CN102031271A - Simple and high-efficiency genetic transformation system of sweet wormwood - Google Patents
Simple and high-efficiency genetic transformation system of sweet wormwood Download PDFInfo
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- CN102031271A CN102031271A CN2010102318006A CN201010231800A CN102031271A CN 102031271 A CN102031271 A CN 102031271A CN 2010102318006 A CN2010102318006 A CN 2010102318006A CN 201010231800 A CN201010231800 A CN 201010231800A CN 102031271 A CN102031271 A CN 102031271A
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Abstract
The invention discloses a simple and high-efficiency genetic transformation system of sweet wormwood. A construction method in the invention comprises the following steps: 1) planting sweet wormwood plants; 2) preparing transformation fluid; 3) culturing transformation bacteria; 4) carrying ouy genetic transformation on plants; 5) screening the transformed plants; and 6) identifying T1 representative types. By utilizing the simple and high-efficiency genetic transformation system of the sweet wormwood, the yield of the transgenic plants can be improved.
Description
Affiliated technical field
The present invention relates to the genetic transformation field in the biotechnology, specifically is a kind of construction process of advantages of simplicity and high efficiency plant sweet wormwood genetic conversion system.
Background technology
The Chinese medicine sweet wormwood, promptly Herba Artemisiae annuae (Artemisia annua L.) has another name called smelly wormwood artemisia, and smelly sweet wormwood is composite family artemisia annual herb plant.Artemisinine that extracts from blue or green punt-pole and derivative thereof are the new antimalarial agents of a class after quinoline and acridine.Find that Artemisinin has anti-schistosome function the beginning of the eighties in last century, effect such as in addition, that sweet wormwood class medicine also has is antitumor, immunomodulatory, anti-arrhythmia.World's Artemisinin drug manufacture mainly relies on China to extract from sweet wormwood, past is mainly utilized wild sweet wormwood resource, artificial culture seldom, in recent years because demand is increasing, wild Chinese medicine sweet wormwood resource is exhausted day by day, must rely on the artificial culture plantation, and artificial culture Artemisinin kind artemislnin content is generally lower, the output blade and the artemislnin content that improve Cultivar are the major objectives of current sweet wormwood breeding.The human improvement plant variety that develops into of current plant genetic transformation technology has been brought unprecedented speed and ability, the output, the quality that realize the improvement plant variety have at short notice obtained obvious effects, and obtaining transgene abrotanum high yield strain system by genetically engineered is one of the most potential approach that improves Artemisinin output.
Plant genetic transforms and starts from the seventies in 20th century, and over more than 30 year, plant genetic engineering is obtained tremendous development, genetically modified crops large scale application on producing.At present, it is agrobacterium-mediated transformation and particle bombardment that plant genetic transforms two kinds of the most frequently used methods, and the receptor tissue of conversion can be that callus, protoplastis etc. separate cell, also can be complete vegetable cell.These method for transformation have following deficiency: (1) depends on tissue culture, complicated operation, and the operator needs professional knowledge base and experience preferably; (2) too much processing often causes the sudden change of receptor tissue, purpose and verity that influence transforms; (3) often occur gene silencing in the transformed plant, thereby reduce the expression efficiency of goal gene greatly.Therefore, in many plant genetics transformed, transformation efficiency was low, long molecular breeding and the gene functional research of seriously restricting of operational cycle.At present, in Arabidopis thaliana isotype Study on plants, non-tissue culture method has become the main means of genetic transformation, and these methods also have broad application prospects in other plant.
Summary of the invention
The purpose of this invention is to provide a kind of advantages of simplicity and high efficiency sweet wormwood genetic conversion system, improve the yield of transfer-gen plant.
