CN101831448A - Plant glyceraldehyde 3-phosphate dehydrogenase gene, preparation method and application thereof - Google Patents
Plant glyceraldehyde 3-phosphate dehydrogenase gene, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a plant glyceraldehyde 3-phosphate dehydrogenase gene, a preparation method and an application thereof. The preparation method comprises the following steps of: A. designing a pair of specific primers by using the total cDNA of a two-week-old rice seedling as a template, and carrying out PCR amplification by using B-Taq as a DNA polymerase to obtain a rice glyceraldehyde 3-phosphate dehydrogenase gene; B. connecting the gene segment obtained in step A to an intermediate vector, and carrying out sequence verification; C. connecting the gene fragment obtained after the verification in step B to the vector to arrange the rice glyceraldehyde 3-phosphate dehydrogenase gene between a corn ubiquitin promoter and an NOS tail to form transformation; and D. transferring the prepared overexpression vector pUbiO containing the target gene into Agrobacterium rhizogenes EHA105, and introducing into a rice callus to obtain a rice transgenic plant. In the application of the gene to rice, the obtaind gene is under the regulation and control of a monocotyledonous potent promoter-ubiquitin promoter, can be expressed without being disturbed by external environment factors, and has genetic stability.
Description
Technical field
The invention belongs to the transgenic plant field, more specifically relate to a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene, the preparation method who also relates to a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene simultaneously also relates to the application of plant glyceraldehyde 3-phosphate dehydrogenase gene in paddy rice anti contravariance.
Background technology
Triphosphoric acid-glyceraldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase; GAPDH) be one of requisite key enzyme in the glycolytic pathway, its catalysis D-glyceraldehyde-3-phosphate (G-3P) reversible oxidation and phosphorylation generates 1,3-diphosphoglyceric acid (DPGA), be one of the most basic enzyme of the activity of earning a bare living, extensively in existence and the various biomass cellss.Nearest correlative study shows, eucaryon and procaryotic glyceraldehyde 3-phosphate dehydro-genase are except playing an important role in glycolysis-, and it is in film dissolving, microtubule boundling, phosphate transferase activity, DNA reparation, apoptosis, nRNA output and all play an important role in the cell cycle.In addition, the GAPDH gene also has the stronger ability of resisting environment-stress, especially under stress conditions such as high temperature, high salt, low temperature, utilizing verified this gene of yeast expression technology to have the function of anti-salt, alkali and high temperature stress, is a kind of important degeneration-resistant border stress gene.The research of this enzyme in plant is not as going deep in Mammals, but research proves successively, glyceraldehyde 3-phosphate dehydro-genase has many undiscovered functions equally in plant, reported that at present this enzyme may bring into play important effect in anaerobism, heat shock, injury and energy supply.3 one glyceraldehyde phosphate dehydrogenase genes from numerous plants, have been cloned at present, and in the unifacial leaf grass, kytoplasm 3 one glyceraldehyde phosphate dehydrogenase genes of barley, corn, millet, paddy rice are also successively cloned out, studies show that in plant, anoxic and heat shock can be induced the GAPDH expression of gene, previously when the tobacco callus was cultivated, the sucrose of substratum middle and high concentration also can make the expression amount of GapC genoid that rising is also arranged.The stress reaction of plant self plays an important role aspect physical abuse, insect and the pathogenic agent invasion and attack reducing, and the GAPDH gene also has the expression of higher level in these shock reactions.Recently, participated in the apoptosis of H2O2 regulation and control with Arabidopis thaliana protoplastis and the yeast GAPDH gene that is studies have shown that of material, at H
2O
2Under the treatment condition, Arabidopis thaliana GAPDH expression of gene raises, and can suppress the H that caused by heat shock
2O
2GAPDH family member new in the paddy rice is isolated in the raising of content and necrocytosis among the present invention, this gene inhibition express transgenic plant shows as salt tolerant period at rice seedling, for the screening and the acquisition of paddy rice anti contravariance strain provides a kind of new method.
Summary of the invention
The objective of the invention is to be to provide a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene, the transfer-gen plant inheritance stability that is obtained, this breadboard overexpression carrier pUbiO is the expression vector of composing type efficiently, can obtain the transfer-gen plant of goal gene high expression level amount; Spend 11 relative other kinds that very high transformation efficiency is also arranged in the application of rice conversion in the used converting material in this laboratory in addition.
