CN103540608A - Method for rapidly obtaining transgenic wheat homozygous plant - Google Patents
Method for rapidly obtaining transgenic wheat homozygous plant Download PDFInfo
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- CN103540608A CN103540608A CN201310463156.9A CN201310463156A CN103540608A CN 103540608 A CN103540608 A CN 103540608A CN 201310463156 A CN201310463156 A CN 201310463156A CN 103540608 A CN103540608 A CN 103540608A
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- 241000209140 Triticum Species 0.000 title claims abstract description 61
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 61
- 241000196324 Embryophyta Species 0.000 title claims abstract description 50
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 101150103518 bar gene Proteins 0.000 claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 230000002363 herbicidal effect Effects 0.000 claims abstract description 8
- 239000004009 herbicide Substances 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 6
- 101150054900 gus gene Proteins 0.000 claims abstract description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 claims description 6
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- 210000001161 mammalian embryo Anatomy 0.000 abstract 1
- 238000011426 transformation method Methods 0.000 abstract 1
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- 101100133720 Arabidopsis thaliana NPR1 gene Proteins 0.000 description 9
- 244000037671 genetically modified crops Species 0.000 description 6
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- 102000003960 Ligases Human genes 0.000 description 1
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Abstract
The invention relates to a method for rapidly obtaining a transgenic wheat homozygous plant. The method comprises the following steps: using a target gene to substitute a gus gene of basic plasmid pAHC25, wherein the target gene is closely linked with a herbicide resistance bar gene; leading the obtained recombinant plasmid into wheat through a wheat gene gun transformation method; placing a wheat transgenic gene plant and a young embryo, which is obtained from a plant descendant blooming for 15 days, in 1/2 MS culture medium containing 1mg/LL-PPT herbicide for continuous culture and screening until the herbicide resistance bar gene of the transgenic plant is homozygous and the descendant can be separated no longer; performing PCR (polymerase chain reaction) detection and verification on the target gene of the transgenic plant with the homozygous herbicide resistance bar gene, thus obtaining the transgenic plant with the homozygous target gene. The method disclosed by the invention has the advantages that a mass of manpower and material resources are saved, and the time for the homozygosis of transgenic wheat is shortened.
Description
Technical field
The present invention relates to wheat biotechnology breeding field, adopt rataria cultivation, herbicide screening and PCR to detect the method combining and obtain fast transgenic wheat homozygous plants.
Background technology
Transgenic technology is since being born, because the advantages such as crop breeding efficiency, extremely breeder's concern are isolated, improved in pointed reproduction strong, that can break between species.According to existing statistical information, since first case genetically modified crops commercialization in 1996 plantation, genetically modified crops are that economy, environment and social security have been brought lasting considerable interests, the cultivated area of genetically modified crops in 2012 is by originally 1,700,000 hectares, expand to 1.7 hundred million hectares, increase by 100 times, made transgenosis become in modern agriculture history application crop technology the most rapidly.The national number of plantation genetically modified crops is increased to 30 by 6, wherein 19Ge Wei developing country; The number of plantation genetically modified crops reaches 1,730 ten thousand; It is 15,000,000,000 dollars that the world market of transgenic seed in 2012 is worth, and the economic benefit that genetically modified crops in 2011 bring is 19,700,000,000 dollars; Transgenic technology for ensureing global grain security and foodgrain self-sufficiency, sustainable development, alleviate poverty and hungry, help to alleviate the challenge that climate change and Global warming bring and make significant contribution [Clive James. Chinese biological engineering magazine, 2013,33 (2): 1-8].
