CN104946631A - Corn transformation event 'double resistance 12-5' and specificity identification method thereof - Google Patents
Corn transformation event 'double resistance 12-5' and specificity identification method thereof Download PDFInfo
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Abstract
The invention discloses a corn transformation event 'double resistance 12-5' and a specificity identification method thereof. The corn transformation event 'double resistance 12-5' is characterized in that a nucleotide sequence shown in SEQ ID No.1 is taken as a left side wing region of a foreign gene and a nucleotide sequence shown in SEQ ID No.3 is taken as a right side wing region of the foreign gene. The transformation event '12-5' provided by the invention is good, the foreign gene can be specifically introduced into a corn line, and insect resistant and glyphosate resistant capability is given to a corn acceptor; an insect-resistant and glyphosate-resistant gene can be stably inherited in the corn acceptor; and expression of the insect-resistant and glyphosate-resistant gene can not produce adverse effects on agronomic characters of the corn acceptor.
Description
(1) technical field
The present invention relates to a kind of corn transformation event and specificity identification method thereof, particularly a kind of corn transformation event " dual anti-12-5 " and method for detecting specificity thereof.
(2) background technology
Corn (Zea mays) is a kind of important crop, is the main food source in a lot of area in the world.Along with the development of plant genetic engineering, carry out one of genetic modification Main Means becoming genetic breeding by importing foreign gene.In Maize Production, pest-resistant and antiweed is all in demand economical character.Insect-resistant transgenic corn can reduce the usage quantity of chemical insecticide significantly, thus reduces production cost and reduce the pollution of agricultural chemicals to environment and crop products.Antiweed corn significantly can reduce the agricultural workforce required for controlling weeds, reduces the input of labor force, reduces weeds to the impact of corn yield.In addition, utilize weedicide controlling weeds also advantageously in popularization no-tillage technology, reduce the loss of soil and fertilizer.Therefore pest-resistant and antiweed performance has outstanding importance in Maize Production.
Transformation event is the molecular structure be made up of at upstream and downstream flanking region and the foreign gene of genomic insertion site foreign gene.Usually, gene transformation plant can obtain a transformant colony, and this transformant colony comprises a large amount of independently event, and wherein each event is unique.The expression of foreign gene in plant is subject to the impact of the chromosome position that foreign gene inserts.This may be derived from the impact of transcriptional regulatory element near chromatin Structure or integration site.The expression level of homologous genes in different transformation event has very large difference, and the space of expressing or temporal mode also may there are differences.And the insertion of foreign gene also may affect the expression of native gene.Therefore, the impact of each separate transformation events on recipient plant is different.Obtain energy effective expression foreign gene, the Plant Transformation event simultaneously not affecting the economical character of plant own has important using value in cultivation genetically modified crops new variety.
At present and do not know that the insertion point of foreign gene in plant integrates rule, therefore need in R&D process produce a large amount of transformant and therefrom screen desirable transformation event.Usually need to produce hundreds of and even thousands of different transformation events, and in these transformation events, filter out the single transformation event with expection transgene expression level and pattern.Utilize breeding method, somatoplasm integration technology this transformation event can be imported in the Maize genome of different genetic background, thus give the ability of the pest-resistant resistance glyphosate of corn.
(3) summary of the invention
The object of the invention is to provide a kind of excellent transformation event " dual anti-12-5 ", and this transformation event can realize foreign gene to be incorporated in corn strain, gives the ability that recipient corn has pest-resistant resistance glyphosate; Pest-resistant Antiglyphosate gene can genetic stability in recipient corn; The expression of pest-resistant Antiglyphosate gene can not produce detrimentally affect to the economical character of recipient corn.
The technical solution used in the present invention is:
The invention provides a kind of corn transformation event " dual anti-12-5 " of single copy insertion exogenous gene sequence in Maize genome specific site, described transformation event arranges the pterion, left side into foreign gene with the nucleotides sequence shown in SEQ ID NO.1, arranges the pterion, right side into foreign gene with the nucleotides sequence shown in SEQ ID NO.3.Transformation event of the present invention utilizes Agrobacterium infestation method, pest-resistant for external source Antiglyphosate gene is imported in maize cell, obtain corn gene colony by the maize cell of regeneration containing foreign gene, and utilize the method for molecular biology and biological assay to screen the corn transformation event obtaining meeting need of production.
Further, described foreign gene comprises anti insect gene and Antiglyphosate gene, described anti insect gene is by two Bt genomic constitutions, preferred anti insect gene gene C ry1Ab and gene C ry2Aj fusion forms, nucleotides sequence is classified as shown in SEQ ID NO.4, and the nucleotides sequence of preferred described Antiglyphosate gene is classified as shown in SEQ ID NO.5.
Further, the nucleotides sequence of preferred foreign gene of the present invention is classified as shown in SEQ ID NO.2.
