CN103320348A - Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer - Google Patents
Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer Download PDFInfo
- Publication number
- CN103320348A CN103320348A CN2013101614400A CN201310161440A CN103320348A CN 103320348 A CN103320348 A CN 103320348A CN 2013101614400 A CN2013101614400 A CN 2013101614400A CN 201310161440 A CN201310161440 A CN 201310161440A CN 103320348 A CN103320348 A CN 103320348A
- Authority
- CN
- China
- Prior art keywords
- aphid
- transgenosis
- sequence
- gene transfer
- mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The present invention provides a Chinese gall aphid-mediated mixture capable of inducing gene transfer, and an identification method thereof. The method comprises: separating secretion from Chinese gall, carrying out grinding, filtration separation, microscopic examination, permeate culture, an Arabidopsis thaliana culture topping-off treatment, dipping transfection, genome DNA extraction, positive sample screening through PCR amplification, sequencing determination, data analysis and other steps to identify occurrence of gene transfer, and comparing with parasitic Chinese gall aphid and the related sequence in GenBank to determine homology, such that it can be initially determined that the mixture can induce horizontal gene transfer between the parasitic aphid and the host plant and is a new transgenic mixture capable of being developed and utilized according to the comparing result.
Description
Technical field
The present invention relates to the Horizontal Gene Transfer between melaphis chinensis Baker and the host plant, specifically belong to a kind of inducible gene and shift mixture and the concrete authentication method thereof that occurs.
Background technology
Horizontal Gene Transfer (Horizontal gene transfer, HGT) propose with respect to vertical transgenosis (parental generation passes to filial generation), mainly occur between the interaction species gene, broken the boundary of sibship, what make that gene flows may become more complicated.Nineteen fifty-nine reported first intestinal bacteria high frequency transduction bacterial strain can pass to genetic information specific Salmonella typhimurium mutant strain.At present, HGT has been introduced into molecular evolution and macroevolution field, is considered to the important motivity that promotes to evolve.Along with in succession finishing of human and some other Model organism genome examining orders, finding has a large amount of homogenic existence on the genome between the far biology of sibship, analyzing these genes of demonstration is to obtain by the nature gene horizontal transfer, has confirmed ubiquity and the edge far away of natural Horizontal Gene Transfer.The horizontal transfer of existing research reporter gene can occur between microorganism, between bacterium and the plant, between bacterium and the animal, between plant, between fungi and the plant, between insect and the fungal component, between insect, can by Host transfer to parasitic body, also can transfer to the host by parasite.
Melaphis chinensis Baker is Aphidiadae Eriosomatinae times aphid family aphid, except distribution one single genus in North America is planted, and all the other distribution East Asia, China is that it mainly distributes ground.The different host of such Aphis complete period type insect, experiencing sexupara the whole life history becomes aphid, male and female sexuale, fundatrix, aptery fundatrigenia, the worm types such as wing fundatrigenia and alienicola is arranged, specialization to host plant is very strong, the Primary host plant only is Anacardiaceae Rhus 5 kind of plant, the parasitic insect gall general designation Turkey-galls that forms, be important medicine and industrial chemicals, be widely used in the fields such as organic synthesis, oil, national defence, chemical industry, electronics, pharmacy, ink, dyestuff, food and light industry.
In to times aphid and the research of host plant coevolution relation, find to have the Horizontal Gene Transfer phenomenon between them, further to mensuration and the analysis of host plant seed, seedling, one-tenth leaf sheet material genome and melaphis chinensis Baker genome sequence, verified really to have mtDNA transgenosis phenomenon between melaphis chinensis Baker and the host plant.Aphid and host plant tissue system difference are larger, and directly iuntercellular transfer difficulty is larger, to the biological property analysis of melaphis chinensis Baker and host plant and formation insect gall thereof, infer that the mixture that aphid forms in the insect gall may play mediation.Through cultivate processing early stage and Arabidopis thaliana is contaminated experiment, preliminary identification this conclusion, found a kind of mixture of inducible gene transfer of melaphis chinensis Baker mediation.
