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DNA introduction method independent of corn genotype

A genotype and maize technology, applied in the field of plant genetic engineering, can solve the problems of difficulty in meeting the needs of molecular breeding in the maize seed industry, low tissue culture efficiency of commercial varieties, and dependence, etc. The effect of efficient import

Active Publication Date: 2019-10-15
BEIJING AGRO BIOTECH RES CENT
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  • Abstract
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AI Technical Summary

Problems solved by technology

High-efficiency DNA introduction technology in maize is an important means of modern molecular breeding. At present, existing DNA introduction technologies such as gene gun method, Agrobacterium method, PEG mediation, etc. are all overly dependent on efficient maize tissue culture system, and the tissue culture of maize is serious. Limited by the maize genotype, the tissue culture efficiency of commercial varieties is very low, and it is difficult to meet the needs of modern molecular breeding in the maize seed industry

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  • DNA introduction method independent of corn genotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]A method of DNA introduction independent of maize genotype:

[0038] Positively charged Fe modified with polyethyleneimine (PEI) 3 o 4 Magnetic nanoparticles (MNPs, Cat. No. 9006, Chemicell, Germany) are used as gene carriers to bind and protect negatively charged DNA. Under the action of a magnetic field, the nano-magnetic beads transfer DNA to corn pollen through the pollen pores, and through the natural reproductive process of pollination and fruiting, the introduction of the target gene is realized.

[0039] The corn materials used are backbone inbred lines: 178, B73, HZ178, Jing 92 and Zheng 58.

[0040] The vector used is the expression vector pYBA1132 containing green fluorescent protein (Liu et al., PlantGenomics in China XIV, 2013, 169) constructed by the unit, and the sequence of the pYBA1132 vector is shown in SEQ ID NO.1, including the CaMV shown in SEQ ID NO.2 The 35S promoter (p35S) and the enhanced green fluorescent protein gene EGFP (Enhance green fluo...

Embodiment 2

[0051] Fluorescent tracking of the entire import process (taking Beijing 92 as an example)

[0052] 1) Expression of EGFP gene in maize pollen. An appropriate amount of transfected pollen was dipped and inoculated on a corn pollen solid medium (15 g / L agar powder was added to the corn pollen culture solution), and cultured at 25° C. in the dark for 24 hours. Dip an appropriate amount of cultivated corn with a brush, gently disperse it in the corn pollen culture solution, and observe the green fluorescence with 488nm excitation light under a laser confocal microscope. Fluorescence observation results such as image 3 shown. image 3 Middle, MNP+EGFP: maize pollen magnetically transfected with EGFP gene; MNP: maize pollen treated with magnetic beads only; CK: non-transfected maize pollen; Merge: green fluorescence and bright field overlay; GFP: green fluorescence; Bright : bright field. according to image 3 It can be seen that 92% (491 / 536) of the pollen transfected with t...

Embodiment 3

[0056] Molecular detection of plants transfected with EGFP Jing92T1

[0057] At the three-leaf stage, the young leaves of Jing 92T1 generation plants transfected with EGFP were taken to extract genomic DNA, total RNA and total protein. Perform PCR detection with the primers in Table 2, and then perform RT-PCR detection on PCR positive plants. In table 1: EGFP-F is the upstream primer (20bp) that is used to detect EGFP gene, and sequence is as shown in SEQ ID No.4; EGFP-R is the downstream primer (20bp) that is used to detect EGFP gene, and sequence is as shown in SEQ ID Shown in No.5; ZmActin1-F is the upstream primer (22bp) that is used to detect ZmActin1 internal reference gene, and sequence is as shown in SEQ ID No.6; ZmActin1-R is the downstream primer (20bp) that is used to detect ZmActin1 internal reference gene, The sequence is shown in SEQ ID No.7. Subsequently, the RT-PCR positive plants were analyzed by Western blot.

[0058] Image 6 It is the graph of molecular...

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Abstract

The invention discloses a DNA introduction method independent of a corn genotype. Magnetic nanoparticles with positive charges are adopted as a gene carrier to be combined with DNA with negative charges, under the action of a magnetic field, the magnetic nanoparticles combined with the DNA transfer the DNA into corn pollen through pollen holes, and introduction of a target gene is realized throughnatural reproduction processes of pollination and fruiting. According to the invention, efficient introduction and transient and stable expression of the target gene in all corn can be realized, andthe bottleneck problems that current mainstream corn gene introduction methods based on tissue culture are limited by a few acceptor materials and have low introduction efficiency and complicated operation are solved.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to an efficient nano-magnetic bead-mediated pollen transfection system and a DNA introduction method applicable to all corn genotypes. Background technique [0002] Corn is a very important crop in the world, and it is also the largest crop in my country. The annual planting area in China exceeds 400 million mu. my country is the second largest producer and consumer of corn, and corn plays an extremely important role in grain production and the national economy. Corn is the main feed and an important raw material for industrial processing. With the rapid increase in the consumption of feed, starch, fuel ethanol, etc., and the demand is strongly driven, my country's corn supply and demand relationship has undergone significant changes, from a basic balance of self-sufficiency with a little export to a tight balance with tight supply and a small amount of imports....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 王作平吴忠义张中保李向龙张春
Owner BEIJING AGRO BIOTECH RES CENT
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