The present invention also provides a kind of corn transformation event " dual anti-12-5 " specific PCR authentication method, the described primer for the qualification of corn transformation event " dual anti-12-5 " specific PCR (whether SP1 with RB-Test detects on the right side of foreign gene to be connected with Maize genome specific position, and whether R1 with LB-test detects on the left of foreign gene to be connected with Maize genome specific position) is:
SP1：5’-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3’；
RB-Test：5’-CTCGTCATCGACCAAGTCATGAAG-3’。
R1：5’-CGTCGTTTTACAACGTCGTGACTGG-3’；
LB-test：5’-AAGACGTCCGGGGGAACCGTTGTTC-3’；
PCR reaction system of the present invention is:
PCR reaction conditions is: 32 circulations, and each circulation is 95 DEG C, 45 seconds; 65 DEG C, 50 seconds; 72 DEG C, 30 seconds.
" dual anti-12-5 " provided by the invention transformation event is made up of the maize genomic sequence SEQ ID NO.3 of maize genomic sequence SEQ ID NO.1, foreign gene T-DNA sequence (not comprising carrier frame) the SEQ ID NO.2 and insertion T-DNA right arm flank that insert T-DNA left arm flank, and this transformation event is obtained by genetic transforming method.In genetic transformation process, foreign gene Nucleotide is incorporated in Plant Genome randomly, and the transformation event of acquisition is unique and can genetic stability.This transformation event can be recombinated in different corn germ plasm resources by sexual hybridization or somatocyte hybriding technology, realizes the improvement to corn.Those skilled in the art will appreciate that by natural or artificial mode, the basis of described transformation event obtains by inserting, lacking, replace, replace, add one or more base the substructure of functional equivalent, also belongs within scope of the present invention.
Transformation event provided by the invention contains can give the pest-resistant expression cassette and resistance glyphosate expression cassette that transgenic corns has pest-resistant resistance glyphosate ability.
Anti insect gene provided by the invention is the fusion gene of cry1Ab and cry2Aj, and nucleotides sequence is classified as SEQ ID NO.4, and wherein, 1-1947bp is the nucleotide sequence of cry1Ab; 1948-1965bp nucleotides sequence is classified as CCCGGGAAGGGTGGAGGA, the connection peptides between coding Cry1Ab and Cry2Aj, and connection peptides sequence is PGKGGG; 1966-3861bp is the nucleotide sequence that cry2Aj improves gene.Fusion gene total length is 3861bp, and the aminoacid sequence of coding is SEQ ID NO.6.Coded protein is made up of 1287 amino-acid residues, and molecular weight of albumen size is 142.8kDa.Cry1 and Cry2 is the anti insect gene that two classes are widely used, wherein Cry1Ab, Cry1Ac, the large-scale promotion application in corn, cotton such as Cry1F and Cry2Ab, Cry1Ab is the Bt crystal insect-killing protein that a kind of killing ability is stronger, and particularly it is particularly high to the insecticidal activity of Pyrausta nubilalis (Hubern)..Cry2Aj has higher killing ability to staple crops lepidoptera pest, with the insecticidal spectrum of the Cry2Ab of current production large-scale popularization relatively, aminoacid sequence is also more similar.Security and the insect resistance capacity of these genes obtain sufficient proof.Present invention uses the fusion gene of cry1Ab and cry2Aj, be characterized in utilizing the pest-resistant Bt gene that two kinds different, and their expression amounts in corn are identical simultaneously.Current research is thought, the generation (Zhao et al., 2003, Nat.Biotechnol.21:1493-1497) that two dissimilar anti insect genes express likely to slow down pest resistance simultaneously, is conducive to the permanently effective utilization of insect-resistant transgenic corn.
The invention provides a kind of pest-resistant expression cassette, this expression cassette is made up of corn polyubiquitin-1 gene promoter (pZmUbi-1), insect-resistant fusion gene cry1Ab-cry2Aj and corn PEP carboxylase gene (pepc) terminator.Wherein corn polyubiquitin-1 gene promoter (pZmUbi-1) size is 2.1kb, and nucleotides sequence is classified as SEQ ID NO.7, is constitutive promoter, and goal gene can be driven in the middle expression in a organized way of corn institute.PEP carboxylase gene (pepc) terminator, derives from corn, and size is 0.2kb, and nucleotides sequence is classified as SEQ ID NO.8.
