CN110229930A - A kind of screening technique of banana oil channel protein gene promoter transcription factor - Google Patents
A kind of screening technique of banana oil channel protein gene promoter transcription factor Download PDFInfo
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Abstract
The invention discloses a kind of screening techniques of banana oil channel protein gene promoter transcription factor, include the following steps, judge the nucleus of promoter, it is expanded according to the primer of banana A genome design promoter, amplification length is 1362bp, its cis-acting elements is analyzed by plantcare and PLACE, carry out the active detection of promoter GUS, the nucleus B of promoter is judged by staining conditions, the transcription factor of yeast one-hybrid screening and promoter interaction, it is verified with the method for yeast one-hybrid, bait carrier is constructed using promoter nucleus B as bait section, screen the banana single crosses library of yeast Osmotic treatment.The present invention identifies under drought stress conditions, with MaPIP1；The transcription factor that 1 promoter is bound directly, the research of more convenient pair of banana water channel protein gene facilitate the drought-resistant ability for promoting transgenic banana, help to further understand MaPIP1；1 drought resisting mechanism.
Description
Technical field
The present invention relates to promoter transcription factor technique fields more particularly to a kind of banana water channel protein gene to start
The screening technique of sub- transcription factor.
Background technique
Banana plant early period is smaller, and root system is shallowly given birth to, and vulnerable to drought, leaf area is big, and transpiration rate is big, especially in Gao Wenji
Section, after banana is by arid, the serious dehydration of blade causes the Permeability of Leaf of banana to increase, greatly injury banana
Normal physiological metabolism activity, causes the banana underproduction and quality decline.In banana cultivation production, water consumption is big, every production 500
Gram dry matter, needs to absorb 300 kilograms of moisture.Banana meets arid in vegetative growth phase, and banana nutrition organs can be made raw
Long depauperation, the speed of growth and increment are remarkably decreased, and when suffering from drought serious, blade is sagging, withered and yellow wilting, and stomata is closed, light
Closing efficiency reduces；Drought is met before bud differentiation, nutrition organs can be made early ageing occur, nutrient accumulation is few, and bud differentiation is by shadow
It rings, the comb number and single fruit number of fruit significantly reduce, to cause the underproduction.Aquaporin is a kind of is located on various cell membranes
Small molecule transmembrane protein, moisture and the transport of other small-molecule substances can be adjusted, plant growth and development and adapt to it is inverse
It plays an important role in the stress procedure of border.Screening for banana oil channel protein gene promoter transcription factor, is related to
Success prepares the good transgenic banana of drought resistance, so now proposing a kind of banana oil channel protein gene promoter
The screening technique of transcription factor.
Summary of the invention
Technical problems based on background technology, the invention proposes a kind of banana oil channel protein gene promoters
The screening technique of transcription factor.
A kind of screening technique of banana oil channel protein gene promoter transcription factor proposed by the present invention, including it is following
Step:
S1: judging the nucleus of promoter, is expanded according to the primer of banana A genome design promoter, amplification
Length is 1362bp, analyzes its cis-acting elements by plantcare and PLACE, carries out the active detection of promoter GUS,
The nucleus B of promoter is judged by staining conditions；
S2: the transcription factor of yeast one-hybrid screening and promoter interaction is verified with the method for yeast one-hybrid,
Bait carrier is constructed using promoter nucleus B as bait section, screens the banana single crosses library of yeast Osmotic treatment,
The determining binding protein with the interaction of nucleus B sequence, obtains rate of rotation factor C information；
S3: the further interaction of verifying rate of rotation factor C and nucleus B, qRT-PCR detect drought stress processing
Under binding protein, obtain can cooperate with nucleus 1 B gene response drought stress binding protein D；
S4: further proving that rate of rotation factor C in conjunction with the promoter region of nucleus B, carries out external interaction verifying,
The measurement of Dual-Luciferase is carried out.
Preferably, to convert the transgenic arabidopsis of CaMV35S promoter as control in the S1, to 100 ㎜, 200
The blade and root system of 15 days transgenic arabidopsis seedlings of ㎜, 300 ㎜ 18%PEG 6000 processing carry out promoter GUS respectively
Active detection.