The present invention's technical scheme that is adopted of dealing with problems is: a kind of advantages of simplicity and high efficiency sweet wormwood genetic conversion system, and its construction process comprises that step is as follows:
1) plantation of sweet wormwood plant;
2) preparation of conversion fluid;
3) cultivation of transformed bacteria;
4) plant genetic transformation;
5) transformed plant screening;
The plantation of described sweet wormwood plant comprises: seeds of southernwood is seeded in black loam a year mid-November in earlier: vermiculite: potted plant in the composite soil of fine sand (4: 2: 1), and in February, second, be put in the greenhouse 20～22 ℃ of temperature, relative humidity 60%～70%, illumination 16h, dark 8h, cultivate about 2 months after, be put in outdoor, when growing to the high 100cm of the stem left and right sides, go its terminal inflorescence, behind continued growth 3～5d, promptly can be used for transforming;
The preparation of described conversion fluid: be to prepare conversion fluid as requested, the cultivation of described transformed bacteria: from the Agrobacterium substratum that is added with kantlex, positive Agrobacterium is inoculated in the YEB liquid nutrient medium that contains Rifampin and kantlex picking, 28 ℃, the 220r/m shaking culture to OD600 be 1.2～1.8 (needing 48h approximately), the centrifugal supernatant of abandoning is collected thalline and is resuspended in the conversion fluid;
Described plant genetic transformation comprises: ready plant is inverted in contains in the plastic tank that transforms damping fluid, cover black plastic bag lucifuge is placed 24h, cultivates plant then according to a conventional method to solid, the results mature seed;
Described transformed plant screening: will obtain T
0For seed, through selecting the substratum screening, obtain positive plant, extract positive plant DNA, by round pcr positive plant is further identified;
Present method can also comprise the evaluation of T1 representative type: according to the transgene characteristics, T1 is carried out phenotypic evaluation for transfer-gen plant.
Comprise among the every 1000ml of conversion fluid described in present method: 1/2MS macroelement (50 *) 10ml, 1/2MS trace element (50 *) 10ml, sucrose 30g, Syringylethanone (40mg/ml) 0.1ml, 6-BA (40mg/ml), 10 μ l, tensio-active agent Silwet L-77,1ml;
The present invention is by agriculture bacillus mediated, the conversion fluid method of directly soaking bud with the band target gene, the ipt gene that to clone from sweet wormwood changes in the sweet wormwood, changing into power is 5%-8%, detect and find ipt gene energy overexpression in sweet wormwood high yield strain system, in the transgene abrotanum enzymic activity of ipt be non-transgene abrotanum 3-4 doubly, cause the transfer-gen plant tiller number obviously to increase; Artemislnin content reaches as high as 0.92% (DW) in the transfer-gen plant, is 1.42 times of artemislnin content in the non-transgene abrotanum.
This genetic conversion system only need does not need to carry out vacuum-treat with Agrobacterium bacterium liquid with colored contact of plant.This system is dipped at bud and is added tensio-active agent SilwetL-77 in the conversion process, compare with the processing that does not add Silwet L-77, transformation efficiency significantly increases, in Agrobacterium liquid substratum, add sucrose, sucrose can be converted into glucose, glucose is main energy substance, for the pollen granule growth provides sufficient nutrient, causes transformation efficiency obviously to improve.This system has been simplified the Transformation Program of sweet wormwood greatly, has improved transformation efficiency, and simple to operate, is suitable for that this class plant of sweet wormwood is tall and big, the cross-pollination species carry out genetic transformation, and this genetic transformation to other species also has good reference to be worth.
The EHA105 Agrobacterium bacterium liquid that will contain pentenyl transferring enzyme (ipt) gene is dipped method and is changed sweet wormwood over to, T
0For seed, anti-Kan screen transfer-gen plant.Detect through PCR, prove that tentatively external source ipt gene has been incorporated in the sweet wormwood genome.T
1Begin performance for plant from the seedling phase and tiller manyly, generally have 5-7 to tiller, reach utmost point conspicuous level with the difference of check variety, the later stage luxuriant growth, branch is many, and prolong flowering period, and resistance to cold strengthens.Prenyltransferase (ipt) gene can cause that phytokinin is synthetic in a large number, to plant degeneration-resistant, anti-aging, tiller etc. produces vital role more.The transfer-gen plant phenotype is the result of ipt gene overexpression just.
