Method for transient expression of wheat leaf single cell based on efficient gene gun conversion and application of method

A biolistic transformation and single-cell technology, applied in biochemical equipment and methods, applications, botany equipment and methods, etc., can solve problems such as low efficiency, unstable expression efficiency of selected reporter genes, lack of biolistic transformation parameters, etc. To achieve the effect of enhancing resistance

Inactive Publication Date: 2013-03-20
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

In recent years, single-cell transient expression technology has also been initially applied in wheat (Schweizer, Pokorny et al. A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat. Molecular Plant-MicrobeInteractions. ), but there are still some problems in this technology, such as the efficiency of biolistic transformation in wheat is low due to the subjective influence of human operation, the lack of efficient screening of biolistic transformation parameters in wheat, and the instability of reporter gene expression efficiency.

Method used

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  • Method for transient expression of wheat leaf single cell based on efficient gene gun conversion and application of method
  • Method for transient expression of wheat leaf single cell based on efficient gene gun conversion and application of method
  • Method for transient expression of wheat leaf single cell based on efficient gene gun conversion and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Establishment of High Gene Gun Transformation Wheat Detached Leaf System

[0027] 1.1 Materials and methods

[0028] Expression vectors with different reporter genes: reporter gene anthocyanin synthesis regulatory gene C1-Lc expression vector pBecks400.Red (McCormac AC, Wu HX, Bao MZ, Wang YB, Xu RJ, Elliott MC, Chen DF. The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery towheat (Triticum aes tivum L.) and barley (Hordeum vulgare L.). Euphytica, 1998, 99 (1): 17-25.). The expression vector psGFP of the green fluorescent protein gene GFP (Takashi Tamura, KojiroHara, Yube Yamaguchi, Nozomu Koizumi and Hiroshi Sano. Osmotic stress tolerance of transgenic tobacco expressing a gene encoding a membrane-located receptor-likeprotein from tobacco plants. PlantPhysiol, 2003, 131: 454-462.). The expression vector pAHC25 of β-1,3-glucuronidase gene GUS (Christensen A H, Quail P H, Ubiquitin promoter-based vectors for high-...

Embodiment 2

[0045] Example 2 Construction of overexpression vector of SGT1 gene Hv-SGT1

[0046] The SGT1 gene cDNA (SEQ ID NO.1) was used as a template, and the primer pair SGT1ATG-BamHIF (TT GGATCC TCGACGCAGACATGG, SEQ ID NO.2) and SGT1-TGA-KpnI-R (GC GGTACC TCATTAATACTCCCAC, SEQ ID NO.3) was amplified by PCR, and the amplified fragment was recovered. The amplified product was double digested with BamHI and KpnI, and the digested product was inserted into the vector pBI220 (Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J.1987,6:3901-3907.)), put Hv-SGT1 at the multiple cloning site behind the 35S promoter. Thus, the target gene Hv-SGT1 was cloned to the downstream of the strong promoter 35S to obtain the expression vector pBI220:Hv-SGT1 ( Figure 4 ). It was verified by sequencing that the vector was constructed successfully.

Embodiment 3

[0047] Example 3 Transition of Hv-SGT1 gene into susceptible wheat leaves by transient expression method to study its interaction with wheat powdery mildew

[0048] 3.1 Materials and methods

[0049] The expression vector of the target gene Hv-SGT1 and the GUS expression vector pAHC25 (Christensen A H, Quail P H. Ubiquitin promoter-based vectors for high-level expression of selectable and / orscreenable marker genes in monocotyledonous plants. Transgenic Research, 1996, 5: 213-218.) Plasmid DNA was mixed and coated with tungsten powder (the molar ratio of the target gene carrier to the GUS gene carrier concentration was 2:1), and the wheat leaves were bombarded with a gene gun for co-transformation.

[0050] The powdery mildew spores were inoculated 4 hours after the leaves were bombarded. Inoculation method: roll a plastic sheet into a drum shape and place it on a porcelain plate, and evenly shake off fresh powdery mildew spores from above. The inoculation density should be hi...

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Abstract

The invention discloses a method for transient expression of a wheat leaf single cell based on efficient gene gun conversion and application of the method. The method for transient expression of the wheat leaf single cell based on the efficient gene gun conversion comprises the following steps of: performing gene gun conversion by using a green fluorescent protein gene as a report gene and a detached leaf of a first wheat leaf as a conversion receptor; immobilizing the wheat leaf by a solid fresh-keeping culture medium, wherein tungsten powder with the diameter of 1 micron is used for gene gun conversion, the using amount is 300ug/gun and the using amount of plasmid is 750ng/gun; and an attacking distance is measured based on 1350psi of cracking membrane helium pressure, a distance between the cracking film and a receptor film is 6 mm, and a distance between the receptor and a stopping net is 6 cm. By the method, haynaldia villosa SGT1 genes are expressed excessively in the detached leaf epidermis cells of the Yangmai 158 of the disease-sensing wheat categories; and a result shows that the index of powdery mildew haustorium in interaction cells can be reduced by the gene transient expression and the invasion of powdery mildew and the formation of the haustorium can be inhibited by the gene so as to increase the resistance of the powdery mildew.

Description

technical field [0001] The invention belongs to the field of biotechnology, and discloses a method for instantaneously expressing single cells of wheat leaves transformed by a high-efficiency gene gun and an application thereof. Background technique [0002] Powdery mildew is one of the main fungal diseases of wheat, and cultivating disease-resistant varieties is currently the most economical and effective method. Conventional breeding has a long cycle, and genetically modified biotechnology breeding is an effective strategy for efficiently cultivating new disease-resistant wheat varieties. However, the large wheat genome, long tissue culture cycle and low transgenic frequency limit the high-throughput and rapid identification of powdery mildew resistance candidate genes and related gene functions using stable transformation methods, hindering the discovery of effective powdery mildew resistance genes. Therefore, it is particularly important to establish a rapid and effecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/02A01H5/00
Inventor 邢莉萍曹爱忠钱晨王秀娥陈佩度
Owner NANJING AGRICULTURAL UNIVERSITY
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