Method for deleting selective marker gene of transgenic poplar

A selection marker and transgenic technology, applied in the field of genetic engineering, can solve problems such as low deletion efficiency, and achieve the effect of improving deletion efficiency

Inactive Publication Date: 2011-09-14
NANJING FORESTRY UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0004] Wang Yonggang (2007) used the Cre / lox system to study the genetic transformation system of poplar without selection markers in the article "Research on Genetic Transformation System without Selectable Marker" (Master's Thesis of Nanjing Forestry University), but did not obtain the transgene that deleted the selectable marker gene. Poplar; Fu Jian (2007) also used this system to conduct research on non-selectable marker transgenic poplar in "Nanlin 895 Poplar Genetic Transformation without Selectable Marker" (Master's Thesis of Nanjing Forestry University), but in 21 transgenic poplar Only one strain was detected to delete the selectable marker gene, only about 5%, and the deletion efficiency was low

Method used

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  • Method for deleting selective marker gene of transgenic poplar

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Experimental program
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Embodiment 1

[0032] Example 1 Preparation of Agrobacterium

[0033] A single colony of Agrobacterium EHA105 was picked, inoculated in 5ml LB medium, and cultured at 28°C and 220rpm for 24h. Add 400??l bacterial solution to 20ml LB medium, culture at 28℃, 220rpm for 3h, until OD 600 Around 0.3. Divide the bacterial solution into 10ml sterile centrifuge tubes and place in ice bath for 10min. Put the centrifuge tube into a centrifuge, centrifuge at 4000rpm for 10min at 4°C. Discard the supernatant, add 5ml of pre-cooled 0.15mol / L NaCl, and gently resuspend. Cool in ice for 30 minutes, centrifuge at 4000 rpm for 10 minutes at 4°C. Discard the supernatant, add 600??l pre-cooled 0.02mol / L CaCl 2 , and gently resuspend. Add 1??lpX6-GFP plasmid to 200??l of Agrobacterium competent cells, mix gently, and ice-bath for 30min. Freeze in liquid nitrogen for 1 min. 37 ℃ water bath for 3 ~ 5min, quickly placed on ice, ice bath for 1min. join 800??lYEB(str 50 ) culture medium, cultured at 28°C, ...

Embodiment 2

[0034] Example 2 Genetic transformation of Nanlin 895 poplar

[0035] The test-tube seedlings of the transgenic plants of Populus Nanlin 895 were placed on the medium M to subculture by root sprouting and sprouting, and the young and flat 3rd to 5th leaves were selected from the test-tube seedlings that had been subcultured for about 1 month. The size, shape and color are basically the same. Cut off the edge of the leaf and cut it into 0.7~0.8cm 2 The leaf disks are explants, inoculated in such a way that the abaxial surface is in contact with the medium. The culture temperature is 25±2°C, the light is 3000~4000lux, and the light time is 16~18h / d. Pre-culture on T medium for 2~3 days, and the abaxial surface of the leaf disc is in contact with the medium. Pick a single colony of Agrobacterium EHA105 (pX6-GFP plasmid) carrying the GFP gene and inoculate it into YEB liquid medium, culture at 27±1°C, 220-230rpm shaking overnight; transfer 0.8-1mL to 25-30mL YEB liquid medium....

Embodiment 3

[0036] Example 3 Deletion of selection markers in Nanlin 895 poplar transgenic plants

[0037] After subculture of the transgenic plants of Populus Nanlin 895 obtained through screening, the stem segments of the transgenic resistant plants that had been analyzed and identified were clipped and cultured in leaf disk differentiation medium W' with the selection marker deleted until adventitious buds differentiated. The adventitious buds and the original explants were transplanted into the adventitious bud elongation medium Y' with the selection marker deleted, and subcultured once every 2-3 weeks. After the adventitious buds elongated to about 1.5 cm, each adventitious bud was separated and independently inserted into the rooting medium G' with the selection marker deleted until a complete implant was formed.

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Abstract

The invention discloses a method for deleting a selective marker gene of a transgenic poplar. The method comprises the steps of first placing a transgenic poplar test tube plantlet on a culture medium M to carry out secondary propagation in a root sprouting manner; scissoring leaf discs to be cultured; and then immerging into an Agrobacterium tumefaciens EHA105 (Ethyl Hexyl Acrylate 105) bacteria liquid with pX6-GFP (p-Xylene 6-Green Fluorescent Protein)) plasmids to be dipped for 10-15 minutes; culturing to obtain a complete plant after removing the bacteria liquid; and culturing in a culture medium containing beta-estradiol until the plant is reproduced after finishing the scissoring the whole plant stem. In the method disclosed by the invention, by using the beta-estradiol added to the culture medium, a callus is formed by inducing the transgenic plant stem until the selective marker gene is deleted in the plant reproducing process; and a poplar transgenic system without the selective marker gene is established by using a recombinant deleting function of the Cre/lox (locus of X-over) system to provide a new way for culturing a new variety of the transgenic poplar with biosafety and lay the basis for safe and high efficient forest transgenic breeding.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for deleting selection marker genes of transgenic poplar trees. Background technique [0002] Poplar is one of the important tree species for afforestation in the world, and poplar also has a large proportion in my country's artificial forests. Among forest plants, poplar is one of the tree species that carried out transgenic research earlier. It is reported that through genetic engineering technology, it has successfully transferred genes such as resistance to diseases and insect pests, salt and alkali resistance, air pollution resistance, lower lignin content, and increased growth. , which provides a basis for breeding new varieties of transgenic poplar with excellent traits. However, due to the safety of transgenic plants and other issues, the environmental release and popularization and application of transgenic poplar are limited. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/09
Inventor 诸葛强陶玉峰周洁王立科潘惠新黄敏仁陈英王光萍
Owner NANJING FORESTRY UNIV
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