CN106222182B - The IbERF5 genes of coding sweet potato ERF transcription and application - Google Patents
The IbERF5 genes of coding sweet potato ERF transcription and application Download PDFInfo
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Abstract
Application the invention discloses IbERF5 in sweet potato and its encoding gene and in regulating and controlling plant stress tolerance.The wherein IbERF5 genes of coding sweet potato ERF transcription are nucleotide sequences shown in SEQ ID NO.1.The protein of IbERF5 gene codes is amino acid sequence shown in SEQ ID No.2.The present invention isolates the global cDNA of encoding transcription factors IbERF5 from sweet potato, is connected on plant expression vector, converts plant using Agrobacterium infestation method, obtains transfer-gen plant, and improve the drought tolerance and salt tolerance of genetically modified plants.This gene can be applied to genetic modification of plants.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to a kind of IbERF5 genes of coding sweet potato ERF transcription, should
The degeneration-resistant application of the protein of gene code and recombinant vector and gene containing the gene.
Background technology
It is big to be all broadly divided into two according to the difference of the mode of action by environment stress induced expression for many genes in plant
Class：Adjust gene and functional gene.Adjust gene be mainly the transcription that is played regulatory role in signal transduction and gene expression because
Son；Functional gene main code some adjust osmotic potentials, remove free radical and active oxygen enzyme etc..It is generally believed that plant is to dry
The resistance of the adverse circumstances such as non-irrigated and with high salt is by controlled by multiple genes, therefore, although importing individual feature gene energy using technique for gene engineering
Increase certain single resistance of plant, but the resistance for improving plant can not be integrated on the whole.Transcription factor is as function
The regulating switch of gene expression can accurately be adjusted different genes, be sent out in plant adverse circumstance signal transduction process
Crucial effect is waved, so the effect by enhancing some transcription factor, can promote multiple and degeneration-resistant related functional gene
Expression, this is a very effective approach for making plant stress-resistance character obtain comprehensive improvement.
Plant AP2/ERF is a huge transcription factor gene family, contains what is be made of 60~70 amino acid
AP2/ERF structural domains and gain the name, be present in all plants.AP2/ERF transcription factors participate in various biological process, including
Environment-stress response such as plant growth, flower development, fruit development, seed development, damage, germ defence, with high salt, arid etc..
AP2/ERF class transcription factors participate in the multi-signals transduction pathway such as salicylic acid, jasmonic, ethylene, abscisic acid, and are adverse circumstance letters
Connection exception in number cross-pathway.According to the number of the structural domain containing AP2/ERF, AP2/ERF transcription factors contain 5 subgroups, packet
Include AP2 (APETALA 2), RAV (related to ABI3/VP1), DREB (dehydration-responsive element
Binding protein), ERF and other.AP2 families contain 2 AP2/EREBP (ethylene-responsive element
Binding protein) structural domain, ERF and DREB families contain only 1 AP2/EREBP structural domain, and RAV families remove one
Also contain 1 B3 structural domain outside AP2/EREBP structural domains.ERF families are the main sub- families of one of AP2/ERF transcription factor large families
Race plays a significant role in adjusting plant biological and abiotic stress reaction.Contain respectively in arabidopsis and rice genome
There are 122 and 139 ERF transcription family members, according to the features such as gene order genealogical tree and albumen conserved domain, intends south
Mustard and rice ERF genes separately constitute 12 and 15 different groups.
Sweet potato is not only important cereal crops, but also is important industrial crops and energy crop.Extensive kind of sweet potato
It is implanted in the country of 100 in the world more, China is largest production state in the world, and sweet potato production accounts for about the 80% of world sweet potato.