At present, the generation main theory that shifts of gene level is thought and is realized by 3 kinds of different approach: transduction-carry out transgenosis as medium between its organism of infecting by virus; Unite-be " directly " horizontal transfer of gene, the direct DNA exchange of species iuntercellular in close relations; Transform-directly from environment, absorb DNA, have been found that in soil, water and the digestive tube to have DNA.Yet there are no the research report of Horizontal Gene Transfer between animal and the plant, new route of metastasis and the mode that provide of gene level transfer originally is provided.
Summary of the invention
But what the object of the present invention is to provide a kind of induced gene horizontal transfer by melaphis chinensis Baker mediation contains microbial mixture and detection method thereof, this mixture has played inducing action in the Horizontal Gene Transfer of aphid and host plant, can be used as a kind of novel transgene component and develop research.
The invention provides a kind of mixture and detection method thereof of inducing melaphis chinensis Baker and host plant gene level to shift, concrete grammar is to isolate the secretory product mixture from Turkey-galls, soaks the gene order that colored experiment, seed culture, extracting genome DNA, pcr amplification, sequencing and data analysis etc. detect transfer through grinding, filtering separation, microscopy, infiltration cultivation, Arabidopis thaliana.
Working method is as follows:
1) times ripening stage collect specimen at the angle is selected larger, the intact and free of contamination sample of individuality from the gall nut that gathers, sterile state proceeds as follows.
2) cut gall nut open, carefully with swab stick separation angle times aphid and Secretory product wherein, an angle times aphid saves backup with dehydrated alcohol.Secretory product is wherein weighed, and gets 5g in the mortar of in advance sterilization, remaining freezing preservation.Fig. 1 is doubly seen with movement wherein in the angle.
3) firmly grind in the secretory product mortar that takes by weighing, thin as far as possible.Add the 30mL sterilized water, continue to grind to form pasty state, then be handled as follows respectively:
A. get the 10mL lapping liquid in the sterilization culture dish, be labeled as liquid No. 1;
B. the remaining 20mL sterilized water that adds, mixing, packing, the trim of in centrifuge tube, weighing, 1000r/m is centrifugal, removes slag.
C. get supernatant liquor through 100 μ m membrane filtrations, get 10mL filtrate in new sterilization culture dish, be labeled as liquid No. 2.
D. remaining filtrate through 100 μ m membrane filtrations is got 10mL in new sterilization culture dish again through 10 μ m membrane filtrations, is labeled as liquid No. 3.
3) add 10% sucrose solution that 10mL prepares in advance in each culture dish as settling agent and 5 μ L Silwet L-77 tensio-active agents, make respectively the steeping fluid of 3 kinds of different treatment.Room temperature leaves standstill 1h.
Before above sample preparation, cultivate in advance arabidopsis thaliana and grow into and bloom and to pinch period.Arabidopis thaliana began plantation in about 50 days before gathering gall nut secretory product cultivates, and pinches before dip treating twice.
4) pinch Arabidopis thaliana after twice removes the colored part of blooming fully before dipping, the Arabidopis thaliana of each Pot soaks respectively flower and writing brush and dips in liquid and be coated with flower and process, two Pot of each steeping fluid processing, and each Pot has 5 strain Arabidopis thalianas.The flower of general major branch floods in nutrient solution, and the time is 2min; Harsh sprout or the short branch that contains bud adopt writing brush dipping method.
5) first dark cultivation of the Arabidopis thaliana after dip treating is 2 days, and then fixed temperature and humidity is cultivated, and collects respectively seed by different processing after the maturation.
6) the Arabidopis thaliana seed of collecting concentrates respectively plantation to cultivate, and the seed of each processing begins to gather fresh Arabidopsis leaf material respectively at the cultivation of sowing seeds at random among 30 Pot about one month.
