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Method for culturing and identifying CmWRKY48 trans-genetic chrysanthemum morifolium and application of trans CmWRKY48 genetic chrysanthemum morifolium

A technology of breeding method and identification method, which is applied in the field of plant genetic engineering and transgenic breeding, to achieve the effect of improving aphid resistance, improving aphid resistance ability, and significant aphid resistance

Inactive Publication Date: 2015-02-25
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

However, there is no relevant report on the improvement of the aphid resistance of plants by the WRKY gene driven by the 35S promoter.

Method used

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  • Method for culturing and identifying CmWRKY48 trans-genetic chrysanthemum morifolium and application of trans CmWRKY48 genetic chrysanthemum morifolium
  • Method for culturing and identifying CmWRKY48 trans-genetic chrysanthemum morifolium and application of trans CmWRKY48 genetic chrysanthemum morifolium
  • Method for culturing and identifying CmWRKY48 trans-genetic chrysanthemum morifolium and application of trans CmWRKY48 genetic chrysanthemum morifolium

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Experimental program
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Embodiment 1

[0080] (1) Cloning of chrysanthemum CmWRKY48

[0081] Using the high aphid resistance variety 'Nannong Xunping' as the material [He Junping. Discussion on the identification and mechanism of aphid resistance of cut chrysanthemum and LLA transgenic research. Nanjing Agricultural University (2010).], take 0.15g of leaves, refer to Trizol RNA extraction kit (TaKaRa) manual operation method to extract the total RNA of leaves, according to M-MLV reverse transcription kit (TaKaRa) reverse transcription to obtain cDNA, according to the sequence information of the gene in the chrysanthemum library, using primer 5 software to design specific primers to amplify Add CmWRKY48;

[0082] Upstream primer CmWRKY48-F: the sequence is shown in SEQ ID NO.2,

[0083] Downstream primer CmWRKY48-R: the sequence is shown in SEQ ID NO.3;

[0084] Using the cDNA of the leaves of ‘Nannong Medal’ as a template, carry out PCR reaction, 50 μL reaction system: 10×PCR Buffer 5.0 μL, CmWRKY48-F, CmWRKY48-R...

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Abstract

The invention belongs to the field of plant genetic engineering and transgenic breeding, and provides a method for culturing and identifying a recombinant vector containing a CmWRKY48 gene, a transformed cell and CmWRKY48 trans-genetic chrysanthemum morifolium and application thereof. The method for culturing CmWRKY48 trans-genetic chrysanthemum morifolium comprises the following steps: (1) cloning a CmWRKY48 gene of chrysanthemum morifolium; (2) constructing a plant expression vector pMDC43-CmWRKY48; (3) transforming the chrysanthemum morifolium by an agrobacterium EHA105 mediated leaf disc method. PCR (Polymerase Chain Reaction) and quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detections on the transformed plant prove that the CmWRKY48 gene is integrated to the DNA of the genome of the transgentic plant and performs transcription. An anti-aphis detection is performed on the transgenetic plant, and the anti-aphis capability of the transgenetic plant is greatly improved, so that a novel and practical method is provided to breeding of stress tolerance variety of chrysanthemum morifolium by utilizing a gene engineering technology, and the progress of chrysanthemum morifolium biotechnological breeding can be effectively promoted.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to a method for improving the aphid resistance of cut chrysanthemums through transgenic CmWRKY48 gene, and specifically relates to a recombinant vector including CmWRKY48 gene, transformed cells, and a method for cultivating and identifying transgenic cut chrysanthemums with CmWRKY48 gene and applications. Background technique [0002] Chrysanthemum (Chrysanthemum morifolium) is one of the top ten famous flowers in my country and one of the four major cut flowers in the world. It has a long history of cultivation. Chrysanthemum has characteristics such as rich flower color, various flower positions, is deeply liked by people, and is widely cultivated in the world. WRKY is a family of transcription factors unique to plants, named for its highly conserved WRKYGQK polypeptide sequence at the N-terminal. WRKY transcription factors participate in various p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10A01H5/00C12Q1/68
Inventor 陈发棣李佩玲宋爱萍王海滨陈素梅张飞赵爽蒋甲福
Owner NANJING AGRICULTURAL UNIVERSITY
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