Compared with other cereal crops, sweet potato is comparatively drought-enduring, but Differences are larger, in the world the sweet potato plantation of significant proportion
In arid environments, and the world is arid, semiarid zone has accounted for the one third of land area or more, shadow of the arid to plant
It rings and ranks first in many natural stress factors.About 2,000,000,000 mu of the existing marginal land resource (bare place) in China is mainly dry
Non-irrigated, saline and alkaline beach.China's agricultural water is in short supply, under cultivated area intense situation, is planted in these regional developments drought-enduring
Sweet potato can reasonably utilize land resource and partly alleviate grain and energy deficiency.Pass through orderly improvement sweet potato
Degeneration-resistant border ability, improve sweet potato marginal land area growth adaptability, to ensure China grain security have it is important
Strategic importance.Therefore new ERF class transcription factor genes are cloned in sweet potato, study its basic biological characteristics and function,
It can provide fundamental basis for entire plant stress-resistance gene regulatory network and stress response reaction mechanism, and be Crop Improvement resistance
Certain material base is provided.
Applicant discloses a kind of coding sweet potato ERF transcription in patent of invention (application No. is 2015102103933)
IbERF4 genes and application, herein study on the basis of, applicant has continued further research, continually looks for sweet
The relevant gene of potato resistance provides basis for sweet potato improvement.
Invention content
The purpose of the present invention is to provide a kind of IbERF5 genes of coding sweet potato ERF transcription.
Second object of the present invention is to provide a kind of protein of the gene code.
Third object of the present invention is to provide a kind of expression vectors containing the gene.
Fourth object of the present invention is to provide a kind of host cell containing the expression vector.
Final object of the present invention is to provide the purposes of the gene.
Technical scheme of the present invention is summarized as follows：
A kind of IbERF5 genes of coding sweet potato ERF transcription, it is nucleotide sequence shown in SEQ ID NO.1.
The nucleotide sequence is by 705 base compositions.
The protein of said gene coding, it is amino acid sequence shown in SEQ ID No.2.The sequence is by 234
A amino acid residue composition.
A kind of expression vector pCAMBIA1305-IbERF5 containing said gene, it contains shown in SEQ ID NO.1
Nucleotide sequence.
One kind containing expression vector Agrobacterium host cell EHA105:The structure of pCAMBIA1305-2 × 35s-IbERF5.
Application of the said gene in conversion plant obtains transfer-gen plant, especially in prepare transgenosis arabidopsis
Using.
The IbERF5 genes have the function of enhancing plant salt endurance and drought tolerance.
Advantages of the present invention：
The present invention isolates the global cDNA of coding ERF transcription gene from sweet potato, is connected to plant expression vector
On, plant is converted using Agrobacterium infestation method, the transfer-gen plant of acquisition has carried out resistance analysis to transfer-gen plant, ties
Fruit shows that IbERF5 genes can enhance the drought-resistance ability and salt resistance ability of positive regulation plant.This gene can be applied to plant
Genetic improvement.
Description of the drawings
The Phylogenetic analysis result of Fig. 1 .IbERF5 amino acid sequences
Fig. 2 .IbERF5 genes arid, salt stress induced expression
Fig. 3 .pCAMBIA1305-2 × 35s-IbERF5 carrier schematic diagrames
The unconverted arabidopsis strains of Fig. 4 and turns IbERF5 gene plant drought resistings and compare
The unconverted arabidopsis strains of Fig. 5 and turns IbERF5 gene plant salt resistances and compare
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But involved specific experiment method is conventional method or according to system unless otherwise specified in following embodiments
The condition for making manufacturers instruction suggestion is implemented.
Embodiment 1
ERF transcription IbERF5 gene orders obtain in sweet potato, specific as follows：
Using OMEGA RNA kits, total serum IgE is extracted from the fresh Sweet Potato Leaf of 100mg, utilizes Reverse Transcription
Box (Bioteke) synthesizes cDNA.
Specific reaction system is as follows：
In PCR instrument reverse transcription reaction is carried out by the following conditions：1,50 DEG C, 45min；2,70 DEG C, 10min；Later on ice
It is cooling.