7) adopt the sky to take root in the thing genome and extract test kit extraction arabidopsis thaliana genomic dna, carry out pcr amplification and sequencing and analysis with insect universal primer (gene-cytochrome b gene that in melaphis chinensis Baker and host plant transgenosis research, is verified).Design of primers is with reference to Simon (1994), and primer sequence is:
P1?5’-TATGTACTACCATGAGGACAAATATC-3’；
P2?5’-ATTACACCTCCTAATTTATTAGGAAT-3’
8) the pcr amplification system is 50 μ l, includes 5U TaKaRa Taq, 1 * PCR Buffer, 2mM MgCl
2, 0.25mMdNTP, each 0.4 μ M of upstream and downstream primer, 20-50ng dna profiling solution.
9) reaction conditions of pcr amplification is 94 ℃ of denaturations 3 minutes; Then carry out 39 circulations, each circulation comprises 94 ℃ of 40s of sex change, and the 50 ℃ of 1m that anneal extend 72 ℃ of 1m; Then fully extend 7m at 72 ℃.
10) pcr amplification product adopts the two-way survey of 3730 sequenators logical, and the gene order length that obtains is respectively 433bp.
11) we finally screen two samples and contain the goal gene sequence the test of increasing one by one of the sample of three kinds of different treatment of cultivating in the sample of 10 μ m processing.The sequence that obtains is searched for through Blast, adopt dna sequence analysis software such as Sequencher, Clustal, Paup and Bayes software etc., with the melaphis chinensis Baker that has recorded and from times aphid host plant of angle, obtain corresponding sequence compare analysis, the Gene Partial sequence that shifts may be for having occured in the sequence that definite screening obtains, and the sequencing results is seen Fig. 2.
We confirm that the secretory product mixture in the Turkey-galls may be a kind of engineered medium of inducing thus, the microorganism that wherein contains (the molecular data Analysis and Identification is fungal component-Buchnera of times aphid, and concrete authentication method is as described below) should play medium in transgenosis.The discovery of the microbial mixture that new induced transgenosis occurs is a large achievement of biotechnology, has higher development and application and is worth.
The authentication method of microorganism in the secretory product that separates in the Turkey-galls:
After secretory product fully grinds, protease digestion, adopt tissue DNA to extract the DNA that test kit extracts mixture, the design primer, with reference to the microorganism universal primer, the DNA that extracts is carried out the pcr amplification of 16s rDNA fragment, amplified production entrusts biotech firm to carry out two-way order-checking, the sequence that obtains is carried out data analysis by blast search with software Sequencher, Clustal, Mega etc., constructing system is grown relation, determine that it is the 16s rDNA sequence of aphid endosymbiosis bacterium Buchnera, thereby determine to contain Buchnera or its histocyte composition in the mixture.
Compared with the prior art the present invention has following characteristics:
The present invention be the mediation of a kind of melaphis chinensis Baker can modificator gene shift contain microbial mixture and detection method thereof.The angle that this mixture forms from angle times aphid doubly in, be a kind of natural mixture that is formed by aphid secretory product and fungal component; Preliminary research finds, this may be that a natural mixture that can carry out transgenosis is arranged, for the transfer of gene provides another kind of possible medium.
Embodiment
Embodiment 1
1) times ripening stage collect specimen at the angle is selected larger, the intact and free of contamination sample of individuality from the gall nut that gathers, sterile state proceeds as follows.
2) cut gall nut open, carefully with swab stick separation Secretory product wherein, get 2g in the mortar of in advance sterilization.
3) secretory product grinds, and is thin as far as possible, adds the 10mL sterilized water, continues to grind to form pasty state, first by 100 μ m, 10 μ m membrane filtrations.
3) add 10% sucrose solution that 10mL prepares in advance in the filtered liquid as settling agent and 5 μ L Silwet L-77 tensio-active agents, make steeping fluid.Room temperature leaves standstill 1h.
4) the Arabidopis thaliana after twice of pinching being soaked flower and writing brush with the steeping fluid of making dips in liquid and is coated with the flower processing.The flower of general major branch floods in nutrient solution, and the time is 2min; Harsh sprout or the short branch that contains bud adopt writing brush dipping method.
