CN101691573A - Gene and protein of secretory peroxidase of calyx canthus and application thereof - Google Patents
Gene and protein of secretory peroxidase of calyx canthus and application thereof Download PDFInfo
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Abstract
The invention relates to genes and proteins of secretory peroxidase of calyx canthus and applications thereof, belonging to the field of molecular biology and gene engineering. The invention provides the gene sequence of secretory peroxidase of calyx canthus, the amino acid sequence of coding protein thereof, and applications of gene transfer plants of secretory peroxidase of calyx canthus. In the invention, the gene of secretory peroxidase of calyx canthus can protect cells of transgenic plants from being damaged by oxygen stress, improve the ability to resist adverse conditions of plants, and improve the transformation efficiency of calluses of transgenic plants.
Description
Technical field:
The present invention relates to molecular biology and genetically engineered field, more specifically must be to the present invention relates to the gene clone of wintersweet Class III type secretory peroxidase, albumen and application thereof.
Background technology:
Farm crop often are subjected to the influence of various adverse circumstances in process of growth, under multiple biology and abiotic stress, plant the multiple variation of form, physiology, biochemical each side can be taken place and produced a large amount of active oxygens.From plant itself, can use the defense mechanism of self to go to overcome the harm of coercing effectively, be the key that influences plant life.
Peroxidase [peroxidase; EC 1.11.1.7] be extensively to be present in the intravital oxydo-reductase of various animals, plant and microorganism; can participate in the active oxygen metabolism process, be the important protective enzyme of plant anti-oxidation protection enzyme system, is considered one of biomarker that the plant response environment coerces.Peroxidase is divided into three classes: ClassI usually based on their sequence and catalysis characteristics, Class II, Class III.Two classes are arranged in the plant materials: Class I and Class III peroxidase, and Class II peroxidase is present among the fungal cell.
The peroxidase of our common indication is a Class III peroxidase (POD), and nearly all terrestrial plant all contains Class III peroxidase (F.Passardi et al.Phytochemistry, 2004) except unicell green alga.These Class III peroxidase are that the Lu Sheng life of plant adaptation hyperoxia facilitates, and the diversity of the complicacy of the evolution of this multigene family and organization structure of the plant, living environment and pathogen is all closely related.
POD is relevant with the resistance of plant, and plant its normal oxygen metabolism under environment stress is interfered, and has improved active oxygen on the one hand and has produced speed, destroys the cytoprotective system on the other hand again.Taking precautions against a vital step of oxidative damage is with O
2 -And H
2O
2, toxicity remove fast, POD can utilize the various substrate can be with H as electron acceptor(EA)
2O
2Change water into.In the antioxidant system system of plant, the synergy of SOD, POD, CAT can reduce the accumulation of oxyradical, and has checked O
2 -H
2O
2Transform to OH by Fenton type Haber-Weiss reaction, the degraded that can avoid or alleviate radical pair biomacromolecule such as nucleic acid, protein (enzyme) etc. destroys and to biomembranous infringement, thus the resistance of raising plant.This shows that POD has important role (H.M.Jespersen et al, Biochem J, 1997 for the removing of ROS toxic action, the reparation of membrane damage; Susumu Hiraga et al, Plant and Cell Physiology, 2004).
A kind of as pathogenesis-related proteins, POD can also be by various disease and pest abduction deliverings (van Loon et al.PlantMol.Biol.Rep, 1994).The effect of POD relates to and coerces that factor inductive cell walls is strengthened, the generation of phenols pisatin, with ROS as (B.K.Kristensen et al.Phytochem Rev such as signal intermediary and biocides, 2004.), resist pathogen and plant parasite (P.F.Dowd et al.Agric Food Chem, 2005.).The tyrosine residues that polysaccharide bonded ferulate and other polymer link, xylogen and suberin monomer are cross-linked to form network curing cell walls (Fry et al.Plant Physiol, 1986 of a complexity by the peroxidation dehydrogenation under the POD mediation; Filippo Passardi et al.Trends inPlant Science), hard cell walls helps to resist further searching for food of insect and animal, when disease and pest is attacked, pass through simultaneously to produce a large amount of active oxygens, form the microorganism that deleterious environment kills or suppresses to invade.At H
2O
2Exist down, POD can also the many single phenol of catalysis, diphenol and aromatic amine reaction generate has highly toxic quinone (Shi X H etal, Viticulture﹠amp to microorganism; Enology, 1997).
