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A method for making scion varieties acquire virus resistance, rna interference vector pcambia2300-3a and transgenic method

An RNA interference and transgenic technology, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as no research reports on pathogenic bacteria or virus genes.

Inactive Publication Date: 2016-11-23
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, in the research on the gene silencing effect of scion varieties obtained by grafting, the target genes are some genes of the plant itself, and there are no research reports on pathogenic bacteria or virus genes as targets.

Method used

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  • A method for making scion varieties acquire virus resistance, rna interference vector pcambia2300-3a and transgenic method
  • A method for making scion varieties acquire virus resistance, rna interference vector pcambia2300-3a and transgenic method
  • A method for making scion varieties acquire virus resistance, rna interference vector pcambia2300-3a and transgenic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. For the cucumber mosaic virus (CMV) gene, 6 ihpRNA plant silencing expression vectors were constructed by comparing the tomato EST database;

[0073] Cucumber mosaic virus (CMV) is a member of the genus Cucumovirus in the family Bromoviridae. CMV is a single-stranded positive-sense RNA virus. The genome is divided into RNA1, RNA2 and RNA3. The genome size is 3389nt, 3035nt and 2197nt, respectively, and they are respectively encapsulated in three virions. Among them, RNA3 contains subgenomic RNA4 with a total length of 1000nt. RNA1 encodes nucleic acid replicase; RNA2 encodes two proteins: RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRP) and 2b protein, RdRP is related to virus infection, 2b protein is similar to a viral motor protein, there are experiments It is proved that 2b protein can weaken RNA-mediated viral gene silencing; RNA3 encodes a motor protein 3A protein, whose specific function is unknown but related to virus cell-to-cell tra...

Embodiment 2

[0156] Example 2. Functional verification of RNA interference carrier

[0157] Material:

[0158] ZT (Trans-Zeatin-Riboside, trans-zeatin riboside), Tim (Timentin, Timentin), folic acid, As (acetobutanone), Kan (kanamycin), IAA (3-indole acetic acid) , IBA (3-indolebutyric acid), and MS salt (M0404) were purchased from Sigma.

[0159] Tomato variety: MoneyMaker, which is susceptible to CMV.

[0160] Medium preparation: (unit: L)

[0161] (1) 1 / 2MS: pH5.8 1 / 2MS salt+15g|sucrose+6.5g|agar

[0162] (2) Medium A: pH5.8 MS salt+30g | sucrose

[0163] (3) Medium B: pH5.8 MS salt+30g|sucrose+6.5g|agar+1mg|ZT

[0164] (4) Medium Base: pH6.0 MS salt+20g|sucrose+6.5g|agar+100mg|inositol+0.1mL|10000×folic acid

[0165] (5) Medium C: pH6.0 Medium Base+2mg|ZT+200mg|Tim+100mg|Kan

[0166] (6) Medium C1: pH6.0 Medium Base+1mg|ZT+80mg|Kan+200mg|Tim

[0167] (7) Medium C3: pH6.0 Medium Base+0.5mg|ZT+1.5mg|IAA+80mg|Kan+200mg|Tim

[0168] (8) Medium D: pH6.0 Medium Base+2mg|IBA+200mg|Ti...

Embodiment 3

[0268] Embodiment 3. Transgenic plants were propagated by cuttings to determine the resistance of each target gene transgenic line.

[0269] Since the T0 generation of transgenic positive plants needs to be preserved after being transferred from the sterile room to the field for survival, it is not suitable for direct virus inoculation. Therefore the present invention adopts the mode of cuttage to carry out vegetative propagation, can solve this problem. At the same time, due to vegetative propagation, cutting propagation can provide more rootstock materials with the same genetic background for subsequent grafting.

[0270] Step 1. When the plant grows to a certain height, the mother plant is used to obtain seeds, take the lateral buds grown from the leaf axils, or take the stem section with a knot, collect the lateral buds or stem sections in a clean triangular flask, add 0.2 mg / L IBA sterile water 50ml, after 1 to 2 weeks, new roots will emerge from the bottom of the side ...

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Abstract

The invention discloses a method for enabling a scion variety to acquire virus resistance as well as an RNA (Ribonucleic Acid) interference vector pCAMBIA2300-3A and a transgenic method, belonging to a transgenic technology of plants and application thereof. The method comprises the following steps of firstly, preparing a transgenic stock for resisting virus; secondly, enabling scions to be grafted onto the transgenic stock for growing and developing. The method is characterized in that the virus-resisting transgenic stock is obtained by transferring the RNA interference vector; a target sequence of the RNA interference vector is a gene segment in a target virus genome. The method disclosed by the invention is used for a test that a tomato scion variety acquires the virus resistance, and a good effect is obtained; aiming at a specific embodiment of the tomato, the constructed RNA interference vector is pCAMBIA2300-1A, pCAMBIA2300-2A, pCAMBIA2300-2B, pCAMBIA2300-3A, pCAMBIA2300-CP or pCAMBIA2300-CMV5. The invention also provides the improved transgenic method. According to virus-resisting plants obtained by using the method, potential safety hazards of transgenic foods can be effectively avoided; in addition, the acquiring efficiency of virus-resisting transgenic varieties can be greatly increased.

Description

[0001] In the first and second office action notices of the parent case, the examiner pointed out that there were multiple inventions in the parent case, which were not unitary, so the applicant decided to file a divisional application for the unprotected inventions in the parent case. This application is a divisional application of application number 201110315208.9 (name of invention: a method for making scion varieties acquire virus resistance and RNA interference vector and transgenic method). technical field [0002] The invention relates to plant transgenic technology and application thereof, in particular to a method for obtaining virus resistance in tomato scion varieties, an RNA interference carrier and a transgenic method. Background technique [0003] Plant virus is an important cause of yield decline and quality deterioration of fruit trees, vegetables, flowers and other horticultural plants. In agricultural production, virus disease is the second largest type of d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01G1/06C12N15/63C12N1/21C12N15/113
Inventor 白描杨国顺钟晓红熊兴耀邓子牛石雪晖陈文婷周敏
Owner HUNAN AGRICULTURAL UNIV
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