Preferably, in the S3, five leaves banana seedlings wholeheartedly are subjected to Osmotic treatment, using it as template, to screening
Binding protein qRT-PCR detection is carried out under drought stress conditions.
Having the beneficial effect that in the present invention
This method is by the method for yeast one-hybrid, and Direct Identification goes out under drought stress conditions in plant for the first time, with
MaPIP1；The transcription factor that 1 promoter is bound directly, the research of more convenient pair of banana water channel protein gene help to mention
The drought-resistant ability for rising transgenic banana, helps to further understand MaPIP1；1 drought resisting mechanism.
Detailed description of the invention
Fig. 1 is a kind of stream of the screening technique of banana oil channel protein gene promoter transcription factor proposed by the present invention
Journey schematic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Referring to Fig.1, a kind of screening technique of banana oil channel protein gene promoter transcription factor, comprising the following steps:
1, the clone of promoter and component analysis
It is expanded according to the primer of banana A genome website design promoter, amplification length 1362bp.Pass through
Plantcare and PLACE analyzes its cis-acting elements.
72 cis-acting elements are shared in the promoter sequence of 1362bp as the result is shown, include many TATA-
Box and CAAT-box core element, including ABA response element ABRE, MYB element, MYC element, ERE element, MeJA response element
Part, the light response elements such as BOX II, G-box, GT1-motif, I-BOX, separate living tissue response element etc..
2, promoter partitioned representation
For identification of M aPIP1；1 (nucleus B) promoter responds the nucleus of drought stress, according to promoter sequence
Column functional element, by 5 ' end missing technologies, by MaPIP1；1 (nucleus B) promoter be segmented into M-P1, M-P2, M-P3,
M-P4, the length of clone is respectively 1362bp, 1274bp, 813bp, 223bp, through digestion, sequencing, with MaPIP1；1 (core
Region B) each deletion fragment size of gene promoter is consistent, and the 35S they replaced respectively on 1304 carrier of pcambia is opened
Mover and arabidopsis thaliana transformation.
The results show that the promoter of M-P1-M-P4 can start GUS enzymatic activity under normal operation can dye face
Color, and there is also certain differences between each segment.The further progress GUS Enzyme activity assay in these segments, M-P1 and
GUS activity of the GUS activity of M-P2 compared with M-P3 and M-P4 is high, wherein the GUS activity highest of M-P2.
3, expression of the 5 ' end deletion fragment transgenic arabidopsis under drought stress
Early period the experiment proves that MaPIP1；1 (nucleus B) transgenic arabidopsis is able to respond drought stress, however
The cis-acting elements that the known arid of discovery or salt stress do not induce in the component analysis 1362bp promoter element,
In order to further study MaPIP1；1 (nucleus B) is started in the regulatory mechanism of response drought stress with converting CaMV35S
The transgenic arabidopsis of son is as control, and to 100mM, 15 days transgenosis of 200mM, 300mM 18%PEG 6000 processing are quasi-
The blade and root system of southern mustard seedling carry out the active detection of promoter GUS respectively, from phenotype, in GUS dyeing, in addition to
CaMV35S adjoining tree, the promoter rotaring gene plant blade of each segment are unstained or dye very shallowly (whether prompt starts
Contain root system specifically expressing element in son), and in transgenosis root system, at tip of a root position, coloring is deeper, wherein -1274 to -1 area
The transgenic arabidopsis dyeing of section (M-P2) is most deep, and in -813 to -1 (M-P3) section, lighter, the dyeing of remaining segment
It is close, in order to more accurately determine the difference of each segment blade and root system GUS expression under drought stress, determine GUS enzyme activity
Property.
The results show that M-P2 (- 1274 to -1) shows higher enzymatic activity under Stress treatment, when concentration is 300mM
When, enzymatic activity 89, be control 1.48 times, should the result shows that, function element relevant to arid osmotic stress may include
Between M-P2 and M-P3, in drought stress processing, the enzymatic activity of CaMV35S transgenic arabidopsis be it is stable, when dry
Non-irrigated stress concentration be 300mM when, the GUS activity of M-P2 segment is 0.77 times of CaMV35S, be higher than normal condition under M-P2 and
The ratio of CaMV35S, it means that high-caliber gene expression and salinity or the infiltration side of body may be implemented in M-P2 (- 1274 to -1)
Compel induction.The result shows that M-P2 (- 1274 to -1) is the nucleus of the promoter, which contains many nucleus
Element CAAT-box and TATA-box.
