CN104829700A - Corn CCCH-type zinc finger protein, and encoding gene ZmC3H54 and application thereof - Google Patents
Corn CCCH-type zinc finger protein, and encoding gene ZmC3H54 and application thereof Download PDFInfo
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Abstract
The invention discloses a corn CCCH-type zinc finger protein, and an encoding gene ZmC3H54 and an application thereof. An amino acid sequence of the corn CCCH-type zinc finger protein is as shown in SEQ ID NO.1 in a sequence table. The nucleotide acid sequence of the encoding gene ZmC3H54 of the corn CCCH-type zinc finger protein disclosed by the invention is as shown in SEQ ID NO.2 in the sequence table. The invention further discloses a recombinant carrier, a recombinant bacterium, a transgenic cell line or an expression cassette containing the encoding gene ZmC3H54. The ZmC3H54 gene disclosed by the invention participates into stress response, so that the resistance to abiotic adversity stress of a plant can be improved; a transgenic plant has relatively high sensitivity on ABA; the tolerance of transgenic rice on drought is improved; the family gene is separated and cloned from corn; and the developing function of the family gene in the aspect of improving the stress resistance of a target plant is identified.
Description
Technical field
The invention belongs to field of crop genetic breeding, be specifically related to a kind of corn C CCH type zinc finger protein and encoding gene ZmC3H54 thereof and application.
Background technology
Corn is important food crop and important feed resource, is also the food crop that whole world ultimate production is the highest.Corn is the kind most suitable as industrial raw material in Three major grain crops, occupies an important position in national economy.But the abiotic stress such as arid, high salt have a strong impact on the output of corn, and therefore excavate and create strong stress resistance, high yield, high-grade maize new germ plasm and seed selection new variety have become the key point realizing Maize Production sustainable development.Along with the fast development of molecular biology and plant genetic engineering breeding technique, improve the productivity of corn under adverse environmental factor by genetic engineering breeding technology, cultivate the new variety of better resistance, become a study hotspot field of current degeneration-resistant genetic improvement.
This CCCH zinc finger protein is present in eukaryote widely, it is the important regulatory factor of a class, vegeto-animal grow and adverse circumstance to external world reaction process in serve the effect that can not ignore, their great majority all follow the metabolism of RNA relevant, usually the expression to gene can regulate and control at transcriptional level or post-transcriptional level.In plant, the gene of CCCH family is numerous, in recent years by the way of information biology, the gene of 68 and 67 CCCH families is identified respectively in Arabidopis thaliana and paddy rice two kinds of model plants, it is relevant to the environment stress of Arabidopis thaliana that the analysis of express spectra also finds wherein there is the most of member in a subfamily, the first step (Wang et al., 2008a) has been walked out for extensively studying this proteinoid in plant.
Some CCCH zinc finger proteins are by excess or suppress the method such as express transgenic and gene expression analysis to prove to play a role in the growth plant, the responsing reaction to environmental stress etc.CCCH type zinc finger protein is the class in zinc finger protein family, and typical CCCH zinc finger protein is at least containing the one or more zinc finger print bodies rearranged by 3 Cys and 1 His orders, and this structure can be summarized as: C-X
5-14-C-X
4-5-C-X
3-H.X represents arbitrary amino acid residue, (Blackshear, 2002; Wang et al., 2008a).
As OsDOS is found relevant to the aging of rice leaf (Kong et al., 2006); AtSZFl and AtSZF2 is found in Arabidopis thaliana to work in the reaction of salt stress (Sun et al., 2007); GhZFPl is found to participate in the Stress responses (Guo et al., 2009) of tobacco to salt and pathogen.AtTZFl is the albumen of a glucose sensitive, participates in the Plant hormones regulators,gibberellins/plant development process of dormin mediation and the reaction (Pomeranz et al., 2010) in the face of environmental stress.The be correlated with function report of adversity gene of these CCCH type zinc finger proteins is at present many at Arabidopis thaliana, paddy rice, in cotton, in corn seldom.It is enough in the research of abiotic stress, and the abiotic stress such as low temperature, high salt, arid generally can cause the change of some genes or the combination of some transcription factors.
At present, a kind of resisting abiotic environment stress ability strong corn C CCH type zinc finger protein ZmC3H54 and encoding gene ZmC3H54 thereof is lacked.
Summary of the invention
The object of this invention is to provide the strong corn C CCH type zinc finger protein of a kind of resisting abiotic environment stress ability and encoding gene ZmC3H54 thereof and application.
In order to realize above object, the present invention is achieved through the following technical solutions: a kind of corn C CCH type zinc finger protein of the present invention, its aminoacid sequence is as shown in sequence table SEQ ID NO.1.
The encoding gene ZmC3H54 of corn C CCH type zinc finger protein of the present invention, its nucleotide sequence is as shown in sequence table SEQ IDNO.2.
The present invention contains recombinant vectors, recombinant bacterium, the transgenic cell line of described encoding gene.
The application of encoding gene ZmC3H54 in the transgenic plant cultivating resisting abiotic environment stress of corn C CCH type zinc finger protein of the present invention or described corn C CCH type zinc finger protein.
Further, described application, described transgenic plant are paddy rice or Arabidopis thaliana.
