Technical field

The invention belongs to the plant gene engineering technology field, relate to a kind of clone and application thereof of continuous corn flooding stress response zmERF2 gene promoter.

Background technology

China is subjected to 9,560,000 hectares of the farmland area average out to of flood every year, and wherein the serious time, the disaster area reaches more than 1,500 ten thousand hectares.At Asia, flood has caused very large loss to the production of corn, South Asia only, and annual 15% the maize sown area of surpassing suffers in various degree harm.In India, flood causes the annual underproduction 25-30% of corn.South China spring maize and northern summer corn often suffer flood and stain evil in normal cultivation season, maize root system is in hypoxia for a long time, causes corn underproduction 10%-40%.Waterlogging stain evil has become one of main restrictive factor of south China corn with high yield, stable yields.Therefore, corn clone flooding stress responsive genes and promotor thereof to illustrating molecule mechanism that corn flooding stress response forms and the genetic improvement of corn and the waterlogging stain of other living crops of drought, have very important theory value and practice significance.

The ERF gene is one of key gene of involved in plant abiotic stress response, is the key gene of paddy rice, deep water rice and Arabidopis thaliana flooding stress response.Therefore, the promotor of corn clone ERF gene helps to illustrate the molecular mechanism that the corn waterlogging tolerance forms, and the genetic improvement of anti-stain for corn and the living crop of other droughts provides theoretical direction simultaneously.Yet the clone of corn ERF promotor at present, there is not yet report.

Summary of the invention

The object of the present invention is to provide a kind of clone and application thereof of continuous corn flooding stress response zmERF2 gene promoter, contain 1 ARE, 1 GARE, 1 GC motif, 2 Skn-1motif, 1 CGTCA-motif and 1 MBS site cis element, these cis elements respond relevant with plant hormone, Anoxia stress; Be the promotor of an organizing specific type, only in root system, express; Be the promotor of an induction type, expressed by the waterflooding inducement efficient.This promotor is connected with the goal gene such as pest-resistant, disease-resistant, degeneration-resistant, under waterflooding, waterlogging stain condition, can improve pest-resistant, disease-resistant, the degeneration-resistant border ability of transfer-gen plant root.This promotor is only expressed at root simultaneously, does not relate to the food-safety problem of edible transgenic plant over-ground part.The zmERF2 promotor has good application prospect in the xerophytic genetic improvement of anti-the stain.

Its technical scheme is as follows:

1, the acquisition of corn zmERF2 gene

According to known Maize genome information, use the protein sequence of the AP2/ERF structural domain high conservative of tobacco ERF2 albumen, carry out the homology compare of analysis, electronic cloning goes out 121 ERF genes.By with the comparative analysis of the other plant ERF genes such as Arabidopis thaliana, paddy rice, tobacco, reject 48 false positives, obtained 73 ERF genes, respectively called after zmERF1--zmERF73.The waterlogging tolerance of corn inbred line Hz32 is stronger, and the Mo17 waterlogging tolerance a little less than, in 1 heart stage of corn 3 leaves, respectively to Hz32 and Mo17 carry out 0,4, the submerging treatment of 12h, and the expression that utilizes real-time PCR that 73 ERF genes are managed root system is throughout analyzed, found that the zmERF2 gene is expressed by the waterflooding inducement efficient at the Hz32 root system, and expression amount is low in Mo17, the result hints that the promotor of zmERF2 gene is the inducible promoter of flooding stress response.

2, the extraction of the total DNA of corn inbred line Hz32 blade

Get maize leaf 5.0g and in liquid nitrogen, grind, change in the 50ml centrifuge tube.Add CTAB damping fluid (1.17MNaCL, 0.0016M EDTA-8.0,0.835M Ttis-7.5,1.6%CTAB, the 1% β-thin basic ethanol) 10ml that is preheated to 95 ℃, mixing.65 ℃ of water-bath internal reactions 90 minutes, jog centrifuge tube frequently.Take out centrifuge tube, after being chilled to room temperature, add the equal-volume chloroform: different alcohol (24: 1), carefully shook test tube 10 minutes.Centrifugal 10 minutes of 8000rpm goes to supernatant in another 50ml centrifuge tube.Add 2/3 volume Virahol, careful mixing changes flocculence DNA in the 50ml centrifuge tube of containing 10ml70% ethanol over to, soaks 12 hours.Outwell 70% ethanol, DNA is dried, add 1ml TE dissolving.After DNA dissolves fully, change in the 2.0ml centrifuge tube, add 10 μ l10mg/ml RNaseA, mixing; Under 37 ℃, incubation 2 hours.Use again 1ml phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, centrifugal 10 minutes of 8000rpm.Supernatant liquor is changed in the 2.0ml centrifuge tube, add the 3M NaAC (PH5.2) of 1/10 volume, 2/3 volume isopropanol precipitating DNA.Flocculence DNA is changed in the 2.0ml centrifuge tube, with 70% washing with alcohol once of short duration centrifugal, make DNA adherent, remove 70% ethanol, dry DNA.After DNA dries, add 0.5ml T.E solution, complete dissolving DNA is in-20 ℃ of prolonged preservation.