Antiglyphosate gene provided by the invention is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene.Glyphosate is a kind of wide spectrum steriland herbicide, and it is by suppressing the activity of 5-enolpyruvylshikimate-3-phosphate synthase in plant, causes plant can not synthetic aromatic amino acid and dead.Conveniently Weeds distribution, can make crop obtain the ability of resistance glyphosate by the EPSPS proceeded to crop glyphosate has a resistance, realize optionally weeding while crop growth.Resistance glyphosate expression cassette provided by the invention is by Cauliflower mosaic virus (Cauliflower Mosaic Virus, CaMV) 35S promoter and the molecular combined promoter (p35S-pZmUbi-1 of corn polyubiquitin-1 gene promoter, nucleotides sequence is classified as SEQ ID NO.9), the 35S gene terminator (SEQ ID NO.11) of G10evo (EPSPS) (nucleotides sequence is classified as SEQ ID NO.5, and the aminoacid sequence of coding is SEQ ID NO.10) and CaMV that 5 ' end is connected with one section of coding AHAS gene chloroplast signal peptide is formed.
The invention provides the method that whether can exist containing dual anti-12-5 in specific detection sample, comprise at least one in following method:
1) design primer according to SEQ ID NO.12 (i.e. the associating of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3), utilize the method for PCR to detect.Known by professional and technical personnel according to known nucleotide sequences Design specific detection primer, such as: PCR primer SP1:5 '-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3 ' can be designed according to right arm and flank Maize genome; RB-Test:5 '-CTCGTCATCGACCAAGTCATG
AAG-3’。Or design PCR primer R1:5 '-CGTCGTTTTACAACGTCGTGACTGG-3 ' according to left arm and flank Maize genome; LB-test:5 '-AAGACGTCCGGGGGAACCGTTG
TTC-3’。
2) plant genome DNA is extracted through restriction enzyme BamHI or XbaI single endonuclease digestion; Using the Antiglyphosate gene G10eve of digoxigenin labeled (SEQ ID NO.5) sequence DNA as probe, detect Antiglyphosate gene and whether exist.
Extract plant genome DNA through restriction enzyme KpnI or SmaI single endonuclease digestion, the cry1Ab full length DNA of digoxigenin labeled is as probe (1-1947bp in SEQ ID NO.4), hybridize to fluorography after on nylon based plasma membrane, detect anti insect gene and whether exist.DNA extraction, probe manufacturing method and Southern hybridizing method are according to J. Pehanorm Brooker " Molecular Cloning: A Laboratory guide ".
3) from plant, extract protein, be primary antibodie with insect resistance protein or resistance glyphosate protein antibodies, carry out Western analysis, detect the expression level of insect resistance protein or resistance glyphosate albumen.Protein extracting method, Western method are in accordance with the method for J. Pehanorm Brooker " Molecular Cloning: A Laboratory guide ".
The invention provides the method that utilization " dual anti-12-5 " transformation event cultivates pest-resistant resistance glyphosate corn.Corn strain for these methods can be isozygoty or heterozygosis, the progeny plants produced by these methods can be mutation or hybrid.
Vegetable cell containing " dual anti-12-5 " transformation event (i.e. SEQ ID NO.12) of the present invention, described plant cellular sources is in Zea corn (Zea mays L.), described Zea corn is preserved in China typical culture collection center, preserving number CCTCC P201506, preservation date: on April 27th, 2015, preservation address: Wuhan, China Wuhan University, postcode 430072.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides a kind of excellent transformation event " dual anti-12-5 ", this transformation event can realize foreign gene specificity to be incorporated in corn strain, gives the ability that recipient corn has pest-resistant resistance glyphosate; Pest-resistant Antiglyphosate gene can genetic stability in recipient corn; The expression of pest-resistant Antiglyphosate gene can not produce detrimentally affect to the economical character of recipient corn.
(4) accompanying drawing explanation
Fig. 1, for a specific embodiments of the present invention containing the plasmid construct figure of goal gene, the border sequence of LB and RB:T-DNA; PZmUbi-1: corn polyubiqutin-1 promotor; P35S:35S promotor (CaMV virus); G10evo: resistance glyphosate EPSPS gene; Cry1Ab-Cry2Aj: pest-resistant Bt fusion gene cry1Ab/cry2Aj.
Fluorograph after the Southern hybridization of Fig. 2, dual anti-12-5 Antiglyphosate gene; BamHI is that genome is through BamHI single endonuclease digestion; XbaI is that genome is through XbaI single endonuclease digestion; Using the G10eve full length DNA of digoxigenin labeled as probe hybridization to nylon based plasma membrane after fluorography; Figure right side is DNA length standard (bp) mark, and arrow refers to hybridize the specific band produced.
Fluorograph after the Southern hybridization of Fig. 3, dual anti-12-5 anti insect gene; The genomic dna that KpnI is " dual anti-12-5 " is cut through KpnI enzyme; The genomic dna that SmaI is " dual anti-12-5 " is cut through SmaI enzyme, and the Cry1Ab full length DNA of digoxigenin labeled, as probe, hybridizes to fluorography after on nylon based plasma membrane, and figure right side is DNA length standard (bp).
