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Method, composition and application of applying genetic engineering technology to prolong banana fresh-keeping period

A technology for composition and fresh-keeping period, which is applied to the application of genetic engineering technology to prolong the fresh-keeping period of bananas, the composition and the application field thereof, can solve problems such as poor effect, achieve good transgenic efficiency, delay the after-ripening phenomenon, and prolong the fresh-keeping of bananas. period effect

Inactive Publication Date: 2011-12-14
黄鹏林
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the past, the method used to reduce the expression of the target gene was mainly to transfer the antisense strand of the target gene into the plant, and the mRNA produced was complementary to the mRNA sequence of the endogenous cis-sense gene to form a Double-stranded (duplex) structure, and then degrade or interfere with protein translation, to achieve the purpose of inhibiting endogenous gene expression; expression and cause co-suppression (co-suppression), thereby inhibiting gene expression
However, the above two methods are not very effective in silencing genes

Method used

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  • Method, composition and application of applying genetic engineering technology to prolong banana fresh-keeping period
  • Method, composition and application of applying genetic engineering technology to prolong banana fresh-keeping period
  • Method, composition and application of applying genetic engineering technology to prolong banana fresh-keeping period

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Contains the construction of the gene carrier related to suppressing banana post-ripening

[0061] 1. Construction of gene silencing universal vector pRNAi

[0062] (1) Plasmid material

[0063] a. pUC19 plasmid: full-length 2.682kb, containing E. coli screening gene AmpR (GenBank accession no. L09137).

[0064] b. pU35STgfp plasmid: with 2X En CaMV 35S promoter, green fluorescent protein (GFP) gene, and nopaline synthase (NOS) gene terminator. The source of the original plasmid is PNAS (1996) 93: 5888-5893. After cutting out 1854 bp with the restriction enzyme HindIII, it was inserted into the pUC19 vector of HindIII, which became pU35STgfp.

[0065] (2) Gene source

[0066] The intron (intron) used to construct the pRNAi vector is derived from the first intron of the banana ACC oxidase gene MAO1A (GenBank accession no.AF030411) and the gene MAO1B (GenBank accession no.AF030410) (its sequence is as shown in SEQ ID No: 17), the sequences of the first in...

Embodiment 2

[0108] Example 2 Banana gene transfer technology and process

[0109] 1. Agrobacterium-mediated gene transfer

[0110] The methods A and B of the transfer are modified from Ma Suxuan (1988), and the steps are as follows:

[0111] A method

[0112] (1) Plant materials and other materials

[0113] Banana North banana variety (Musa spp. cv. Pei Chiao, AAA group). Suspension culture cells induced by male inflorescences, and their callus. The following materials are all commercially available.

[0114] (2) Transplant medium

[0115] The Agrobacterium strain used was LBA4404 (Hoekema et al., 1983), and the pBI121-2AnS or pBI121-1AnS plasmid constructed in Example 1 was transformed (for transformation, please refer to Molecular Cloning).

[0116] (3) banana gene transfer method

[0117] Step 1: Induction of callus

[0118] The male inflorescences of banana variety (Musa spp.cv.Pei Chiao, AAA group) were taken and placed in induction medium (callus induction medium, as shown...

Embodiment 3

[0293] Example 3 Obtaining Molecular Evidence of Transformed Banana by Southern Hybridization

[0294] Banana transgenic cells were regenerated into seedlings through antibiotic screening, and after the reporter gene was confirmed by GUS active histochemical staining, molecular level analysis could be carried out. In this example, Southern hybridization analysis was used to confirm the integration of the DNA fragment to be transferred. into the banana genome.

[0295] 20 μg of plant genomic DNA was taken, digested with appropriate restriction enzymes, and analyzed by electrophoresis with 0.7% agarose gel. The electrophoresis colloid was treated twice for 15 minutes with 0.25N HCl, and then denatured buffer (1.5M NaCl, 0.5M Tris-HCl, pH 7.2, 1mM Na 2 EDTA) for 15 minutes twice, and then with neutralization buffer (1.5M NaCl, 0.5M Tris-HCl, pH7.2, 1mM Na 2 EDTA) for 15 minutes twice. The DNA in the colloid was transferred to the Hybond N transfer membrane (Amersham) under the...

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Abstract

The invention discloses a method, composition and application for prolonging the fresh-keeping period of bananas by using genetic engineering technology. The method uses a novel banana transgene method to transfer a banana ACC oxidase control composition into bananas, wherein the combination The substance comprises at least one interfering RNA, a gene transfer expression vector and a pharmaceutically acceptable carrier, the interfering RNA is used to inhibit / reduce the mRNA expression of banana ACC oxidase, thereby inhibiting the ethylene biosynthesis of banana, thereby delaying Banana post-ripening phenomenon, therefore, can extend the fresh-keeping period of bananas.

Description

technical field [0001] The present invention relates to the application of genetic engineering technology to prolong the method, composition and application of banana fresh-keeping period, especially refers to the transfer of interfering RNA in banana by gene transfer method to inhibit / reduce the mRNA of banana ACC oxidase gene A method, a composition and an application for expressing and then inhibiting / reducing the biosynthesis of ethylene. Background technique [0002] "Post-ripening" means the process in which climacteric fruits and vegetables complete their own ripening after picking. For transportation or storage, some climacteric fruits and vegetables need to be picked in advance. Its purpose is to prolong the period of transportation and storage through the post-ripening effect of climacteric fruits and vegetables. Appropriate measures (such as low temperature, air conditioning, ethylene absorbers, ethylene inhibitors, etc.) can also be taken according to needs to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N5/10A01H4/00
Inventor 黄鹏林杜宜殷廖育辰林宜佑李盛新黄暐芬
Owner 黄鹏林
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