It send the total serum IgE of extraction to Hua Da gene (Beijing Genomic Institute, BGI), passes through high-flux sequence
Sweet potato transcript profile database is obtained, and sweet potato Unigene databases are obtained by De novo splicings, wherein
CL5071.Contig1_All has found that the gene may participate in stress response after being compared with NR databases.It will
CL5071.Contig1_All passes through OFR FINDER (http://www.ncbi.nlm.nih.gov/projects/gorf/)
The ORF that online software predicted gene is completed, and designed using Primer Premier 5 and expand complete ORF primers：
IbERF5(F):5'ATGGCTTTATCTGAGGAAAG 3'
IbERF5(R):5'TTAAACCCTAATTCCATGAT 3'
It is expanded, is as follows using TAKARA companies Primerstar GXL archaeal dna polymerases：
Reaction condition is as follows：94 DEG C, 4min；98 DEG C, 10Sec；55 DEG C, 15Sec；68 DEG C, l.5min；72 DEG C, 10min；
30 cycles.PCR product is purified using OMEGA DNA purification kits, PCR product after purification is carried with pEASY-Blunt
Body connection (this process uses the pEASY-Blunt Vector Cloning Kit kits of TransGEN), reaction system is such as
Under:The 1 μ L of cDNA segments 4ul, pEASY-Blunt carrier of IbERF5 genes.Reaction condition:20-30 DEG C, 30min.Connection product
Transformed E-Coli.DH5 α competence, is coated on IPTG, 100mg/ of the X-Gal containing 40ul 25mg/ml, 16ul 50mg/ml
It is cultivated on the LB Agar Platings of mL Amp, forms single bacterium colony.White colony is selected, is confirmed using bacterium colony PCR methods
The length scale of Insert Fragment in pEASY-Blunt carriers, it is consistent with expection.The sequencing of Nanjing Jin Sirui biotechnologies company is sent to obtain
Obtain gene order SEQ ID NO.1.
Embodiment 2
IbERF5 protein sequence homogeneous assays, it is specific as follows：
Sequencing obtains cDNA overall lengths 1235bp, ORF 705bp, encodes 234 amino acid SEQ ID NO.2, translation is obtained
It obtains protein sequence and (http is compared with NCBI protein datas://blast.ncbi.nlm.nih.gov/Blast.cgi), it obtains
Obtained plant species homologous gene similar with IbERF5 protein sequences.On the basis of Multiple range test is analyzed, establish each same
The systematic evolution tree of source plant species gene, refers to Fig. 1.Including capsicum (Capsicum annuum, CaEREBP-C4), tomato
(Solanum lycopersicum, SlERF5), carrot (Daucus carota, DcERF1), petunia (Petunia
Hybrida, PhEREBF12), artemisia annua (Artemisia annua), arabidopsis (Arabidopsis thaliana).It utilizes
5.1 softwares of MEGA carry out the structure of systematic evolution tree, and it is closer with capsicum and tomato affiliation to obtain IbERF5 affiliations.
Embodiment 3
Expression (as shown in Figure 2) of the IbERF5 after arid and salt stress, it is specific as follows：
The seedling of clip Soviet Union No. 16 25cm of potato, and retain 3 fully expanded leaves, it is put into 25 DEG C of trainings in 1/2Hogland nutrient solutions
Support 15 days, with 20%PEG6000 processing 12h, for 24 hours, 48h, take 3 repetitions, -80 DEG C of refrigerators be stored in after liquid nitrogen flash freezer.Extract RNA
And it is inverted to cDNA, method is as described in Example 1.Using the fluorescence quantitative kit for being TOYOBO companies.Reaction is in quantitative PCR
It is carried out on instrument (Applied Biosystems stepone plus), the expression quantity of gene is detected according to the method for relative quantification,
Response procedures are carried out according to the operation manual that TOYOBO is provided, and sweet potato Tubulin genes are as the internal reference in reaction, Tubulin
Primer sequence：
F, CAACTACCAGCCACCAACTGT, R, CAAGATCCTCACGAGCTTCAC；
IbERF5 quantifies primer：
F, TTAAGCTGAGAGGCAGCAAA, R, CTTAATCACCGCCACTTCCT；And gene after arid, salt stress
It is expressed by Salt treatment.