Fig. 4, Western analyze the expression level of insect resistance protein matter Cry1Ab/Cry2Aj in different tissues organ; + be positive control;-be negative control (non-transgenic corn); M is protein molecular weight standard (kD); Material: dual anti-12-5, the applied sample amount of blade, stem, root is identical fresh weight, and seed is the amount loading with 20%; Arrow refers to the signal band of insect-killing protein.
Fig. 5, Western analyze the expression level of Antiglyphosate gene G10eve-EPSPS in different tissues organ; + be positive control (Escherichia coli expresses);-be negative control (non-transgenic); M is protein molecular weight standard; Material: dual anti-12-5 and dual anti-12-9, the applied sample amount of blade, stem, root is identical fresh weight, and seed is the amount loading with 20%; Arrow refers to the signal band of insect-killing protein.
The PCR of Fig. 6, insertion point verifies electrophorogram; 1 is transgenic corns left side flap PCR; 2 is non-transgenic reference left side flap PCR; 3 is transgenic corns right side flap PCR; 4 is non-transgenic reference right side flap PCR; M is DNA size criteria (PCR primer is between 200-500bp).
Fig. 7, " dual anti-12-5 " DNA genetic stability in recipient corn detects electrophorogram; M is DNA size criteria sample (100bp gradient);-be negative control (non-transgenic corn); + be positive control (adding the non-transgenic corn sample of the PCR positive products through sequencing checking); 1,2,3,4,5,6,7 is the 1st respectively, 2,3,4,5, and the recipient corn sample in 6,7 generations; The expection size of PCR primer is 145bp, and the estimation on the agarose gel analyzed with PCR primer is basically identical.
Fig. 8, " dual anti-12-5 " detection of expression of insect resistance protein matter in recipient corn.M, protein molecular weight standard (kDa);-, negative (non-transgenic recipient corn); +, the protein that E.coli expresses; 1,2,3,4,5,6,7 is the 1st of transgenic corns respectively, the corn in 2,3,4,5,6 and 7 generations.The nonspecific signal band of the 130kDa in recipient plant and expection are consistent.
Fig. 9, " dual anti-12-5 " resistance glyphosate protein expression in recipient corn detects electrophorogram; M is protein molecular weight standard (kDa);-be negative (non-transgenic recipient corn); 1,2,3,4,5,6,7 is the 1st of recipient corn respectively, and the corn in 2,3,4,5,6 and 7 generations, the nonspecific signal band of the 48kDa in recipient plant and expection are consistent.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the acquisition of transformation event
(1) acquisition of the plasmid vector containing foreign gene
The present invention is used for the Vector map of corn transformation as shown in Figure 1, and transform plastids vector nucleotide sequence is SEQ ID NO.13, and title, the position of concrete carrier element are as shown in table 1.Wherein the nucleotides sequence of T-DNA gene is classified as shown in SEQ ID NO:2.SEQ ID NO:2 comprises complete resistance glyphosate expression cassette and pest-resistant expression cassette, specifically be made up of such as lower part, resistance glyphosate expression cassette: derive from the combined promoter (nucleotides sequence is classified as shown in SEQ ID NO.9) that the 35S promoter of CaMV and corn Polyubiqutin-1 promotor are formed, this combined promoter drives 5 ' end to be connected with the Antiglyphosate gene G10evo (EPSPS) (nucleotides sequence is classified as shown in SEQ ID NO.5) of one section of coding AHAS gene chloroplast signal peptide, terminator is that (length is 0.2kb for the 35S gene terminator of CaMV, its nucleotides sequence is classified as SEQ ID NO.11), pest-resistant expression cassette: Cry1Ab-PGKGGG-Cry2Aj fusion gene, the promotor of Cry1Ab-Cry2Aj fusion gene is driven (to be obtained from Maize genome by PCR for deriving from corn polyubiquitin-1 gene promoter (pZmUbi-1), size is 2.1kb, nucleotides sequence is classified as SEQ ID NO.7, pZmUbi-1 can drive goal gene to express in all plant tissues), terminator is PEP carboxylase gene (pepc) terminator (size is 0.2kb, and nucleotides sequence is classified as SEQ ID NO.8) deriving from corn.Utilize electric shocking method (2500V) to import in Agrobacterium LBA4404 by the transform plastids obtained, obtain the Agrobacterium containing conversion carrier.
The title of table 1 corn transformation carrier element, position and function
(2) maize genetic transforms
Obtaining corn transformation event method used in this research is agriculture bacillus mediated method, the method reported according to Frame etc. and culture medium prescription (Plant Physiol, 2002,129:13-22) transform, use glyphosate is screening reagent, and concrete steps are as follows:
(1) get the pollination parental maize fringe of latter 8 ~ 10 days, collecting size is 1.0-1.5mm immature embryo.By the Agrobacterium containing conversion carrier and immature embryo at 22 DEG C of Dual culture 2-3 days.