Wheat is the important food crop of the world's second largest that are only second to corn at present, 2,010 65.3 hundred million tons of gross annual outputs.China is the maximum Wheat Production state in the whole world, and 2010 gross annual outputs reach 11.2 hundred million tons.The same with other countries, climatope and disease and pest are to affect the important factor that Wheat in China is produced stable and Sustainable development.In the last few years, conventional breeding was being made slow progress aspect raising yield and quality of wheat, and the animal nutritions such as transgenosis are to address these problems the new approach of opening up.At present, although transgenic wheat is not also at China Business, China scientific research personnel has carried out disease-resistant, pest-resistant, herbicide-resistant, drought-enduring and improve the research of the transgenic wheat of quality, and obtain good progress [Xia Lanqin, Ma Youzhi, He Yi, Huw D. Jones. Journal of Experimental Botany, 2011, doi:10.1093/jxb/err342].
The methods such as the transgenic method of wheat still be take particle gun mediated method as main at present, agriculture bacillus mediated are auxiliary.The common multiple copied of goal gene that particle bombardment imports is in the majority, and offspring is separated serious, needs how for Continuous Tracking, to detect, and could obtain the transfer-gen plant isozygotying.The transfer-gen plant isozygotying is significant for the function of research purpose gene and the stability of expression analysis and heredity.Because the genome of wheat is larger, PCR etc. detect comparatively difficulty, and the workload how to detect for Continuous Tracking is very huge, need to drop into a large amount of man power and materials.Industry personnel seeks a kind ofly can improve cultivation efficiency for a long time always, reduces the method for cultivating cost, finds the approach that obtains fast the transfer-gen plant isozygotying.
Summary of the invention
The object of the invention is: for the acquisition of transgenic wheat homozygous plants in the past, need carry out to transgenic wheat offspring the continuous multi-generation PCR detection of goal gene, workload is huge, and can only carry out the series of problems such as 1 generation detection every year, a kind of method of quick acquisition transgenic wheat homozygous plants is provided.
The present invention seeks to realize like this: a kind of method of quick acquisition transgenic wheat homozygous plants, it is characterized in that: the recombinant plasmid that contains goal gene is imported to wheat, by the rataria cultivation to transgenic wheat, herbicide screening and PCR, detected and obtained fast transgenic wheat gene pure plant, concrete steps are:
A, take pAHC25 as basic plasmid, goal gene being inserted to SmaI and the SacI restriction enzyme of pAHC25 cuts between site, the gus gene that replaces pAHC25 with goal gene, makes the antiweed bar gene close linkage in goal gene and pAHC25 basis plasmid;
B, the recombinant plasmid that upper step is obtained adopt gene gun conversion method import wheat and obtain T
0for Transgenic plant of wheat;
C, get T
0the rataria of blooming after 15 days for Transgenic plant of wheat, is placed on the 1/2MS substratum containing 1mg/L L-PPT weedicide and cultivates, and discards the not seedling of antiweed, treats that seedling grows to 6cm left and right and transplants to basin alms bowl, and conventional cultivation is managed to plant blossom;
D, take away the rataria of spending after 15 days, at least repeat c step one time, until offspring seedling is no longer dead, this seedling is the Transgenic plant of wheat of antiweed bar gene pure;
Whether e, the homology of getting the antiweed bar gene pure that d step obtains contain goal gene with carrying out goal gene PCR detection for 1 strain transgenic wheat leaf DNA in plant, as contain goal gene, again the PCR that carries out goal gene with all plant leaf DNA in generation with this strain homology is detected to verify whether goal gene isozygotys, as goal gene isozygotys, these plant are transgenic wheat homozygous plants.
The invention has the advantages that: because the present invention adopts the Screening of Media containing weedicide in earlier stage, in conjunction with rataria increasing generation technique, last is carried out PCR detection for homology is same for the strain in plant, whether checking contains target gene, again to carry out the PCR detection validation of goal gene with all plant in generation with this strain homology, save a large amount of man power and materials, while rataria increasing generation technique has also been saved goal gene and has been isozygotied the required time of process.
