CN107142267A - The mulberry tree ethylene responses factor and its application - Google Patents
The mulberry tree ethylene responses factor and its application Download PDFInfo
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- CN107142267A CN107142267A CN201710530055.7A CN201710530055A CN107142267A CN 107142267 A CN107142267 A CN 107142267A CN 201710530055 A CN201710530055 A CN 201710530055A CN 107142267 A CN107142267 A CN 107142267A
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Abstract
The invention discloses the mulberry tree ethylene responses factor and its application.Wherein the sequence of gene, albumen is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.Whether the present invention can identify plant in growing environments such as arid, high salt, low temperature, ABA.
Description
Technical field
The present invention relates to plant genetic engineering field, more particularly to a kind of mulberry tree ethylene responses factor (MmERF) gene
Clone and its expression under stress conditions.
Background technology
Plant can run into low temperature in growth and development process, some arid and saline and alkaline etc. abiotic stress, and they are mesh
It is preceding influence plant growth and crop yield main limiting factor, plant in order to adapt to the environment of surrounding, when by low temperature, do
When drought, high salt etc. are coerced, cell perceives rapidly stress from outside signal and by a series of signal transmittance process, and generation stress signal
And be pooled in nucleus, specific transcription regulatory factor is finally activated, and then trigger a series of physiology, biochemical reaction, form high
The orderly signals-modulating network of effect, so as to produce responsing reaction to stress.
As a kind of simple plant gas hormone, ethene grows and performance weight in Stress response plant
Act on.Ethene participate in the regulation and control seed sprouting of plant, plant strain growth, bloom, the Sex Determination of fruit maturation, unisexual flower, device
Official's aging and the physiology course such as come off, in addition second it is dilute also in plant biological with being played a significant role in abiotic stress response.Plant
Object adjusts the biosynthesis of ethene and opening for signal transduction pathway by the coordinated regulation of transcriptional level and post-transcriptional level
Close, coordinate the cross action of ethylene signaling pathway and other hormone signal approach, complete various vital movements and resist various inverse
Coerce in border.
The ethylene responses factor (Ethylene responsive factor, ERF) is a kind of distinctive transcription factor of plant,
Positioned at Ethylene Signal passage downstream, the expression of controllable stress-related genes is played in terms of response of the plant to environment-stress
Important effect.ERF can not only adjust their expression with one in pathogenesis-related gene promoter so as to carry by combining
The disease resistance of high plant, moreover it is possible to by the response for recognizing the abiotic stress that plants are participated in other non-cis-acting elements.
At present, the research of erf gene is more in the plants such as paddy rice, wheat, tomato, but to the ERF class bases in mulberry tree
It is also fewer because studying.
China is the centre of origin of mulberry tree, and germ plasm resource is extremely enriched.It is several by Mulberry Germplasm Resources and breeder
The effort of 10 years, national Collection and conservation has nearly 3000 parts of Mulberry Germplasm Resources now, wherein many resources all have fruit valency
Value, medical value, economic value etc..The Morus multicaulis kind (Morus multicaulis) that we are obtained with clone educates 71-1MmERF
Gene ORF sequences, pass through its sequence signature of bioinformatic analysis.Meanwhile, it have detected using the method for quantitative fluorescent PCR
The changing rule of transcriptional level of the MmERF genes under arid, high salt, low temperature, ABA stress conditions.By being ground to the gene
Study carefully, us is recognized expression of the mulberry tree MmERF genes under the conditions of Different stress from molecular level.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of mulberry tree ethylene responses factor (MmERF) gene cloning and its
Expression under Abiotic stress conditions, expression regulation of the ethylene responses factor gene under environment-stress that can be seen in mulberry tree.
For mulberry tree ethylene responses factor gene, the technical solution adopted by the present invention is, its nucleotide sequence such as SEQ ID
Shown in NO.1.
Above-mentioned mulberry tree ethylene responses factor gene coerces the application in expression mark as plant abiotic.
Preferably, the plant is mulberry tree, its Latin name is Morus multicaulis.
Preferably, described abiotic stress is arid, high salt, low temperature, ABA stress.
For the albumen of mulberry tree ethylene responses factor gene coding, the technical solution adopted by the present invention is, its amino acid sequence
Row are as shown in SEQ ID N0.2.
The albumen of above-mentioned mulberry tree ethylene responses factor gene coding coerces answering in expression mark as plant abiotic
With.
Preferably, the plant is mulberry tree, its Latin name is Morus multicaulis.