Description of drawings
Fig. 1 is that transfer-gen plant and wild-type compare photo.
Fig. 2 is that transfer-gen plant PCR detects photo.
Embodiment
A kind of advantages of simplicity and high efficiency sweet wormwood genetic conversion system, its construction process comprises that step is as follows:
1) plantation of sweet wormwood plant: seeds of southernwood is seeded in the composite soil of black loam, vermiculite, fine sand potted plant in year mid-November in earlier, and wherein black loam, vermiculite, fine sand part by weight are 4: 2: 1, in February, second, be put in the greenhouse 20～22 ℃ of temperature, relative humidity 60%～70%, illumination 16h, dark 8h, cultivate about 2 months after, be put in outdoor, when growing to the high 100cm of the stem left and right sides, go its terminal inflorescence, behind continued growth 3～5d, promptly can be used for transforming;
2) preparation of conversion fluid: prepare conversion fluid as requested, comprise among the every 1000ml of conversion fluid: 1/2MS macroelement (50 *) 10ml, 1/2MS trace element (50 *) 10ml, sucrose 30g, Syringylethanone (40mg/ml) 0.1ml, 6-BA (40mg/ml), 10 μ l, tensio-active agent Silwet L-77,1ml.
3) cultivation of transformed bacteria: from the Agrobacterium substratum that is added with kantlex, positive Agrobacterium is inoculated in the YEB liquid nutrient medium that contains Rifampin and kantlex picking, 28 ℃, the 220r/m shaking culture to OD600 be 1.2～1.8 (needing 48h approximately), the centrifugal supernatant of abandoning is collected thalline and is resuspended in the conversion fluid;
4) plant genetic transformation: ready plant is inverted in contains in the plastic tank that transforms damping fluid, cover black plastic bag lucifuge is placed 24h, cultivates plant then according to a conventional method to solid, the results mature seed;
5) transformed plant screening: will obtain T
0For seed, the MS through containing Rifampin and kantlex selects the substratum screening, obtains positive plant, extracts positive plant DNA, by round pcr positive plant is further identified;
6) T1 representative type is identified: the function with transgene is reference, and T1 is carried out phenotypic evaluation for transfer-gen plant.
Claims (3)
1. advantages of simplicity and high efficiency sweet wormwood genetic conversion system, its construction process comprises that step is as follows:
1) plantation of sweet wormwood plant;
2) preparation of conversion fluid;
3) cultivation of transformed bacteria;
4) plant genetic transformation;
5) transformed plant screening;
The plantation of described sweet wormwood plant comprises: seeds of southernwood is seeded in the composite soil of black loam, vermiculite, fine sand potted plant in year mid-November in earlier, and wherein black loam, vermiculite, fine sand part by weight are 4: 2: 1, in February, second, be put in the greenhouse 20～22 ℃ of temperature, relative humidity 60%～70%, illumination 16h, dark 8h, cultivate about 2 months after, be put in outdoor, when growing to the high 100cm of the stem left and right sides, go its terminal inflorescence, behind continued growth 3～5d, promptly can be used for transforming;
The preparation of described conversion fluid: be to prepare conversion fluid as requested;
The cultivation of described transformed bacteria: from the Agrobacterium substratum that is added with kantlex, the positive Agrobacterium of picking is inoculated in the YEB liquid nutrient medium that contains Rifampin and kantlex, 28 ℃, the 220r/m shaking culture to OD600 be 1.2～1.8, the centrifugal supernatant of abandoning is collected thalline and is resuspended in the conversion fluid;
Described plant genetic transformation comprises: ready plant is inverted in contains in the plastic tank that transforms damping fluid, cover black plastic bag lucifuge is placed 24h, cultivates plant then according to a conventional method to solid, the results mature seed;
Described transformed plant screening: will obtain T
0For seed, through selecting the substratum screening, obtain positive plant, extract positive plant DNA, by round pcr positive plant is further identified;
2. advantages of simplicity and high efficiency sweet wormwood genetic conversion system construction process according to claim 1, its step also comprise the evaluation of T1 representative type: according to the transgene characteristics, T1 is carried out phenotypic evaluation for transfer-gen plant.