The expression of peroxidase mainly comprises constructive expression and inducible expression.Except with the plant eubolism with growing the structure-type peroxidase relevant, the synthetic abduction delivering type that belongs to of very most of peroxidase.This coerces Processing Test and the gene regulating institute on molecular level confirms for a large amount of.Action of gravity, ultraviolet lighting, arid, saline and alkaline, temperature, aging, variation of ecology and environment, infection process and damage all can be induced the synthetic expression of multiple phenolic compound such as phenyl propyl compound, peroxidase is coerced response (Dixon et al.Plant Cell, 1995) by participating in this type of secondary metabolism generation.
The aminoacid sequence alterable height of POD, isozyme is of a great variety, in the arabidopsis gene group, 73
Individual gene has obtained evaluation, and great majority are expressed at root.These 73 genes account for the total est sequence 2.2% of root, but have only sub-fraction to show tissue specificity (Welinder KG et al.Eur J Biochem, 2002).138 gene distribution of paddy rice are in almost all (Passardi F et al.Phytochemistry, 2004.) on the karyomit(e)s.The defective of any peroxidase enzymes or reticent tending to produce complicated phenotypic characteristic owing to the interference of other isozyme.In fact, Arabidopis thaliana also has only two kinds of peroxidase enzymes defective mutant to obtain research (F.Passardi et al.Planta, 2006.) at present.More than the POD isozyme kind, textural difference is big, and the assorted specific function that causes being difficult to specific POD of substrate is systematically determined.Because the uncertainty of these gene functions, present research still concentrates on the evaluation of each individual gene specific function.
Up to now, relation research about POD and Plant diseases resistance is the most extensive, Svalheind etc. studies show that, cucumber susceptible variety and disease-resistant variety can both induce POD synthetic to infecting of germ, both differences mainly are the increase more rapid (Svalhein of disease-resistant variety than susceptible variety POD, et, al.physiol plant, 1990).People's such as Baga proof, wheat leaf blade peroxidase enzymes POX
2Gene infects the back by abduction delivering (BajaM.et, al.Plant MolecularBiology, 1995.) in Powdery Mildew.POD also participates in apoptosis, and Scialabba etc. studies show that peroxidase is induced in a large number to resist old and feeble relevant lipid peroxidation (A.Scialabba et al.Eur.J.Histochem, 2002.) in artificial old and feeble radish seed.The POD gene that utilizations such as Ipelcl are cloned from leguminous plants oppositely changes willow over to, make content of lignin reduce 10%-20% (IPEKCI Z et al.Plant Biotechnol journal, 1999.), Mansouri etc. find in the transgenic Fructus Lycopersici esculenti plant of overexpression POD, the content of xylogen risen (Mansouri I.E et al Physiologia.Plantarum, 1999).These have proved that POD is also relevant with xylogen or corky generation.
In numerous research, do not see the relevant report of relevant POD in how anti-plant wintersweet.And wintersweet [Chimonanthus praecox] is as a kind of species with how anti-characteristic, and the adaptive faculty of environment is more intense to external world.And wintersweet is drought-enduring, cold-resistant, disease and pest is less, the how anti-gene of certain existence in wintersweet.The Wintersweet Flower ESTs sequence that the contriver utilizes this laboratory to make up has been cloned peroxidase gene from Wintersweet Flower cDNA library.Made up the carrier that wintersweet POD gene is suitable for expression of plants, made the proteins encoded of this gene of expression of plants by agriculture bacillus mediated approach, thereby improve the anti-adversity of these species.
Summary of the invention:
The objective of the invention is: (1) provides a kind of dna sequence dna, its a kind of cured enzyme secretion peroxidase of encoding, and called after CpPOD (2) provides a kind of gene engineering method of utilizing to obtain the CpPOD encoded protein; (3) provide the application of secretory peroxidase of calyx canthus gene aspect the cultivation stress resistance plant.
Technical scheme:
The invention provides a kind of cured enzyme secretion peroxidase gene, it has as the described nucleotide sequence of SEQ ID NO:1 in the sequence table.
The present invention is from wintersweet corolla cDNA library, utilize the PCR method screening and cloning to go out the secretory peroxidase of calyx canthus gene: to utilize RNAiso Reagent to extract the total RNA of wintersweet corolla, according to the synthetic wintersweet corolla cDNA library of SMARTTM cDNA Library ConstructionKit.1000 library clone order-checkings of picking and bioinformatic analysis obtain wintersweet corolla resistance ESTs sequence at random.From Wintersweet Flower ESTs sequence, find the cDNA sequence of coding wintersweet POD.According to this sequences Design upstream and downstream primer, and the Wintersweet Flower cDNA library of setting up with this laboratory is that template is carried out pcr amplification, clones the secretory peroxidase of calyx canthus full-length gene.