Preferably, described abiotic stress is arid, high salt, low temperature, ABA stress.
The beneficial effects of the invention are as follows：
The present invention improves mulberry tree genome while probing into the mulberry tree ethylene responses factor (MmERF) gene in abiotic stress bar
The expression analysis of part, mulberry tree MmERF clone will establish base further to cultivate resistance New Mulberry Variety by transgenic technology
Plinth, can effectively make up the deficiency of traditional breeding method, so as to accelerate the seed selection process of mulberry tree resistance new varieties.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is that fluorescence quantitative RT-RCR detects relative expression spirogram of the MmERF genes under high-salt stress in mulberry sapling tender leaf；
CK：Control；1d：High salt treatment 1 day；2d：High salt treatment 2 days；4d：High salt treatment 4 days；7d：High salt treatment 7 days；10d：High salt
Processing 10 days.
Fig. 2 is that fluorescence quantitative RT-RCR detects relative expression spirogram of the MmERF genes under drought stress in mulberry sapling tender leaf；
CK：Control；1d：Osmotic treatment 1 day；3d：Osmotic treatment 3 days；5d：Osmotic treatment 5 days；10d：Osmotic treatment 10 days；15d：It is dry
Drought processing 15 days.
Fig. 3 is that fluorescence quantitative RT-RCR detects relative expression quantity of the MmERF genes under 4 DEG C of low temperature stress in mulberry sapling tender leaf
Figure；CK：Control；12h：Osmotic treatment 12 hours；1d：Osmotic treatment 1 day；3d：Osmotic treatment 3 days；6d：Osmotic treatment 6 days；
10d：Osmotic treatment 10 days；RN2d：Normal temperature renaturation 2 days；RN5d：Normal temperature renaturation 5 days.
Fig. 4 is that fluorescence quantitative RT-RCR detects relative expression spirogram of the MmERF genes under ABA stress in mulberry sapling tender leaf；
CK：Control；2h：ABA is handled 2 hours；6h：ABA is handled 6 hours；12h：ABA is handled 12 hours；1d：ABA is handled 1 day；3d：
ABA is handled 3 days；5d：ABA is handled 5 days.
Embodiment
The clone of the mulberry tree ethylene responses factor M mERF genes of embodiment 1
1.1 design primers
For examination mulberry tree breed be stored in the national germplasm Zhenjiang mulberry tree garden of Inst. of Silkworm, Chinese Academy of Agricultural Sciences educate 71-
1.Using the acquired ESTs sequences for including MmERF gene cDNA fragments, design primer clone, and verified, primer sequence
Row are as follows：
MmMmERF-F：5′-AGGAAAACAAATTTCTCGTCA-3′；
MmMmERF-R：5′-AACGCAAGTTTTCAAGGTC-3′
The primer of quantitative fluorescence analysis is carried out according to acquired gene ORF sequences Designs, primer sequence is as follows：
q MmERF-F：5′-TTCCAGGGTTTCAAGGATGAC-3′
q MmERF-R：5′-GTTGTTGAGCCACGAGAGAA-3′
According to β-actin gene (the GeneBank accession number of stable expression in mulberry tree searched on NCBI：
DQ785808) the upstream and downstream primer of sequences Design reference gene, upstream primer sequence β-actin-F be 5 '-
AGCAACTGGGATGACATGGAGA-3 ', downstream primer sequence β-actin-R are 5 '-CGACCACTGGCGTAAAGGGA-3 '.
The clone of 1.2 mulberry tree ethylene responses factor M mERF genes
Mulberry tree tender leaf RNA is extracted using Takara kits, extraction step is carried out according to kit specification, electrophoresis detection
RNA bands are complete；And according to Takara reverse transcription reagent box specification by RNA reverse transcriptions into cDNA.With the chains of cDNA the 1st of synthesis
It is that primer enters performing PCR amplification using MmERF-F/R for template, wherein, the Ex Taq enzymes that PCR enzymes are produced using Takara companies,
PCR response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 35s, 72 DEG C of extension 40s, 37 circulations；72
DEG C renaturation 5min；4 DEG C of preservations.Electrophoresis detection purpose fragment size, uses Takara companies Agarose Ge1 DNA
Purification Kit kits reclaim PCR primer, obtain mulberry tree MmERF genetic fragments, purification process is with reference to reagent
Box specification.Purpose fragment DNA ligase is connected with pMDTM18-T carriers.Connection product is transformed into Host Strains E.coli
TOP10 bacterial strain competent cells, are coated on the LB solid plates containing Amp.Sample presentation after picking single bacterium colony culture, by raw work
Bioengineering (Shanghai) Co., Ltd. completes sequencing, and sequencing result carries out blast analyses, and obtain 1500bp includes complete ORF
MmERF gene orders (GenBank accession number：MF188168), wherein, include 140bp 5 '-UTR,
193bp 3 '-UTR and 1167bp complete ORF.