Embodiment
embodiment 1: turn the acquisition of Arabidopsis thaliana NPR1 DNA triticum homozygous plants
Material: pAHC25 basis plasmid is provided by the Xu Huijun researcher of Institute of Crop Science, Chinese Academy of Agricultural Science, Arabidopsis thaliana NPR1 gene You Ben project team clones by PCR method, Restriction enzyme Sma I and SacI enzyme are provided by the precious biotechnology (Dalian) of TaKaRa company limited, and weedicide L-PPT is provided by Sigma company.Wheat is raised wheat and by Lixiahe region in Jiangsu institute of agricultural sciences wheat chamber, is provided for No. 12.1/2MS substratum is by reducing by half the macroelement in standard MS substratum to obtain.
Adopt PCR method clone Arabidopsis thaliana NPR1 gene, and add respectively Restriction enzyme Sma I and SacI restriction enzyme site in the upstream and downstream of gene; Take pAHC25 as basic plasmid, adopt Restriction enzyme Sma I and SacI that pAHC25 plasmid (this plasmid contains bar screening-gene and the gus reporter gene of antiweed L-PPT simultaneously) DNA is carried out enzyme and cut, excision gus gene, reclaims 7.8Kb left and right DNA enzyme and cuts large fragment, with T
4dNA ligase is connected with clone's interpolation SmaI and the Arabidopsis thaliana NPR1 gene of SacI restriction enzyme site, builds Arabidopsis thaliana NPR1 gene and the closely linked wheat expression vector of bar gene.
Adopt particle bombardment (reference literature [wheat crops journal, 2008,28 (6): 935-940]) that the expression vector of the Arabidopsis thaliana NPR1 gene of structure is imported to wheat and raise wheat No. 12, obtain transgenosis T
0for plant 21 strains, after transfer-gen plant blossoms and bears fruit, strip the rataria of blooming after 15, be placed in containing on the 1/2MS substratum of 1mg/L L-PPT weedicide and cultivate, transfer-gen plant offspring occurs that Herbicid resistant is separated, and the transfer-gen plant offspring of antiweed is transplanted, after solid, continue to repeat screening, until transfer-gen plant offspring Herbicid resistant is no longer separated, this is antiweed bar gene pure plant, from T
1in generation, starts to derive from different T
0for there being successively bar gene pure transfer-gen plant to occur in the offspring of transfer-gen plant, until T
6generation, 21 transgenosis T
0for all obtaining bar gene pure transfer-gen plant in the offspring of plant.
The PCR that the bar gene pure transfer-gen plant obtaining is carried out to Arabidopsis thaliana NPR1 detects (reference literature [Molecular Plant Breeding, 2012,10(6): 655-661].
To 21 of above acquisition bar gene pure transgenic wheat strains, respectively select the DNA of 1 strain blade to carry out goal gene PCR detection, as contain goal gene, again to carry out the PCR detection of goal gene with all plant from generation to generation with this strain homology, to verify whether goal gene isozygotys.
By detecting, from 21 bar gene pure transgenic lines, screen altogether in two years the transgenic line of 15 Arabidopsis thaliana NPR1 gene pures.
embodiment 2
Material: basic plasmid pAHC25, Restriction enzyme Sma I and SacI enzyme, weedicide L-PPT and substratum are all identical with embodiment 1.
TaPIMP1 gene is provided by the Zhang Zengyan researcher of Institute of Crop Science, Chinese Academy of Agricultural Science, and wheat Yangmai No.158 and Yang Mai are provided by Lixiahe region in Jiangsu institute of agricultural sciences wheat chamber for No. 12, and Alondra Wei Ben project team preserves germplasm.
Adopt method and the step identical with embodiment 1, by wheat TaPIMP1 Gene Replacement for the gus reporter gene of basic plasmid pAHC25, build wheat TaPIMP1 gene overexpression carrier, this gene and antiweed bar gene close linkage.
This Overexpression vector is imported to wheat Yangmai No.158, raised wheat No. 12 and Alondra by particle bombardment, adopt the homozygous method for genetic identical with embodiment 1, to T
5in generation, in two years, obtains the transgenic line of 23 bar gene pures.