5) dark cultivation is after 2 days first through the Arabidopis thaliana after the dip treating, and then fixed temperature and humidity is cultivated, and collects respectively seed by different processing after the maturation.
6) the Arabidopis thaliana seed of collecting concentrates respectively plantation to cultivate, and begins to gather fresh Arabidopsis leaf material about one month.
7) adopt the sky to take root in the thing genome and extract test kit extraction arabidopsis thaliana genomic dna, carry out pcr amplification and sequencing and analysis with insect universal primer (gene-cytochrome b gene that in melaphis chinensis Baker and host plant transgenosis research, is verified).Design of primers is with reference to Simon (1994), and primer sequence is:
P1?5’-TATGTACTACCATGAGGACAAATATC-3’；
P2?5’-ATTACACCTCCTAATTTATTAGGAAT-3’
8) the pcr amplification system is 50 μ l, includes 5U TaKaRa Taq, 1 * PCR Buffer, 2mM MgCl
2, 0.25mMdNTP, each 0.4 μ M of upstream and downstream primer, 20-50ng dna profiling solution.
9) reaction conditions of pcr amplification is 94 ℃ of denaturations 3 minutes; Then carry out 39 circulations, each circulation comprises 94 ℃ of 40s of sex change, and the 50 ℃ of 1m that anneal extend 72 ℃ of 1m; Then fully extend 7m at 72 ℃.
10) pcr amplification product adopts 3730 sequenators to carry out two-way order-checking.
11) sequence that obtains is searched for through Blast, adopt dna sequence analysis software such as Sequencher, Clustal, Paup and Bayes software etc., with the melaphis chinensis Baker that has recorded and from times aphid host plant of angle, obtain corresponding sequence compare analysis, determine according to homology whether the sequence that screening obtains is that the Gene Partial sequence that shifts has occured.
Embodiment 2
1) with embodiment 1.
1) with embodiment 1.
2) with embodiment 1.
3) with embodiment 1.
4) with embodiment 1.
5) with embodiment 1.
6) with embodiment 1.
7) genomic dna of the Arabidopis thaliana of extraction dip treating, adopt above-mentioned primer to carry out pcr amplification, if can obtain the sequence of 433bp, and very high by the homology of the computed in software such as Blast, Mega and Paup itself and melaphis chinensis Baker, can illustrate the gene level transfer phenomena has occured.
Description of drawings
Fig. 1. the angle is doubly and inner angle times aphid and Secretory product composition
Fig. 2. the relation between the gene order that detects in the Arabidopis thaliana and melaphis chinensis Baker and other aphids. the sample of group I is the melaphis chinensis Baker kind among the figure, group II is the sample of the aphid of downloading from GenBank, the Arabidopis thaliana sample of transgenosis that has been the generation that filters out of the sample in the square frame wherein, group O is outgroup.
Sequence table
Claims (2)
1. one kind contains aphid fungal component Buchnera mixture by the brought out transgenic engineering of melaphis chinensis Baker mediation, it is characterized in that melaphis chinensis Baker colonizes on the Primary host plant forms insect gall, be full of melaphis chinensis Baker and its secretory product in the insect gall, separate these materials, cultivate the processing of pinching, dipping transfection, extracting genome DNA, pcr amplification, sequencing, data analysis etc. through grinding, filtering separation, mirror mirror, infiltration cultivation, Arabidopis thaliana and detect the sequence that transgenosis has occured.
2. as claimed in claim 1ly a kind ofly can shift the working method that microbial bacteria is induced for the identification of melaphis chinensis Baker secretory component producer, concrete operations are as follows:
1) gather the Turkey-galls sample in the melaphis chinensis Baker ripening stage, select larger, not have cracking, intact gall nut, cut open, separation white movement is wherein weighed.