Another object of the present invention is the preparation method who has been to provide a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene, the full length cDNA sequence of the paddy rice glyceraldehyde 3-phosphate dehydrogenase gene that this method obtains, but not the genome sequence of total length, because eukaryotic gene group DNA ten minutes is huge, its complexity is protein and mRNA about 100 times, and contain a large amount of tumor-necrosis factor glycoproteinss, therefore the cDNA that is set out by mRNA clones, and its complexity is than directly much simple from genomic clone; In addition, this gene that present method obtains is under the regulation and control of monocotyledonous potent promotor-ubiquitin promoter, but makes and the expression of this gene composing type be not subjected to the interference of external environment factor, and inheritance stability.A kind of plant of this glyceraldehyde 3-phosphate dehydro-genase gene of energy overexpression, this transfer-gen plant shows as salt tolerant.
A further object of the present invention is to be to provide the application of kind of plant glyceraldehyde 3-phosphate dehydrogenase gene in paddy rice anti contravariance, this enzyme is catalysis D-glyceraldehyde-3-phosphate (G-3P) reversible oxidation and phosphorylation in glycolytic pathway, generate 1,3-diphosphoglyceric acid (DPGA), it is one of the most basic enzyme of the activity of earning a bare living, simultaneously, recent research shows that also this gene has participated in the stress response of plant, because glycan molecule also is a kind of important signaling molecule, so, will understand by nutritive substance to the applicant to the research of this gene, the plant signal approach of the complexity that bio-hormone and some chemical moleculars are formed and growth and development of plants and the adverse circumstance that they are regulated and control have been replied crucial meaning.
In order to achieve the above object, the present invention adopts following technological method:
The preparation method of one kind of plant glyceraldehyde 3-phosphate dehydrogenase gene, this method may further comprise the steps:
A, utilize round pcr, total cDNA with two-week rice seedlings (extracts the RNA of two all rice seedlings, and be template with this RNA, obtain the double-stranded total cDNA of paddy rice through reverse transcription) be template, and the cDNA sequence (its sequence is the nucleotide sequence shown in the SEQID NO:3) of the gene that provides according to ncbi database (http://www.ncbi.nlm.nih.gov/), design a pair of special primer that comprises this full length gene ORF (open reading frame), with B-Taq (TOYOBA company) is archaeal dna polymerase pcr amplification (94 ℃ of 3min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 30cycles, 72 ℃ of 10min); The product electrophoresis is also cut glue and is reclaimed (An Biao company glue reclaims test kit), thereby acquisition comprises the cDNA sequence (1200bp) of the total length open reading frame of this paddy rice glyceraldehyde 3-phosphate dehydrogenase gene, and its sequence is the nucleotide sequence shown in the SEQ ID NO:4.
B, the gene fragment that the A step is obtained are connected among the commercial intermediate carrier PBS-T II (TIANGEN company) (carrier collection of illustrative plates such as Fig. 1), sequence verification, obtain right-on this paddy rice glyceraldehyde 3-phosphate dehydrogenase gene fragment of base sequence, a kind of separating plant glyceraldehyde 3-phosphate dehydrogenase gene, its sequence are the nucleotide sequence shown in the SEQ ID NO:4.
C, the correct gene segment that B step card is obtained is connected to carrier pUbiO (doctor's Wang Ying Diplomarbeit: the gene expression analysis in the living process of EMBRYO IN RICE fetal hair of transforming acquisition, 2004) in, the pUbiO carrier is at carrier pAHC17 (ALAN H.CHRISTENSEN and PETER H.QUAIL.Ubiquitinpromoter-based vectors for high-level expression of selectable and/or screenablemarker genes in monocotyledonous plants.Transgenic Research, 1996, transform 5:213-218) and on the basis of pCAMBIA1301 (http://www.patentlens.net/daisy/bios/585.html) and come, carrier pAHC17 contains transformed corn ubiquitin promoter and NOS tail, and carrier pCAMBIA1300 (http://www.patentlens.net/daisy/bios/585.html) series to be present plant change very because of in one of more plant expression vector of use.The construction step of a kind of carrier pUbiO is: at first introduce four multiple clone site-BamH I, Kpn I, Sac I, Smal I before the NOS tail with carrier pAHC17.Subsequently transformed pAHC17 is utilized double digestion to obtain comprising the fragment of corn ubiquitin promoter and NOS tail, handle pCAMBIA1301 with identical double digestion simultaneously, and be connected with above-mentioned fragment, thereby obtain containing the carrier that can be used for the paddy rice overexpression of corn ubiquitin promoter and NOS tail, as shown in Figure 2, make the paddy rice glyceraldehyde 3-phosphate dehydrogenase gene place between corn ubiquitin promoter and the NOS tail carrier that formation can transform or express;
D, the overexpression carrier pUbiO that contains goal gene that will prepare change Agrobacterium EHA105 over to (from China typical culture collection center,), import in the mature embryo inductive rice callus tissue, and through breaking up the final paddy rice transfer-gen plant that obtains this gene of overexpression again.
