CN106047890A - Key ethylene response factor for regulating chlorophyll degradation of citrus peels - Google Patents
Key ethylene response factor for regulating chlorophyll degradation of citrus peels Download PDFInfo
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Abstract
The invention provides a key ethylene response factor CitERF6 (SEQ: NO. 1) for regulating the chlorophyll degradation of citrus peels. The key ethylene response factor comprises a conservative AP2/ERF structural domain and a Pat4(RKRK) nuclear localization signal. During the ethylene treatment of harvested citrus fruits, the expression of CitERF6 is enhanced under external ethylene induction. When the CitERF6 instantly overexpresses in the citrus peels, the chlorophyll degradation of the citrus peels can be promoted and accelerated. The key ethylene response factor has the advantages that by the PCR, real-time and quantitative PCR and citrus fruit instant expression technologies, the citrus ethylene response factor CitERF6 is cloned, and identification of the CitERF6 shows that the CitERF6 can participate in the chlorophyll degradation; the CitERF6 is applicable to genetic engineering breeding of plant color modification and especially to the regulation of the chlorophyll degradation of the citrus peels, and a theoretical basis is provided for the further researches of the peel coloring technology and the acting mechanism of the CitERF6.
Description
Technical field
The invention belongs to plant molecular biotechnology and genetic engineering field, relate to the pass of a regulation and control orange peel removing green
The key ethylene responses factor (CitERF6), and at the genetic engineering breeding of plant color and luster modification with in regulation and control orange peel removing green
In application.
Background technology
Citrus belong to Rutaceae (Rutaceae) Aurantioideae (Aurantioideae), are divided into Poncirus (Poncirus
Raf), Fortunella (Fortnoella Swing) and both citrus (Citrus L.), be the big fruit variety of the first in the world, its plantation
Area and yield all rank first, and Ye Shi China originates in one of fruit.Citrus fruit meat flavor is unique and has anticancer and antitumor etc.
Function, and peel is rich in aromatic substance and natural pigment (Chlorophylls and Carotenoids).Citrusfruit is often adopted at green ripe stage
Receive, for promoting fruit appearance uniform coloring, improve fruit commodity and value, commercial frequently with ethylene fumigation or ethephon
Method of dipping accelerates peel chlorophyll degradation.
Chlorophyll is pigment the abundantest in global biosphere, and it had both participated in photosynthesis, provided for animals and humans again
Food, also has hemopoietic, cures the disease and the function such as removing toxic substances.Although chlorophyll is the most important, but its degraded occurs to decline plant
During old and fruit maturation is whole, and also by environmental stimulus, such as extreme path photograph, low temperature and ethylene etc..Just because of this, leaf
The degraded of verdazulene not only creates this in riotous profusion colorful world, and adds the commodity of most of fruit, such as Citrus, perfume (or spice)
Any of several broadleaf plants, Fructus actinidiae chinensis, Fructus Lycopersici esculenti and pears etc..
Ethylene, as a kind of important phytohormone, the whole process of involved in plant growth promoter, is also that fruit becomes simultaneously
One of most important regulation and control person in ripe process.It can promote the accumulation of fruit softening, sugar and the degraded of acid, the distributing of odoramentum
With peel removing green.The ethylene responses factor (ERF) is positioned at the end of ethylene signaling approach, containing an AP2/ in its sequence
ERF domain, the target gene of the various ethylene responses of transcribed regulation and control, and then produce ethylene biological effect widely.But
During orange peel removing green, the biological mechanism of ERFs family member's controlling chlorophyll degraded is unclear.
Summary of the invention
It is an object of the invention to provide the crucial ethylene responses factor (CitERF6) of a regulation and control orange peel removing green, its
Nucleotide sequence is as shown in SEQ:NO.1.
It is a further object to provide the base that the described ethylene responses factor (CitERF6) is modified at plant color and luster
Because of the application in Engineering Breeding, the especially application in regulation and control orange peel removing green.Research shows, the Citrus that the present invention provides
Ethylene responses factor CitERF6 (SEQ:NO.1) controllable chlorophyll degradation, therefore may be used on the gene that plant color and luster is modified
In Engineering Breeding and regulation and control orange peel removing green.