The preparation method of the transgenic plant of a kind of described resisting abiotic environment stress of the present invention, comprises the steps:
(1) nucleotide sequence and the aminoacid sequence of the encoding gene ZmC3H54 of corn C CCH type zinc finger protein is obtained;
(2) corn ZmC3H54 gene fragment is obtained with PCR;
With the methods analyst corn gene ZmC3H54 of the quantitative fluorescent PCR express spectra when environment stress, ZmC3H54 gene fragment is building up in plasmid vector;
(3) the Plastid transformation Agrobacterium with ZmC3H54 step (2) obtained;
By the Agrobacterium-mediated Transformation target plant with transform plastids;
(4) selection systems of target plant transgenic positive seedling; The resisting abiotic environment stress of transgenic homozygous plant is analyzed.
Further, in step (1), by the cloning vector plasmids containing ZmC3H54 gene after BamHI+XbaI double digestion, DNA is utilized to reclaim the DNA fragmentation of the SEQ ID NO.2 of test kit recovery 1119bp, this fragment and corresponding enzyme are cut pCAMBIA1301a carrier be connected, the carrier called after p1301a-ZmC3H54 recombinant vectors of acquisition;
With being the primer sequence that template PCR clones this gene from corn cDNA, the primer of this amplification ZmC3H54 gene is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.5, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.6;
In step (3), described target plant is paddy rice or Arabidopis thaliana.
In step (4), the analysis of resisting abiotic environment stress comprises drought-enduring analysis and plant seed germination to the sensitivity analysis of plant hormone ABA.
Of the present invention a kind of for vitro detection ZmC3H54 gene after induction in plant expression amount and in plant the method for expression amount difference in each tissue, comprise the steps:
(1) the blade treatment samples of each time period and plant is several different organizes sample after getting the corresponding induction of plant;
(2) RNA of each sample is extracted by Trizol method;
(3) use DnaseI DNA digestion, then reverse transcription forms cDNA, with it for template;
(4) analyze with quantitative fluorescent PCR, the detection primer sequence of quantitative fluorescent PCR, described primer is for being divided into upstream primer and downstream primer;
The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.3, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.4.
Further, in step (1), described plant is corn;
Stress treatment samples: vegetable material is corn B73 self-mating system, when corn seedling grows to tri-leaf period, compare with the plant of normal irrigation, carry out 10%PEG solution respectively, the ABA solution of 100uM and 200mmol/L NaCl solution Stress treatment, sample after process different time sections, the time is respectively 0h, 1h, 3h, 6h, 12h, 24h; With scissors clip test group and control group corn seedling same area blade respectively, the treatment stage of each, sample three parts; After sampling, sample liquid nitrogen freezing is stored in-80 DEG C;
Sample of tissue: in order to analyze the expression level difference of corn ZmC3H54 gene in each tissue, choose 8 representational tissues of corn, described representational tissue is respectively root, stem, spire, filigree, tassel, front fruit ear of not pollinating, bract, Fetal liver cells, is stored in-80 DEG C with liquid nitrogen freezing after sampling;
In step (4), the detection of total serum IgE purity and content: get 1 μ L total serum IgE sample and be added on micro-spectrophotometer probe, detect total serum IgE purity and content;
Get normal corn to compare, corn reference gene Actin primer, described primer is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.8.
Further, in step (4), get normal Arabidopis thaliana and compare, Arabidopis thaliana reference gene Actin primer, described primer is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.9, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.10.
Beneficial effect: ZmC3H54 gene of the present invention take part in stress response, can improve the resisting abiotic environment stress ability of plant.Transgenic paddy rice and transgenic arabidopsis have higher susceptibility to ABA, transgenic paddy rice improves the tolerance of arid, the present invention's this family gene of separating clone identify that it is improving the function played in object stress resistance of plant from corn, is of great significance for the degeneration-resistant novel crop varieties tool of cultivation.
The present invention is relative to prior art, and tool has the following advantages:
(1) the present invention is separated, clones and obtain ZmC3H54 gene from corn, abduction delivering analysis finds that the expression of ZmC3H54 is subject to the induction of Exogenous ABA, PEG and high salt, by Agrobacterium-medialed transformation method respectively by its Introduced into Rice and Arabidopis thaliana, and obtain corresponding transfer-gen plant.Transfer-gen plant improves the susceptibility of ABA and ability in drought-enduring strengthens.
(2) the present invention is based on bioinformatic analysis, according to its evolutionary relationship and stress-inducing expression pattern, screen the encoding gene of the CCCH type zinc finger protein that is expressed by arid and ABA induced strong, regulate its expression level in plant materials by transgenic technology, the resistance of plant can be improved.The expression amount situation of ZmC3H54 gene in the methods analyst transgenic arabidopsis of semiquantitive PCR can be used in the present invention.Specifically according to transcription factor ZmC3H54 gene order design primer, utilize round pcr to increase from the total cDNA of corn inbred line B73 and obtain ZmC3H54 gene.Abduction delivering analysis finds that the expression of ZmC3H54 is subject to the induction of Exogenous ABA, PEG and high salt.
(3) the present invention is that plant stress-resistance genetically engineered provides important genetic resources, is conducive to the research of degeneration-resistant new variety, significant to raising crop yield.