3. advantages of simplicity and high efficiency sweet wormwood genetic conversion system construction process, it is characterized in that: comprise among the every 1000ml of described conversion fluid: 1/2MS macroelement (50 *) 10ml, 1/2MS trace element (50 *) 10ml, sucrose 30g, Syringylethanone (40mg/ml) 0.1ml, 6-BA (40mg/ml) 10 μ l, tensio-active agent
Silwet L-77, 1ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104542295A (en) * | 2015-01-16 | 2015-04-29 | 山西大学 | Transgenic breeding method of sweet wormwood |
| CN111763689A (en) * | 2020-05-22 | 2020-10-13 | 浙江大学 | Method for improving transgenic efficiency of upland cotton standard line TM-1 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616664A (en) * | 1995-03-29 | 2005-05-18 | 威斯康星校友研究基金会 | Transgenic plants with altered senescence characteristics |
| WO2007090143A2 (en) * | 2006-02-01 | 2007-08-09 | Pioneer Hi-Bred International, Inc. | Soybean isopentenyl transferase genes and methods of use |
| CN101016553A (en) * | 2007-01-22 | 2007-08-15 | 大连理工大学 | Composition used for plants direct gene conversion |
| CN101225384A (en) * | 2008-02-04 | 2008-07-23 | 山西省农业生物技术研究中心 | Agrobacterium-mediated woody plants leaf mud in-situ gene transformation method |
| CN101748148A (en) * | 2010-01-28 | 2010-06-23 | 湖南大学 | Transgenic method of agrobacterium-mediated rape field |
-
2010
- 2010-07-17 CN CN2010102318006A patent/CN102031271A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616664A (en) * | 1995-03-29 | 2005-05-18 | 威斯康星校友研究基金会 | Transgenic plants with altered senescence characteristics |
| WO2007090143A2 (en) * | 2006-02-01 | 2007-08-09 | Pioneer Hi-Bred International, Inc. | Soybean isopentenyl transferase genes and methods of use |
| CN101016553A (en) * | 2007-01-22 | 2007-08-15 | 大连理工大学 | Composition used for plants direct gene conversion |
| CN101225384A (en) * | 2008-02-04 | 2008-07-23 | 山西省农业生物技术研究中心 | Agrobacterium-mediated woody plants leaf mud in-situ gene transformation method |
| CN101748148A (en) * | 2010-01-28 | 2010-06-23 | 湖南大学 | Transgenic method of agrobacterium-mediated rape field |
Non-Patent Citations (4)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库》 20090215 张明婧 小油桐组织培养及ipt基因遗传转化研究 全文 1-3 , 2 * |
| 《园艺学报》 20001231 张赛群等 异戊烯基转移酶基因导入番茄及转基因植株再生 全文 1-3 第26卷, 第6期 2 * |
| 《西南师范大学学报（自然科学版）》 20090630 秦淑丽等 异戊烯基转移酶基因的克隆与转化烟草的研究 全文 1-3 第34卷, 第3期 2 * |
| 刘凡等: "农杆菌介导的植物原位转基因方法研究进展", 《分子植物育种》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104542295A (en) * | 2015-01-16 | 2015-04-29 | 山西大学 | Transgenic breeding method of sweet wormwood |
| CN104542295B (en) * | 2015-01-16 | 2016-06-01 | 山西大学 | A kind of sweet wormwood transgenic breeding method |
| CN111763689A (en) * | 2020-05-22 | 2020-10-13 | 浙江大学 | Method for improving transgenic efficiency of upland cotton standard line TM-1 |
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Application publication date: 20110427 |