The feature functionality of the ethylene responses factor (CitERF6) that the present invention provides is as follows:
1. gene sequence characteristic
From the arabidopsis AP2/ERF gene reported and Citrus genome database (http: //
Www.citrusgenomedb.org) carry out BLAST (TBLASTN and BLASTP) and analyze acquisition CitERF6 (SEQ:NO.1).Profit
With round pcr, in conjunction with primer, SEQ:NO.3 and SEQ:NO.4 amplification is obtained this gene, and use order-checking means to test further
Card.The sequencing results shows, this gene contains a conservative AP2/ERF domain, and this domain has DNA and combines merit
Can, belong to ERF transcription family, possibly together with Pat4 (RKRK) nuclear localization signal, illustrate that it positions cell in cell
In core.
In gene clone, by the arabidopsis AP2/ERF gene reported and Citrus genome database (http: //
Www.citrusgenomedb.org) carry out BLAST (TBLASTN and BLASTP) analysis acquisition CitERF6, CitERF6 to contain
The AP2/ERF domain that one Pat4 (RKRK) nuclear location domain is made up of 60-70 aminoacid, has DNA combined function.
2. allelic expression
Use real-time quantitative PCR (QPCR) technology, using Citrus β-actin (SEQ:NO.7) as internal reference (QPCR primer pair
For SEQ:NO.8 and SEQ:NO.9), utilize primer that SEQ:NO.10 with SEQ:NO.11 is analyzed CitERF6 in different Citrus samples
Expression pattern.Experimental result shows, during after Niu Heer Navel Orange Fruits is adopted, ethylene processes material, compared with air-treatment, through second
It is real that what alkene processed adopts rear hesperidium aurantium, and after 48h, Citrus color index (CCI) substantially rises, and chlorophyll content then significantly reduces.With
Consistent, the expression of CitERF6 is obviously enhanced by exogenous ethylene, and peak expression occurs when processing 4h, first
In the chlorophyllous degraded of peel.
In gene expression analysis, the real-time quantitative PCR detection data acquisition method of relative quantification △ △ Ct, by Niu Heer umbilicus
Orange ethylene process initial point (0h) expression be set to 1, result show compared with air-treatment, through ethylene process adopt after Citrus chachiensis Hort.
After fruit 48h, Citrus color index (CCI) substantially rises, and chlorophyll content then significantly reduces, and the expression of CitERF6 is by ethylene
Induce and be obviously enhanced, and peak expression occurs, prior to the chlorophyllous degraded of peel when processing 4h.
Said gene expression of results explanation CitERF6 take part in the chlorophyllous degradation process of orange peel, controllable Citrus
The removing green of peel.
3. gene function feature
Utilize primer to SEQ:NO.3 and SEQ:NO.4, in conjunction with the opening of pcr clone CitERF6 (SEQ:NO.1)
Reading frame, is carried on pGreen II 0029 62-SK carrier, is built into CitERF6-SK recombinant expression carrier, and by electricity
Striking is conducted in Agrobacterium GV3101::pSoup.With Citrus chachiensis Hort. for examination material, will be containing CitERF6-SK and empty expression vector
Agrobacterium strains be expelled to respectively in orange peel, after cultivating 5 days result display CitERF6 instantaneous excess in hesperidium aurantium skin
During expression, its chlorophyllous degraded can be promoted, accelerate peel removing green, show the rising of CCI value and the reduction of chlorophyll content.
This function of CitERF6 can be applicable in genetic engineering breeding and the regulation and control orange peel removing green of modified plant color and luster.
In gene function analysis, the structure of expression vector is that the open reading frame of CitERF6 is used Roche company Gao Bao
True enzymatic amplification is carried on pGreen II 0029 62-SK carrier, and is conducted into Agrobacterium GV3101: by electric shocking method:
For subsequent experimental in pSoup.