The gene of secretory peroxidase of calyx canthus of the present invention contains a complete open reading frame by 1047 based compositions, is the SEQ ID NO:1 sequence in the sequence table, called after CpPOD.The initiator codon of opening code-reading frame is ATG, and terminator codon is TGA.
The invention still further relates to the protein of wintersweet POD genes encoding.It has the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table, is to contain 348 amino-acid residues, and its theoretical molecular size is 37.6kDa, and the prediction iso-electric point is 9.63.
Wintersweet POD gene of the present invention can insert expression vector and express.Therefore, another aspect of the present invention provides can express wintersweet POD expression carrier in host cell, and described expression vector can be the carrier that is used at bacterial expression, also can be to be used for the carrier of expressing plant.
As described in embodiment hereinafter, can also utilize engineered method, in efficient escherichia expression system, give expression to the wintersweet POD albumen of biologically active.
The invention provides wintersweet POD gene in the application of cultivating on the resistance plant, comprise with the expression vector transformed plant cells that makes up; The plant transformed cell culture is become plant.CpPOD gene coding region of the present invention is cloned among the plant expression vector pETV7, it is imported tobacco leaf, obtain the tobacco strain system of overexpression, improve the anti-oxidant and degeneration-resistant ability of tobacco by the Agrobacterium infestation method.
The present invention carries out the water-retentivity experiment to Nicotiana gossei and transgene tobacco, and the tobacco of finding to change CpPOD can greatly reduce the loss of moisture, and percentage of water loss is compared Nicotiana gossei and reduced by 50% behind its stomatal closure.
The present invention coerces the mensuration of damage to chlorophyll content by oxygen, finds that the chlorophyll loss of transgene tobacco is starkly lower than Nicotiana gossei, and the relative Nicotiana gossei of transgene tobacco has shown the ability that stronger antioxygen is coerced damage.
The present invention also provides a kind of method of screening transgenic plant, uses the wintersweet POD gene gene that serves as a mark, and is selective pressure with the hydrogen peroxide, screens transgenic plant.
The invention provides the application of wintersweet POD gene aspect raising callus transformation efficiency.
Beneficial effect:
The invention provides a kind of secretory peroxidase of calyx canthus gene and albumen, made up plant expression vector, and obtain transgenic plant.The tobacco that changes CpPOD can greatly reduce the loss of moisture, and percentage of water loss is compared Nicotiana gossei and reduced by 50% behind its stomatal closure.Change the relative Nicotiana gossei of CpPOD tobacco and shown the ability that stronger antioxygen is coerced damage, with the H of 50mM
2O
2The aqueous solution was handled after 8 days, and the chlorophyll rate of loss of transgene tobacco is 39%, and the chlorophyllous rate of loss of wild-type tobacco is 96%, and the loss of the chlorophyll of transgene tobacco is starkly lower than Nicotiana gossei.Can use the CpPOD gene that serves as a mark, be selective pressure with the hydrogen peroxide, screens transgenic plant.The CpPOD gene can also improve the transformation efficiency of maize calli.
Describe the present invention hereinafter with reference to accompanying drawing with embodiment, be understood that embodiment only describes the present invention for example, and unrestricted the present invention.
Description of drawings:
Fig. 1: with wintersweet corolla cDNA is template, the pcr amplification electrophorogram.
M:marker size from top to bottom is: 2000bp, 1000bp, 750bp, 500bp, 250bp
1: the purpose band;
Fig. 2: SDS-PAGE prokaryotic expression whole protein;
M:Marker is respectively 99,66,43,31,20,14 (KDa);
1: the BL21 host bacterium whole protein that contains recombinant clone plasmid pET-32a::CpPOD;
CK: the BL21 host bacterium whole protein that contains empty carrier plasmid pET-32a;
Fig. 3: the building process of CpPOD plant expression vector;
Fig. 4: the positive seedling RT-PCR of transgene tobacco identifies
M:marker size from top to bottom is: 2000bp, 1000bp, 750bp, 500bp, 250bp
1, the positive seedling RT-PCR of 2 transgene tobaccos 3,4 wild-type tobacco RT-PCR
Fig. 5: the transgene tobacco water-retentivity is analyzed;
Fig. 6: different concns H
2O
2Wild-type and transgene tobacco blade that the aqueous solution is handled;
Fig. 7: oxygen is coerced the influence of damage to the transgene tobacco chlorophyll content;
Embodiment;
Embodiment one: the clone of wintersweet POD gene
1.RNA extraction
Get 500mg wintersweet corolla (removal holder) and extract the total RNA of Wintersweet Flower with RNAiso Reagent.