This paddy rice glyceraldehyde 3-phosphate dehydrogenase gene that relates among the present invention participates in the salt stress reaction path of rice seedling phase.Analysis of protein (http://www.ncbi.nlm.nih.gov/) to this desaturase shows, it contains the conservative structural domain (as Fig. 6) of glyceraldehyde 3-phosphate dehydrogenase, to other species, as corn, Arabidopis thaliana, studies show that, the plant glyceraldehyde 3-phosphate dehydrogenase gene has participated in a series of stress response reactions, therefore, the analysis that the genetic expression of glyceraldehyde 3-phosphate dehydrogenase gene under various stress conditions of this paddy rice is carried out is gone in application, spend 11 in the wild-type after one week of growth on the normal MS substratum, place 42 ℃ respectively, 200mM salt, among 20% PEG, and 0,1,2,4,8,12, sampling after 24 hours, extract RNA, reverse transcription obtains to carry out the sxemiquantitative pcr analysis behind the cDNA, the result shows, this gene is at high salt, arid, (as Fig. 7) all appears raising in the expression amount under the hot shock condition, simultaneously, the applicant has also carried out the salt tolerant analysis to the overexpression transfer-gen plant of this paddy gene, with the T2 of transgenic over expression plant for spending 11 rice paddy seeds to peel off in seed and the wild-type, aseptically process, seed is seeded on the MS substratum, 28 ℃, 16hr light/8hr is dark, the plant height of statistics transfer-gen plant and wild-type after one week of growth, select the overexpression transfer-gen plant consistent with wild-type plant growing way, with the wild-type is contrast, again after the disinfection, be inoculated in the MS substratum that contains 200mM NaCl, 28 ℃, 16hr light/8hr is dark, add up plant height after cultivating for two weeks, the result shows, 4.71 centimetres of the plant height average out to of the paddy rice of wild-type after two weeks of growth on the high salt culture medium, the average plant height of two overexpression strain systems has then reached 6.10 centimetres and 5.77 centimetres respectively, wild-type is high relatively, show that this glyceraldehyde 3-phosphate dehydro-genase gene overexpression transformed plant is on the substratum of high salt concentration, relative wild-type, the overexpression transfer-gen plant of this gene is grown plant height after two weeks obviously than wild-type height under high-salt stress, prove that this gene of overexpression has improved paddy rice anti contravariance.
One application of kind of plant glyceraldehyde 3-phosphate dehydrogenase gene in paddy rice anti contravariance, its application process is:
In the present invention, this serine-tryptophan protein kinase gene sequence gene order in ncbi database number is Os02g0601300, and this gene order is a sequence shown in the SEQ ID NO:3.By two kinds of primer GAPF (CGGGATCCCGTTTGCACTCCTCTCGATCTC) and GAPR (CGGGATCCTCTGCAGGCCTCCATGAGGAG), its nucleotide sequence is respectively SEQ ID NO:1 and SEQ IDNO:2, with the rice cDNA library that obtained as template, the synthetic and segment of this kinase gene total length open reading frame that obtained comprising of one section 1200bp length by the PCR mode.This segment is connected among the commercial middle transition carrier PBS-T II it to be inserted segment and identifies correct through nucleic acid sequencing, cut by restriction enzyme BamH I enzyme then and obtain this fragment, and it is connected among the same overexpression carrier pUbiO that handled with BamH I the insertion of acquisition forward.To contain the pulsating carrier transfection of purpose Agrobacterium EHA105, cultivate Agrobacterium in the YM substratum, and mature embryo inductive rice callus is organized in the bacterium liquid soaks, last in adding the selection substratum of Totomycin screening positive clone.In a specific embodiment of the present invention, callus is selected from paddy rice, obtains the transfer-gen plant of glyceraldehyde 3-phosphate dehydro-genase gene overexpression by selecting the last screening of substratum, and this plant shows as salt-tolerant trait period seedling.
