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Method for constructing particle gun mediated liriodendron hybrids transformation system

A technology for hybridizing Liriodendron chinensis and construction methods, which is applied in the field of genetic engineering and can solve problems such as high cost, low transformation efficiency, and gene silencing

Inactive Publication Date: 2018-03-06
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Agrobacterium-mediated genetic transformation is the most widely used and is in a dominant position, but its disadvantage is that it is not suitable for monocotyledonous plants. However, the gene gun bombardment method can overcome this defect, not only is not limited by the transformation receptor, Moreover, the operation is simple, so the application research of gene gun transformation method in forest tree genetic breeding will become more and more extensive.
However, there are still some shortcomings in gene gun transformation technology that need to be further explored and improved, mainly including: high cost; low transformation efficiency; low proportion of stable inheritance; complex structural changes of foreign DNA after transformation, such as DNA circularization, basement and fragment separation, loss or rearrangement, etc.; foreign gene sequences are often inserted in multiple copies, and there are many chimeras, which are not easy to rule out; genetic stability is poor, and gene silencing is easy to occur; theoretical issues such as the integration mechanism of foreign genes are also not very clear
Therefore, using this method for genetic transformation still needs to solve many problems, especially the problem of low transformation efficiency.

Method used

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  • Method for constructing particle gun mediated liriodendron hybrids transformation system
  • Method for constructing particle gun mediated liriodendron hybrids transformation system
  • Method for constructing particle gun mediated liriodendron hybrids transformation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The material used in this example is: embryogenic callus of Liriodendron tulipifera, and the plasmid vector is pJIT166-GFP.

[0038] 1. Use the QIAGEN Plasmid Midi and Maxi Kits kit to extract and purify the plasmid.

[0039] 1) Preparation before extraction: Add RNase A to Buffer P1 to make the final concentration 100ug / mL, and store at 2-8°C. Add LyseBlue to Buffer P1, shake the bottle gently before use to ensure that LyseBlue is well mixed. Check the SDS status in Buffer P2. Buffer P3 should be pre-cooled first.

[0040] 2) Plasmid extraction process: (1) Pick a single colony from the colony medium with a pipette tip, inoculate it into 2 mL of LB medium containing kanamycin, and culture it at 37° C. and 300 rpm for 8 hours. Wherein, the capacity of the used flask is at least 4 times the capacity of the culture medium. (2) Dilute the starting culture 1 / 1000 to 1 / 500, and then inoculate it into LB medium containing kanamycin. The amount of medium used for inoculati...

Embodiment 2

[0065] Example 2 Construction of GUS Gene Transformation Hybrid Liriodendron System Mediated by Biolistic

[0066] In this example, the embryogenic callus of Liriodendron chinensis was used as the recipient material for gene gun transformation. The plasmid vector is pBI121-GUS, and the QIAGEN Plasmid Midi and Maxi Kits kit is used to extract and purify the plasmid, and the extraction process is the same as in Example 1. The main media used are as follows:

[0067] Embryogenic callus subculture and recovery medium: 3 / 4MS+6-BA 0.25mg / L+2,4-D 2.0mg / L+VC5mg / L+LH 0.5g / L+ sucrose 30g / L+ crystal agar 2.4g / L.

[0068] Embryogenic callus hyperosmotic medium: 3 / 4MS+6-BA 0.25mg / L+2,4-D 2.0mg / L+VC 5mg / L+LH0.5g / L+sucrose 30g / L+Crystal agaric 2.4 g / L+Mannitol 0.4M.

[0069] Suspension cell culture medium: 3 / 4MS+6-BA 0.25mg / L+2,4-D 2.0mg / L+VC 5mg / L+LH0.5g / L+sucrose 30g / L.

[0070] Suspension cell somatic embryo induction medium: 3 / 4MS+ABA2.0mg / L+VC 5mg / L+CH0.2g / L+ activated carbon 2g / L+...

Embodiment 3

[0088] Example 3 Construction of Gene Gun-Mediated LhWox1 Gene Transformation Hybrid Liriodendron Liriodendron System

[0089] The results of Examples 1 and 2 show that the GFP gene and the GUS gene can be successfully transformed into the hybrid Liriodendron tulipifera system using the gene gun-mediated genetic transformation technology, and not only the optimal bombardment parameters of this transformation system have been obtained , and the regenerated plants transgenic for GUS gene were obtained through resistance screening.

[0090] The construction method of the LhWox1 gene transformation hybrid Liriodendron system of this embodiment is the same as in Example 2, wherein the plasmid used in this embodiment is LhWOX1, and the LhWox1 gene transformation hybrid Liriodendron system was successfully obtained, and the specific results are as follows:

[0091]The callus after bombardment grows somatic embryos in a good growth state after recovery culture and induction culture, a...

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Abstract

The invention discloses a method for constructing a particle gun mediated liriodendron hybrids transformation system. The method comprises the following steps: 1) taking embryonic callus of liriodendron hybrids, and performing high osmotic treatment; 2) extracting plasmids, preparing gold dust, and preparing micro-particles; 3) performing particle bombardment; 4) transferring the callus to restoreto a normal level to perform dark recovery culture; 5) performing embryoid induction culture, performing light culture after embryoid grows out, and inoculating into a rooting medium to perform screening culture after seedlings grow out; and 6) performing molecular detection, thereby obtaining the liriodendron hybrids transformation system. According to the method for constructing the particle gun mediated liriodendron hybrids transformation system, disclosed by the invention, the feasibility of the particle gun mediated liriodendron hybrids system is explored, and a reliable experimental foundation is provided for transforming liriodendron hybrids by mediating target genes with a particle gun in future, so that a novel technology is created for functional gene transformation of the liriodendron hybrids, and a technical support is provided for researching the effects of various functional genes in the liriodendron hybrids.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a hybrid tulip tree transformation system mediated by a gene gun. Background technique [0002] At present, plant transgenic technologies mainly include Agrobacterium transformation method, gene gun bombardment method, pollen tube channeling method, PEG-mediated method, electric shock transformation method, liposome transformation method, low-energy ion beam-mediated method, ultrasonic-mediated method, laser microtransformation method, etc. Beam perforation, etc. Among them, Agrobacterium-mediated genetic transformation is the most widely used and is in a dominant position, but its disadvantage is that it is not suitable for monocotyledonous plants. However, the gene gun bombardment method can overcome this defect, not only is not limited by the transformation receptor, Moreover, the operation is simple. Therefore, the research o...

Claims

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Application Information

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IPC IPC(8): C12N15/89A01H4/00A01H5/00A01H6/00
Inventor 陈金慧施季森顾婧李美平王丹成铁龙杨立明
Owner NANJING FORESTRY UNIV
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