2.cDNA the structure in library
Get total RNA, according to the synthetic Wintersweet Flower cDNA library of SMARTTM cDNA Library Construction Kit.1000 library clone order-checkings of picking and bioinformatic analysis obtain Wintersweet Flower resistance ESTs sequence at random.
3. the clone of wintersweet POD gene fragment
From the Wintersweet Flower ESTs sequence that this experiment provides, find the cDNA sequence of coding Wintersweet Flower POD.According to this sequences Design upstream and downstream primer, and the Wintersweet Flower cDNA library of setting up with this laboratory is that template is carried out pcr amplification.
Auele Specific Primer is as follows:
CpPOD-P1:5 '-GGAATTCAAAACCATGGCCGCTACTGCT-' 3, the italic base is the EcoRI restriction enzyme site;
CpPOD-P2:5 '-CCGCTCGAGTCTTTCATTCATTCATCACAA-' 3, the italic base is an Xho I restriction enzyme site.
The clone PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 50sec; 50 ℃ of annealing 50sec; 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min.Amplified production detects through 1% agarose gel electrophoresis, as shown in Figure 1, a specific band is arranged on the 1000bp position greatly.And then according to a conventional method fragment cloning is arrived among the pMD18-T Vector (available from Dalian Takara company), and check order through Shanghai bio-engineering corporation.
Embodiment two: the CpPOD Gene Sequence Analysis
Comprise 1047bp through this gene cDNA sequence open reading frame that checks order, 348 amino acid of encoding, the molecular weight size is 37.6kDa, the prediction iso-electric point is 9.63.The result shows the cDNA sequence of the POD gene that has obtained total length, is CpPOD with this unnamed gene.The sequence of the POD gene of its amino acid sequence coded and known other species has higher homology, all more than 61%.
According to preceding 30 left and right sides amino acid of psort analysis CpPOD is that signal peptide and its expression product are positioned outside the born of the same parents.Through comparing with the structural performance analysis of other species class III peroxidase, prediction CpPOD avtive spot is positioned at the 71-82 amino acids, is the AAALLRIHFHDC sequence, and wherein the 80th His is a key amino acid.First calcium ion binding site is positioned at the 81st, 84,86,88,90 amino acids, and second calcium ion binding site is positioned at the 210th, 255,258,263 amino acids.The near-end heme group is present in the 201-211 position, and structure sequence is DLVALSGGHTI, and the prediction iron ion is incorporated into the 209th His amino acid, and disulfide linkage is present in 49 and 130,82 and 87,136 and 331,216 and 243 amino acids.The substrate binding site of CpPOD is positioned at the 179th.In addition, the 39th amino acids is possible posttranslational modification site.
Embodiment three: CpPOD efficiently expressing in intestinal bacteria
1. construction of prokaryotic expression vector
In order to show the encoding function of CpPOD gene, the CpPOD gene clone is arrived between the EcoRI and XhoI site of pET-32a (+), and transformed into escherichia coli BL21, obtain to efficiently express.
Design and synthesize a pair of Auele Specific Primer (with embodiment 1), press molecular cloning normal experiment program, amplify the gene coded sequence that two ends have specific restriction enzyme site with the method for PCR, gene is connected with carrier pMD18-T, checking is correct.Get pMD18::CpPOD plasmid and pET-32a (+) plasmid (purchasing) respectively with EcoRI, XhoI double digestion, be connected again in Shanghai ancient cooking vessel state biotech firm, to connect in the product transformed into escherichia coli BL21 bacterial strain (purchasing biotech firm) in Kai Ji, and with the LB plate screening recon that contains the 100ug/ml penbritin.After plasmid enzyme restriction evaluation and PCR evaluation, the correct recon of connection that sifts out is checked order, prove that the reading frame of the dna sequence dna that inserts carrier is correct.Have the CpPOD expression carrier being built into, name pET-32a::CpPOD is used for the abduction delivering analysis.