(2) immature embryo after step (1) being cultivated is transferred on the callus inducing medium containing final concentration 200mg/L Ticarcillin/Clavulanate Acid microbiotic (GlaxoSmithKline PLC, the U.S.), and 28 DEG C of light culture 10-14 days kill Agrobacterium.
(3) all callus after step (2) inducing culture are forwarded in the screening culture medium containing final concentration 2mM glyphosate, 28 DEG C of light culture 2-3 weeks.
(4) callus that transfer step (3) is all is in the fresh screening culture medium containing (2mM) glyphosate, and 28 DEG C of light culture 2-3 are all.
(5) then, the embryonal connective tissue that transfer step (4) survives on regeneration culture medium, 28 DEG C of light culture 10-14 days, every ware strain.
(6) transfer step (5) embryonal connective tissue is on fresh regeneration culture medium, 26 DEG C of illumination cultivation 10-14 days.
(7) transfer step (6) all full-grown plants are on root media, and the regrowth after taking root, until root development is complete, is transplanted in greenhouse and is grown breeding, for screening strength by 26 DEG C of illumination cultivation.
Embodiment 2: the screening of transformation event
240 pest-resistant resistance glyphosate corn independent transformants (embodiment 1) are obtained by agriculture bacillus mediated.According to carrier sequence, (SEQ ID NO.13 designs primer, with PCR method screening containing Antiglyphosate gene G10eve (nucleotides sequence is classified as shown in SEQ ID NO.5) and anti insect gene cry1Ab-cry2Aj (nucleotides sequence is classified as shown in SEQ ID NO.4), simultaneously not containing the corn transformation body of vector backbone sequence, and in greenhouse, carry out the preliminary test of pest-resistant resistance glyphosate performance: by spraying the glyphosate of 0.4wt%, remove the transformant of glyphosate resistance difference, remaining transformant connects Pyrausta nubilalis (Hubern)., screen the transformant not having Pyrausta nubilalis (Hubern). to cause harm, amount to the corn transformation body with stronger pest-resistant resistance glyphosate ability of acquisition 12 containing transformation event " dual anti-12-1 " ~ transformation event " dual anti-12-12 ".Respectively the transformant and corn inbred line B73 that contain dual anti-separate transformation events are hybridized and reserve seed for planting, carry out screening strength subsequently.
(1) glyphosate resistance screening
12 T3 and T5 containing the transformant of transformation event " dual anti-12-1 "-transformation event " dual anti-12-12 " are selected to carry out glyphosate resistance contrast for transgenic corns.20-30 days after transgenic corns and parent's non-transgenic reference seed germination, grow to the 4-5 leaf phase, spray the glyphosate (Monsanto Company, the U.S.) that final concentration is 0.4wt%, dosage is 25L/ mu, within 7 days, records corn growth situation and mortality ratio afterwards.Glyphosate spraying test result is as shown in table 2, and transgenic strain all has obvious resistance glyphosate ability.Corn resistance level wherein containing transformation event " dual anti-12-1 ", " dual anti-12-3 ", " dual anti-12-4 ", " dual anti-12-5 ", " dual anti-12-7 ", " dual anti-12-9 ", " dual anti-12-10 " and " dual anti-12-12 " is higher.
Table 2. dual anti-corn glyphosate resistant proof
(2) insect resistance capacity test
1) field insect resistance capacity test
12 T3 and T5 containing the transformant of transformation event " dual anti-12-1 "-transformation event " dual anti-12-12 " are selected to carry out pest-resistant performance analysis for transgenic corns respectively.Germinateing in transgenic corns and parent's non-transgenic reference greenhouse and within latter 20 days, spraying final concentration is that the glyphosate of 0.4wt% determines it is after transgenosis; respectively get 10 strains; every strain connect 1 age Ostrinia furnacalis 10, Ostrinia furnacalis is from Plant Protection institute, Chinese Academy of Agricultral Sciences's Corn Pests group.By pieces of an egg as 28 ± 1 DEG C, hatch under RH 70 ± 5%, 16h:8h (L:D) condition, the larva chosen in hatching 12 hours surveys experiment for raw.Connect worm 6 days " Invest, Then Investigate " Pyrausta nubilalis (Hubern). situation of the harms, carry out pest-resistant classification.Pest-resistant classification adopts 9 grade standards (Marcon et al., 1999): 1 ~ 3 grade: worm channel needle prick shape (1 grade: rare, dispersion; 2 grades: moderate quatity; 3 grades: a large amount of).4 ~ 6 grades: worm channel match end size (4 grades: rare, dispersion; 5 grades: moderate quatity; 6 grades: a large amount of).7 ~ 9 grades: worm channel is greater than match end (7 grades: rare dispersion; 8 grades: moderate quatity; 9 grades: a large amount of).Resistance grade classification: 1 ~ 2 grade (high resistance), 3 ~ 4 grades (pest-resistant), 5 ~ 6 grades (sense worm), 7 ~ 9 grades (high sense).Result is as shown in table 3, and " dual anti-12-1 ", " dual anti-12-5 ", " dual anti-12-9 ", " dual anti-12-10 " and " dual anti-12-11 " have high pest-resistant performance.