Accompanying drawing explanation
Fig. 1 is the amino acid sequence homology comparison chart of ZmC3H54 of the present invention and other species CCCH family zinc finger protein conserved regions;
Fig. 2 is the evolutionary tree after having reported the CCCH zinc finger protein family amino acid alignment of function in ZmC3H54 of the present invention and Arabidopis thaliana and other plant;
Fig. 3 is the abduction delivering pattern analysis of ZmC3H54 gene of the present invention when ABA, arid, high salt adverse circumstance process and the tissue expression pattern figure of ZmC3H54 gene; A:10%PEG solution-treated; The ABA solution-treated of B:100uM; C:200mmol/LNaCl solution-treated;
Fig. 4 of the present inventionly turns the raising comparison diagram of ZmC3H54 trans-genetic hybrid rice to the tolerance of arid; A: Osmotic treatment 15 days; B: Osmotic treatment 30 days; C: recover to water 20 days; D: the survival rate of restoration ecosystem wild-type and transfer-gen plant after 20 days;
Fig. 5 of the present inventionly turns the raising comparison diagram of ZmC3H54 trans-genetic hybrid rice to the susceptibility of ABA; A-C: the length of wild-type and growth phenotype, seedling height and the root of transgenic seedlings in the MS nutrient solution of different concns ABA;
Fig. 6 is ZmC3H54 of the present invention at wild-type (WT) Arabidopis thaliana and T1 for the expression analysis figure in transfer-gen plant; A: quantitative fluorescence analysis; B: semi-quantitative analysis;
Fig. 7 of the present inventionly turns the raising comparison diagram of ZmC3H54 gene Arabidopis thaliana to ABA susceptibility; Phenotype on A:MS substratum; Phenotype on B:MS+0.2ABA substratum; Phenotype on C MS+0.4ABA substratum; Germination rate on D: three plates;
Fig. 8 is the comparison diagram of pCAMBIA1301a carrier after pCAMBIA1301 carrier and transformation before transformation involved in the present invention.
Embodiment
The present invention is described further with specific embodiment by reference to the accompanying drawings.Should be appreciated that, these embodiments only for illustration of object, and are not used in the restriction scope of the invention.
In embodiment, method therefor is ordinary method if no special instructions, and the primer is by the synthesis of Shanghai raw work biological company limited; Order-checking is undertaken by Invitrogen company; The various restriction enzymes used in experiment, ligase enzyme, DNA Marker, Taq archaeal dna polymerase, dNTPs etc. are purchased from Takara company; Reverse Transcription box is purchased from Promega company; Plasmid extraction kit, glue reclaim test kit all and genome extract test kit and be purchased from Quan Shijin Bioisystech Co., Ltd, method is all carried out with reference to specification sheets.
A kind of corn C CCH type zinc finger protein of the present invention, its aminoacid sequence is as shown in sequence table SEQ ID NO.1.
Described sequence table SEQ ID NO.1 is that two more typical CCCH type zinc refer to CX from aminoterminal 44-95 amino acids residue sequence
8cX
5cX
3h and CX
5cX
4cX
3the conserved structure of H.
The encoding gene of described corn C CCH type zinc finger protein, its nucleotide sequence is as shown in sequence table SEQ ID NO.2.The invention provides the nucleotide sequence of a new corn zinc finger protein encoding gene, its coding one is by ZmC3H54 gene that is arid and ABA abduction delivering, GRMZM2G117007, main manifestations is that its corresponding transgenic seed sprouting has higher susceptibility to ABA, and plant has certain drought resistance.Corn C 3H54 gene, the long 1119bp of GRMZM2G117007 total length 1466bp, cDNA coding region sequence, as shown in SEQ ID NO.2, this genes encoding 372 amino acid, and containing two more typical CCCH type zinc fingers CX
8cX
5cX
3h and CX
5cX
4cX
3h, as shown in SEQ ID NO.1.
The encoding gene ZmC3H54 of corn C CCH type zinc finger protein of the present invention, its nucleotide sequence is as shown in sequence table SEQ IDNO.2.
The present invention contains recombinant vectors, recombinant bacterium, the transgenic cell line of described encoding gene ZmC3H54.
The application of encoding gene ZmC3H54 in the transgenic plant cultivating resisting abiotic environment stress of corn C CCH type zinc finger protein of the present invention or described corn C CCH type zinc finger protein.
Described application, described transgenic plant are paddy rice or Arabidopis thaliana.
Embodiment 1
The acquisition of corn ZmC3H54 gene
Grow to after tri-leaf period until corn seedling, extract total serum IgE, and reverse transcription is cDNA.Retrieval MAIZEGENOME and ncbi database, obtain the supposition encoding sequence of ZmC3H54, with Primer Premier 5.0 software design special primer, primer is synthesized by Sheng Gong biotech firm.Primer sequence is as follows:
ZmC3H54-F 5′-CG
GGATCCATGTACAACTTCAAAGTGAAGC-3′SEQ ID NO.5
BamHI
ZmC3H54-R 5′-GC
TCTAGATCAGGCCACCATCTGCTC-3′SEQ ID NO.6
XbaI
With the total cDNA of above-mentioned acquisition corn for template, obtain by PCR the coding region that includes entire open reading frame, length is 1119bp, reclaims, is connected on pEASY-T1 carrier, checks order.Gained sequence is corn ZmC3H54 gene.
The transformation of plant expression vector pCAMBIA1301
Plant expression vector pCAMBIA1301 is transformed into pCAMBIA1301a carrier, and Fig. 8 is the collection of illustrative plates of pCAMBIA1301a carrier after pCAMBIA1301 carrier and transformation before transformation involved in the present invention.
As shown in Figure 8, concrete steps are as follows:
(1) with restriction enzyme EcoRI and SacI double digestion again T4 ligase enzyme be connected, add a 35S fragment at the multiple clone site place of pCAMBIA1301 carrier, direction is consistent with GUS direction, is built into pCAMBIA1301-35S carrier;
(2) then by PstI and HindIII double digestion, then connect with T4 ligase enzyme, and then add a bit of polyA after 35S fragment, be built into pCAMBIA1301-35S-polyA carrier (being called for short pCAMBIA1301a carrier below).