2. prokaryotic expression
The bacterial strain that will contain the pET-32a::CpPOD recon is inoculated in (the automatic abduction delivering substratum of ZYM5052 (1L): peptone 10g, yeast extract 5g, glucose 0.5g, alpha-lactose 2g, Na in the automatic abduction delivering substratum of ZYM5052
2HPO
45.372g, KH
2PO
42.041g, NH
4Cl 1.604g, Na
2SO
40.426g, MgSO
40.2958g, glycerine 5mL, pH 7.0), 37 ℃ are cultured to bacterium liquid OD
600=2.1-3.3.The recombinant expressed bacterium of BL21 is ground and ultrasonication, and we have obtained containing the BL21 host bacterium crude enzyme liquid of recombinant clone plasmid pET-32a::CpPOD.Carry out the 12%SDS-PAGE electrophoresis detection, as can be seen from Figure 2, the CpPOD gene efficiently expresses in BL21.
Embodiment four: contain the structure of plant expression vector of CpPOD gene and the preparation of transgene tobacco.
CpPOD gene coding region of the present invention is cloned into plant expression vector pTEV7, and (preserve in this laboratory, Yin Junhua, clone and the functional study of Wintersweet Flower aquaporin CpTIP cDNA, middle National IP Network includes, 2008.) in, by the Agrobacterium infestation method it is imported in the tobacco, make the tobacco strain system of overexpression, improve the anti-oxidant and degeneration-resistant ability of tobacco.
1. the structure of plant expression vector
Design a pair of primer, introduce the restriction enzyme site of XbaI and XhoI respectively.
CpPOD-P3 (italic is the XbaI enzyme cutting site):
5′-GCTCTAGAACCATGGCCGCTACT-′3
CpPOD-P2 (italic XhoI restriction enzyme site):
5′-CCGCTCGAGTCTTTCATTCATTCATCACAA-′3
Press molecular cloning normal experiment program, amplify the purpose fragment with the method for PCR, with fragment cloning to pMD18-TVector, order-checking.To identify that correct positive recombinant plasmid pMD18::CpPOD and plant expression vector pETV7 carries out XbaI and XhoI double digestion through order-checking.And enzyme is cut product reclaim, connect, be built into purpose carrier pTEV7::CpZPOD (construction strategy is seen Fig. 3) and be transformed among the agrobacterium tumefaciens GV3101.
2. utilize leaf dish infestation method transformation of tobacco
(1) Agrobacterium of picking through identifying is inoculated in the liquid nutrient medium, 28 ℃, the about 48h of 250rpm shaking culture, to the logarithmic growth later stage, centrifugal, abandon supernatant, thalline 1/2MS liquid medium (1/2 * MS macroelement, 1 * MS trace element, 1 * MS VITAMIN, 1 * molysite, pH5.7-5.8) suspension is diluted to about OD600=0.5.
(2) get aseptic tobacco leaf, remove the main lobe arteries and veins, it is cut into small pieces (1cm * 1cm).
(3) tobacco leaf that shears is placed the MS division culture medium (1 * MS macroelement, 1 * MS trace element, 1 * MS VITAMIN, 1 * molysite, sucrose 30g/L, 6-BA 3mg/L, NAA O.2mg/L, agar powder 8g/L, pH5.7-5.8.), 28 ℃, light application time 16h/ days, intensity of illumination 2000LX cultivated 2 days.
(4) tobacco leaf that will cultivate 2 days immerses 10min in the described firm ready bacterium liquid of the first step, blots bacterium liquid on aseptic filter paper, blade is placed on the MS substratum 28 ℃ of dark cultivations 2 days.
(5) blade that is total to after cultivating washs 3 times with the sterilized water that contains cephamycin 300mg/L earlier after microcolony occurring around it, wash 1 time with the MS nutrient solution that contains cephamycin 300mg/L again, blot with aseptic filter paper then, change on the MS screening culture medium that contains Totomycin, constant temperature culture (condition is cultivated with pre-), per 15 days replacing one subcultures.
When (6) treating that bud grows to the 1cm left and right sides, downcut and move in the root media, short its taken root.After treating root system development, be numbered, and move into and fill in the flowerpot of sterile soil, preserved moisture 2 days the room temperature Routine Management with plastics film transforming seedling.
Embodiment five: with RT-PCR method validation transgene tobacco
1. from the tobacco leaf that has imported the CpPOD gene, extract total RNA with ordinary method, with the negative contrast of the tobacco of wild-type, the result shows that 28S and 18S strip-type are neat, and the 28S band is obviously bright in the 18S band, illustrate that total RNA integrity is fine, do not degrade, can identify as further reverse transcription.With the tobacco RNA of the commentaries on classics CpPOD gene that extracts is that primer obtains strand cDNA through reverse transcription with CpPOD-P2.