The present invention compared with prior art, have the following advantages and effect: the invention provides the preparation method and the application of this gene in paddy rice of a paddy rice glyceraldehyde 3-phosphate dehydrogenase gene, utilize agriculture bacillus mediated rice callus genetic transformation technology to obtain the paddy rice overexpression transfer-gen plant of this gene, rice callus in the invention is to be formed by kind of an embryonal induction, the transfer-gen plant inheritance stability that is obtained, this breadboard overexpression carrier pUbiO is the expression vector of composing type efficiently, can obtain the transfer-gen plant of goal gene high expression level amount; Spend 11 relative other kinds that very high transformation efficiency is also arranged in the application of rice conversion in the used converting material in this laboratory in addition.Among the present invention overexpression a key gene glyceraldehyde 3-phosphate dehydrogenase gene in growth and development of plants, and find that the overexpression transfer-gen plant of this gene shows as salt tolerant, found that this gene is in the new function of plant stress-resistance reverse side.This gene that present method obtains is under the regulation and control of monocotyledonous potent promotor-ubiquitin promoter, but makes and the expression of this gene composing type be not subjected to the interference of external environment factor, and inheritance stability.A kind of plant of this glyceraldehyde 3-phosphate dehydro-genase gene of energy overexpression, this transfer-gen plant shows as salt tolerant.
Description of drawings
Fig. 1 is a kind of plant expression vector synoptic diagram
Intermediate carrier PBS-T II
Fig. 2 is a kind of plant expression vector synoptic diagram
Overexpression carrier pUbiO
Hygromycin is a hygromycin resistance, and promoter is a ubiquitin promoter, and the NOS tail is a transcription terminator.
Fig. 3 is a kind of transformed plant genome PCR qualification result
Wherein 1 is contrast wild-type plant, and 2-10 is a transfer-gen plant, and the result shows that the 1-5# transfer-gen plant all has insertion.
Fig. 4 is a kind of transformed plant sxemiquantitative pcr analysis qualification result
Wherein 7 are contrast wild-type plant, and 1-6 is a transfer-gen plant, and the result shows that 1,2,3 transfer-gen plant purpose segment expression amounts have rise.
Fig. 5 is the statistical study of a kind of transfer-gen plant salt tolerant phenotype
X-coordinate is the strain set type, be followed successively by wild-type, overexpression strain from left to right and be 3, the overexpression strain is 2, ordinate zou is the plant height statistics after two weeks of growth on the high salt culture medium, and it is that relative wild-type plant height is higher that the result is presented at the two weeks back overexpression strains of growing on the high salt culture medium.
Fig. 6 is a kind of vegetable-protein domain analyses
Wherein Gp_dh_C superfamily is the C-end structure territory of glyceraldehyde 3-phosphate dehydrogenase, and Gp_dh_Nsuperfamily is the NAD binding domains of the N-end of glyceraldehyde 3-phosphate dehydrogenase, and GAPDH-I is the conserved domain of glyceraldehyde 3-phosphate dehydrogenase I.
Fig. 7 is the expression amount sxemiquantitative pcr analysis of a kind of gene under stress conditions
Wherein be the variation of the expression amount of gene under high temperature, high salt and drought condition from left to right, on be designated as and handle the thick time, centre is expression of gene, is interior target expression below, the result shows that the expression of this gene under these stress conditions raised to some extent.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as: Chen Zhenghua chief editor, Higher Education Publishing House, 1986, woody plant tissure is cultivated and is used; (Germany) rely nanotesla (REINERT, J.), (English) graceful approximately (YEOMAN, M.M.) work; You Ruilin, Wang Mo is kind to translate BJ University Press, 1988, vegetable cell and tissue culture laboratory manual; And (moral) Ba Erci chief editors such as (W.BARZ); Xia Zhen Australia etc. translates, Science Press, 1983, the condition described in plant tissue culture and the books such as application on biotechnology thereof, or the condition of advising according to manufacturer.
