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RNA interference vector and use thereof

An RNA interference and carrier technology, applied in applications, recombinant DNA technology, and introduction of foreign genetic material using vectors, etc., can solve the problems of nematode race variation, lack of antigen material sources, and inability to meet production needs, and achieve root knots and egg masses. Effects of population reduction, reliable nematode control strategies, and effective nematode control strategies

Inactive Publication Date: 2009-02-04
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the commonly used methods of nematode control include crop rotation, use of nematicides, biological control and breeding of nematode-resistant varieties, etc. Since crop rotation is not suitable for controlling root-knot nematodes with a wide range of hosts, the extensive use of nematodes is expensive and expensive. The environment is seriously polluted, drug resistance is easy to develop, and biological control is slow to take effect. The most economical and effective way is to cultivate and utilize new varieties resistant to nematodes
Conventional disease resistance breeding currently has two problems: one is the use of a single resistance gene to make nematodes that are not affected by resistance multiply and develop into new diseases, or cause nematode race variation; on the other hand, the lack of sources of antigenic materials also makes Conventional breeding is far from meeting production needs
Like other organisms, nematodes need to constantly feel various changes and stimuli in their living environment, transmit them to the cells through a series of signal cascade processes, and adjust the corresponding physiological processes and gene expression in the cells, thereby generating a response to the stimuli. Various adaptive responses, it is speculated that the MAPK gene encoding the signal transduction enzyme may play a key role in the interaction between the parasitic nematode and the host, but there is no report in this regard

Method used

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  • RNA interference vector and use thereof
  • RNA interference vector and use thereof
  • RNA interference vector and use thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: in vitro verification experiment

[0045] The dsRNA of MiMPK1 gene was synthesized in vitro, the egg masses and 2nd instar larvae of M. incognita were treated simultaneously by soaking method, and octopamine was used to stimulate the phagocytosis of 2nd instar larvae of M. incognita. The interference effect was verified in vitro. After the ovum block was immersed in the dsRNA of 2mg / ml for 1 day, it was observed that the number of 2 instar larvae hatched was significantly more than that of the control group, but its mortality rate reached more than 85%, while the mortality rate of the control group was not higher than 5%; The hatched nematodes and unhatched egg masses were inoculated into the roots of tomato plants. After 45 days, no root knots and egg masses were found in the tomato roots, indicating that the nematode larvae whose MiMPK1 gene was silenced lost their infectivity. After the egg mass was soaked in 2mg / ml dsRNA for 3 days, the number of hatch...

Embodiment 2

[0046] Embodiment 2: the acquisition of transfer RNA interference vector tomato plant

[0047] Step 1: Construction of RNA interference inducing vector

[0048] The RNA interference inducing vector pFGC5941 (gifted by the University of Leeds, UK, is a commonly used vector and can be purchased in the market) has a herbicide resistance (BAR) gene, and the restriction site is repeated with the insert, so it is necessary to construct a hygromycet-containing Insert the RNA interference inducing vector of pFGC5941 (3577bp) into the cloning vector pCAMBIA1300 with hygromycin selection marker (preserved by the Laboratory of Vegetable and Flower Research Institute, Chinese Academy of Agricultural Sciences, and can be distributed to the public)

[0049] pFGC5941 and pCAMBIA1300 were digested with EcoRI / PstI respectively ( figure 1 ), digested overnight at 37°C, the digested product of pCAMBIA1300 was electrophoresed and the 8.9kb fragment was recovered by gel cutting, and the 3.5kb fr...

Embodiment 3

[0071] Example 3: Detection of insect resistance of transgenic plants

[0072] Step 1 Preparation of explants for tomato transformation

[0073] (1) Soak the tomato seeds with 70% ethanol for 3min;

[0074] (2) Discard ethanol, add sterile water to wash twice;

[0075] (3) discard the sterile water, add 20% sodium hypochlorite for disinfection for 20 minutes;

[0076] (4) Discard sodium hypochlorite, blot excess water after rinsing with sterile water for 5-6 times;

[0077] (5) The seeds are moved into a triangular flask equipped with MS medium, 30 seeds in each bottle, and placed evenly;

[0078] (6) Culture at 26-28°C in the dark and light culture for 6-7 days after germination until the cotyledons open at an angle of 120°;

[0079] (7) Put the tomato seedlings to be cut on the filter paper, clamp the hypocotyl part of the seedlings with tweezers, make the two cotyledons overlap and lay flat on the bottom of the petri dish, cut off the top of the cotyledons, and then cut...

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Abstract

The present invention provides a RNA interference vector and an application thereof on genetically modified nematode-resistant plants, which belong to the technical field of plant protection. The RNA interference vector is characterized in that a plant expression vector contains a nucleotide sequence which is more than 74 percent similar to a sequence shown by Sequence No:1 and has the same function as the protein expressed by the Sequence No:1 sequence does, and the nucleotide sequence is inserted into the plant expression vector in the positive and negative directions. The constructed vector transforms a receptor plant; the transcribed exogenous gene can form a foldback double-stranded hairpin loop RNA structure; after the genetically modified plant is eaten by nematode, the target gene in the body of the nematode, MiMPK1 gene, interacts with the dsRNA expressed by the exogenous gene in the body of the plant, so that a silencing effect occurs; and as a result, the nematode cannot normally develop and breed. Thus the effect of inhibiting the dissemination of nematode to protect crops is achieved.

Description

technical field [0001] The invention belongs to the technical field of plant protection, in particular to an RNA interference vector and its application in nematode-resistant transgenic plants. Background technique [0002] At present, commonly used nematode control methods include crop rotation, use of nematicides, biological control and breeding of nematode-resistant varieties, etc. Since crop rotation is not suitable for controlling root-knot nematodes with a wide range of hosts, extensive use of nematodes is expensive and expensive. The environment is seriously polluted, drug resistance is easy to develop, and biological control is slow to take effect. The most economical and effective way is to cultivate and utilize new nematode-resistant varieties. Conventional disease resistance breeding currently has two problems: one is the use of a single resistance gene to make nematodes that are not affected by resistance multiply and develop into new diseases, or cause nematode ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N1/15C12N1/19C12N1/21A01H1/00A01H5/00
Inventor 谢丙炎陈国华杨宇红茆振川冯东昕冯兰香
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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