CN104630228A - Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter - Google Patents
Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter Download PDFInfo
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- CN104630228A CN104630228A CN201510073572.7A CN201510073572A CN104630228A CN 104630228 A CN104630228 A CN 104630228A CN 201510073572 A CN201510073572 A CN 201510073572A CN 104630228 A CN104630228 A CN 104630228A
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Abstract
The invention belongs to the fields of genetic engineering and molecular breeding and discloses a promoter of cotton heat shock transcription factor GhHsf 39 genes and application of the promoter. The nucleotide sequence of the promoter is shown as SEQ ID NO:1. When a plant is subjected to abiotic stress, the expression of target genes can be regulated by pghhsf39. When the pghhsf39 sequence cloned from upland cotton is subjected to different abiotic stress stimulus, the expression quantity of the GhHSF 39 genes can be increased. The pghhsf39 promoter disclosed by the invention is connected with the target genes so as to form an expression cassette, and transgenic plants subjected to adversity stress to regulate the expression of the target genes can be obtained. The invented pghhsf39 provides an important promoter element for researching plant stress resistance and disease resistance. Therefore, the promoter has wide application values.
Description
Technical field
What the present invention relates to is gene promoter of a gene engineering technology field and uses thereof, belongs to biological technical field, is specifically related to one and grows cotton the promotor of thermal excited transcryption factor gene GhHsf39 and application thereof.
Background technology
Majority of plants can not autonomous growth selection condition, therefore needs to adjust various physiological activity according to the envrionment conditions of constantly change, to complete necessary vital process.The expression regulation of gene mainly realizes the change of gene on transcriptional level by promotor, thus realizes growth and development of plants and Metabolism regulation.Promotor is the nucleotide sequence of the one section of specific region being positioned at upstream region of gene, general containing multiple cis-acting elements, these cis-acting elements and various trans-acting factor synergy, rna regulation polysaccharase carries out transcribing of different levels to gene on specific Time and place.
According to the mode that plant materials Natural promoter promotor gene is transcribed, three kinds can be divided into: composing type, tissue or organ Idiotype, induction type.The mode of first two promotor promotor gene is persistence, unqualified, and RNA and protein expression are stable, and therefore this two classes promotor exists certain drawback when utilization.They cannot be special from space-time the expression of regulatory gene, started gene continuous expression in plant materials can be caused, consume the matter and energy of cell excessively.Such as tobacco mosaic virus (TMV) 35S promoter (CaMV35).It can drive foreign gene to express with continual high levels in organ in a organized way in the institute of plant, causes certain harm possibly to the normal growth of plant.
The feature of inducible promoter is the expression only just starting or improve gene when some specific physical chemistry or biostimulation occur, this kind of promotor can fast and effeciently be induced " open and close " of goal gene, can accept inducement signal as required under plant specific etap, histoorgan or growing environment, promotor gene is expressed; Also can remove induction along with the elimination stimulated, stop the expression of gene.Effectively can not only play the effect of gene like this, can not cause again the waste of matter and energy in plant materials, it is significant for plant genetic engineering breeding therefore to clone various different inducible promoter.
At present, in different plant about the report of abduction delivering promotor.But the promotor about being subject to the adverse circumstance abduction deliverings such as high temperature, arid and high salt concentration on cotton is not also reported.This patent is that pghhsf39 promotor cloned by material with cotton, and this promotor can start when cotton plants runs into different abiotic stress or raise the expression of GhHSF39 gene fast.In addition, the present invention establishes the expression cassette of pghhsf39 promotor and reporter gene by molecular biology method, by genetically modified functional analysis, confirm that it can control the expression of different goal gene as effectively start of plant genetic engineering, thus complete the present invention.
Summary of the invention
The object of the present invention is to provide one by the promotor of gene of abiotic stress induction, be specially one and grow cotton the promotor of thermal excited transcryption factor gene GhHsf39 and application thereof, for plant stress-resistance genetic engineering breeding provides support.This promotor has multiple feature: (1) can respond multiple different stress factors, such as high temperature, arid, high salt and high pH; (2) promotor has corresponding feature fast: when cotton is subject to various abiotic stress, and GhHSF39 gene promoter can raise rapidly the expression amount of goal gene; When coercing disappearance, the expression level of goal gene can reduce rapidly again, returns to original level.Because pghhsf39 promotor has quick response, the energy expenditure of plant thus effectively can be reduced.