Embodiment 4
The structure of binary plant expression vector pCAMBIA1305-IbERF5, it is specific as follows：
PCAMBIA1305-2 × 35s-IbERF5 carriers schematic diagram is as shown in figure 3, first with pEASY-Blunt-IbERF5
Plasmid is template, using primer
IbERF5-PstI(F):5'aaCTGCAG ATGGCTTTATCTGAGGAAAG 3'
IbERF5-BamHI(R):5'ccGGATCC TTAAACCCTAATTCCATGAT 3'
Introduce restriction enzyme site PstI and BamHI respectively before and after IbERF5, institute in reaction system and condition such as embodiment 1
It states.Then PCR product and pCAMBIA1305 empty carriers plasmid use BamHI and PstI double digestions respectively, by the digestion products of the two
Connection, linked system are as follows：
Connection product Transformed E-Coli.DH5 α, be coated on the card of concentration containing 100mg/ml receive chloramphenicol resistance LB tablets on.37
DEG C culture, picking single bacterium colony carries out bacterium colony PCR verifications after 12h, by the positive bacterium of bacterium colony PCR verifications, shakes bacterium extraction plasmid, digestion
Identification obtains purpose band, finally send Jin Sirui sequencing companies to be sequenced, the results showed that carrier pCAMBIA1305-2 × 35s-
IbERF5 structures are correct.
Embodiment 5
Agrobacterium strain EHA105 for plant transgene:The structure of pCAMBIA1305-2 × 35s-IbERF5, specifically
It is as follows：
The agrobacterium strains that the present invention uses are EHA105.The expression vector built is turned using frozen-thawed method
Enter Agrobacterium.Detailed process is：1) ice bath melted EHA105 competent cells, the expression that at least 100ng recovery purifyings are added carry
Constitution grain, gently mixing, 20~30min of ice bath；2) liquid nitrogen flash freezer 5min, 37 DEG C of thermal shock 5min, be immediately placed on ice 1~
2min；3) the LB culture mediums of 800 μ L antibiotic-frees of addition, 28 DEG C, 200rpm recoveries 3.5h；4) 4000rpm centrifuges 3min, sops up
Culture medium；5) mixing residue bacterium solution is applied to addition 100mg/ml cards and receives the solid LB of mycin and 100mg/ml rifampins and trains base
On；6) it is inverted 30~48h of culture for 28 DEG C；7) PCR detects positive colony, and 4 DEG C save backup.
Embodiment 6
IbERF5 converts Col wildtype Arabidopsis thalianas, specific as follows：
Positive colony in embodiment 5 is inoculated into 50ml YEP (containing 100 μ g/ml Rif, 100 μ g/ml Kan) liquid training
It supports in base, 28 DEG C of 180rpm continue culture to OD600=0.8.4000rpm centrifuges 10min, abandons culture medium, collects thalline.With quasi-
Southern mustard penetrates into buffer solution by mycelium dilution OD600=0.6, is prepared into arabidopsis and infects liquid.As arabidopsis bolting 4-5cm
Preparation is infected, and the preceding 3d infected removes its terminal inflorescence, to utilize the growth of axillary inflorescence.After growing axillary inflorescence, under
The flowers are in blossom in portion begins to be converted when pollination.A large amount of waterings before conversion, and the bud pollinated and pod are extractd.It prepares altogether
50ml arabidopsis infects liquid (1/2MS, 5% sucrose, 0.05%SilwetL-77), pours into culture dish, and the inflorescence of arabidopsis is soaked
Enter wherein 30s, notices that lotus throne leaf not be infected with bacterium solution.Plant is taken out, is disposed across in pallet, dark treatment is for 24 hours.It uprightly puts later
Set square plate normal illumination culture.T0 is harvested when fruit pod maturation for seed.