Embodiment 1:
The preparation method of one kind of plant glyceraldehyde 3-phosphate dehydrogenase gene the steps include:
A utilizes round pcr, total cDNA (extracts the RNA of two all rice seedlings with two-week rice seedlings, and be template with this RNA, obtain the double-stranded total cDNA of paddy rice through reverse transcription) be template, and a pair of special primer that comprises this full length gene ORF of the cDNA sequence (SEQ ID NO:3) of the gene that provides according to ncbi database design, its nucleotide sequence is respectively SEQ ID NO:1 and SEQ ID NO:2, with B-Taq (TOYOBA company) is archaeal dna polymerase pcr amplification (94 ℃ of 3min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 30cycles, 72 ℃ of 10min); The product electrophoresis is also cut glue and is reclaimed (An Biao company glue reclaims test kit), thereby obtains this paddy rice glyceraldehyde 3-phosphate dehydrogenase gene cDNA sequence (1200bp); Its sequence is the aminoacid sequence shown in the SEQ ID NO:4.
The gene fragment that B obtains the A step is connected among the commercial intermediate carrier PBS-T II (TIANGEN company), sequence verification, obtain the fragment of right-on this paddy rice glyceraldehyde 3-phosphate dehydrogenase gene of base sequence, a kind of separating plant glyceraldehyde 3-phosphate dehydrogenase gene, its sequence are the nucleotide sequence shown in the SEQ ID NO:4.
C cuts the correct gene segment that B step card obtains by restriction enzyme BamH I enzyme, the fragment that obtains is connected among the carrier pUbiO of same this laboratory transformation acquisition of cutting with BamH I enzyme, the pUbiO carrier is to transform on the basis of carrier pAHC17 and pCAMBIA1301 and come, carrier pAHC17 contains transformed corn ubiquitin promoter and NOS tail, and carrier pCAMBIA1300 series is to use one of more plant expression vector in the present plant transgene.A kind of its building process of carrier pUbiO is: at first introduce four multiple clone site-BamH I, KpnI, Sac I, Smal I before the NOS tail with carrier pAHC17.Subsequently transformed pAHC17 is utilized double digestion to obtain comprising the fragment of corn ubiquitin promoter and NOS tail, handle pCAMBIA1301 with identical double digestion simultaneously, and be connected with above-mentioned fragment, thereby obtain containing the carrier that can be used for the paddy rice overexpression of corn ubiquitin promoter and NOS tail, as shown in Figure 2, make the paddy rice glyceraldehyde 3-phosphate dehydrogenase gene place between corn ubiquitin promoter and the NOS tail carrier that formation can transform or express;
D changes the carrier of preparation over to Agrobacterium EHA105 (from China typical culture collection center), imports in the mature embryo inductive rice callus tissue, and through breaking up the final paddy rice transfer-gen plant that obtains this gene of overexpression again.
Embodiment 2: conversion of this glyceraldehyde 3-phosphate dehydro-genase gene overexpression plant and the conversion of Screening of Rice Agrobacterium
Sophisticated rice paddy seed shells, and used product are to spend 11 (from Institute of Crop Science, Chinese Academy of Agricultural Science) in the japonica rice.Aseptic washing is 3 times on the super clean bench, in the rinse of 70% (volume ratio) ethanol, and back sterilization 5 minutes, sterile water wash seed 3 times, 5% (mass volume ratio) clorox disinfection seed 45 minutes.Sterile water wash seed 5-6 time is poured seed into sterilization and is blotted on the filter paper, after drying up seed is seeded in the N6 substratum (with reference to " vegetable cell and tissue culture laboratory manual "), 28 ℃, secretly cultivates for 4 weeks.Observe the inductive callus, faint yellow, fine and close embryo callus subculture with after small shield separates, is changed in the new N6 substratum over to 2 weeks of succeeding transfer culture.Select embryo callus subculture fine and close, relatively dry to be transferred on the pre-culture medium (a large amount of trace of N6 substratum reduce by half, and add the AS (Syringylethanone) of 200uM), secretly cultivated 2-4 days down for 28 ℃.Picking Agrobacterium EHA105 (from China typical culture collection center) positive colony simultaneously is seeded in an