2) grind under the sample sterile state, thin as far as possible.Add the 30mL sterilized water, the furnishing pasty state is handled as follows:
A. get the 10mL lapping liquid in the sterilization culture dish, make steeping fluid No. 1;
B. the remaining 20mL sterilized water that adds, packing, the trim of weighing, 1000r/m is centrifugal, removes slag.
C. supernatant liquor filters through 100 μ m strainers, gets 10mL in new sterilization culture dish, makes steeping fluid No. 2.
D. remaining mistake 10 μ m strainers are got 10mL in new sterilization culture dish, make steeping fluid No. 3.
3) each culture dish sucrose solution and 5 μ L Silwet L-77 auxiliarys of 10% of adding that 10mL prepares in advance, room temperature leaves standstill 1h.
Before above sample preparation, cultivate in advance arabidopsis thaliana and grow into the state of to pinch of blooming.
4) Arabidopis thaliana is pinched, and removes the part of blooming, and the Arabidopis thaliana of each Pot soaks respectively flower and writing brush and dips in liquid and be coated with flower processing.The flower of general major branch floods in nutrient solution, and the time is 2min; Harsh sprout or the short branch that contains bud adopt the writing brush coating process.
5) process first dark the cultivation 2 days of rear Arabidopis thaliana, then fixed temperature and humidity is cultivated, and collects seed by different processing after the maturation.
6) the Arabidopis thaliana seed of collecting is planted respectively cultivation, gathers fresh Arabidopsis leaf material behind the cultivation one and a half months.
7) adopt the Tigen Plant Genome to extract test kit and extract arabidopsis thaliana genomic dna, carry out pcr amplification and sequencing and analysis with insect universal primer (gene that in melaphis chinensis Baker and host plant transgenosis research, is verified).
8) adopt dna sequence analysis software, by comparing analysis with the corresponding sequence of melaphis chinensis Baker after measured, determine that the sequence that screening obtains is the partial sequence that the gene of transfer has occured.
Secretory product mixture in the Turkey-galls may be a kind of medium of inducing of transgenosis, and the microorganism that wherein contains (the molecular data Analysis and Identification is the fungal component of times aphid) has played medium in transgenosis.
The discovery of the microbial mixture that new induced transgenosis occurs is a large achievement of biotechnology, has higher development and application and is worth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101614400A CN103320348A (en) | 2013-05-06 | 2013-05-06 | Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101614400A CN103320348A (en) | 2013-05-06 | 2013-05-06 | Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103320348A true CN103320348A (en) | 2013-09-25 |
Family
ID=49189394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101614400A Pending CN103320348A (en) | 2013-05-06 | 2013-05-06 | Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103320348A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1934937B (en) * | 2005-09-19 | 2010-05-12 | 刘声树 | Gallnut aphid gall life stage artificial culture method |
| US20110086118A1 (en) * | 2009-10-14 | 2011-04-14 | Woongjin Coway Co., Ltd. | Composition for prevention of influenza viral infection comprising sumac extract, air filter comprising the same and air cleaning device comprising the filter |
| CN102524183A (en) * | 2011-12-12 | 2012-07-04 | 中国林业科学研究院资源昆虫研究所 | Method for improving yield of chinensis gallnuts by adjusting occurrence time of stem mothers by using accumulated temperature |
| KR20120140281A (en) * | 2011-06-21 | 2012-12-31 | 김영균 | Method of manufacturing natural anticoccidial drug |
-
2013
- 2013-05-06 CN CN2013101614400A patent/CN103320348A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1934937B (en) * | 2005-09-19 | 2010-05-12 | 刘声树 | Gallnut aphid gall life stage artificial culture method |