4, yeast one-hybrid screening and MaPIP1；The transcription factor of 1 (nucleus B) promoter interaction
In order to further analyze MaPIP1；1 (nucleus B) responds the molecular mechanism of drought stress, miscellaneous using yeast list
The method of friendship is verified, and is constructed bait carrier for promoter nucleus M-P2 as bait section, is screened at yeast arid
The banana cDNA single crosses library of reason, the determining albumen with the interaction of nucleus sequence.
3AT is the competitive inhibitor of yeast HIS3 albumen synthesis, for inhibiting the leakage of His3 gene to express, by certainly
Activation testing result is found out, on the SD-TLH plate of addition 25mM, 50mM, MaMaPIP1；1 (nucleus B) transformant is raw
Length is all obviously inhibited by different degrees of, and growth ratio is less than the growth ratio of negative control, shows un-activation HIS3
Reporter gene, therefore carry out yeast one-hybrid on the basis of 50mM 3AT and sieve library.
With contain correct pHIS2-MaPIP1；The Y187 yeast transformant of 1 (nucleus B) bait plasmid is as receptor
Bacterium prepares competence, and Library plasmid is transferred to wherein, SD-Trp-Leu-His+50mM 3AT plate is applied, records transformation efficiency
Are as follows: Total number oftransformants=(1376/20+172/2+25/0.2) × 1/3 × 8000=7.46 ×
105, according to the transformant quantity of efficiency plate, calculate sieve library efficiency are as follows: Transformation efficiency=7.46
× 105/25ug=2.98 × 104/ug.
The detection of positive colony His reporter gene, the 33 initial positive colonies that will be grown on SD-TL deficiency plate
Contact plate is to SD-TL and SD-TLH+150mM 3AT defect plate after transformant is diluted with sterile water respectively, it can be seen that positive
To impinge upon do not add 3AT and add in the screening flat board of 3AT can normal growth, and negative control is due to that will not activate HIS3
Reporter gene, therefore cannot grow in the screening flat board for lacking histidine and adding 3AT or undergrowth, therefore,
In 33 initial positive colonies, can addition 3AT screening flat board on it is eugonic be to have activated HIS3 reporter gene.
Bacterium colony is carried out to further revolution verifying in the screening flat board of addition 3AT.
There are 29 bacterium colonies that can activate HIS3 reporter gene to some extent as the result is shown, this 29 positive bacterias are dropped into row
DNA sequencing and blast compare analysis, obtain 23 different and MaPIP1；The albumen of 1 (nucleus B) interaction,
Verifying further is turned round to this 23 different albumen further progresss.
23 kinds of positive colonies can all pass through HIS3 reporter gene as the result is shown.These functional proteins include beta-
Amylase (BMY) gene, acidic chitinase gene, pectate lyase 2 (PL2), neutral
Ceramidase, glucan endo-1,3-beta-glucosidase, V-type proton ATPase subunit d2,
Germin-like protein, KH domain-containing protein, heat shock cognate 70kDa
Protein, 3'-N-debenzoyl-2'-deoxytaxol N-benzoyltransferase, tubulin alpha-
3chain, alpha-1,4glucan phosphorylase L isozyme, pyrrolidone-carboxylate
Peptidase, ADP-ribosylation factor, pyruvate kinase, levodione reductase, MADS
Box protein (MADS3) etc..