Table 3: different transformation event is identified the resistance class of Pyrausta nubilalis (Hubern).
2) the pest-resistant performance test in laboratory
Select 12 T3 and T5 containing the transformant of transformation event " dual anti-12-1 "-transformation event " dual anti-12-12 " for transgenic corns respectively, select the lobus cardiacus blade do not launched completely to carry out com-borer resistant mensuration in laboratory at transgenic corns and contrast corn lobus cardiacus mid-term (6-8 leaf launches completely).Connect Pyrausta nubilalis (Hubern). 2 days " Invest, Then Investigate "s and take food area and Pyrausta nubilalis (Hubern). death condition.The results are shown in Table shown in 4, it is good that result shows most of transgenic corns resistance.Their blade is taken food considerably less, particularly dual anti-12-5, dual anti-12-9 and dual anti-12-11 connect worm after 2 days Pyrausta nubilalis (Hubern). take food area less than 10mm
2.After 2 days, Pyrausta nubilalis (Hubern). mortality ratio is more than 80% to connect worm, and after 4 days, most of transgenic corns snout moth's larva mortality ratio arrives 100%.
Table 4: the transgenic corns lobus cardiacus phase is to the insect-resistance of Ostrinia furnacalis
Note: in table 4, a, b represent significant difference
(3) foreign gene inserts copy number qualification
Comprehensively pest-resistant and resistance glyphosate measurement result, selects pest-resistant and that glyphosate tolerant ability is all stronger dual anti-12-1, dual anti-12-5, dual anti-12-9 and dual anti-12-10 to carry out foreign gene and inserts copy number qualification.Use Roche Holding Ag (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche) to carry out Southern analysis, the operation steps provided in accordance with test kit is carried out hybridization probe and is made and DNA hybridization detection.Namely corn gene group DNA is extracted, respectively through restriction enzyme BamHI and XbaI enzyme cutting (these restriction enzymes have single recognition site in foreign gene), electrophoretic separation endonuclease bamhi on agarose gel, then DNA is transferred on nylon based plasma membrane, utilize G10eve (the nucleotide sequence SEQ ID NO.5) full length DNA of digoxigenin labeled as fluorography later on probe hybridization to nylon based plasma membrane.Result display only has " dual anti-12-5 " to demonstrate single slice, " dual anti-12-5 " obtains the signal band of an about 14kb when BamHI enzyme is cut, obtain the signal band (Fig. 2) of about 5.0kb when XbaI enzyme cutting, prove that in " dual anti-12-5 ", Antiglyphosate gene singly copies insertion.
The Cry1Ab of the insect-resistant fusion gene on the right in T-DNA is utilized to carry out Southern hybridization analysis as probe to " dual anti-12-5 " anti insect gene copy number, the genomic dna of restriction enzyme KpnI and SmaI to " dual anti-12-5 " is used to carry out single endonuclease digestion respectively, after agarose gel is separated, transfer on nylon based plasma membrane, then utilize the Cry1Ab (in nucleotide sequence SEQ ID NO.4 1-1947bp) of digoxigenin labeled as fluorography later on probe hybridization to nylon based plasma membrane.Result shows, and obtains the signal band of an about 7kb, and obtain the signal band (Fig. 3) of about 6.5kb when SmaI enzyme is cut when cutting with KpnI enzyme.
Therefore above-mentioned experiment proves " dual anti-12-5 " to be an anti insect gene and Antiglyphosate gene is all the transformation event that single copy inserts.
(4) exogenous gene expression component analysis
Utilize the tolerant gene expression situation of molecular biology ordinary method to " dual anti-12-5 " to carry out Western method to measure.Take the sample of different tissues in the 5-6 leaf phase, utilize Cry1Ab antibody and G10eve-EPSPS to detect the expression level (Western detection method is with reference to " the Molecular Cloning: A Laboratory guide " of J. Pehanorm Brooker) of killing gene and Antiglyphosate gene respectively.Result shows, insect resistance protein matter, at various different tissues, comprises in blade, stem, root and seed and all has expression, but expresses relatively low in root and seed, as Fig. 4.Resistance glyphosate protein has higher expression level on Zea mays root, stem and blade, as shown in Figure 5.
(5) economical character of transgenic corns
Insert copy number according to pest-resistant performance, resistance glyphosate ability and foreign gene, screening acquisition has high pest-resistant resistance glyphosate ability, and " dual anti-12-5 " that foreign gene list copy inserts carries out the analysis on economical character.It is variant that result shows transformation event " dual anti-12-5 " vegetative period, florescence and non-transgenic parent, transgenic corns uses the output of Gyphosate herbicice on " dual anti-12-5 " do not affect.Non-transgenic parental maize grain number per spike and 100-grain weight, significantly lower than " dual anti-12-5 ", may be because the harm of Pyrausta nubilalis (Hubern). causes (table 5).