The present invention is achieved by the following technical solutions,
First aspect, the present invention relates to a kind of Nucleotide pghhsf39, and the sequence of described Nucleotide is as shown in SEQ ID NO.1.
Second aspect, the present invention relates to a pair primer pair for the described Nucleotide pghhsf39 that increases, sequence is respectively as shown in SEQ ID NO.2, SEQ ID NO.3.
The third aspect, the present invention relates to a kind of recombinant vectors, comprises described Nucleotide pghhsf39, as pMD19-T cloning vector, recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP etc.
Fourth aspect, the present invention relates to a kind of transformant, comprises Nucleotide pghhsf39 according to claim 1.
Preferably, the host that described transformant adopts is intestinal bacteria or Agrobacterium.
5th aspect, the present invention relates to the purposes of a kind of described Nucleotide pghhsf39 as promotor; The source of the gene order that described promotor connects can be same species, also can be different plant species.
Preferably, described purposes is specially Nucleotide pghhsf39 as the application in promoter regulation abiotic stress inducible gene expression.
Preferably, described purposes is specially, using Nucleotide pghhsf39 as promotor, and the expression of regulatory gene, and then realize regulating plant resistance or regulation and control resistance against diseases.
Preferably, described purposes is specially the application that Nucleotide pghhsf39 carries out gene expression regulation in abiotic stress, and described application comprises the steps:
Step one, connects goal gene encoding sequence by pghhsf39 promotor and is connected into expression vector, obtaining recombinant vectors;
Step 2, by described recombinant vectors transformation Agrobacterium, obtains Agrobacterium engineering bacteria;
Step 3, transforms target plant by described Agrobacterium engineering bacteria, can realize promotor pghhsf39 regulation and control to genetic expression in abiotic stress.
Preferably, described target plant comprises tobacco.
Preferably, described purposes is specially Nucleotide pghhsf39 as the purposes of promotor in plant breeding.
Preferably, the expression that described purposes is specially regulatory gene is specially the expression amount of regulation and control GhHSF39 gene.
6th aspect, the present invention relates to the preparation method of a kind of described Nucleotide pghhsf39, it is characterized in that, comprise the steps: to clone the promoter region of thermal excited transcryption factor gene GhHsf39 from cotton gene group and transformation of E. coli, screening positive clone carries out DNA sequencing, deduct the sequence of GhHsf39 gene by order-checking gained sequence, obtain the nucleotide sequence of described promotor pghhsf39.
Preferably, described cotton is upland cotton.
The present invention has following beneficial effect: GhHSF39 gene promoter provided by the invention, can start rapidly the expression of goal gene when plant is subject to abiotic stress; When environment stress disappears, the expression amount of goal gene can recover rapidly again, therefore substantially can not affect the whole growth and development process of plant, can be widely used in plant stress-resistance genetic engineering breeding.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the electrophorogram of the clone of pghhsf39 promotor;
Wherein, 1 is the pcr amplification product that cotton thermal excited transcryption factor GhHSF39 gene promoter is cloned;
Fig. 2 is cotton when being subject to different abiotic stress, and the difference of GhHSF39 gene under promotor pghhsf39 regulation and control expresses change;
Fig. 3 is expression after the expression of GhHSF39-YFP in the tobacco of instantaneous conversion recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP and heat shock;
Wherein, A: before heat shock, details in a play not acted out on stage, but told through dialogues; B: before heat shock, light field; C: before heat shock, dark bright merging; D: after heat shock, details in a play not acted out on stage, but told through dialogues; E: after heat shock, light field; F: after heat shock, dark bright merging.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
The experimental technique of unreceipted actual conditions in following embodiment, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual is shown in the condition described in versions in 1989 of New York:Cold Spring Harbor Laboratory Press, or according to the condition that manufacturer advises.
The Agrobacterium EHA105 that the present invention relates to is at " Huang Yali, Jiang Xiliang, Yunlong, field, Guo Ping, Zhu Changxiong; The research of agriculture bacillus mediated trichoderma harzianum genetic transformation, Chinese biological engineering magazine, 2008,28 (3): 38-43 " open in document; Plasmid vector pEarleyGate101 obtains by openly commercially available commercial channel.
the promotor Cloning and sequence analysis of embodiment 1 thermal excited transcryption factor GhHSF39 gene
(1) pcr amplification object fragment
According to the primers of the Lei Mengde cotton of having announced (Gossypium raimondii) thermal excited transcryption factor GhHSF39 gene promoter area, amplified production size is 1711bp.