The bioinformatic analysis of the mulberry tree ethylene responses factor M mERF genes of embodiment 2
Utilize on-line analysis instrument PSORT and SMART (http://smart.embl-heidelberg.de/), according to
The document of report carries out the prediction of nuclear localization sequence and the analysis of protein domain, the mulberry tree ethylene responses factor to target gene
MmERF albumen is by an AP2/ERF domain being made up of 64 amino acid residues, positioned at the middle part of albumen, wherein, the 14th
It is respectively alanine (A) and aspartic acid (D) with the 19th amino acids residue, it is typical ERF transcription to illustrate it.
Utilize DNAStar softwares and on-line analysis software ExPASy (http:∥prosite.expasy.org/
Prosite.html/) mulberry tree ethylene responses factor M mERF genes are more comprehensively analyzed, the coded by said gene is obtained
Amino acid sequence SEQ ID NO.2, the isoelectric point of gene code mulberry tree ethylene responses factor M mERF protein is 5.02, molecule
Quality is 43.30kDa.
Expression pattern of the mulberry tree ethylene responses factor M mERF genes of embodiment 3 under various stress conditions
When the young sprout for examination mulberry sapling grows to that about 20cm is long, carry out arid, high salt, low temperature, ABA respectively to mulberry sapling and coerce
Processing.In whole stress-inducing processing procedure, the temperature of all experiment seedlings, intensity of illumination, photoperiod and humidity etc. are non-
Biotic factor keeps constant.When mulberry sapling shows obvious damage symptoms under various Stress treatments, control is taken respectively and each
The mulberry tree spire under stress conditions is planted, after being wrapped up with aluminium-foil paper in rapid input liquid nitrogen, is put in -80 DEG C of refrigerator and preserves.Adopt
With the method for quantitative fluorescent PCR, the actin gene (β-actin) using the stable expression of mulberry tree determines mulberry tree and existed as internal reference
The expression of target gene under stress conditions.All stress experiments are that 3 biology repeat 3 technologies repetitions.
High-salt stress：The NaCl solution for being 300mM for examination mulberry sapling concentration carries out salt stress induction processing.Test mulberry sapling and
Control mulberry sapling is positioned over same illumination box, 25 DEG C of temperature, daily illumination in 16 hours, 8 hours dark.Respectively at 1 day, 2
My god, 4 days, 7 days and the mulberry tree spire (1-3 leaves position) under condition of salt stress is taken at 10 days as experiment material.
Drought stress：25 DEG C of illumination boxs of temperature are positioned over for examination mulberry sapling, are not watered always, daily illumination in 16 hours, 8
Hour is dark.Mulberry tree spire (1-3 leaves position) was taken respectively at 1 day, 3 days, 5 days, 10 days and 15 days as experiment material.
Low temperature stress：4 DEG C of illumination boxs of temperature, daily illumination in 16 hours, 8 hours dark are positioned over for examination mulberry sapling.Point
Mulberry tree spire (1-3 leaves position) was not taken in 12 hours, 1 day, 3 days, 6 days and 10 days as experiment material, after be positioned over temperature 25
DEG C illumination box carries out normal temperature renaturation, and daily illumination in 16 hours, 8 hours dark, renaturation takes mulberry tree spire (1- in 2 days and 5 days
3 leaves position) it is used as experiment material.
ABA is coerced：The ABA solution for being 0.1mol/L for examination mulberry sapling concentration carries out ABA stress-inducing processing, is positioned over temperature
Spend 25 DEG C of illumination boxs, daily illumination in 16 hours, 8 hours dark.Respectively at 2 hours, 6 hours, 12 hours, 1 day, 3 days and 5
It takes mulberry tree spire (1-3 leaves position) as experiment material.
