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Method for enhancing black streaked dwarf resistance of paddy rice by using artificial microRNA (micro Ribonucleic Acid) and special double chain RNA thereof

A technology for black-streaked dwarf disease and rice, applied in the direction of DNA/RNA fragments, recombinant DNA technology, botanical equipment and methods, etc., to achieve high biological safety, wide range of prevention and control, and reduce uncertain phenotypes

Inactive Publication Date: 2013-12-18
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, in the searchable prior art, the method of using artificial microRNA technology to improve rice resistance to black-streaked dwarf virus has not been reported

Method used

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  • Method for enhancing black streaked dwarf resistance of paddy rice by using artificial microRNA (micro Ribonucleic Acid) and special double chain RNA thereof
  • Method for enhancing black streaked dwarf resistance of paddy rice by using artificial microRNA (micro Ribonucleic Acid) and special double chain RNA thereof
  • Method for enhancing black streaked dwarf resistance of paddy rice by using artificial microRNA (micro Ribonucleic Acid) and special double chain RNA thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Obtaining the dedicated double-stranded RNA sequence for transforming microRNA

[0024] The forward primer primer3: 5'-AAGTTTTTTGAGTCTGAGATA-3' (SEQ NO.27) and the reverse primer primer4: 5'-GACATCAGCTGATTTGAGTC-3' ( SEQ NO.28); extract the total RNA of the diseased leaves of rice black-streaked dwarf disease, and clone the S6 fragment sequence of rice black-streaked dwarf virus genome by RT-PCR method, and submit the sequence to the online software WMD3 (http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi?page=Home;project=stdwmd), according to the energy value of double-stranded RNA, through the comparison with the whole rice genome and mRNA sequence, the energy value of double-stranded RNA , the secondary structure of engineered microRNAs determines dedicated RNA sequences.

[0025] The method for extracting and purifying the total RNA of rice black-streaked dwarf diseased leaves is as follows:

[0026] 1. Weigh 0.2g of fresh tissue and grind it fully in liq...

Embodiment 2

[0046] Embodiment 2: Design and synthesis of artificial microRNA carrier

[0047] Use rice endogenous microRNA as the backbone structure, such as osa-MIR528, osa-MIR319, osa-MIR159, osa-MIR171, etc. The mature sequence (mature sequence) and the reverse complementary sequence (mature*sequence) of the rice endogenous miccroRNA were replaced with sequenceA and sequenceB, respectively. Among them, sequenceA and sequenceB are the sequence in the dedicated double-stranded RNA sequence and its reverse complementary sequence (mismatch<3); at the same time, the enzyme cutting site and the protective base are respectively designed at the 5' and 3' of the microRNA; obtained by artificial synthesis Artificial microRNA vector.

[0048] The following will take the design and synthesis of the artificial microRNA vector Art-528-S6-1 as an example in detail:

[0049] Using the rice osa-MIR528 (accession number: MI0003201) in the microRNA database as the backbone structure, replace the mature...

Embodiment 3

[0050] Embodiment 3: Construction of plant expression vector

[0051] The preparation and transformation process of Escherichia coli DH5α competent cells: pick up E.coli DH5α stored in glycerol at -70°C with a sterile inoculation loop, and obtain DH5α on the plate after cultured at 37°C for 16-20 hours by streak dilution method single colony; pick a single colony in 5ml LB liquid medium, 180rpm shaking culture for 12h; take 1ml DH5αLB bacterial liquid in 100ml LB liquid medium, 180rpm shaking culture to OD value 0.4; place on ice for 15 minutes, sterile Pour into a 50ml Beckman centrifuge tube under the conditions, centrifuge at 3500rpm for 10min at 4°C, discard the supernatant; use 30ml ice-cooled 0.1M CaCl for precipitation 2 Resuspend the solution; centrifuge at 3500rpm for 10min at 4°C, pour off the supernatant carefully; resuspend the pellet in 2ml ice-cold 0.1M CaCl2 solution containing 15% glycerol; divide these competent cells into 100μl portions.

[0052] Preparation...

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Abstract

The invention discloses a method for enhancing black streaked dwarf resistance of paddy rice by using an artificial microRNA (micro Ribonucleic Acid) and a special double chain RNA thereof. The double chain RNA consists of a sequence A and a sequence B. The method comprises the following steps of: replacing a mature body sequence of an entogenous microRNA of paddy rice and an inverse complementary sequence of a mature body thereof respectively by the sequence A and the sequence B; simultaneously, designing a restriction enzyme cutting site and a protecting basic group on 5' and 3' of the microRNA, and obtaining an artificial microRNA vector through artificial synthesis; and transferring the vector into a plant expression vector with a strong promoter through restriction enzyme ligation, and introducing the plant expression vector into paddy rice to obtain transgenic paddy rice which is resistant to black streaked dwarf. The disease resistance of paddy rice is enhanced through an artificial microRNA technology, target missing is prevented, high safety is achieved, and the disease-resistant property can be inherited stably.

Description

technical field [0001] The invention relates to a method for improving rice resistance to black-streaked dwarf disease and its special double-stranded RNA, in particular to the use of artificial microRNA technology and its double-stranded special RNA, belonging to the field of biotechnology. Background technique [0002] Rice is the largest food crop in my country, and the production of rice has important strategic significance for ensuring food security in my country. Although my country has made great progress in improving the yield and quality of rice through long-term efforts and using traditional breeding techniques, the pests and diseases of rice have not been effectively controlled. Especially in recent years, the occurrence of rice black-streaked dwarf virus has caused huge losses to rice production in my country. [0003] Rice black-streaked dwarf disease is caused by Rice black-streaked dwarf viral disease (RBSDV), which belongs to the Fijivirus genus of the Reovi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/82A01H5/00
Inventor 王兴军赵传志杨连群夏晗李长生李爱芹
Owner 山东省农业科学院高新技术研究中心
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