2. being template again with cDNA, is that primer carries out pcr amplification (primer sequence is with embodiment four) with CpPOD-P3, CpPOD-P2 then.Amplified production detects through 1% agarose gel electrophoresis, and a specific band is arranged on the 1000bp position greatly, and is consistent with CpPOD gene size, and on the swimming lane of wild-type plant negative control this band not, illustrate that the transfer-gen plant of detection is positive.This has proved that tentatively the CpPOD gene has been integrated in the tobacco gene group, and has obtained the expression (see figure 4) on rna level.
Embodiment six: the transgene tobacco water-retentivity is measured
It is close to get under the equal culture condition size, and Nicotiana gossei identical in quality and transgene tobacco blade are put into 28 ℃ of incubators after taking by weighing fresh weight.For the blade pore is closed fully, after 150min, begin to measure moisture and reduce situation, after this quality that takes by weighing blade every 1h is measured 5 times altogether, calculates the percentage of water loss (as Fig. 5) of two kinds of blades.
Percentage of water loss=(weight after fresh weight-dehydration)/fresh weight
The interior relative Nicotiana gossei of percentage of water loss of transgene tobacco unit time had reduced by 50% after the result showed stomatal closure.
Embodiment seven: oxygen is coerced damage the influence of transgene tobacco chlorophyll content is measured
Get the Nicotiana gossei and the transgene tobacco blade of close leaf age, punching is handled.Two kinds of blades are suspended in the H that contains 0mM, 10mM, 20mM, 30mM, 40mM, 50mM respectively
2O
2On the aqueous solution (Fig. 6), 28 ℃, normal illumination were cultivated after 8 days, with the alcoholic extraction of 5mL96% (v/v) to bleaching.Measure OD
665, OD
649, each handles 5 variations (Fig. 7) that repeat and measure chlorophyll content.
Chlorophyll-a concentration Ca=13.95OD wherein
665-6.88OD
649,
Chlorophyll b concentration C b=24.96OD
649-7.32OD
665,
Chlorophyll total concn C=Ca+Cb
Chlorophyllous content (mg/g)=[chlorophyllous concentration * extracting liquid volume * extension rate]/sample fresh weight (or dry weight).
Oxidative stress is to the mensuration of transgene tobacco and Nicotiana gossei damage, and the result shows that the chlorophyll loss of transgene tobacco is starkly lower than Nicotiana gossei.The relative Nicotiana gossei of transgene tobacco has shown the ability that stronger antioxygen is coerced damage, and this may be to come from the H of the CpPOD of overexpression for the external world
2O
2The removing ability of toxic action, and the variation of chlorophyll content shows that CpPOD has the certain protection effect to photosynthetical system.Can utilize the wintersweet POD gene gene that serves as a mark, be selective pressure with the hydrogen peroxide, screens transgenic plant.In addition, the reinforcement of the wild relatively plant cell walls of transfer-gen plant has also reduced extraneous H to a certain extent
2O
2Toxic action.
Embodiment eight: the maize transformation callus, improve transformation efficiency
1. the Agrobacterium that contain pTEV7::CpPOD plasmid of picking through identifying is inoculated in the liquid nutrient medium, 28 ℃, the about 48h of 250rpm shaking culture, to the logarithmic growth later stage, centrifugal, abandon supernatant, thalline suspends with the 1/2MS liquid medium and is diluted to about OD600=0.5.
2. get 10～14 days the female young fringe of corn inbred line H99 of pollination, the rataria of picking 1.5～2.0mm under the aseptic condition, scultellum places N up
6(N on the inducing culture
6+ 2,4-D3mg/L), secretly cultivate about 15 days, produce I type initial callus, every 15 days subcultures 1 time, produce steady I I type callus behind the subculture 2 times.Get the acceptor material of the II type callus of subculture 4 times as Agrobacterium-mediated Transformation.
3. the corn II type callus of cultivating is immersed 20min in the described firm ready bacterium liquid of the first step, every the 5min vibration once, on aseptic filter paper, blot bacterium liquid, callus is placed on the MS substratum, 28 ℃ of dark cultivations 3 days.
