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RNA induced silencing complex mediated shearing site and uses thereof

A site and DNA molecule technology, applied in the field of cut sites, can solve the problems of application barriers, difficulty in predicting the true high-level structure of viral RNA, etc.

Inactive Publication Date: 2010-12-15
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although bioinformatics methods can be used to predict the advanced structure of target RNA by computer simulation and evaluate the impact of the structure on the accessibility of the target, however, due to the lack of methods that can accurately and stably predict the advanced structure of long-chain RNA, it is difficult Predict the true high-level structure of viral RNA during natural infection, because viral RNA cannot have a single stable structure, which brings great potential for the further application of siRNA-mediated RNA silencing in medicine and agricultural production obstacle

Method used

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  • RNA induced silencing complex mediated shearing site and uses thereof
  • RNA induced silencing complex mediated shearing site and uses thereof
  • RNA induced silencing complex mediated shearing site and uses thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the design of the amiRNA targeting the 2051 / 2052 site in the 3'UTR region of the cucumber mosaic virus genome

[0033] 1) Cucumber mosaic virus (CMV) 3′UTR can be recognized by plant endogenous RNA silencing pathway

[0034] In order to study whether the 3′UTR region can be recognized and targeted for degradation by the plant RNA silencing system during CMV infection, in view of the fact that the CMV genome is too long, in order to better maintain the natural folding structure of the 3′UTR region, a Q-CMV Expression vectors 35S-R4A and 35S-R4 for complete subgenomic RNA4A and RNA4. The construction method of expression vector 35S-R4A and 35S-R4 is as follows: pQCD2 (Ding, S.W., J.P.Rathjen, W.X.Li, R.Swanson, H.Healy, and R.H.Symons.1995.Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasma vector.J.Gen.Virol.76(Pt 2):459-464) (Institute of Microbiology, Chinese Academy of '-GAGCTCTGGTCTCCTTATGGAGAAC-3') primers for PCR...

Embodiment 2

[0048] Embodiment 2, cultivating transgenic plants resistant to cucumber mosaic virus with the amiRNA of embodiment 1

[0049] One, the amiRNA of embodiment 1 cultivates the transgenic plant resistant to cucumber mosaic virus

[0050] a) Construction of amiRNA precursor with miR159a precursor as the backbone

[0051] The Arabidopsis miR159a precursor 266bp fragment was amplified by RT-PCR using the Arabidopsis genomic DNA as a template. In addition to the complete pre-miR159a (184bp), the 5′ end of the fragment also contained 82bp genome sequence. The sequences of primers miR159-For and miR159-Rev used in RT-PCR are as follows:

[0052] miR159-For:5′- CCACAGTTTGCTTATGTCGGATCC-3′ and miR159-Rev:5′-ATG TAGAGCTCCCTTCAATCC -3'.

[0053] The italic part at the 5' end of the miR159-For primer is the introduced XbaI site, and the underlined part of the miR159-Rev primer is 18 nucleotides in the 21 nucleotide sequence of the mature miR159.

[0054] The fragment obtained after ...

Embodiment 3

[0110] Specificity analysis of the amiRNA of embodiment 3, embodiment 1 in transgenic plants

[0111] In order to exclude the non-specific silencing of plant endogenous genes by the stable expression of amiRNA transgenes, that is, the so-called "off-target" phenomenon, the sequences of all amiRNAs were entered into the NCBI BLAST website for comparison and analysis. It was found that in the 21nt sequences of amiRNAs, there was no Any amiRNA can pair with the continuous complementarity of Arabidopsis endogenous transcripts beyond 15 nt. In order to further exclude the possibility of amiRNA targeting plant endogenous genes, at the same time input the amiRNA sequence into "Plant miRNA Target Finder"

[0112] ( http: / / bioinfo3.noble.org / miRNA / miRU.htm ) "TIGR Arabidopsis GeneIndex 1" and "TIGR Tobacco Gene Index 1" databases for potential target gene searches. The results showed that only one endogenous transcript - AT3G29250 - could form less than 3 incomplete pairs with amiR-...

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Abstract

The invention discloses a shear locus mediated by RNA induced silencing complex and application thereof. The locus can be applied in designing a factitial tiny RNA. In a DNA molecule coding a factitial tiny RNA precursor, the factitial tiny RNA precursor is a single-chain RNA capable of generating a tiny RNA in plant; the single-chain RNA forms a stem-loop structure; a nucleotide sequence containing sequence 1 in a sequence list is arranged in a fragment of the stem part of the stem-loop structure; two base pairs which are not complementary are arranged on the stem part of the stem-loop structure; and the two uncomplementary base pairs correspond to the ninth position and the tenth position from tail end of 5' in the sequence 1. The invention also discloses a method for culturing plant resisting cucumber mosaic virus. With the DNA molecule coding the factitial tiny RNA precursor, the plant with high efficient virus resistance can be obtained, so that the method is a biological engineering method with economy and efficiency.

Description

technical field [0001] The present invention relates to RNA-induced silencing complex-mediated cleavage sites and applications thereof. Background technique [0002] The plant RNA silencing system is a conserved mechanism that exists widely in eukaryotes. Its mode of action is to produce small molecule RNA through a series of complex synthetic pathways and make the target RNA complementary to its sequence by site-specific shearing. degradation. Most of these small RNAs are 21-24nt in length, mainly including microRNA (microRNA) and small interfering RNA (siRNA). Shorter microRNA precursors have a single-strand hairpin structure, and artificial microRNA (artificial microRNA, amiRNA) similar in structure to microRNA precursors can efficiently and specifically induce endogenous gene silencing in plants. Recent studies have shown that amiRNA, as an effective gene silencing tool, also has good application potential in plant antiviral research. [0003] Small RNA-mediated RNA s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/11C12N15/63C12N15/82C12N5/10C12N1/15C12N1/19C12N1/21A01H1/00A01H5/00C12N15/113
Inventor 郭惠珊段成国
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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