In gene function analysis, apply penetrating fluid (10mM MgCl2, 10mM MES, 150mM acetosyringone, pH be
5.6) OD will be adjusted to containing the agrobacterium strains of CitERF6-SK or empty expression vector600=0.75, with the syringe of band syringe needle
Being expelled to the equatoriat plane opposing parts of same citrusfruit peel respectively, its result display overexpression is after 5 days, and injection contains
After CitERF6 gene bacterium solution, its fruit colour CCI value is higher, and the more inclined yellow of color, chlorophyll content is lower.
The application in other respects of this gene also belongs to the protection category of patent of the present invention.
The present invention utilizes Citrus genome database (http://www.citrusgenomedb.org) information, in conjunction with knob
He Er Citrus sinensis Osbeck is adopted rear ethylene and is processed the change of ERF gene expression amount in material, and instantaneous overexpression in orange peel
Phenotype, draws 1 ERF gene C itERF6 (SEQ:NO.1) relevant to orange peel removing green, for its effect machine of research further
System and fruit coloration technology provide theoretical foundation.
Accompanying drawing explanation
Fig. 1 is that Niu Heer Citrus sinensis Osbeck adopts rear ethylene process CCI and chlorophyll content change and CitERF6 and CitERF8 exists
Expression pattern under its process.Niu Heer Citrus sinensis Osbeck is adopted rear ethylene and is processed CCI (A), chlorophyll content change (B), CitERF6
(SEQ:NO.1) expression pattern (C) and CitERF8 (SEQ:NO.2) under managing at which manage lower expression pattern (D) at which.
LSD represents least significant difference method.
Fig. 2 is that CitERF6 overexpression and CCI and chlorophyll content thereof in hesperidium aurantium skin change.CitERF6 (SEQ:
NO.1) overexpression phenotype (A), CCI (B) and chlorophyll content change (C) in hesperidium aurantium skin.* P < 0.05, * * P <
0.01。
Detailed description of the invention
Below in conjunction with the accompanying drawings and specific embodiment, not have the CitERF8 (SEQ:NO.2) of regulation and control orange peel removing green,
And as a example by CitERF6 (SEQ:NO.1), be further elaborated the present invention, but embodiment is not intended to the protection model of the present invention
Enclose.
Genetic manipulation method conventional in following embodiment is with reference to " Molecular Cloning: A Laboratory guide " (third edition).
Embodiment 1: gene is cloned
Obtain based on Citrus Fructus Citri sinensis and crin graceful joining genes group data base (http://www.citrusgenomedb.org)
CitAP2/ERF gene family member.The gene that may belong to AP2/ERF is the most directly downloaded according to annotation, the most logical
Cross the arabidopsis AP2/ERF gene reported and carry out BLAST (TBLASTN and BLASTP) analysis, both modes are obtained
Sequence utilize AP2/ERF conserved domain to screen one by one, then with CAP3 sequence assembly program (http: //
Pbil.univ-lyon1.fr/cap3.php) repetitive sequence is removed, it is thus achieved that CitERF6 (SEQ:NO.1) and CitERF8 (SEQ:
NO.2)。
In conjunction with primer to SEQ:NO.3 and SEQ:NO.4, and SEQ:NO.5 and SEQ:NO.6, utilize round pcr, expand respectively
Increasing two genes of gained, wherein 30 μ l PCR system are: 10 × Buffer 3 μ l, dNTP 2.4 μ l, up/down trip each 0.6 μ of primer
L, enzyme 0.15 μ l, DEPC-H2O 21.3 μ l, 50mM MgSO41.2 μ l, cDNA 0.75 μ l；PCR program is: 95 DEG C of denaturations
5min；95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 1min, 35 thermal cycles；72 DEG C extend 10min, 4 DEG C of guarantors
Deposit.PCR primer uses order-checking means (Invitrogen) to verify further.