GhHSF39-pro F(SEQ ID NO.2)：5’-TAAAAATAAAATTCGGTTCGAGTAAATG-3’
GhHSF39-pro R(SEQ ID NO.3)：5’-AGTCAAGAACGGCGGCGGACCAACCTCG-3’
Extract upland cotton (Gossypium hirsutum) gene DNA, and be diluted to 100ng/ μ l as template, utilize above-mentioned primer to carry out pcr amplification.
(2) object fragment recovery and build intermediate carrier
Reclaim target DNA fragment (Fig. 1) by agarose gel electrophoresis, employing health is that the quick sepharose DNA of ShiJi Co., Ltd reclaims test kit, and concrete operation step is shown in test kit specification sheets.
As shown in Figure 1, Marker:DL2000DNA Marker; The pcr amplification product of 1: cotton thermal excited transcryption factor GhHSF39 gene promoter clone.
Be connected with the pMD19-T cloning vector of TaKaRa company by the target DNA fragment reclaimed, concrete operation step is shown in test kit specification sheets.By above-mentioned connection product conversion competent escherichia coli cell DH5 α, even spread, on the LB culture medium flat plate containing Pyocianil, is inverted for 37 DEG C and is cultivated 16-20 hour.Bacterium colony on picking flat board, shakes bacterium and cultivates, get bacterium liquid and do template and carry out PCR positive identification.
(3) sequence verification
Through the positive colony of qualification, deliver bacterium liquid and carry out DNA sequencing to Shanghai Bo Shang Bioisystech Co., Ltd, gained sequence is deducted the part belonging to GhHSF39 gene, obtain promoter sequence, its nucleotide sequence as shown in SEQ ID NO.1, called after pghhsf39.
the GhHSF39 gene of embodiment 2:pghhsf39 promoters driven in cotton by expression during Different stress
analyze
The environment stress process of 2.1 cotton seedlings and RNA extracting
(1) different adverse circumstance process cotton seedling
Get the upland cotton Coker312 of five leaf phases, with 42 DEG C of environmental simulation heat stresses, process whole plant one hour, then recover four hours at 30 DEG C, time segment collects blade; With MS substratum, 100mM sodium chloride solution Drought stress simulation, alkaline stress, salt stress respectively that 200mM mannitol solution, pH are 8.8, continue process three hours, time segment collects root.The material of all collections puts into Liquid nitrogen storage at once, for the extracting of RNA.
(2) extracting above-mentioned materials RNA
Get the organization material of control group and various different experiments group, adopt the polysaccharide polyphenol plant total RNA extraction reagent box of TIANGEN Biotech (Beijing) Co., Ltd. to extract RNA, concrete operation step is shown in test kit specification sheets.
The synthesis of 2.2 1 chain cDNA and quantitative fluorescent PCR analysis
(1) by RNA reverse transcription be the cDNA of a chain
Adopt the PrimeScript RT reagent Kit with gDNA Eraser Reverse Transcription box of TaKaRa company RNA to be inverted to the cDNA of a chain, concrete operation step is shown in test kit specification sheets.
(2) quantitative fluorescent PCR analysis
Encoding sequence (coding sequence, CDS) according to GhHSF39 gene designs Auele Specific Primer for quantitative fluorescent PCR, and sequence is as follows:
GhHSF39RT F(SEQ ID NO.4)：5’-GCCTTTACTCAATGAGCACCAGATGATG-3’
GhHSF39RT R(SEQ ID NO.5)：5’-TTAATTGAGCAGCAGCTGTTGACAGACTAGA-3’
Adopt ubiquitin gene as internal reference, according to ubiquitin gene order design Auele Specific Primer, sequence is as follows:
ubiquitin F(SEQ ID NO.6)：5’-CCAGAAGGAATCCACTTTGC-3’
ubiquitin R(SEQ ID NO.7)：5’-CCAGCTCACATCAGCATACG-3’
Employing TaKaRa company
premix Ex TaqTM II test kit carries out quantitative fluorescent PCR, and concrete operation step is shown in test kit specification sheets.Use EXCEL software to carry out data analysis, analyzing employing method is 2 (-Δ Δ Ct).