The structure of corn ZmC3H54 expression vector
The ZmC3H54+pEASY-T1 carrier of acquisition and pCAMBIA1301a carrier are used BamHI and XbaI double digestion simultaneously, and it is as shown in table 1 that enzyme cuts system:
Table 1
According to double digestion system application of sample above, enzyme reclaims object fragment and pCAMBIA1301a carrier large fragment respectively after cutting 3 hours.Then the ZmC3H54 goal gene fragment after being cut back to close by enzyme is connected with pCAMBIA1301a carrier large fragment, and ligation system is as shown in table 2,
Table 2
16 DEG C of connections are after 3-5 hour, and after a series of conversion with checking, obtain p1301a-ZmC3H54 recombinant vectors, be the expression vector of ZmC3H54.
The conversion of the Agrobacterium of corn ZmC3H54 expression vector
To build above and the pCAMBIA1301-35S-ZmC3H54-polyA carrier freeze-thaw method verified imports in Agrobacterium EH105, concrete steps are as follows:
(1) from-80 DEG C of Ultralow Temperature Freezers, take out Agrobacterium competent cell, thaw on ice, add the Overexpression vector plasmid of 3-5 μ L, flick mixing, place 5 minutes on ice;
(2) quick-frozen 1 minute in liquid nitrogen immediately, to be placed on rapidly in water-bath 37 DEG C of heat shocks 5 minutes;
(3) add the YEP liquid nutrient medium of the antibiotic-free of 300-500 μ L, be placed in 28 DEG C of shaking tables, cultivate 5 hours under 220rpm training condition;
(4) under 10000rpm condition centrifugal 30 seconds, abandon supernatant, add the resuspended thalline of 100 μ L, coat on the YEP solid plate containing 50mg/L rifomycin (Rif) and 50mg/L kantlex (Kan), be inverted at sealing latter 28 DEG C and cultivate 2-3 days.
(5) get single bacterium colony and shake bacterium, protect bacterium, extract plasmid, then carry out bacterium colony PCR or digestion verification.
(6) the correct bacterium liquid of checking is put into-80 DEG C of preservations stand-by.
Embodiment 2
The expression pattern analysis of corn ZmC3H54 gene under PEG, ABA, NaCl condition and enterprise schema analyze
As shown in Figure 3, be the abduction delivering pattern analysis of ZmC3H54 gene of the present invention when ABA, arid, high salt adverse circumstance process and the tissue expression pattern of ZmC3H54 gene; A:10%PEG solution-treated; The ABA solution-treated of B:100uM; The process of C:200mmol/L NaCl solution;
1. Stress treatment sampling: vegetable material is corn B73 self-mating system, when corn seedling grows to tri-leaf period, compare with the plant of normal irrigation, carry out 10%PEG solution respectively, the ABA solution of 100uM and 200mmol/L NaCl solution Stress treatment, sample after process different time sections, the time is respectively 0h, 1h, 3h, 6h, 12h, 24h; With scissors clip test group and control group corn seedling same area blade respectively, the treatment stage of each, sample three parts.After sampling, sample is stored in the refrigerator of-80 DEG C immediately with liquid nitrogen freezing;
Sample of tissue: in order to analyze the expression level difference of corn ZmC3H54 gene in each tissue, choose 8 representational tissues of corn, described representational tissue is respectively root, stem, spire, filigree, tassel, front fruit ear of not pollinating, bract, Fetal liver cells, is stored in the refrigerator of-80 DEG C immediately with liquid nitrogen freezing after sampling;
2. the RNA of each sample is extracted by Trizol method;
RNA extracts: extract total serum IgE according to improved Trizol method, concrete steps are as follows:
(1) take out materials from-80 DEG C of refrigerators, put into grinding, put on one's gloves, add liquid nitrogen grinding, then by the powder transfer of about 100mg to 1.5mL centrifuge tube, in Liquid nitrogen precooler;
(2) add 1mL Trizol reagent, mixing of firmly turning upside down, for 15s, places 10min under room temperature condition; 4 DEG C, the centrifugal 20min of 13,000rpm;
(3) draw supernatant in a new centrifuge tube, add 5M NaCl and the 250 μ L chloroforms of 250 μ L; 4 DEG C, the centrifugal 20min of 10,000rpm;
(4) Aspirate supernatant is in new centrifuge tube, adds 200 μ L Virahols, and room temperature places 10min; 4 DEG C, the centrifugal 15min of 13,000rpm;
(5) careful supernatant discarded, adds 75% ethanol of 500 μ L, uses DEPC water preparation ethanol; 4 DEG C, the centrifugal 5min of 7,500rpm, outwells ethanol;
(6) repeating step step (5), 4 DEG C, the centrifugal 5min of 7,500rpm, outwells ethanol, is placed upside down on paper, and ambient temperatare is done, and time of drying is 5-10min;
(7) gently get rid of, tube wall liquid is got rid of at the bottom of pipe, preserve and get 2 μ L detected results for-70 DEG C.
3. use DnaseI DNA digestion, then reverse transcription forms cDNA, with it for template;
4. the detection of total serum IgE purity and content: get 1 μ L total serum IgE sample and be added on micro-spectrophotometer probe, detect total serum IgE purity and content.A
260/ A
280between 1.8-2.0, illustrate that the total serum IgE quality of extraction is higher.
Analyze with quantitative fluorescent PCR, the detection primer sequence of quantitative fluorescent PCR, described primer is for being divided into upstream primer and downstream primer;
The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.3, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.4.
