Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof

A technology for plants and constructs, applied in the fields of biotechnology and botany, which can solve problems such as judging genetic laws

Inactive Publication Date: 2012-07-11
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the constraints of the level of discipline development and research methods, as well as the complex genetic phenomena

Method used

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  • Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof
  • Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof
  • Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0194] 5. Preparation and transformation of Agrobacterium competent cells by freeze-thaw method

[0195] a) Pick a single colony of GV3101 from a fresh plate cultured at 28°C for 48 hours, transfer it to 20ml LB liquid medium (rif 50mg / l, GM 5050mg / l), and culture overnight at 28°C 250rpm with shaking (not too thick) . (All operations below are carried out under sterile conditions).

[0196] b) After ice bathing for 20 minutes, divide the bacteria solution into 5 ml centrifuge tubes (each tube 4 ml), and ice bath for 10 minutes.

[0197] c) Centrifuge at 4000 rpm (5-10° C.) for 10 minutes, and discard the supernatant.

[0198] d) Add 1ml of fully pre-cooled 20mM CaCl to each tube 2 To resuspend the bacteria. Ice bath for 10 minutes.

[0199] e) Centrifuge at 4000 rpm (5-10° C.) for 10 minutes, and discard the supernatant.

[0200] f) Add 300μl 20mM CaCl to each tube 2 (Depending on the concentration of the bacteria), after merging, divide into 1.5ml centrifuge tubes.

...

Embodiment 1

[0237] Embodiment 1, fine design of antiviral miRNA

[0238] The inventors collected 56 genome sequences of turnip mosaic virus TuMV from different sources, compared and analyzed the full-length sequence of its silencing suppressor HC-Pro, and also collected 11 genome sequences of cucumber mosaic virus CMV from different sources Sequence, comparative analysis of the full-length sequence of its gene silencing suppressor 2b. For the results of sequence comparison, see image 3 and Figure 4 . Through sequence comparison analysis, small fragments with strong conservation were selected from gene silencing suppressor HC-Pro and 2b sequences as possible candidate artificial miRNA target sequences.

[0239] In order to more effectively design artificial miRNA plant expression vectors, based on the full-length sequence of HC-Pro of TuMV (SEQ ID NO: 35) and the full-length sequence of 2b of CMV (SEQ ID NO: 36), the inventors silenced these two genes The secondary structure of the r...

Embodiment 2

[0253] Embodiment 2, artificial miRNA plant expression vector construction

[0254] Reference is made to the method provided in The Plant Cell (2006), Vol. 18, 1121-1133.

[0255] The construction of the artificial miRNA expression vector uses the vector pRS300 (obtained from the Max Planck Institute of Developmental Biology, Germany, the vector comes with pre-miR319a) as a template, and its map is shown in figure 1 .

[0256] Cloning methods such as figure 2 As shown, the main route is to replace the mature small fragment of miR319 present in the pre-miR319a of the vector pRS300 with artificial miRNA in the manner of site-directed mutagenesis to construct a plant gene expression vector for antiviral artificial miRNA:

[0257] 1) Input each sequence in the aforementioned "DNA sequence corresponding to the artificial miRNA nucleotide sequence" into the online design tool WMD-Web MicroRNA Designer (http: / / wmd.weigelworld.org / cgi-bin / mirnatools.pl ?page=4), produce the follow...

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Abstract

The present invention relates to a plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof. The invention firstly designs oligonucleotides with excellent silencing effect according to the nucleic acid sequence of a gene silencing suppressor of a plant virus infecting plants. When the virus infects a plant, these oligonucleotides transfected into the plant can form miRNA in the plant which specially binds to the corresponding parts of mRNA (massager RNA) of the virus gene silencing suppressor to affect the transcription of the mRNA, thereby achieving virus inhibitory effect. In addition, oligonucleotides of the invention are optimized and designed on the basis of locating the most conservative positions after comparing the virus gene silencing suppressors derived from different virus subtypes, the oligonucleotides designed based on these positions has broad-spectrum resistant effect on related viruses after being introduced into the plant.

Description

technical field [0001] The invention belongs to the fields of biotechnology and botany; more specifically, the invention relates to an artificial miRNA for inhibiting plant viruses and its construction and use. Background technique [0002] Cabbage vegetables mainly include Chinese cabbage (Brassica campestris L. ssp. pekinensis) and Chinese cabbage (Brassica campestris L. ssp. chinensis). Chinese cabbage is called green cabbage for short, and it is called rapeseed in the north. It has strong adaptability, fast growth, high yield, good nutrition, and its consumption ranks first among all kinds of vegetables. There are many types and varieties of pakchoi, short growth period, wide adaptability, high yield, labor saving, easy planting, and annual production and supply. The products are tender and nutritious, and are loved by consumers. Its annual output accounts for 30%-40% of the total vegetable output, making a great contribution to supplementing the off-season vegetables ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/82C12N5/10
Inventor 孙传宝何玉科
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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