Result shows, as Fig. 2, after heat-shock stress one hour, under the control of promotor pghhsf39, the expression amount of GhHSF39 gene improves more than 400 times, envrionment temperature recover normal after, expression amount be reduced to heat shock rapidly before level; Under drought stress and alkaline stress, the expression amount of GhHSF39 gene is passed in time and is improved gradually, but increase rate is only had an appointment, 1-3 doubly; When plant is subject to salt stress, the expression level of GhHSF39 presents the situation of falling after rising, and process to 1 constantly little, expression amount rises gradually, and the little expression amount constantly of 1-3 declines gradually.These expression changes of gene GhHSF39 illustrate that promotor pghhsf39 is stress induced, particularly have strong reaction to heat stress, and after heat stress disappears, can reduce rapidly the expression amount of gene.
embodiment 3: recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP builds and tobacco wink
time transformation assay
3.1 recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP build
(1) the CDS sequence of promotor pghhsf39 and gene GhHSF39 is connected
According to the CDS sequence of promotor pghhsf39 and gene GhHSF39, design primer:
pghhsf39FStuⅠ(SEQ ID NO.8)5’-AAAAGGCCTTTTTAAAAATAAAATTCGGTTCGAGTAAATG-3’
pghhsf39R(SEQ ID NO.9)5’-CCGGAATTCCGGTTGAGACTTCGTTTTCACTTCAC-3’
GhHSF39CDS F(SEQ ID NO.10)5’-CCGGAATTCCGGATGGAAGGAGTTGTAGTAAAAGAAGAG-3’
GhHSF39CDS R SpeⅠ(SEQ ID NO.11)5’-CGGACTAGTCCGTGGATTTGACCTGAGATAACCC-3’
GhHSF39CDS F, GhHSF39CDS R Spe I is used to clone out by the CDS of gene GhHSF39 from cotton cDNA, concrete grammar reference example 1.Then utilize overlapping PCR method, use primer pghhsf39F Stu I, pghhsf39R, GhHSF39F, GhHSF39R Spe I, connects the CDS of pghhsf39 and GhHSF39, obtains the PCR primer of two ends with the pghhsf39-GhHSF39 of restriction enzyme site Stu I and Spe I.
(2) digestion with restriction enzyme is used
Electrophoresis is carried out to PCR primer, reclaims purifying object fragment.Carry out enzyme with Stu I and Spe I to recovery fragment to cut, carry out enzyme with Stu I and Avr II couple of plant expression vector pEarleygate101 and cut, reclaim digestion products.
(3) ligase enzyme is used to connect above-mentioned digestion products
Use T4 ligase enzyme to be connected by above-mentioned digestion products, obtain recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP, the gene expression product of this carrier is with yellow fluorescence protein label.
3.2 instantaneous conversion tobacco and heat shock analysis
(1) by recombinant vectors transformation Agrobacterium
With freeze-thaw method, recombinant vectors pEarleygate101-pghhsf39::GhHSF39-YFP is transformed in Agrobacterium EHA105, the Agrobacterium mono-clonal on picking flat board, is inoculated in 1ml liquid resistance culture base, cultivate 24 hours in 28 DEG C of shaking tables.
(2) instantaneous conversion tobacco
Bacterium liquid is transferred to (AS of MES and 40uM containing 10mM) in 10ml liquid nutrient medium, cultivates 16 hours in 28 DEG C of shaking tables.Centrifugal bacterium liquid collects thalline, with the MgCl of 10mM
2the resuspended thalline of solution, to OD600=1, finally adds AS and is 200uM to final concentration and leaves standstill 3 hours.Get tobacco in three week age, be close to tobacco blade back with the syringe of removing syringe needle and above-mentioned bacterium liquid is expelled in mesophyll.
(3) expression conditions and Heat thermostability is observed
Above-mentioned tobacco light culture is after three days, and clip fritter blade, observes expression conditions under laser confocal microscope.Be placed on by tobacco plant in 42 DEG C of environment, heat shock one hour, uses confocal laser scanning microscope expression conditions, afterwards at once again as Fig. 3.Above-mentioned tobacco light culture is after three days, and clip fritter blade, observes expression conditions under laser confocal microscope.Be placed on by tobacco plant in 42 DEG C of environment, heat shock one hour, uses confocal laser scanning microscope expression conditions afterwards at once again.As shown in the figure: after heat shock, gene expression amount is apparently higher than before heat shock, illustrates when adverse circumstance stimulates, promotor pghhsf39 really can the expression amount of up-regulated gene.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a Nucleotide pghhsf39, is characterized in that, the sequence of described Nucleotide is as shown in SEQ ID NO.1.