amount of Agrobacterium coating and contains on the corresponding antibiotic YM solid medium 28 ℃ of dark down cultivations 36 hours.Collect Agrobacterium and be resuspended in the 1/2N6+AS substratum, make the OD600 of bacterial suspension reach 0.8-1.0.To change over to through the callus of preceding cultivation wherein and soak about 15-25 minute.Bacterium liquid is poured out, and on aseptic paper, assurance bacterium liquid is washed dried callus, changes on the common substratum in 20 ℃ of dark cultivations 3 days again.With callus sterile water wash 3 times of cultivating altogether, add the N6 liquid nutrient medium that contains cephamycin (500mg/L) again and clean, repeat 2-3 time.Change the exsiccant callus over to contain cephamycin (250mg/L) and Totomycin (50mg/L) N6 substratum, 28 ℃, secretly cultivated for two weeks, change new two weeks of selection culture medium culturing again over to.The callus cultured tissue of screening is for the last time changed in the pre-differentiation substratum that contains Totomycin (50mg/L), 28 ℃, secretly cultivate a week.Select the faint yellow callus that grows fine, move into and to contain in the MS substratum of Totomycin (50mg/L), light was cultivated 15～20 days, can see having green bud to grow, and changed a subculture in 15 days.When the length of green bud reaches 2 centimetres of left and right sides, move in the 1/2 MS root media.After growing root system, regeneration plant changed in the culturing pot cultivate.Seedling after the transplanting is placed on hot-house culture Cheng Chengmiao.
The foranalysis of nucleic acids of embodiment 3 these glyceraldehyde 3-phosphate dehydro-genase gene overexpression transformed plants
1, be used for the extraction of the total DNA of transformed plant of PCR:
Adopt CTAB (cetyl trimethylammonium bromide) method to extract total DNA of transfer-gen plant, at first, get the young tender rice leaf that is about 2 centimetres, be put in the mortar, add 1.5 * CTAB extracting solution: (pH 8.0 for CTAB 15g, 1M TrisCl, 75ml, 0.5M EDTA 30ml, NaCl 61.4g add water and are settled to 1000ml) solution 300 μ l, be ground to pulpous state, add 1.5 * CTAB extracting solution of 300 μ l again, be transferred in the 1.5ml centrifuge tube.65 ℃ of temperature were bathed 30 minutes at least.Be cooled to room temperature (identical below 20-25 ℃), add 600 μ l volume ratios and be 24: 1 chloroform: primary isoamyl alcohol (volume ratio 24: 1), put upside down mixing after, 12000rpm, centrifugal 5 minutes of room temperature.Draw supernatant, added isopyknic Virahol precipitation at room temperature 10 minutes.4 ℃, 12000rpm, centrifugal 10 minutes.Abandon supernatant, 70% (volume ratio) washing with alcohol precipitates once.After drying precipitated, add 50ul distilled water or TE (10mmol/l Tris-Cl, 1mmol/lEDTA) solution dissolving.
2, the genome PCR of transformed plant identifies:
The PCR reaction system is:
Dna profiling 1.0 μ l
10 * Buffer (containing Mg2+), 3.0 μ l
2mM?dNTP 0.6μl
The upstream primer 0.6 μ l of 10mM
The downstream primer 0.6 μ l of 10mM
Taq enzyme (2.5U) 0.3 μ l
Add ddH2O to 30 μ l
The pcr amplification condition is: 94 ℃, and 3 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute; 35 cycle numbers; 72 ℃ 10 minutes.The result as shown in Figure 3.
The sxemiquantitative pcr analysis of embodiment 4 these glyceraldehyde 3-phosphate dehydro-genase gene overexpression transformed plants
1, the extraction of transformed plant mRNA:
(50mg) placed liquid nitrogen after the paddy rice spire cut rapid weighing with scissors, added 1ml RNA extracting solution (Trizol) after being ground into powder, and room temperature was placed 5 minutes; Adding 0.2ml chloroform (chloroform: Trizol=1: 5), fiercely shook 15 seconds, room temperature was placed about 2 minutes, and 4 ℃, centrifugal 15 minutes of 12000rpm; Get the colourless supernatant in upper strata, (Virahol: Trizol=1: 2), room temperature is placed after 10 minutes 4 ℃, centrifugal 15 minutes of 12000rpm to add the 0.5ml Virahol; Add 75% (volume ratio) ethanol 1ml washing precipitation once, make its suspension with rifle piping and druming precipitation, 4 ℃, centrifugal 5 minutes of 7500rpm; Top liquid is removed in suction, dries up on air drying 5 minutes or the super clean bench; Molten being deposited in the DEPC water.