T0 is preceding after 4 DEG C of refrigerator vernalization 3d, 0.5%NaClO surface sterilizations for seed sowing, it is seeded in 1/2MS solids
(a great number of elements, trace element halve, hygromycin containing 20mg/L) is cultivated on culture medium.Transformant is selected after 7~14d, and there is tide
The transformed plant physical efficiency of chloramphenicol resistance is grown on containing hygromycin culture medium, and root obviously extends rather than transformant yellow gradually
It is dead.When plant to be planted grows 4-5 piece leaves, extraction leaf DNA carries out PCR identifications, and primer and PCR programs are the same as in embodiment 1
The clone of segment among IbERF5 genes.1% agarose gel electrophoresis detects PCR product.Purpose piece can be obtained by crossing PCR amplification
Disconnected is arabidopsis positive transformant, is continued culture and waits for that fruit pod maturation harvests T1 for seed.PCR is accredited as to positive plant
The T1 that strain is harvested continues to sow for seed, also passes through hygromycin resistance screening, counts its trait segregation ratio.Through Chi-square Test
Meet 3 in mendel's law:1 segregation ratio.Partial resistance seedling is implanted into soil, the transgenosis kind of harvest T2 generation homozygosis
Son.
(1) it identifies turning IbERF5 gene arabidopsis drought resistances
IbERF5 is taken to be overexpressed arabidopsis strain (OV-1, OV-2) and be sowed at 1/ for wild type (WT) plant point of conversion
One week on 2MS culture mediums, seedling is transplanted to containing vermiculite and Nutrition Soil 2:1 (v/v) is filled to modeling of the same size after mixing
Compost weight is consistent in material culturing pot and in each alms bowl of holding of weighing, and alms bowl face is horizontal and size is consistent.Stop watering after 4 weeks
It carries out drought stress, phenotype, one day observation phenotype of subsequent rehydration is observed after one week (see Fig. 4).It can obtain, in arabidopsis
It is overexpressed the response that IbERF5 can be with positive regulation arabidopsis to arid.
(2) it identifies turning IbERF5 gene arabidopsis salt-resistances
IbERF5 is taken to be overexpressed arabidopsis strain (OV-1, OV-2) and be sowed at 1/ for wild type (WT) plant point of conversion
One week on 2MS culture mediums, seedling is transplanted to containing vermiculite and Nutrition Soil 2:1 (v/v) is filled to modeling of the same size after mixing
Compost weight is consistent in material culturing pot and in each alms bowl of holding of weighing, and alms bowl face is horizontal and size is consistent.After 4 weeks, saturation is poured
The NaCl submerging treatments that 300mM is used after water, observe phenotype, and measure relative chlorophyll content after 10 days (see Fig. 5).It can obtain
Go out, the response that IbERF5 can be with positive regulation arabidopsis to salt is overexpressed in arabidopsis.
Claims (6)
1. a kind of IbERF5 genes of coding sweet potato ERF transcription, which is characterized in that the IbERF5 genes are SEQ ID
Nucleotide sequence shown in NO.1, the IbERF5 genes have the function of enhancing plant salt endurance and drought tolerance.
2. the protein of IbERF5 gene codes described in claim 1, which is characterized in that the protein is SEQ ID No.2
Shown in amino acid sequence.
3. a kind of expression vector pCAMBIA1305-2 × 35s-IbERF5 containing gene described in claim 1.
4. a kind of Agrobacterium host cell EHA105 containing expression vector described in claim 3:pCAMBIA1305-2×35s-
The construction method of IbERF5.
5. application of the expression vector in conversion plant obtains transfer-gen plant described in claim 3.
6. application according to claim 5, which is characterized in that the plant is arabidopsis.
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| 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 |
| CN114134158B (en) * | 2021-12-17 | 2022-10-28 | 广东省科学院南繁种业研究所 | IbDRM gene of purple sweet potato and application thereof |
| CN115927361B (en) * | 2022-03-18 | 2024-05-24 | 华中农业大学 | Gene TgFTS1 related to flowering regulation and petal aging of bulbous flower and application thereof |
| CN114395566B (en) * | 2022-03-28 | 2022-05-24 | 江苏省农业科学院 | Application of sweet potato ERF transcription factor IbERF4 in promoting synthesis of plant chlorogenic acid substances |
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