Get normal corn to compare, corn reference gene Actin primer, described primer is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.8.
5. remove genomic dna process
(1) first according to the RNA concentration levels of each sample of previous step detection, when calculating unified final concentration, the RNA amount that each sample will add, in Eppendorf tube, reaction system is as shown in table 3, adds following reagent successively:
Table 3
(2) crawl mixing after, 42 DEG C temperature bath 2 minutes or room temperature place after 30 minutes stand-by.
6. reverse transcription: the step operation provided according to precious biotech firm Reverse Transcription box, reverse transcription system is as follows:
(1) in the centrifuge tube of trace, reverse transcription reaction system is as shown in table 4, adds following reagent successively:
Table 4
(2), after crawl mixing, 37 DEG C are incubated 15 minutes;
(3) 85 DEG C process 5 minutes after, 4 DEG C hatch several minutes after;
(4) centrifugal 5 seconds of room temperature, to collect at the bottom of all solution to pipe, with RNase Free dH
2o dilutes 10 multiples, and to be placed on-20 DEG C of Refrigerator stores stand-by.
7. quantitative fluorescent PCR reaction
(1) design of primers
Primer uses Primer Express 3.0 (ABI) software to design, and by Shanghai, bio-engineering corporation synthesizes.
The nucleotides sequence that goal gene ZmC3H54 detects primer is classified as:
qPCR-ZmC3H54-F 5′-GGACGCGCCTCTGCAAG-3′SEQ ID NO.3
qPCR-ZmC3H54-R 5′-ATCTGCATGCCGACGGACA-3′SEQ ID NO.4
The nucleotides sequence of corn reference gene Actin primer is classified as:
qPCR-ZmActin-F 5′-GGGATTGCCGATCGTATGAG-3′SEQ ID NO.7
qPCR-ZmActin-R 5′-GAGCCACCGATCCAGACACT-3′SEQ ID NO.8
(2) quantitative fluorescent PCR reaction system is as shown in table 5,
Table 5
(3) PCR response procedures
PCR response procedures is as follows: 95 DEG C of 10min; 95 DEG C of 15sec, 60 DEG C of 1min, totally 40 circulations, after response procedures sets, dose solubility curve, and reaction starts.After reaction terminates, copy out data, adopt 2
– Δ Δ CTmethod processes obtained data.Each Gene Experiments repeats 3 times, does 3 Duplicate Samples at every turn.
Embodiment 3
The sequence homology of corn ZmC3H54 gene and homology analysis
As shown in Figure 1, be the amino acid sequence homology comparison chart of ZmC3H54 of the present invention and other species CCCH family zinc finger protein conserved regions; According to sequence result, in ncbi database, carry out sequence alignment, find the gene order that is cloned into and CCCH zinc finger protein family kinship nearest.Compared by the protein sequence of this transcription factor and known other CCCH type zinc finger protein family member, analyzing its zinc fingers type is CCCH type, and according to the constitutional features of this DNA binding domain, the structure of this ZmC3H54 albumen is CX
8cX
5cX
3h.
As shown in Figure 2, for reported function in ZmC3H54 of the present invention and Arabidopis thaliana and other plant CCCH zinc finger protein family amino acid alignment after evolutionary tree; In order to analyze the Phylogenetic that ZmC3H54 has reported the CCCH type zinc finger protein of function with other further, the CCCH type zinc finger protein of different plant and ZmC3H54 are carried out the analysis of Phylogenetic.Found by sequence alignment and cluster analysis: ZmC3H54 and wherein sub-fraction zinc finger protein are very near on Phylogenetic, and they
Unique one is formed in whole system evolutionary tree, namely a unique subfamily is become, member's structure comparison in this subfamily is guarded, and the most of members wherein reported are relevant to environment stress, to be in the news the abiotic stress of involved in plant with the AtC3H47 (AtSZF1) of ZmC3H54 in a branch, AtC3H29 (AtSZF1) and CaKR1.
Embodiment 4
The acquisition of transgenic paddy rice
By the plant expression vector p1301a-ZmC3H54 built in embodiment 1, by agriculture bacillus mediated rice transformation method by spending in its Introduced into Rice kind, through preculture, dip-dye, Dual culture, screening have hygromycin resistance callus, break up, take root, hardening, transplanting, obtain 25 seedling plants.In order to filter out positive transgenic seedling, seedling leaves being cut into 0.5-1cm length, being soaked in Gus staining fluid, dye 12 hours in 37 DEG C of incubators, the ethanol decolorization with 75% removes chlorophyll, then observes, final acquisition 16 strain Transgenic Rice seedlings.
Embodiment 5
The acquisition of transgenic arabidopsis
(1) inflorescence soaking method arabidopsis thaliana transformation
Shake bacterium: the Agrobacterium bacterium liquid getting over-express vector in 100 μ l embodiments 1, is added in 100ml LB or YEP liquid nutrient medium, adds corresponding resistance, 28 DEG C of incubated overnight.
1.Buffer configures:
With NaOH adjust pH to 5.8, add tensio-active agent: often liter of Buffer adds 300 microlitres
2. prepare conversion fluid:
Centrifugal 28 DEG C of bacterium liquid shaken that spend the night collect thalline, and use 50ml centrifuge tube, the centrifugal 10min of 4000r/min, adds 25mlBuffer, and piping and druming precipitation makes it resuspended.