2, obtain rice cDNA library by the RT-PCR reverse transcription.
3, PCR reaction
Make template with the cDNA product that above-mentioned reaction obtains, carry out the PCR reaction, system is as follows:
The PCR reaction system is:
CDNA template 1.0 μ l
10 * Buffer (containing Mg2+), 3.0 μ l
2mM?dNTP 0.6μl
The upstream primer 0.6 μ l of 10mM
The downstream primer 0.6 μ l of 10mM
Taq enzyme (2.5U) 0.3 μ l
Add ddH2O to 30 μ l
The pcr amplification condition is: 94 ℃, and 3 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute; 30 cycle numbers; 72 ℃ 10 minutes; 4 ℃ of preservations.The result as shown in Figure 4
T with the transgenic over expression plant
2Peel off for spending 11 rice paddy seeds in seed and the wild-type, aseptically process, operate on the Bechtop, step is as follows: at first use the sterile water wash seed three times, the dust of the seed-coat on flush away surface, and then with sterilizing 5 minutes sterile water wash seed 3 times, 5% (mass volume ratio) clorox disinfection seed 45 minutes in 70% (volume ratio) ethanol.Behind sterile water wash seed 5-6 time, seed is seeded on the MS substratum, 28 ℃, 16hr light/8hr is dark, the plant height of statistics transfer-gen plant and wild-type after one week of growth, selecting the overexpression transfer-gen plant consistent with wild-type plant growing way, is contrast with the wild-type, again after the disinfection, be inoculated in the MS substratum that contains 200mM NaCl, 28 ℃, 16hr light/8hr is dark, cultivates two all backs statistics plant heights, the result shows, 4.71 centimetres of the plant height average out to of the paddy rice of wild-type after two weeks of growth on the high salt culture medium, the average plant height of two overexpression strain systems has then reached 6.10 centimetres and 5.77 centimetres respectively, and the result shows, this glyceraldehyde 3-phosphate dehydro-genase gene overexpression transformed plant is on the substratum of high salt concentration, compare with wild-type, plant height is higher, as shown in Figure 5.
SEQUENCE?LISTING
＜110〉Wuhan University
＜120〉a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene and preparation method and application
＜130〉a kind of plant glyceraldehyde 3-phosphate dehydrogenase gene and preparation method and application
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ctgttaggct?tgagaagccc?gccagctatg?accagatcaa?ggctgcaatc?aaggaggagg 1020
ctgagggcaa?gctcaagggc?atccttggat?acgttgagga?ggaccttgtt?tccactgact 1080
tccagggtga?cagcaggtcc?agcatctttg?atgccaaggc?tggcattgct?ttgagcgaca 1140
cgttcgtgaa?gcttgtgtcc?tggtacgaca?acgaatgggg?atacagcacc?cgtgtgatcg 1200
acctgatccg?tcacatgcac?agcaccaact?agacgagccc?tcctcatgga?ggcctgcaga 1260
tacaggggag?ttgtgttttg?ccccagagaa?gagtagatga?agcctcttcc?gagaataaat 1320
tttaaattct?gtatggtttt?atgtccgtcg?aaacctaaaa?ctatacttgg?ttgtatcatg 1380
gtggttggtt?gggcctggtc?atggctcata?ttttgtgtct?aattttcttg?cgcttaatct 1440
aaatcgaagt?gttgcttcgc 1460
<210>4
<211>1108
<212>DNA
＜213〉synthetic
<400>4
cgtttgcact?cctctcgatc?tccattcgtt?tttgagttcc?tcgtttgctc?cgcctctctt 60
tcactcatgg?gcaagattaa?gatcggaatc?aatgggttcg?gccgcatcgg?caggctggtg 120
gccagggtgg?cgctgcagag?cgaggatgtc?gagctcgttg?ccgtcaacga?tcccttcatc 180
accaccgagt?acatgacata?catgttcaag?tatgacaccg?tccacggcca?gtggaagcat 240
catgaggtca?aggtcaagga?ctccaagacc?ctcatctttg?gcacgaaaga?ggttgcggtg 300
ttcggctgca?ggaaccctga?ggagatccca?tgggctgcgg?ctggtgctga?atacgttgtt 360
gagtctactg?gtgttttcac?cgacaaggac?aaggcagcag?ctcacttgaa?gggtggtgcc 420
aagaaggtcg?tcatttctgc?tcccagcaaa?gacgccccca?tgttcgttgt?tggtgtcaac 480