4. the callus after cultivating altogether with the sterilized water washing that contains cephamycin 100mg/L 3 times, is washed 1 time with the MS nutrient solution that contains cephamycin 100mg/L earlier again, blots with aseptic filter paper then, changes the N that contains Totomycin over to
6On the screening culture medium, constant temperature culture, screening culture medium of replacing in per 13 days.
(6) after the screening 3 times, the callus with survival changes subculture medium over to again, calculates the callus of survival.The survival rate that transforms the maize calli of pTEV7::CpZPOD plasmid is 6%, apparently higher than pTEV7 empty plasmid that transforms and the survival rate 3% that contains the maize calli of other plasmids, shows that the cpPOD gene can improve the transformation efficiency of maize calli.
Sequence table
The sequence of SEQID NO.1
(i) sequence signature:
(A) length: 1047bp
(B) type: Nucleotide
(C) chain: strand
(ii) molecule type: Nucleotide
(iii) sequence description: SEQID NO.1
1 ATGGCCGCTA?CTGCTGCTTC?TTCTTCCTCG?TCCTCCACGG?CGGTGGTGGT?GGTGCTTGTT
61 CTTGCTTGCC?TTGCTTTCTT?GTCAGAAGCG?GAGAGTTCTC?CTCCTGTTGT?GAAGGGACTT
121 TCATGGAGTT?TCTACAAGTC?TAGCTGCCCA?AAGGTGGAAA?GTATTGTACG?GAGCCACCTT
181 AAGAAGGTTT?TCAAGAAGGA?TATTGGCCAG?GCTGCTGCCC?TGCTTCGCAT?CCATTTCCAT
241 GACTGCTTTG?TTCAGGGGTG?TGATGGGTCA?GTGCTGTTGG?ATGGATCAAC?TGCCGGTCCA
301 GGTGAGCAAG?ACGCACCACC?AAACCTAACA?CTGAGGAAGG?AGGCATTCAA?GGTCATCAAC
361 CAACTCCGTG?CTCTTATCCA?CAAACAATGT?GGCCAGGTCG?TCTCCTGCTC?GGACATTGTT
421 GCTCTTGCTG?CTCGTGACCC?AGTCGCTCTG?TCGGGCGGTC?CAAATTACAG?AGTGCCACTG
481 GGTCGCCGAG?ATAGCCTGAA?GTTTGCCACA?AGAGATGCGA?CGCTCGCTAA?CCTTCCGGGA
541 CCATCCTCAA?ATGCCGCTGT?TCTCATTGAA?GCTCTGAGCA?AAAAGAAGCT?TGACGCCACC
601 GATCTTGTGG?CCCTCTCTGG?TGGTCACACC?ATTGGAATCG?GCCATTGCTC?ATCGTTCACC
661 GACCATCTCT?ACCCATCTCA?AGACTCCACC?ATGGACCAGA?CCTTTGCAAA?GAACCTCTAC
721 AAGACCTGCC?CCGAGGAAAA?CACTAACAAC?ACCACGGTGT?TGGACATCCG?ATCTCCGAAC
781 AAGTTCGACA?ACAAGTACTA?TGTTGACCTC?ATGAACCGAC?AGGGACTCTT?CACGTCCGAC
841 CAGGACTTGT?ACACTGACAA?GAGGACACGA?CCGATCGTCA?CGAGCTTCGC?GATCAACGAG
901 ACCTTGTTCT?TTGAGAAATT?TGCTCTGTCA?ATGGTGAAGA?TGGGTCAGCT?GAGTGTATTG
961 ACTGGGAAAG?AGGGTGAGAT?ACGTGCAAAT?TGCTCTGCTC?GCAACTCCGC?AAGGTCTTCA
1021 CTGTGGTCTA?TTGTGATGAA?TGAATGA
The sequence of SEQID NO.2
(i) sequence signature:
(A) length: 348 amino acid
(B) type: amino acid
(C) chain: strand
(ii) molecule type: polypeptide
(iii) sequence description: SEQID NO.2
1 MAATAASSSS?SSTAVVVVLV?LACLAFLSEA?ESSPPVVKGL
41 SWSFYKSSCP?KVESIVRSHL?KKVFKKDIGQ?AAALLRIHFH
81D CFVQGCDGS VLLDGSTAGP?GEQDAPPNLT?LRKEAFKVIN
121 QLRALIHKQC?GQVVSCSDIV?ALAARDPVAL?SGGPNYRVPL
161 GRRDSLKFAT?RDATLANLPG?PSSNAAVLIE?ALSKKKLDAT
201 DLVALSGGHT?IGIGHCSSFT?DHLYPSQDST?MDQTFAKNLY
241 KTCPEENTNN?TTVLDIRSPN?KFDNKYYVDL?MNRQGLFTSD
281 QDLYTDKRTR?PIVTSFAINE?TLFFEKFALS?MVKMGQLSVL
321 TGKEGEIRAN?CSARNSARSS?LWSIVMNE＊
Claims (10)
1. a dna sequence dna is characterized in that it has the described nucleotide sequence of SEQ ID N0:1 in the sequence table.