5, qRT-PCR detects the binding protein under drought stress processing
The banana seedlings of five leaves wholeheartedly carry out Osmotic treatment, using it as template, to 23 binding proteins screened dry
QRT-PCR detection is carried out under non-irrigated stress conditions, the results show that 23 genes are in perfume (or spice) under different degrees of drought stress conditions
Expression pattern in any of several broadleaf plants blade and root system is approximately as having 10 in the gene for expression pattern of falling after rising in blade, present
The gene for rising expression pattern has 7, and the gene in decline expression pattern has 4, has 2 in the gene for rising expression pattern afterwards is first dropped
It is a, there are 9 in the gene for expression pattern of falling after rising in root system, there are 5 in the gene for rising expression pattern, is in the lower petition of surrender
The gene of expression patterns has 4, has 2 in the gene for rising expression pattern afterwards is first dropped, the lifting unconspicuous gene of expression trend has 3
A, according to research purpose, in conjunction with expression pattern of the gene in Leaf of banana and root system, we pick one of transcription
Factor M ADS3 (transcription factor C) is conducted further research, inducing expression of the MADS3 (transcription factor C) by drought stress,
In blade, when drought stress soil moisture be 45% when, MADS3 (transcription factor C) gene Leaf of banana expression quantity most
Greatly, and in root system, expression trend of MADS3 (transcription factor C) gene under drought stress is similar in blade, this
As a result MaPIP1 can be cooperateed with by further demonstrating MADS3 (transcription factor C) transcription factor；1 (nucleus B) gene response
Drought stress.
6, the external interaction detection of Dual-Luciferase
In order to further prove MADS3 (transcription factor C) and MaPIP1；The promoter region of 1 (nucleus B) combines,
External interaction verifying is carried out, the measurement of Dual-Luciferase has been carried out, by MaPIP1；1 (nucleus B) promoter M-P2 segment
It is building up to 0800 carrier of reporter vector pGreen II and is named as pMaPIP1；1 (nucleus B):: LUC will turn
Record factor M ADS3 (transcription factor C) overall length ORF, which is building up on 1301 carrier of effector vector pCambia, to be named as
35S::MADS3 (transcription factor C) is transformed into GV3101 Agrobacterium and expresses in tobacco leaf, as a result as shown
pMaPIP1；1 (nucleus B):: the relative fluorescence element activity of LUC is than 35S::MADS3 (transcription factor C)-pMaPIP1；1
(nucleus B):: LUC is low, illustrates that MADS3 (transcription factor C) can be with MaPIP1 in banana；1 (nucleus B) is opened
Mover combination positive regulation MaPIP1；The transcription of 1 (nucleus B).
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited to
This, anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention
And its inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (3)
1. a kind of screening technique of banana oil channel protein gene promoter transcription factor, which comprises the following steps:
S1: judging the nucleus of promoter, is expanded according to the primer of banana A genome design promoter, amplification length
For 1362bp, its cis-acting elements is analyzed by plantcare and PLACE, the active detection of promoter GUS is carried out, passes through
Staining conditions judge the nucleus B of promoter；
S2: the transcription factor of yeast one-hybrid screening and promoter interaction is verified with the method for yeast one-hybrid, will be started
Sub- nucleus B constructs bait carrier as bait section, screens the banana single crosses library of yeast Osmotic treatment, determining and core
The binding protein of heart district domain B sequence interaction, obtains rate of rotation factor C information；
S3: the further interaction of verifying rate of rotation factor C and nucleus B, qRT-PCR detect the knot under drought stress processing
Hop protein obtains the binding protein D that can cooperate with nucleus 1 B gene response drought stress；
S4: further prove that rate of rotation factor C in conjunction with the promoter region of nucleus B, carries out external interaction verifying, carries out
The measurement of Dual-Luciferase.
2. a kind of screening technique of banana oil channel protein gene promoter transcription factor according to claim 1, special
Sign is, to convert the transgenic arabidopsis of CaMV35S promoter as control in the S1, to 100 ㎜, 200 ㎜, 300 ㎜
The blade and root system of 15 days transgenic arabidopsis seedlings of the processing of 18%PEG 6000 carry out the active inspection of promoter GUS respectively
It surveys.
3. a kind of screening technique of banana oil channel protein gene promoter transcription factor according to claim 1, special
Sign is, in the S3, the banana seedlings of five leaves wholeheartedly is carried out Osmotic treatment, using it as template, to the combination egg screened
It is white that qRT-PCR detection is carried out under drought stress conditions.
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| CN114107363A (en) * | 2021-11-24 | 2022-03-01 | 南京瑞源生物技术有限公司 | Method for screening motif interacting with transcription factor A |
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| CN104313026A (en) * | 2014-09-23 | 2015-01-28 | 中国热带农业科学院海口实验站 | Banana aquaporin gene promoter and applications thereof |
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