Embodiment 2: gene expression analysis
(1) experimental technique
1. fruit material is collected
After full-bloom stage, the Navel Orange Fruits of 150 days is adopted and was supported fortune laboratory the rear same day, selects size uniform, and Maturity is relatively uniform
Fruit carry out test.Fruit being randomly divided into two groups (often groups 100), often organizes and be averagely positioned over 3 hermetic containers, one group leads to
40μL·L-1Ethylene gas 12h, and another group blowing air 12h, then turn shelf and be positioned over 20 DEG C of environment.Respectively at 0,4,8,12
Sample with 48h.Take 9 fruits every time, be divided into repeat 3 biologys.
2. the mensuration of orange peel color index (CCI): use colour difference meter MiniScan XE Plus (HunterLab, U.S.
State) measure, computing formula is CCI=1000 × a*/L*×b*。
3. measuring chlorophyll content: chlorophyll extracts and uses 80% acetone to extract in three times, every time in 4 DEG C after extracting,
12000 × g is centrifuged 5 minutes Aspirate supernatant, is finally settled to 10ml volume, with itself 663nm and 645nm of spectrophotometric determination
Absorbance, computing formula is: Chl a (mg/ml)=9.78 × A663–0.99×A645；Chl b (mg/mL)=21.4 ×
A645–4.65×A663。
4.RNA extract: orange peel diced after rapid liquid nitrogen freezing, be stored in-80 DEG C standby.When extracting RNA, claim
Take 1g to freeze sample and add after liquid nitrogen is fully ground, join 4ml 65 DEG C preheating CTAB/ beta-mercaptoethanol Extraction buffer from
In heart pipe, vortex mixed makes cell thoroughly rupture, 65 DEG C of heating 3-5min；Then addition 4ml chloroform in centrifuge tube: isoamyl alcohol
(24:1) extract, abundant vortex mixed；15 DEG C of 10000rpm are centrifuged 10min, and Aspirate supernatant, to new centrifuge tube, is taken out again
Carry once；The supernatant obtained carefully is drawn in new centrifuge tube, adds the LiCl of the 10mol/l of 1/4 volume, 4 DEG C of placements
Overnight；Second day, 4 DEG C of 10000rpm were centrifuged 20min, outwell supernatant, and it is unnecessary to be inverted on napkin by centrifuge tube to remove
Solution；Add 65 DEG C of SSTE of 400 μ l, dissolution precipitation；Add 400 μ l chloroforms: isoamyl alcohol (24:1) extract, vortex mixed；
Liquid is thoroughly sucked 20 DEG C of 10000rpm in 1.5ml centrifuge tube and is centrifuged 10min, suct clear liquid and add 2 in new centrifuge tube
The dehydrated alcohol of-20 DEG C of pre-coolings of times volume, mixing of turning upside down, place more than 30min for-80 DEG C；4 DEG C of 10000rpm are centrifuged
25min, outwells supernatant, of short duration centrifugal rear sucking-off residual liquid, will be deposited in fume hood and dry (about 5-10min)；Often pipe adds
Enter 20 μ l DEPC water dissolution precipitations, obtain total serum IgE sample；With 1% agarose gel electrophoresis detection gained sample integrity, knot
Fruit display sample has two bands, and brightness is upper bar: lower bar=2:1, illustrates that RNA sample is complete without degraded；Use uv-spectrophotometric
Meter detection gained sample purity, draw 1 μ l RNA sample with 49 μ l DEPC water dilution after, detection OD260And OD280, analysis obtains
OD260/OD280=1.8-2.0, illustrates RNA purity conformance with standard.According to formula OD260× 40 (RNA concentration factor) × 50 (samples
Extension rate) concentration (ng/ μ l) of sample RNA can be calculated, carry out next step research.
5.cDNA synthesizes
The total serum IgE extracted uses TURBO DNase Kit (Ambion, the U.S.) to remove contaminating genomic DNA, takes 1.0 μ g
RNA presses iScript cDNA Synthesis Kit (Bio-Rad, the U.S.) reverse transcription and becomes cDNA.