Table 5: the number of grain per ear of transgenic corns, 100 weight and breeding time
Note: * represents because serious insect pest causes grain to weigh and the reduction of grain number.
Embodiment 3: the qualification of foreign gene insertion point flanking sequence
Use TAIL-PCR (the Thermal asymmetric interlaced PCR) method (Liu of the reports such as Liu, Plant Journal 1995,8 (3): 457-463) regional sequence of both sides, excellent transformation event dual anti-12-5 foreign transgenes DNA insertion point is measured.The method carries out continuous print pcr amplification with degenerated primer combination respectively by 3 nested Auele Specific Primers, utilizes different annealing temperature optionally to increase target fragment.The DNA fragmentation obtained amplification is cloned, check order also and corn internet database (http://www.maizegdb.org) compares.
Pterion on the left of T-DNA
Be accredited as the fragment comprising 3 ' flanking region and carry out (the SEQ ID NO.1 that checks order.Sequence between Nucleotide 1-576bp corresponds to corn gene group DNA, and the sequence between Nucleotide 577-826bp corresponds to foreign DNA.
Pterion on the right side of T-DNA
(SEQ ID NO.3) is checked order to the fragment comprising 5 ' flanking region that is accredited as obtained by TAIL-PCR method.The sequence of Nucleotide 1-210bp corresponds to foreign DNA, and the sequence of Nucleotide 211-1007bp corresponds to corn gene group DNA.
Form transformation event of the present invention by above-mentioned through order-checking comparison and authenticated insertion point upstream and downstream flanking sequence, Extrinsic Anti-insect Genes expression cassette and anti-herbicide gene expression cassette sequence assembly, nucleotides sequence is classified as SEQ ID NO.12 (i.e. the associating of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3).
(2) foreign gene specific detection
The PCR primer (as shown in table 6) of the dual anti-12-5 of specific detection is may be used for according to nucleotide sequence (the SEQ ID NO.12) design of transformation event.
The dual anti-12-5 specific detection primer of table 6.
Described PCR reaction system is: 10 × amplification buffer 5 μ L, dNTP mixture 200 μm of ol/L, forward primer 10pmol, reverse primer 10pmol, genomic dna 0.1 ~ 2 μ g, Taq archaeal dna polymerase 2.5 μ L, MgCl
21.5mmol/L, adds distilled water to 50 μ L.
PCR condition is: 32 circulations, and each circulation is 95 DEG C, 45 seconds; 65 DEG C, 50 seconds; 72 DEG C, 30 seconds.The condition of PCR can adjust according to the enzyme used and reaction system.Agarose electrophoretic analysis is carried out to acquisition PCR primer.The freeze-draw method of right boundary is 304bp and 350bp (Fig. 6) respectively.PCR primer can be carried out sequencing time necessary to verify further.
Embodiment 4: genetic stability detects
1) Detection of Stability of transformation event " dual anti-12-5 " T-DNA in recipient corn
Design primer according to the recombinant DNA molecules (SEQ ID NO.12) of transformation event " dual anti-12-5 " and carry out the specific PCR detection of insertion point, the recipient corn getting 1-7 generation respectively extracts genome, utilizes PCR to identify T-DNA integration.Primer is: BR-1 (5 ' GGCGAATGCTAGAGCAGCTTG
AGCT-3 ') and GN-1 (5 ' CCTACTGCGATGACGTTCGGTGCC-3 '),
Reaction system: 10 × amplification buffer 5 μ L, dNTP mixture 200 μm of ol/L, BR-110pmol, 10pmolGN-1, genomic dna 0.1 ~ 2 μ g, Taq archaeal dna polymerase 2.5 μ L, MgCl
21.5mmol/L, adds distilled water to 50 μ L.
Reaction conditions: 95 DEG C 1 minute, 60 DEG C 40 seconds, 72 DEG C 30 seconds, 30 circulations.From Shanghai, raw work obtains TAQ enzyme.Result shows, and the transgenic corns PCR result of all generations is all positive.Agarose electrophoretic analysis shows, and PCR primer size is in the same size with expection is 145bp (Fig. 7).
2) " dual anti-12-5 " exogenous gene expression Detection of Stability
Sow the corn that 1-7 generation contains " dual anti-12-5 " transformation event respectively, with parent's non-transgenic corn for contrast, the 5-6 leaf phase is grown to from corn, about 10mg maize leaf is obtained from the top of corn the 3rd leaf, pulverize in the sample extracting solution (sample extracting solution is the 10wt%SDS aqueous solution) of 200 microlitres, and 100 DEG C are heated 6 minutes, after centrifugal, 20 microlitre supernatant electrophoretic separation (SDS-PAGE).Protein transduction moves on on cellulose membrane by electrophoresis later, carries out Western analysis.Primary antibodie is the anti-Cry1Ab of rabbit or the anti-G10evo of rabbit, the two anti-goat anti-rabbit iggs being horseradish peroxidase and marking.Result display insect-resistance fusion protein (Fig. 8) and resistance glyphosate albumen (Fig. 9) are at each generation stably express.
