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Method for constructing and expressing micro ribonucleic acid (miRNA) target simulation sequence by using plant virus vector

A technology of sequence listing sequences and sequences, which is applied in the field of miRNA target mimic sequence construction and expression, can solve the problems of long experimental period, prone to mutation, and difficult to achieve, and achieves fast and convenient expression level, reduced expression level, and wide host range. Effect

Active Publication Date: 2014-03-12
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This cloning method is cumbersome to operate and is prone to mutations in the PCR reaction, which will affect the next step of the experimental operation; The vector is transferred to the plant through Agrobacterium to construct a stable transgenic plant. This process takes a long experimental period in some plants, requires high technical requirements and is difficult to achieve.
The above two problems limit the application of miRNA target simulation technology in the study of plant miRNA function

Method used

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  • Method for constructing and expressing micro ribonucleic acid (miRNA) target simulation sequence by using plant virus vector
  • Method for constructing and expressing micro ribonucleic acid (miRNA) target simulation sequence by using plant virus vector
  • Method for constructing and expressing micro ribonucleic acid (miRNA) target simulation sequence by using plant virus vector

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1. Construction and application of miRNA158 target simulation sequence recombinant expression vector

[0028] 1. Construction of miRNA158 target mimic sequence recombinant expression vector (pTY102-MIM158) ( figure 2 )

[0029] 1) According to the miRNA158 sequence shown in Sequence 2 of the Sequence Listing, two single-stranded DNAs of double-stranded DNA molecules encoding the miRNA158 target simulation sequence were chemically synthesized (shown in sequences I and II, where the capital letter bases represent the added cohesive ends sequence, the underlined bases are 3 random bases added):

[0030] Sequence I: 5'-CTAGAAAagctttgtca cta acatttggga-3',

[0031] Sequence II: 5'-AGCTtcccaaatgt tag tgacaaagctTTT-3';

[0032] 2) The plant virus expression vector pTY102 (its sequence is shown in Sequence Listing Sequence 1, and the structural diagram of its T-DNA region is shown in figure 1 As shown) performing double digestion with restriction endonucleases...

Embodiment 2

[0056] Example 2. Construction and application of miRNA172 target simulation sequence recombinant expression vector

[0057] 1. Construction of miRNA172 target mimic sequence recombinant expression vector (pTY102-MIM172) ( figure 2 )

[0058] 1) According to the miRNA172 sequence shown in Sequence 3 of the Sequence Listing, chemically synthesize two single-stranded DNAs of double-stranded DNA molecules encoding the miRNA172 target simulation sequence (shown in sequences III and IV, where the capital letter bases represent the added cohesive ends sequence, the underlined bases are 3 random bases added):

[0059] Sequence III: 5'-CTAGAAAatgcagcatcg agt tcaagattct-3',

[0060] Sequence IV: 5'-AGCTagaatcttga act cgatgctgcatTTT-3';

[0061] 2) The plant virus expression vector pTY102 (its sequence is shown in Sequence Listing Sequence 1, and the structural diagram of its T-DNA region is shown in figure 1 As shown) performing double digestion with restriction endonucleases...

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Abstract

The invention discloses a method for constructing and expressing a micro ribonucleic acid (miRNA) target simulation sequence by using a plant virus vector. Experiments prove that deoxyribonucleic acid (DNA) molecules which are chemically synthesized and encoded and have the miRNA158 or miRNA172 target simulation sequences are inserted into the plant virus expression vector pTY102 to obtain a recombinant expression vector, the recombinant expression vector is imported into tobaccos leaf, and after three weeks, the phenotypic modulation of tobacco and the raising of the expression of the miRNA target genes can be detected. According to the method, bridge polymerase chain reaction (PCR) is not needed; the method is simple; and by using the characteristics of high expression, wide host range and high speed of spreading in the whole plant of virus, the expression level of plant endogenous miRNA can be reduced quickly and conveniently, and a new way is provided for researching the plant endogenous miRNA function.

Description

technical field [0001] The invention relates to a method for constructing and expressing a miRNA target simulation sequence using a plant virus vector. Background technique [0002] In 2007, Franco-Zorrilla et al. reported a gene IPS1 (Induced by Phosphate Starvation1) in Arabidopsis. This gene belongs to a member of the TPSI family. The expression of IPS1 gene is induced by low phosphorus stress, and its transcript is a non-coding RNA, which contains a region complementary to miR399. However, unlike other members of this family, this region of IPS1 contains mutations in the key binding site, so although the transcribed RNA can be recognized and bound by miR399, it cannot be cut and degraded by the RISC complex containing miR399 , and more importantly, the interaction between miRNA and mRNA will affect the normal function of miR399, such as inhibiting miR399-mediated degradation of PHO2 gene transcripts, thereby shutting down the function of miRNA. By modifying the miRNA ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/83C12N15/66A01H5/00
Inventor 刘玉乐汤旸沙爱华
Owner TSINGHUA UNIV
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