Mulberry tree ethylene responses factor M mERF genes relative expression quantity change display under different abiotic stress：High-salt stress
Under the conditions of, MmERF genes relative expression quantity is lower than under normal growing conditions, and overall trend is first slow decline, in processing 4
It when be minimized value (0.52 times of control), it is then slow again to rise, 0.85 times of (figure of control was finally risen to by the 10th day
1).Under drought stress conditions, the performance up-regulation of MmERF gene relative expression quantities overall trend, with the extension of Osmotic treatment time,
MmERF gene relative expression quantities are gradually risen, and maximum was reached at the 15th day, for 3.69 times (Fig. 2) of control.Low temperature stress
Under the conditions of, the obvious up-regulation of the relative expression quantity overall trends of MmERF genes performance, after persistently handling 1 day on to be transferred to high value (right
According to 2.53 times), then it is slow decline after rapidly rise to the maximum (3.89 times of control) in these stages, it is multiple through normal temperature
Property 2 days, expression quantity is fallen after rise, continues renaturation after 5 days, and expression quantity is returned to close to control value (Fig. 3).Under ABA stress conditions,
The performance of MmERF gene relative expression quantities overall trend is lowered, and with the extension of ABA Stress treatment times, MmERF genes are with respect to table
It was gradually reduced before this up to amount, value was minimized at the 12nd hour, for 0.33 times of control, rear slow rise was maintained at by the 5th day
0.78 times or so (Fig. 4) of control.MmERF genes have obvious fluctuation status under various stress conditions, and this shows MmERF
Its function may be played, under Abiotic stress conditions, the expression of MmERF genes is induced, makes plant active response stress from outside.
Analyzed by quantitative fluorescent PCR, using MmERF genes under drought stress, high-salt stress, low temperature stress and ABA stress conditions
The lifting situation of overall expression, the Relative gene expression figureofmerit in different stress times, and MmERF genes are in plant
Conservative, can identify whether plant the growing environment such as coerces in high salt, low temperature, arid or ABA with this.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within the modifications, equivalent substitutions and improvements made etc., should be included in the claim protection model of the present invention
Within enclosing.
SEQUENCE LISTING
<110>Institute of Sericulture, Anhui Academy of Agricultural Sciences
<120>Mulberry tree ethylene responses factor M mERF and its application
<130> NO
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1500
<212> DNA
<213> MmERF
<400> 1
aggaaaacaa atttctcgtc aacaaaaaaa aaaaaaaaaa aaaaaaaaaa ctttcttctc 60
tgaaaaatct caaacctttt ttcttgattc tctgcaagag gtgttttgga tccgggttgt 120
ttgttgtgca gtgaaggaga atgtgtggag gtgctattat ctccgacttc atctcgacgc 180
cgcggtccac gcgtctgacc gcggactacc tctggccgga tctgaagaaa tccggctccg 240
gcaagcgctt ctgcaagcct gtgagatcgg tgatcgtcga catcgacgat gacttcgagg 300
ctgatttcca gggtttcaag gatgactccg acgtcgacaa cgacgacgac gttatagatg 360
tcaagccttt cgctttctct gctcgcaagc ccaccttctc tcgtggctca acaactgtga 420
aatataccga atctgatggg caagctgaga aatctgcaaa gagaaagaga aagaaccagt 480
ataggggaat tcggcagcgc ccatggggta agtgggctgc tgagatccgc gaccctagga 540
aaggtgtccg ggtttggctc ggaactttca acactgctga agaagctgca agagcttatg 600
atgctgaggc acgacgaatc cgtggtaaga aagccaaggt gaatttccca gatgaaactc 660
cgcgtgcttt gcctaaacat cctgttaagg aaagtcctaa gagatcactg cccaaggaaa 720
attcgaactc caccgagtcg aatttgaaca atcaaagttt caattttgtg aataactctg 780
atctggacta ctacaatgct atgggttttc tggaagagaa accgctgact aatcaatatg 840
agcatgtgga gactctccct gctaaagcgg atgttggact caaatccaat gctccagctg 900
ctactactcc gctgtatttc agttctgatc agggtagcaa ttcatttgag tgttccgact 960
ttgggttggg agaacatggc tcaaagactc cagaaatttc ttctgttttc tcagccactt 1020
cagaaaatga tgactcgcta tttcttgagg atactaaccc aacaaagaag ctgaagtctg 1080