3) " dual anti-12-5 " pest-resistant resistance glyphosate biological assay
Pyrausta nubilalis (Hubern). measures (Marcon et al., 1999) and shows the 1st, 2,3,4,5,6, the transgenic corns in 7 generations to one age Pyrausta nubilalis (Hubern). insecticidal effect be all 100%, do not find the problem that pest-resistant level is lower.Spray glyphosate test to show, the transgenic corns in the 1st, 2,3,4,5,6,7 generations can resist the glyphosate pesticide of 100 grams every mu active glyphosate content.Do not find the problem that obvious resistance glyphosate ability reduces.Result proves pest-resistant and resistance glyphosate ability genetic stability.
Embodiment 5: the sexual hybridization transformation of " dual anti-12-5 " transformation event
1) " dual anti-12-5 " sexual hybridization transformation
With the corn containing " dual anti-12-5 " transformation event for donor parents, with corn inbred line B73 for receptor parent is once hybridized, backcross for 4 times and 3 selfings.In backcross process, use glyphosate remove not containing the separation strain of dual anti-12-5 transgenosis composite structure.The stable self-mating system B735 containing compound transgenic structure of the present invention is obtained in BC4F3 generation (after 4 generations that backcrossed selfing 3 generation).DNA is extracted from this strain leaf tissue, amplify the DNA segment that external source inserts gene and flank thereof, confirm through sequencing analysis, from consistent with the compound transgenic structure sequence of donor parents, illustrate that " dual anti-12-5 " transformation event stablizes transformation in new acceptor material.
2) " dual anti-12-5 " transformation event is used for hybrid seeding
Take B735 as female parent, Mo17 is that male parent assembly obtains cross-fertilize seed B7M, carries out to B7M pcr analysis and the order-checking that external source inserts gene and flanking DNA thereof, and result display B7M contains transformation event " dual anti-12-5 ".Field resistance test display, B7M has good resistance glyphosate ability and pest-resistant performance.
Claims (9)
1. a corn transformation event " dual anti-12-5 ", is characterized in that described transformation event arranges the pterion, left side into foreign gene with the nucleotides sequence shown in SEQ ID NO.1, arranges the pterion, right side into foreign gene with the nucleotides sequence shown in SEQ ID NO.3.
2. corn transformation event " dual anti-12-5 " as claimed in claim 1, is characterized in that described foreign gene comprises anti insect gene and Antiglyphosate gene.
3. corn transformation event " dual anti-12-5 " as claimed in claim 2, is characterized in that described anti insect gene is by two Bt genomic constitutions.
4. corn transformation event " dual anti-12-5 " as claimed in claim 3, is characterized in that the nucleotides sequence of anti insect gene is classified as shown in SEQ ID NO.4.
5. corn transformation event " dual anti-12-5 " as claimed in claim 2, is characterized in that the nucleotides sequence of described Antiglyphosate gene is classified as shown in SEQ ID NO.5.
6. corn transformation event " dual anti-12-5 " as claimed in claim 1, is characterized in that the nucleotides sequence of described foreign gene is classified as shown in SEQ ID NO.2.
7. corn transformation event " dual anti-12-5 " specific PCR authentication method, the primer identified for corn transformation event " dual anti-12-5 " specific PCR described in it is characterized in that is:
SP1：5’-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3’；
RB-Test：5’-CTCGTCATCGACCAAGTCATGAAG-3’。
R1：5’-CGTCGTTTTACAACGTCGTGACTGG-3’；
LB-test：5’-AAGACGTCCGGGGGAACCGTTGTTC-3’。
8. method as claimed in claim 7, is characterized in that described PCR reaction system is:
PCR reaction conditions is: 32 circulations, and each circulation is 95 DEG C, 45 seconds; 65 DEG C, 50 seconds; 72 DEG C, 30 seconds.
9. the vegetable cell containing " dual anti-12-5 " transformation event, described plant cellular sources is in Zea corn (Zea mays L.), described Zea corn is preserved in China typical culture collection center, preserving number CCTCC P201506, preservation date: on April 27th, 2015, preservation address: Wuhan, China Wuhan University, postcode 430072.
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| WO2024051077A1 (en) * | 2022-09-07 | 2024-03-14 | 杭州瑞丰生物科技有限公司 | Transgenic soybean event cal16 and detection method therefor |
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| CN106167818A (en) | 2016-11-30 |
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