3. transform
Choose florescence Arabidopis thaliana six basin, about 25, conversion fluid suction pipe previous step prepared drips on the flower and apical meristem that Arabidopis thaliana do not open, and notices that allow bacterium liquid hang over takes as far as possible, improves transformation efficiency.In order to ensure transformation efficiency, bacterium drop adds twice.After transformed, with preservative film, all plant are wrapped up, avoid the too fast evaporate to dryness of bacterium liquid, affect transformation efficiency.Preservative film can be extractd after 24 hours.General conversion needs 1-2 time, and second time transforms and is selected in latter four to five days of first time conversion.
(2) screening of the positive seedling of Arabidopis thaliana
1. seed primary dcreening operation, plants the seed received in containing selective agent, on the substratum as kantlex/Totomycin etc., filters out resistance seedling 35 strain.
2. get the seedling of partial blade to screening and carry out Gus dyeing, final acquisition 20 strain transgenic Arabidopsis plants.
Embodiment 6
ZmC3H54 transgenic rice plant drought tolerance is identified
Plant expression vector in embodiment 1 is proceeded in Agrobacterium EHA105, then infects rice callus, after through Dual culture, antibiotic-screening, obtains ZmC3H54 gene overexpression transfer-gen plant.
Get ZmC3H54 trans-genetic hybrid rice strain L4, L6 as experimental group and contrast strain (Zhong Hua rice strain) rice paddy seed be placed in culture dish, soaking with deionized water makes its imbibition sprout, when seedling grows to about 10cm, get in experimental group GUS can catch look and the seedling that growing way is consistent is transferred to Nutrition Soil together with control group: in the square basin of vermiculite=1:l, subregion equivalent is planted, and each strain arranges three repetitions.Osmotic treatment is carried out cultivate surrounding in heliogreenhouse after.By the transgenic paddy rice after cultivation surrounding and control group rice seedlings, pour into saturation water, to be now process 0 day, after coercing 30 days dry morning, renewal cultivation 20 days, observes phenotype and also adds up its survival rate.As shown in Figure 4, the ZmC3H54 of turning trans-genetic hybrid rice of the present invention improves the tolerance of plant to arid.
Embodiment 7
ZmC3H54 transgenic rice plant is to the sensitivity analysis of ABA
As shown in Figure 5, for the ZmC3H54 of turning trans-genetic hybrid rice of the present invention improves the susceptibility of plant to ABA; A-C: the length of wild-type and growth phenotype, seedling height and the root of transgenic seedlings in the MS nutrient solution of different concns ABA;
In T2 generation, is turned ZmC3H54 gene L4, L6, L8 rice strain planting seed in containing different concns ABA 0 μM, 1 μM, 3 μMs, on the 1/2MS substratum of 5 μMs, with not genetically modified middle colored paddy rice for contrast, under 28 DEG C of conditions, 16h illumination/8h dark makes its germination and growth, observe the growth phenotype of seedling and compare after cultivating 2 weeks, the height and the root that comprise seedling are long, experiment repetition 3 times.
Can obviously find out from figure, containing in the nutrient solution of ABA, the growth of wild-type and Transgenic Rice Seedlings is all suppressed, and the suppression degree of transgenic seedlings is significantly greater than wild-type, be mainly manifested in transgenosis group seedling stem height and be all shorter than control group with root is long, during raising 5 μMs along with ABA concentration, transgenic seedlings growth-inhibiting is more obvious, transgenosis group seedling long root hardly.These results illustrate that overexpression ZmC3H54 can significantly improve the susceptibility of transfer-gen plant to ABA.
Embodiment 8
ZmC3H54 transgenic Arabidopsis plants is to the sensitivity analysis of ABA
As shown in Figure 7, the raising comparison chart of ZmC3H54 gene Arabidopis thaliana to ABA susceptibility is turned for of the present invention; Phenotype on A:MS substratum; Phenotype on B:MS+0.2ABA substratum; Phenotype on C MS+0.4ABA substratum; Germination rate on D: three plates;
Get Arabidopis thaliana L20, L29, L35 strain T2 of turning ZmC3H54 gene for seed and turn pCAMBIA1301a empty carrier transfer-gen plant T2 for seed, disinfect 15min with the Javelle water of 12%, rear aqua sterilisa cleaning 5-6 time, after having sterilized, under being placed in 4 DEG C of temperature, vernalization 3 days.After spring flower, it clicks and enters containing different concns ABA by subregion respectively, 0 μM, 0.2 μM, on the MS substratum of 0.4 μM, keep flat Arabidopis thaliana after sealing and cultivate in greenhouse, under 22 DEG C of conditions, 16h illumination/8h dark makes its germination and growth, and period observes it and sprouts number and character mutation.Experiment repetition 3 times.As seen from the figure: compared with control group, ZmC3H54 transgenic arabidopsis strain improves the susceptibility to ABA.
Can infer by embodiment 7 and embodiment 8 signal pathway that ZmC3H54 gene may take part in ABA and mediates further, and play a role in the sprouting and seedling development process of seed.
Embodiment 9
The expression amount situation of ZmC3H54 gene in transgenic arabidopsis
Of the present invention a kind of for vitro detection ZmC3H54 gene after induction in plant expression amount and in plant the method for expression amount difference in each tissue, comprise the steps:
The blade treatment samples of each time period and plant is several different organizes sample after getting the corresponding induction of transgenic arabidopsis; In step (1), in the transgenic arabidopsis strain obtained in Example 5, our random choose 4 transformants, are respectively L20, L29, L35, L9;
(2) RNA of each sample is extracted by Trizol method;
(3) use DnaseI DNA digestion, after extracting Arabidopis thaliana RNA, reverse transcription becomes cDNA, with it for template;
(4) detection of total serum IgE purity and content: get 1 μ L total serum IgE sample and be added on micro-spectrophotometer probe, detect total serum IgE purity and content;
Utilize the expression of the method for fluorescent quantitation and semiquantitive PCR to goal gene ZmC3H54 to analyze respectively, wild rice is detected as negative control simultaneously.