| US20110086118A1 (en) * | 2009-10-14 | 2011-04-14 | Woongjin Coway Co., Ltd. | Composition for prevention of influenza viral infection comprising sumac extract, air filter comprising the same and air cleaning device comprising the filter |
| KR20120140281A (en) * | 2011-06-21 | 2012-12-31 | 김영균 | Method of manufacturing natural anticoccidial drug |
| CN102524183A (en) * | 2011-12-12 | 2012-07-04 | 中国林业科学研究院资源昆虫研究所 | Method for improving yield of chinensis gallnuts by adjusting occurrence time of stem mothers by using accumulated temperature |
Non-Patent Citations (5)
| Title |
|---|
| ZHUMEI REN 等: "Comparative population structure of Chinese sumac aphid Schlechtendalia chinensis and its primary host-plant Rhus chinensis", 《GENETICS》 * |
| 任竹梅: "五倍子蚜与寄主植物DNA序列系统发育关系及其协同进化", 《山西大学学报(自然科学版)》 * |
| 吴玉新 等: "桃蚜与其内共生菌Buchnera之间的系统进化关系分析", 《南京农业大学学报》 * |
| 李继变 等: "角倍蚜与其唯一夏寄主植物盐肤木种群遗传多样性比较", 《山西大学学报(自然科学版)》 * |
| 李继变: "角倍蚜mtDNA基因序列遗传多样性与谱系地理研究", 《中国优秀硕士学位论文全文数据库(农业科技辑)》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109370939A (en) | One plant of Bei Laisi bacillus and its separation method and application | |
| CN109988730B (en) | Biocontrol bacterium for mulberry diseases and application thereof | |
| Kavamura et al. | Land management and microbial seed load effect on rhizosphere and endosphere bacterial community assembly in wheat | |
| CN105830760B (en) | A kind of wild rice smut Inoculation Method successfully making the pregnant hay of wild rice stem plant | |
| CN107299105A (en) | Watermelon blight bacteria pathogenic FonAGL3 genes, its missing DNA fragmentation, deletion mutant and its application | |
| CN108998389A (en) | There is pseudomonad and the application of antagonism to rice Xanthomonas campestris and Pyricularia oryzae | |
| CN105838616A (en) | Ustilago esculenta haploid strain UET1 and use thereof | |
| CN113493755B (en) | Pseudomonas gainstaedi GT03, application thereof and obtained control agent | |
| CN110904130A (en) | Construction and application of antibacterial nuclear disease gene GmGST1 and transgenic GmGST1 plant | |
| Zhang et al. | Analysis of endophytic actinobacteria species diversity in the stem of Gynura cusimbua by 16S rRNA gene clone library | |
| CN104694532A (en) | Extracting method of RNA of rubber tree powdery mildew | |
| Holmes et al. | Crown gall development on cannabis (Cannabis sativa L., marijuana) plants caused by Agrobacterium tumefaciens species-complex | |
| CN107858372A (en) | A kind of agriculture bacillus mediated cotton transient transformation methods | |
| Suarez-Montes et al. | Isolation and identification of microalgal strains with potential as carotenoids producers from a municipal solid waste landfill | |
| CN107723381A (en) | Banana crown rot bacterium LAMP detection primer and its detection method | |
| CN107488610A (en) | A kind of isolation and identification method of paddy bacterial fringe rot pathogen | |
| CN107190089A (en) | The method that using molecular labeling 3 kinds of strawberry cultivars are carried out with quick discriminating | |
| CN103320348A (en) | Chinese gall aphid-mediated microorganism-containing mixture capable of inducing gene transfer | |
| CN109706087A (en) | A kind of letter penicillium bacterial strain and its separation method and application | |
| CN103952426A (en) | Double reporter gene contained binary T-DNA (transferred deoxyribonucleic acid) carrier as well as construction method and application of double reporter gene contained binary T-DNA carrier | |
| CN106011004A (en) | Nitrogen-fixing microorganism G96 as well as bacterium agent preparation method and application thereof | |
| IL194085A (en) | Devices and methods for the isolation and cultivation of microorganisms | |
| Shmas et al. | Identification and Pathogenicity of Phytopathogenic Bacteria associated with soft rot disease on some Potato Cultivars | |
| Yayla | New record of Pentalonia nigronervosa Coquerel, 1859 (Hemiptera, Aphididae) in Turkey | |
| CN109929811A (en) | The red middle potato virus X strain in pasture and its construction method and application in a kind of Potato Cultivars |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130925 |
|
| WD01 | Invention patent application deemed withdrawn after publication |