3, the clone of zmERF2 gene promoter

According to the promoter sequence of corn inbred line B73zmERF2 gene, design a pair of special primer, left primer: 5-' ATGGTCGGTGCCTGCTGCGTGGG-3', right primer: 5 '-GCACAAATCCTTTCCCTTTCCTT-3 '.As template, carry out pcr amplification with the DNA of Hz32.The pcr amplification reaction system is: 2.5 μ l10 * PCR damping fluid, 2 μ l10mM dNTPs, 1 μ l10mM special primer, the special right primer of 1 μ l10mM, 1 Taq of unit enzyme, 40ng template DNA, cumulative volume 25 μ l.The pcr amplification program is: 95 ℃ 3 minutes; 95 ℃ 1 minute, 61 ℃ 1 minute, 72 ℃ 2 minutes 30 seconds, circulate 35 times; 72 ℃ 7 minutes; 4 ℃ 30 minutes.Pcr amplification product 120 volts of volts DS electrophoresis 45 minutes in 1% sepharose, use the gel of Beijing Quanshijin Biotechnology Co., Ltd to reclaim test kit, reclaim specific DNA fragment, and this dna fragmentation is connected on the pGEM-T easy carrier, carry out dna sequencing.

4, the structure of conversion carrier

Use is with the primer (left primer adds the HindIII restriction enzyme site, and right primer adds the BamHI restriction enzyme site) of restriction enzyme site, carries out pcr amplification take the DNA of corn inbred line Hz32 as template.The PCR product is connected with pGEM-T easy carrier, Transformed E scherichia coli DH5 α, the White strain that is grown on the LB solid plate that contains penbritin, X-Gal and IPTG is separated.Detect through PCR, select positive colony to carry out dna sequencing, extract the correct plasmid DNA of zmERF2 promoter sequence of cloning.Use simultaneously digested plasmid of HindIII and BamHI restriction enzyme, 1% agarose gel electrophoresis reclaims the zmERF5 promoter fragment.Use HindIII and BamHI restriction enzyme while enzyme to cut the pBI121 plasmid vector, and reclaim carrier segments.ZmERF2 promoter fragment and pBI121 carrier segments are linked external, be built into the pBI121-zmERF2-GUS carrier, be transformed in the Agrobacterium GV3101 bacterial strain.

5, turn the acquisition of zmERF2 promotor Arabidopis thaliana plant

The GV3101 Agrobacterium that will contain the pBI121-zmERF2-GUS carrier is inoculated in the LB liquid nutrient medium, and adding Rifampin and kantlex to final concentration is 100mg/L, 28 ℃ of shaking culture.Centrifugal collection thalline is diluted to OD=0.8 with ddH2O, and every 100ml bacterium liquid adds 5.0g sucrose and 50 μ l Silweet L-77.Use floral dip method is contaminated Arabidopis thaliana, gathers in the crops seed after the maturation, seed is broadcast containing on the MS solid screening culture medium of 100mg/L kantlex.The resistant plant that grows from screening culture medium is defined as turning zmERF2 promotor positive plant after PCR detects.

6, the detection of zmERF2 promoter activity

To turn the zmERF2 Arabidopis thaliana and carry out submerging treatment seedling stage, the transgenic arabidopsis seedling of normal growth and submerging treatment will be carried out the GUS histochemical stain.Found that, under the normal growth condition, turn root and the blade of zmERF2 promotor Arabidopis thaliana plant, the blueness of GUS dyeing do not occur; And under the flooding condition, the root that turns zmERF2 promotor Arabidopis thaliana presents stronger GUS blueness, and blade has no the GUS blueness.Studies show that the zmERF2 promotor is an organizing specific type promotor of expressing in root, also is the inducible promoter of a waterflooding abduction delivering simultaneously.

Compared with prior art, beneficial effect of the present invention is: 1) the zmERF2 promotor directly drives the transgenic plant of foreign gene, under the flooding condition rapidly and efficiently (4h) start foreign gene in the great expression of plant root, can improve the waterlogging tolerance of plant, or the ability such as pest-resistant, disease-resistant; 2) the zmERF2 promotor directly drives the transgenic plant of foreign gene, because foreign gene is abduction delivering under flooding condition only, and expressive site only is confined to root, so for the transgenic plant that eat non-root, do not have Transgenic Food Safety Issue.

Specifically, advantage of the present invention: the 1) inducible promoter of abduction delivering under the flooding condition; 2) tissue-specific promoter at the root specifically expressing; 3) the startup ability of this promotor is stronger, is strong promoter; 4) the waterlogging tolerance genetic improvement for corn and the living crop of other droughts provides new root specifically expressing, the promotor resource of waterflooding induction type; 5) to the analysis of contained cis element in this promotor, can further illustrate the molecular mechanism that the corn waterlogging tolerance forms.

Embodiment

Below in conjunction with embodiment the present invention is described in more detail.

The zmERF2 promotor is cloned on the conversion carrier, connects thereafter a Tnos terminator, be built into a conversion carrier.The goal gene that transforms with correct direction, is cloned between zmERF2 promotor and the Tnos terminator, builds conversion carrier.Then by particle bombardment or agrobacterium co-cultivation mediated method goal gene is transformed in the recipient cell, obtains transfer-gen plant by technique means such as tissue culture.Under waterlogging stain, flooding condition, the zmERF2 promotor can start goal gene in plant root great expression.

The above; only be the better embodiment of the present invention; protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.

Claims (3)

1. a continuous corn flooding stress response zmERF2 gene promoter is characterized in that, its nucleotide sequence is shown in sequence table SEQ ID NO:1.

2. one kind contains the plant expression vector that the described continuous corn flooding stress of claim 1 responds the zmERF2 gene promoter.

3. the as claimed in claim 1 application of continuous corn flooding stress response zmERF2 gene promoter in the plant genetic improvement of anti-the stain.