A kind of genetic transformation method of poplar alba

A genetic transformation method, the technology of Populus alba, which is applied in the field of genetic transformation of Poplar alba, can solve the problems of shortening the transformation time and the inapplicability of genetic transformation of Poplar alba, and achieve the effect of shortening the transformation time and improving the transformation efficiency

Active Publication Date: 2021-01-29
CHINESE ACAD OF FORESTRY
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Problems solved by technology

[0003] In view of this, the purpose of the present invention is to provide a kind of poplar genetic transformation method, solve the inapplicability problem of existing poplar genetic transformation method in the poplar genetic transformation, can greatly improve the transformation efficiency of white poplar, and effectively shorten the conversion time

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  • A kind of genetic transformation method of poplar alba
  • A kind of genetic transformation method of poplar alba
  • A kind of genetic transformation method of poplar alba

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preparation example Construction

[0037] In the present invention, the explants are soaked in the infecting bacteria solution, and the soaking time is preferably 10-15 minutes. The preparation method of the infection bacterium liquid described in the present invention preferably comprises the following steps: A. Agrobacterium carrying the recombinant plasmid is streaked on the YEB plate medium to obtain a single colony; the YEB plate medium contains 50 mg / L of kanamycin and 50mg / L of rifampicin;

[0038]B. Inoculate the single colony according to the ratio of 1 single colony: 5mLYEB liquid medium, and culture with shaking until OD 600 0.7~0.9, to get the expanded strain; the YEB liquid culture medium contains the kanamycin of 50mg / L and the rifampicin of 50mg / L;

[0039] C. Inoculate the expanded strains in YEB anti-antibiotic liquid medium and culture with shaking until OD 600 0.7 to 0.9 to obtain an activated bacterial liquid; the volume ratio of the expanded strain to the YEB non-resistant liquid medium ...

Embodiment 1

[0061] Transferring the GUS gene into Populus alba:

[0062] 1. Connect the GUS gene (GenBank: MG687280.1) to the ΔpCAMBIA1302 vector. The ΔpCAMBIA1302 vector is a new binary expression vector transformed from the traditional pCAMBIA1302. It mainly uses the CaMV35S promoter (CaMV35S-P) (835bp) and multiple The cloning site (42bp) replaced the constitutive CaMV35S promoter (CaMV35S-P) (538bp) and the reporter gene GFP on the original T-DNA segment of the pCAMBIA1302 vector. Transfer the recombinant gene into DH10B Escherichia coli competent. After the sequencing was correct, the plasmid was extracted and transformed into EHA105 Agrobacterium competent by electric shock transformation.

[0063] 2. Streak the Agrobacterium (EHA105) carrying the recombinant plasmid on a YEB (50mg / L Kan+50mg / L Rif) plate, culture at 28°C, and a single colony appears after 2 days.

[0064] 3. Pick a single colony for colony PCR, and inoculate it in 1mL YEB (50mg / L Kan+50mg / LRif) liquid medium, and...

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Abstract

The invention provides a genetic transformation method of poplar alba, which relates to the technical field of genetic transformation. The invention transfers the target gene into the body of poplar alba through the agrobacterium infection method, and obtains positive plants in a relatively short period of time. In the embodiment of the present invention, the β-glucuronidase gene (GUS) is used as a reporter gene to carry out the genetic transformation of Populus alba, and a large amount of transgenic Populus alba can be obtained in a short time. The poplar genetic transformation method of the invention solves the inapplicability problem of the existing poplar genetic transformation method in the genetic transformation of poplar, can greatly improve the transformation efficiency of poplar, and effectively shortens the transformation time.

Description

technical field [0001] The invention belongs to the technical field of genetic transformation, and in particular relates to a genetic transformation method of Populus alba. Background technique [0002] Populus alba (PopμLμs abla L.) is widely distributed in the western Mediterranean basin, Central Europe, Eastern Europe and the valleys of Central Asia. In my country, it is mainly distributed in the south of Liaodong, Shaanxi, Ningxia, Gansu, Shanxi, Qinghai, Xinjiang and Tibet. It is the main afforestation tree species in the Qinghai-Tibet Plateau and the Northwest Loess Plateau. It is often used for farmland protection, road greening, barren sand and wasteland management, building materials and firewood, furniture and carving patterns, etc. In the 1980s, my country began the research on tissue culture and rapid propagation of Populus alba, and made important progress. However, the research on the transformation system of Populus alba is not very mature at present, especia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/65A01H4/00A01H5/00A01H6/00
CPCA01H4/00C12N15/65C12N15/8205
Inventor 曾庆银刘妍婧姜鹏飞王晓霞王一鸣
Owner CHINESE ACAD OF FORESTRY
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