2., for the primer pair of the Nucleotide pghhsf39 as claimed in claim 1 of increasing, it is characterized in that, the sequence of described primer pair is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.
3. a recombinant vectors, is characterized in that, comprises Nucleotide pghhsf39 according to claim 1.
4. a transformant, is characterized in that, comprises Nucleotide pghhsf39 according to claim 1.
5. a Nucleotide pghhsf39 as claimed in claim 1 is as the purposes of promotor.
6. purposes as claimed in claim 5, it is characterized in that, described purposes is specially Nucleotide pghhsf39 as the application in promoter regulation abiotic stress inducible gene expression.
7. purposes as claimed in claim 6, it is characterized in that, described purposes is specially, using Nucleotide pghhsf39 as promotor, the expression of regulatory gene, and then realize regulating plant resistance or regulation and control resistance against diseases.
8. purposes as claimed in claim 6, it is characterized in that, described purposes is specially the application that Nucleotide pghhsf39 carries out gene expression regulation in abiotic stress, and described application comprises the steps:
Step one, connects goal gene encoding sequence by pghhsf39 promotor and is connected into expression vector, obtaining recombinant vectors;
Step 2, by described recombinant vectors transformation Agrobacterium, obtains Agrobacterium engineering bacteria;
Step 3, transforms target plant by described Agrobacterium engineering bacteria, can realize promotor pghhsf39 regulation and control to genetic expression in abiotic stress.
9. purposes as claimed in claim 6, it is characterized in that, described purposes is specially Nucleotide pghhsf39 as the purposes of promotor in plant breeding.
10. prepare the method for Nucleotide pghhsf39 as claimed in claim 1 for one kind, it is characterized in that, comprise the steps: to clone the promoter region of thermal excited transcryption factor gene GhHsf39 from cotton gene group and transformation of E. coli, screening positive clone carries out DNA sequencing, deduct the sequence of GhHsf39 gene by order-checking gained sequence, obtain the nucleotide sequence of described promotor pghhsf39.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112481268A (en) * | 2021-01-25 | 2021-03-12 | 河南大学 | Cotton promoter PGhPGFRecombinant vector and application thereof |
| CN113151349A (en) * | 2020-10-20 | 2021-07-23 | 中国农业科学院生物技术研究所 | Stress-resistant functional system AcSeDcdw for improving biological salt-resistant and drought-resistant performances and application thereof |
| WO2022082864A1 (en) * | 2020-10-20 | 2022-04-28 | 隆平生物技术(海南)有限公司 | Efficient stress-resistant module sydcw for intelligently responding to stress signal and use thereof in crop breeding |
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| CN102199610A (en) * | 2011-03-30 | 2011-09-28 | 上海市农业生物基因中心 | Application of OsHSF01 (oryza sativa heat stress transcription factors 01) gene |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151349A (en) * | 2020-10-20 | 2021-07-23 | 中国农业科学院生物技术研究所 | Stress-resistant functional system AcSeDcdw for improving biological salt-resistant and drought-resistant performances and application thereof |
| WO2022082865A1 (en) * | 2020-10-20 | 2022-04-28 | 隆平生物技术(海南)有限公司 | Stress-resistant functional system acsedcdw for improving biological salt tolerance and drought resistance performance and use thereof |
| WO2022082864A1 (en) * | 2020-10-20 | 2022-04-28 | 隆平生物技术(海南)有限公司 | Efficient stress-resistant module sydcw for intelligently responding to stress signal and use thereof in crop breeding |
| CN113151349B (en) * | 2020-10-20 | 2023-03-07 | 中国农业科学院生物技术研究所 | Stress-resistant functional system AcSeDcDw for improving biological salt-resistant and drought-resistant performances and application thereof |
| CN112481268A (en) * | 2021-01-25 | 2021-03-12 | 河南大学 | Cotton promoter PGhPGFRecombinant vector and application thereof |
| CN112481268B (en) * | 2021-01-25 | 2024-01-30 | 河南大学 | Cotton promoter P GhPGF And recombinant vector and application thereof |
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