2. a protein is characterized in that it is by claim 1 described dna sequence encoding, has the described aminoacid sequence of SEQ ID NO:2.
3. a recombinant prokaryotic expression vector is characterized in that containing gene as claimed in claim 1.
4. a recombinant plant expression vector is characterized in that containing gene as claimed in claim 1.
5. expression vector transformed host cells with claim 3, it is intestinal bacteria.
6. expression vector transformed host cells with claim 4, it is an agrobacterium tumefaciens.
7. the genetically modified host cell that transforms of the carrier of a claim 4, it is tobacco cell, maize cell.
8. the described gene order of claim 1 is in plant disease-resistant, anti-oxidant and adversity gene application in engineering.
9. the described gene order of claim 1 is in the application that improves aspect the callus transformation efficiency.
10. method of screening transgenic plant is characterized in that using the nucleotide sequence of claim 1 gene that serves as a mark, and is selective pressure with the hydrogen peroxide, screens transgenic plant.
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| CN2009100674569A CN101691573B (en) | 2009-08-31 | 2009-08-31 | Gene and protein of secretory peroxidase of calyx canthus and application thereof |
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| CN101691573A true CN101691573A (en) | 2010-04-07 |
| CN101691573B CN101691573B (en) | 2012-05-30 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010869A (en) * | 2010-04-26 | 2011-04-13 | 吉林大学 | Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof |
| CN107312077A (en) * | 2017-08-01 | 2017-11-03 | 西南大学 | Albumen and the application of wax plum CpSOC1 genes and its coding |
| CN107840872A (en) * | 2017-08-16 | 2018-03-27 | 西南大学 | Albumen and the application of wax plum CpWOX13 genes and its coding |
| CN110295183A (en) * | 2019-07-29 | 2019-10-01 | 西南大学 | A method of citrus is improved to canker resistance based on CsPrx25 overexpression |
| CN116676411A (en) * | 2023-05-06 | 2023-09-01 | 吉林农业科技学院 | ZmPRX19 gene mutation detection primer and application thereof in screening of maize inbred line with strong somatic cell regeneration capability |
-
2009
- 2009-08-31 CN CN2009100674569A patent/CN101691573B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010869A (en) * | 2010-04-26 | 2011-04-13 | 吉林大学 | Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof |
| CN102010869B (en) * | 2010-04-26 | 2012-05-30 | 吉林大学 | Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistance application thereof |
| CN107312077A (en) * | 2017-08-01 | 2017-11-03 | 西南大学 | Albumen and the application of wax plum CpSOC1 genes and its coding |
| CN107312077B (en) * | 2017-08-01 | 2019-06-21 | 西南大学 | Albumen and the application of wax plum CpSOC1 gene and its coding |
| CN107840872A (en) * | 2017-08-16 | 2018-03-27 | 西南大学 | Albumen and the application of wax plum CpWOX13 genes and its coding |
| CN107840872B (en) * | 2017-08-16 | 2019-06-21 | 西南大学 | Albumen and the application of wax plum CpWOX13 gene and its coding |
| CN110295183A (en) * | 2019-07-29 | 2019-10-01 | 西南大学 | A method of citrus is improved to canker resistance based on CsPrx25 overexpression |
| CN110295183B (en) * | 2019-07-29 | 2023-05-02 | 西南大学 | Method for improving resistance of citrus to canker based on CsPrx25 overexpression |
| CN116676411A (en) * | 2023-05-06 | 2023-09-01 | 吉林农业科技学院 | ZmPRX19 gene mutation detection primer and application thereof in screening of maize inbred line with strong somatic cell regeneration capability |
| CN116676411B (en) * | 2023-05-06 | 2024-01-02 | 吉林农业科技学院 | ZmPRX19 gene mutation detection primer and application thereof in screening of maize inbred line with strong somatic cell regeneration capability |
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| Publication number | Publication date |
|---|---|
| CN101691573B (en) | 2012-05-30 |
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