actcagagaa tgcggtgctt cctgaagaaa atcatgcaaa gactctgtca gaggagcttt 1140
cggctttcga gtctcagatg aagttctttc agatgccata tcttgaggga agctgggatg 1200
cgttattgga caccttcctc gctggcgatt caactcagga tggtggaaac tctatcaatc 1260
tttggagttt tgatgacttc tccaccgtgt ctggcgaagc cttctaagcc aattccccag 1320
ctttcctaat taatgtaaat aaggctacat gaattgttta tctgcataag gattgcacaa 1380
acagatgaac atgcacaagc cttgtatgga gagtgattcg aggtagctaa tatcggtgta 1440
tggcgagtat atagatatta ggttttactt cttaccttga agaccttgaa aacttgcgtt 1500
<210> 2
<211> 388
<212>Protein 1
<213>MmERF albumen
<400> 1
mcggaiisdf istprstrlt adylwpdlkk sgsgkrfckp vrsvivdidd dfeadfqgfk 60
ddsdvdnddd vidvkpfafs arkptfsrgs ttvkytesdg qaeksakrkr knqyrgirqr 120
pwgkwaaeir dprkgvrvwl gtfntaeeaa raydaearri rgkkakvnfp detpralpkh 180
pvkespkrsl pkensnstes nlnnqsfnfv nnsdldyyna mgfleekplt nqyehvetlp 240
akadvglksn apaattplyf ssdqgsnsfe csdfglgehg sktpeissvf satsenddsl 300
fledtnptkk lksdsenavl peenhaktls eelsafesqm kffqmpyleg swdalldtfl 360
agdstqdggn sinlwsfddf stvsgeaf 388
Claims (8)
1. mulberry tree ethylene responses factor gene, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.1.
2. the albumen of mulberry tree ethylene responses factor gene coding, it is characterised in that its amino acid sequence such as SEQ ID NO.2 institutes
Show.
3. mulberry tree ethylene responses factor gene as claimed in claim 1 coerces answering in expression mark as plant abiotic
With.
4. mulberry tree ethylene responses factor gene coerces answering in expression mark as plant abiotic as claimed in claim 3
With, it is characterised in that the plant is mulberry tree, and its Latin name is Morus multicaulis.
5. mulberry tree ethylene responses factor gene coerces answering in expression mark as plant abiotic as claimed in claim 3
With, it is characterised in that the abiotic stress is arid, high salt, low temperature, ABA stress.
6. the albumen of mulberry tree ethylene responses factor gene coding as claimed in claim 2 is used as plant abiotic stress expression mark
Application in thing.
7. the albumen of mulberry tree ethylene responses factor gene coding is used as plant abiotic stress expression mark as recited in claim 6
Application in will thing, it is characterised in that the plant is mulberry tree, its Latin name is Morus multicaulis.
8. the albumen of mulberry tree ethylene responses factor gene coding is used as plant abiotic stress expression mark as recited in claim 6
Application in will thing, it is characterised in that the abiotic stress is arid, high salt, low temperature, ABA stress.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2017104504311 | 2017-06-14 | ||
| CN201710450431 | 2017-06-14 |
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| Publication Number | Publication Date |
|---|---|
| CN107142267A true CN107142267A (en) | 2017-09-08 |
Family
ID=59785820
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608929A (en) * | 2021-01-05 | 2021-04-06 | 重庆文理学院 | Longan flowering regulation gene DlERF23, protein and application thereof |
| CN114085276A (en) * | 2021-11-17 | 2022-02-25 | 广东省科学院南繁种业研究所 | Upstream regulatory factor IbERF10 and application thereof in regulation of expression of purple sweet potato IbbHLH2 |
| CN114107317A (en) * | 2021-10-22 | 2022-03-01 | 宁波大学 | Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof |
-
2017
- 2017-06-30 CN CN201710530055.7A patent/CN107142267A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608929A (en) * | 2021-01-05 | 2021-04-06 | 重庆文理学院 | Longan flowering regulation gene DlERF23, protein and application thereof |
| CN112608929B (en) * | 2021-01-05 | 2021-07-23 | 重庆文理学院 | Longan flowering regulation gene DlERF23, protein and application thereof |
| CN114107317A (en) * | 2021-10-22 | 2022-03-01 | 宁波大学 | Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof |
| CN114085276A (en) * | 2021-11-17 | 2022-02-25 | 广东省科学院南繁种业研究所 | Upstream regulatory factor IbERF10 and application thereof in regulation of expression of purple sweet potato IbbHLH2 |
| CN114085276B (en) * | 2021-11-17 | 2022-09-16 | 广东省科学院南繁种业研究所 | Upstream regulatory factor IbERF10 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato |
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