The PCR that the bar gene pure transfer-gen plant obtaining is carried out to wheat TaPIMP1 gene detects (reference literature [nuclear agricultural science report, 2011,25(3): 0421-0426]), after testing, in 23 bar gene pure transgenic lines, having 17 strains is the TaPIMP1 transgenosis phytem (table 1) of isozygotying.
Table 1 turns the TaPIMP1 DNA triticum goal gene situation of isozygotying
Each embodiment is not limitation of the present invention above; described target gene is not limited to Arabidopsis thaliana NPR1 gene and TaPIMP1 gene; described wheat breed is also not limited to raise wheat No. 12, Yangmai No.158, raise wheat No. 12 and Alondra; as long as those skilled in the art is under enlightenment of the present invention; target gene is proceeded to wheat breed, all belong to protection category of the present invention.
Claims (1)
1. a method that obtains fast transgenic wheat homozygous plants, it is characterized in that: the recombinant plasmid that contains goal gene is imported to wheat, by the rataria cultivation to transgenic wheat, herbicide screening and PCR, detected and obtained fast transgenic wheat goal gene homozygous plants, concrete steps are:
A) take pAHC25 as basic plasmid, goal gene being inserted to SmaI and the SacI restriction enzyme of pAHC25 cuts between site, the gus gene that replaces pAHC25 with goal gene, makes the antiweed bar gene close linkage in goal gene and pAHC25 basis plasmid;
B) recombinant plasmid upper step being obtained adopts gene gun conversion method import wheat and obtain T
0for Transgenic plant of wheat;
C) get T
0the rataria of blooming after 15 days for Transgenic plant of wheat, is placed on the 1/2MS substratum containing 1mg/L L-PPT weedicide and cultivates, and discards the not seedling of antiweed, treats that seedling grows to 6cm left and right and transplants to basin alms bowl, and conventional cultivation is managed to plant blossom;
D) take away the rataria of spending after 15 days, at least repeat c step one time, until offspring seedling is no longer dead, this seedling is the Transgenic plant of wheat of antiweed bar gene pure;
Whether the homology of e) getting the antiweed bar gene pure that d step obtains contains goal gene with carrying out goal gene PCR detection for 1 strain transgenic wheat leaf DNA in plant, as contain goal gene, again the PCR that carries out goal gene with all plant leaf DNA in generation with this strain homology is detected to verify whether goal gene isozygotys, as goal gene isozygotys, these plant are transgenic wheat homozygous plants.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967746A (en) * | 2017-02-04 | 2017-07-21 | 深圳万智联合科技有限公司 | A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed |
| CN111631130A (en) * | 2020-06-09 | 2020-09-08 | 石河子大学 | Method for accelerating strong winter wheat hybridization breeding process by embryo culture technology for 4 generations in 1 year |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942452A (en) * | 2010-07-30 | 2011-01-12 | 江苏省农业科学院 | Method for improving sheath blight resistance of wheat |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942452A (en) * | 2010-07-30 | 2011-01-12 | 江苏省农业科学院 | Method for improving sheath blight resistance of wheat |
Non-Patent Citations (2)
| Title |
|---|
| 周淼平 等: "转Gastrodianin 基因提高小麦赤霉病和纹枯病的抗性", 《作物学报》, vol. 38, no. 9, 3 July 2012 (2012-07-03), pages 1617 - 1624 * |
| 景寅: "油菜BnGOLS1基因功能鉴定及酶学性质的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 09, 15 September 2012 (2012-09-15), pages 047 - 51 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967746A (en) * | 2017-02-04 | 2017-07-21 | 深圳万智联合科技有限公司 | A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed |
| CN111631130A (en) * | 2020-06-09 | 2020-09-08 | 石河子大学 | Method for accelerating strong winter wheat hybridization breeding process by embryo culture technology for 4 generations in 1 year |
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