6. gene expression analysis: use the online software of Primer3 (http://frodo.wi.mit.edu/primer3/)
Design QPCR primer, using Citrus β-actin (SEQ:NO.7) as internal reference, primer is SEQ:NO.8 and SEQ:NO.9, CitERF6
Primer is SEQ:NO.10 and SEQ:NO.11, and CitERF8 primer is SEQ:NO.12 and SEQ:NO.13.QPCR reaction system is 20
μ l, containing 10 μ l Ssofast EvaGreen Supermix (Bio-Rad, the U.S.), upstream and downstream primer (10 μMs,
Invitrogen) each 1 μ l, 2 μ l cDNA (23 < Ct < 24), 6 μ l DEPC-H2O, each QPCR detection all includes without reaction mould
The negative control of plate.QPCR response procedures is 95 DEG C of 3min；95 DEG C of 10s, 60 DEG C of 30s, 45 circulations；95℃10s；From 65 DEG C to
95 DEG C, often rise 0.5 DEG C and read first order fluorescence signal value.Instrument be CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad,
The U.S.).The data acquisition method of relative quantification △ △ Ct of QPCR detection, processes initial point (0h) by Niu Heer Citrus sinensis Osbeck ethylene
Expression is set to 1.
(2) experimental result
After Niu Heer Citrus sinensis Osbeck is adopted, ethylene processes (40 μ L L-1, 12h, 20 DEG C) and in material, ethylene processes and accelerates fruit
Chlorophyll degradation, 48h fruit colour index CCI and chlorophyll content have respectively reached-8.0 and 59.38g g-1FW, and matched group
It is respectively-10.4 and 108.72g g-1FW (accompanying drawing 1A, B).CitERF8 is not induced by ethylene, expression after ethylene processes
Still keeping relatively uniform with air-treatment, and CitERF6 is to ethylene-sensitive, ethylene is induced about 6.80 times after processing 4h, (accompanying drawing
1C, D), illustrate that it may have regulating effect to orange peel removing green.
Embodiment 3: gene function is verified
(1) experimental technique
1. construction of recombinant vector
According to CitERF6 (SEQ:NO.1) full length sequence design primer (SEQ:NO.3 and the SEQ:NO.4) amplification having verified that
Its open reading frame.With orange peel cDNA as template, changing a little with reference to Roche company high-fidelity enzyme system, preparation is eventually
Volume 30 μ l PCR system: 10 × Buffer 3 μ l, dNTP 2.4 μ l, up/down trip primer 0.6 μ l, enzyme 0.15 μ l, DEPC-H2O
21.3 μ l, 50mM MgSO41.2 μ l, cDNA 0.75 μ l.Reaction condition is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 10sec,
58 DEG C of annealing 5sec, 72 DEG C extend 2.5min, 35 thermal cycles；72 DEG C extend 10min, 4 DEG C of preservations.PCR primer connects after reclaiming
It is connected to pGEM-T easy carrier, recombiant plasmid is imported bacillus coli DH 5 alpha competent cell through cultivating screening positive clone weight
Upgrading grain reconnects on pGreen II 0029 62-SK expression vector after using corresponding restriction enzymes double zyme cutting
Cloning and sequencing.Thin by finally confirming that the expression vector correctly built imports GV3101::pSoup Agrobacterium competence by electric shock
In born of the same parents, bacterium solution is spread evenly across the LB solid training containing 25 μ g/ml gentamycins, 5 μ g/ml tetracyclines and 50 μ g/ml kanamycin
Support screening positive clone on base.With 20% glycerol be stored in-80 DEG C standby.
2. Citrus transient expression
After the glycerol stock depositing in-80 DEG C is inoculated activation, scrape well-grown bacterium colony, use 10mM MgCl2, 10mM
MES (biological buffer), 150mM acetosyringone, pH is the penetrating fluid suspension of 5.6 so that it is OD600It is 0.75.Note with band syringe needle
Penetrating fluid is injected orange peel by emitter, every time in experiment, will design strict negative control, i.e. inject the II Han pGreen
The agrobacterium strains penetrating fluid of 0029 62-SK empty carrier.Liquid is sampled after measuring the CCI of peel bacterium solution permeable areas after injecting 5 days
Nitrogen quick-freezing ,-80 DEG C preserve in case measuring used by chlorophyll content.