The detection primer sequence of quantitative fluorescent PCR, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.3, and the nucleotide sequence of described downstream primer is as shown in sequence table SEQ ID NO.4.
Using Arabidopis thaliana Actin gene as interior source reference, by parameters such as adjustment annealing temperature, cycle index, template amounts, set up suitable semiquantitive PCR reaction system.
The nucleotides sequence of Arabidopis thaliana reference gene Actin primer is classified as
qPCR-AtActin-F 5′-TCGTTGCCCCTCCAGAGA-3′SEQ ID NO.9
qPCR-AtActin-R 5′-TACTCTGCCTTTGCGATCCA-3′SEQ ID NO.10
As shown in Figure 6, Fig. 6 is ZmC3H54 of the present invention at wild-type (WT) Arabidopis thaliana and T1 for the expression analysis in transfer-gen plant; A: quantitative fluorescence analysis; B: semi-quantitative analysis; ZmC3H54 can in transgenic line normal expression, in different strains, expression amount has larger difference, wherein higher comparatively speaking with L29 and the L35 chosen strain expression amount, the expression amount of other transgenic lines is relatively low, may be relevant with the insertion point of goal gene fragment and copy number.And in wild rice, do not have expression goal gene being detected.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and application claims protection domain is defined by appending claims, specification sheets and equivalent thereof.
Claims (10)
1. a corn C CCH type zinc finger protein, its aminoacid sequence is as shown in sequence table SEQ ID NO.1.
2. the encoding gene ZmC3H54 of corn C CCH type zinc finger protein according to claim 1, its nucleotide sequence is as shown in sequence table SEQ ID NO.2.
3. recombinant vectors, recombinant bacterium, transgenic cell line containing encoding gene ZmC3H54 described in claim 2.
4. the application of encoding gene ZmC3H54 in the transgenic plant cultivating resisting abiotic environment stress of corn C CCH type zinc finger protein according to claim 1 or corn C CCH type zinc finger protein according to claim 2.
5. application according to claim 4, described transgenic plant are paddy rice or Arabidopis thaliana.
6. a preparation method for the transgenic plant of resisting abiotic environment stress according to claim 4, is characterized in that comprising the steps:
(1) nucleotide sequence and the aminoacid sequence of the encoding gene ZmC3H54 of corn C CCH type zinc finger protein is obtained;
(2) corn ZmC3H54 gene fragment is obtained with PCR;
With the methods analyst corn gene ZmC3H54 of the quantitative fluorescent PCR express spectra when environment stress, ZmC3H54 gene fragment is building up in plasmid vector;
(3) the Plastid transformation Agrobacterium with ZmC3H54 step (2) obtained; By the Agrobacterium-mediated Transformation target plant with transform plastids;
(4) selection systems of target plant transgenic positive seedling; The resisting abiotic environment stress of transgenic homozygous plant is analyzed.
7. the preparation method of the transgenic plant of resisting abiotic environment stress according to claim 6, it is characterized in that: in step (1), by the cloning vector plasmids containing ZmC3H54 gene after BamHI and XbaI double digestion, DNA is utilized to reclaim the DNA fragmentation of the SEQ ID NO.2 of test kit recovery 1119bp, this fragment and corresponding enzyme are cut pCAMBIA1301a carrier be connected, the carrier called after p1301a-ZmC3H54 recombinant vectors of acquisition;
With being the primer sequence that template PCR clones this gene from corn cDNA, the primer of this amplification ZmC3H54 gene is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.5, and the nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.6;
In step (3), described target plant is paddy rice or Arabidopis thaliana.
In step (4), the analysis of resisting abiotic environment stress comprises drought-enduring analysis and plant seed germination to the sensitivity analysis of plant hormone ABA.
8. for vitro detection ZmC3H54 gene after induction in plant expression amount and in plant the method for expression amount difference in each tissue, it is characterized in that comprising the steps:
(1) the blade treatment samples of each time period and plant is several different organizes sample after getting the corresponding induction of plant;
(2) RNA of each sample is extracted by Trizol method;
(3) use DnaseI DNA digestion, then reverse transcription forms cDNA, with it for template;
(4) analyze with quantitative fluorescent PCR, the detection primer sequence of quantitative fluorescent PCR, described primer is for being divided into upstream primer and downstream primer;
The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.3, and the nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.4.
9. according to claim 8 for vitro detection ZmC3H54 gene after induction in plant expression amount and in plant the method for expression amount difference in each tissue, it is characterized in that: in step (1), described plant is corn;
Stress treatment samples: vegetable material is corn B73 self-mating system, when corn seedling grows to tri-leaf period, compare with the plant of normal irrigation, carry out 10%PEG solution respectively, the ABA solution of 100uM and 200mmol/L NaCl solution Stress treatment, sample after process different time sections, the time is respectively 0h, 1h, 3h, 6h, 12h, 24h; With scissors clip test group and control group corn seedling same area blade respectively, the treatment stage of each, sample three parts; After sampling, sample liquid nitrogen freezing is stored in-80 DEG C;
Sample of tissue: in order to analyze the expression level difference of corn ZmC3H54 gene in each tissue, choose 8 representational tissues of corn, described representational tissue is respectively root, stem, spire, filigree, tassel, front fruit ear of not pollinating, bract, Fetal liver cells, is stored in-80 DEG C with liquid nitrogen freezing after sampling;
In step (4), the detection of total serum IgE purity and content: get 1 μ L total serum IgE sample and be added on micro-spectrophotometer probe, detect total serum IgE purity and content;
Get normal corn to compare, corn reference gene Actin primer, described primer is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, and the nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.8.