gagaaggagt?acaagtctga?cgttaacatt?gtctccaacg?ctagttgcac?caccaactgc 540
ctggctcctc?tcgccaaggt?catcaatgac?agatttggca?tcgttgaggg?tttgatgacc 600
actgtccatg?ccatcactgc?tacccagaag?actgttgatg?ggccctcgat?gaaggactgg 660
agaggtggaa?gggctgctag?cttcaacatc?attcctagca?gcactggagc?tgccaaggct 720
gtcggcaagg?tgcttcctgc?cctcaatgga?aagctgactg?gaatggcttt?ccgtgttccc 780
acagtcgatg?tttccgttgt?tgatctgact?gttaggcttg?agaagcccgc?cagctatgac 840
cagatcaagg?ctgcaatcaa?ggaggaggct?gagggcaagc?tcaagggcat?ccttggatac 900
gttgaggagg?accttgtttc?cactgacttc?cagggtgaca?gcaggtccag?catctttgat 960
gccaaggctg?gcattgcttt?gagcgacacg?ttcgtgaagc?ttgtgtcctg?gtacgacaac 1020
gaatggggat?acagcacccg?tgtgatcgac?ctgatccgtc?acatgcacag?caccaactag 1080
acgagccctc?ctcatggagg?cctgcaga 1108
Claims (3)
1. isolating glyceraldehyde 3-phosphate dehydrogenase gene, its sequence is a nucleotide sequence shown in the SEQ ID NO:4.
2. the preparation method of the described kind of plant glyceraldehyde 3-phosphate dehydrogenase gene of claim 1, this method may further comprise the steps:
A, utilize round pcr, with the total cDNA of two-week rice seedlings is template, and a pair of special primer that comprises this full length gene ORF of cDNA sequences Design of the gene that provides according to ncbi database, is the archaeal dna polymerase pcr amplification with B-Taq, 94 ℃ of 3min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 30cycles, 72 ℃ of 10min, product electrophoresis also cut glue and reclaim, and acquisition comprises the cDNA sequence of the total length open reading frame of this paddy rice glyceraldehyde 3-phosphate dehydrogenase gene;
B, the gene fragment that (A) step is obtained are connected among the intermediate carrier PBS-TII, sequence verification, the full length fragment of the right-on paddy rice glyceraldehyde 3-phosphate dehydrogenase gene of acquisition base sequence;
C, the correct gene segment that (B) step card is obtained are connected among the carrier pUbiO that transforms acquisition, and the paddy rice glyceraldehyde 3-phosphate dehydrogenase gene is placed between corn ubiquitin promoter and the NOS tail, form the carrier that transforms or express;
D, the overexpression carrier pUbiO that contains goal gene that will prepare change Agrobacterium EHA105 over to, import in the mature embryo inductive rice callus tissue, and through breaking up the paddy rice transfer-gen plant that obtains this gene of overexpression eventually again.
3. the application of a kind of glyceraldehyde 3-phosphate dehydrogenase gene in paddy rice anti contravariance shown in the claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436595A (en) * | 2013-04-29 | 2013-12-11 | 冯家望 | LAMP detection primer group of NOS terminator, kit and detection method |
| CN106148391A (en) * | 2015-04-10 | 2016-11-23 | 武汉大学 | Application in cultivating drought-resistant and anti-salt transgenic paddy rice for the mouse nNOS gene |
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| CN1436242A (en) * | 2000-04-13 | 2003-08-13 | 马克·阿龙·埃马尔法尔布 | Expression-regulating sequences and expression products in field of filamentous fungi chrysosporium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436595A (en) * | 2013-04-29 | 2013-12-11 | 冯家望 | LAMP detection primer group of NOS terminator, kit and detection method |
| CN106148391A (en) * | 2015-04-10 | 2016-11-23 | 武汉大学 | Application in cultivating drought-resistant and anti-salt transgenic paddy rice for the mouse nNOS gene |
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