(2) experimental result
Overexpression is after 5 days in orange peel for CitERF6 (SEQ:NO.1), and the injection of same fruit is containing CitERF6
After gene bacterium solution, its fruit colour is more yellow relative to injection empty carrier, and chlorophyll degradation is rapider, its physical signs CCI after measured
With chlorophyll content (accompanying drawing 2), after result also demonstrates that overexpression CitERF6, peel chlorophyll content is lower.Summary is tied
Really, show that CitERF6 has the function of regulation and control orange peel removing green.
The present invention utilizes PCR, real-time quantitative PCR, citrusfruit transient expression technology, clones and identifies Citrus ethylene and ring
Answer factor CitERF6 (SEQ:NO.1) controllable chlorophyll degradation, may be used on vegetation color modify genetic engineering breeding and
In regulation and control orange peel removing green.The application in other respects of this gene also belongs to the protection category of patent of the present invention.
Claims (3)
1. the crucial ethylene responses factor of a regulation and control orange peel removing green, it is characterised in that entitled CitERF6, its nucleoside
Acid sequence is as shown in SEQ:NO.1.
The crucial ethylene responses factor of a regulation and control orange peel removing green the most according to claim 1, it is characterised in that obtain
The characterization step obtaining CitERF6 includes:
(1) gene clone: by the arabidopsis AP2/ERF gene reported and Citrus genome database (http: //
Www.citrusgenomedb.org) carrying out BLAST to analyze and obtain CitERF6 and CitERF8, nucleotide sequence is respectively SEQ:
NO.1 and SEQ:NO.2, in conjunction with primer to SEQ:NO.3 and SEQ:NO.4, and SEQ:NO.5 and SEQ:NO.6 expands gained two
Gene, and use order-checking means to verify further；
(2) gene expression analysis: application Real-time quantitative PCR, using Citrus β-actin as internal reference, sequence is SEQ:NO.7,
QPCR primer to for SEQ:NO.8 and SEQ:NO.9, utilize primer to SEQ:NO.10 and SEQ:NO.11, and SEQ:NO.12 and
SEQ:NO.13, analyzes the expression pattern of CitERF6 and CitERF8 in different Citrus sample, and all QPCR primer specificity are through molten
Point curve analysis, gel electrophoresis analysis and QPCR product sequence verification again；
(3) gene function checking: utilize primer to SEQ:NO.3 and SEQ:NO.4, in conjunction with the opening of pcr clone CitERF6
Property reading frame, is carried on pGreen II 0029 62-SK carrier, is built into CitERF6-SK recombinant expression carrier, and pass through
Electric shocking method is conducted in Agrobacterium GV3101::pSoup, with Citrus chachiensis Hort. for examination material, will carry containing CitERF6-SK and null representation
The agrobacterium strains of body is expelled in orange peel respectively, measures the Citrus chachiensis Hort. of two kinds of peel bacterium solution permeable areas after cultivating 5 days respectively
Fructus Citri tangerinae color index and chlorophyll content.
The crucial ethylene responses factor of a regulation and control orange peel removing green the most according to claim 1 is modified at plant color and luster
Genetic engineering breeding in application, especially regulation and control orange peel removing green in application.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709332A (en) * | 2017-10-25 | 2019-05-03 | 中国农业大学 | The application of serine residue number in apple transcription factor ERF17 |
| CN109709332B (en) * | 2017-10-25 | 2020-10-27 | 中国农业大学 | Application of serine residue number in apple transcription factor ERF17 |
| CN112501182A (en) * | 2020-12-07 | 2021-03-16 | 山西农业大学 | Poplar ERF transcription factor gene and application thereof |
| CN113373161A (en) * | 2021-08-03 | 2021-09-10 | 中国农业科学院棉花研究所 | Application of GhERF017 gene in regulating and controlling plant salt tolerance |
| CN113373161B (en) * | 2021-08-03 | 2022-07-08 | 中国农业科学院棉花研究所 | Application of GhERF017 gene in regulating and controlling plant salt tolerance |
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