10. according to claim 8 for vitro detection ZmC3H54 gene after induction in plant expression amount and in plant the method for expression amount difference in each tissue, it is characterized in that:
In step (4), get normal Arabidopis thaliana and compare, Arabidopis thaliana reference gene Actin primer, described primer is for being divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.9, and the nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.10.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399327A (en) * | 2016-11-23 | 2017-02-15 | 东北林业大学 | Western balsam poplar PtrZFP103 gene as well as coding protein and application thereof |
| CN107164404A (en) * | 2017-06-30 | 2017-09-15 | 浙江农林大学 | Alpine ash EgrZFP6 adapts to the purposes of stress improving plant under osmotic stress |
| CN107746847A (en) * | 2017-11-14 | 2018-03-02 | 中国科学院青岛生物能源与过程研究所 | A kind of application of rape C CCH class transcription factors |
| CN108103075A (en) * | 2018-02-01 | 2018-06-01 | 南京农业大学 | A kind of switchgrass gene PvC3H29 for delaying plant senescence and its application |
| CN108570472A (en) * | 2017-03-13 | 2018-09-25 | 中国科学院遗传与发育生物学研究所 | Applications of the soybean transcription factor GmZF351 in plant stress tolerance regulation and control |
| CN108753752A (en) * | 2018-04-24 | 2018-11-06 | 长江大学 | Phosphomevalonate kinase gene and its coding albumen in one ginkgo terpene compound synthesis path and application |
| CN109553671A (en) * | 2019-01-24 | 2019-04-02 | 华中农业大学 | Trifoliate orange Cold resistant genes PtrTZF1 and its application in plant cold resistance genetic improvement |
| CN109912704A (en) * | 2019-03-22 | 2019-06-21 | 济南大学 | Corn zinc finger binding protein interaction factor gene Actin and its recombinant expression carrier and application |
| CN112501181A (en) * | 2020-12-04 | 2021-03-16 | 福建省亚热带植物研究所 | Rice stress resistance related gene OsTZF7 and encoding protein and application thereof |
| CN112725349A (en) * | 2020-12-14 | 2021-04-30 | 四川农业大学 | Dactylis glomerata zinc finger protein gene DgMYM1, and expression vector and application thereof |
| CN114292943A (en) * | 2021-12-27 | 2022-04-08 | 浙江大学 | Application of tomato SlC3H39 gene as negative regulatory factor in improving low temperature resistance of tomato |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392020A (en) * | 2011-11-16 | 2012-03-28 | 吉林大学 | Cloning and function analysis of Zea mays adverse stress inducible promoter |
| CN104120138A (en) * | 2014-07-26 | 2014-10-29 | 江西农业大学 | Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene |
| CN104140462A (en) * | 2013-05-07 | 2014-11-12 | 中国农业大学 | Plant salt tolerance related protein GhSnRK2-6, and coding gene and applications thereof |
| CN104513823A (en) * | 2014-11-24 | 2015-04-15 | 扬州大学 | Drought and salt tolerant transgenic plant preparation method |
-
2015
- 2015-05-11 CN CN201510236703.9A patent/CN104829700A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392020A (en) * | 2011-11-16 | 2012-03-28 | 吉林大学 | Cloning and function analysis of Zea mays adverse stress inducible promoter |
| CN104140462A (en) * | 2013-05-07 | 2014-11-12 | 中国农业大学 | Plant salt tolerance related protein GhSnRK2-6, and coding gene and applications thereof |
| CN104120138A (en) * | 2014-07-26 | 2014-10-29 | 江西农业大学 | Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene |
| CN104513823A (en) * | 2014-11-24 | 2015-04-15 | 扬州大学 | Drought and salt tolerant transgenic plant preparation method |
Non-Patent Citations (2)
| Title |
|---|
| AIB05486.1: "C3H transcription factor, partial [Zea mays]", 《NCBI》 * |
| 彭晓剑: "玉米全基因组CCCH锌指蛋白基因家族进化分析", 《万方数据知识服务平台》 * |
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| CN108570472A (en) * | 2017-03-13 | 2018-09-25 | 中国科学院遗传与发育生物学研究所 | Applications of the soybean transcription factor GmZF351 in plant stress tolerance regulation and control |
| CN107164404B (en) * | 2017-06-30 | 2020-10-20 | 浙江农林大学 | Application of E.grandis EgrZFP6 in improving stress adaptation of plants under osmotic stress |
| CN107164404A (en) * | 2017-06-30 | 2017-09-15 | 浙江农林大学 | Alpine ash EgrZFP6 adapts to the purposes of stress improving plant under osmotic stress |
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| CN108753752A (en) * | 2018-04-24 | 2018-11-06 | 长江大学 | Phosphomevalonate kinase gene and its coding albumen in one ginkgo terpene compound synthesis path and application |
| CN109553671A (en) * | 2019-01-24 | 2019-04-02 | 华中农业大学 | Trifoliate orange Cold resistant genes PtrTZF1 and its application in plant cold resistance genetic improvement |
| CN109912704A (en) * | 2019-03-22 | 2019-06-21 | 济南大学 | Corn zinc finger binding protein interaction factor gene Actin and its recombinant expression carrier and application |
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