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Method for silencing Arabidopis thaliana endogenous miRNA through utilizing antisense oligonucleotides

An antisense oligonucleotide, Arabidopsis technology, applied in the field of using antisense oligonucleotides to silence Arabidopsis endogenous miRNAs, can solve problems such as application limitations, Arabidopsis drought stress, etc., and achieve a long experimental period. , the interference effect is significant, the effect of avoiding cell drought stress

Active Publication Date: 2014-04-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, sucrose-mediated AMO entry into Arabidopsis cells will cause drought stress in Arabidopsis, and its application is limited
At present, there are no other reports on the use of AMO to silence endogenous miRNAs in Arabidopsis in vivo

Method used

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  • Method for silencing Arabidopis thaliana endogenous miRNA through utilizing antisense oligonucleotides
  • Method for silencing Arabidopis thaliana endogenous miRNA through utilizing antisense oligonucleotides

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 Experimental material and method

[0020] 1. Design and Synthesis of Antisense Oligonucleotides

[0021] Obtain the mature body sequence of the target miRNA from miRbase, and design an antisense oligonucleotide sequence that is completely complementary to the mature body sequence according to the principle of sequence complementarity. The antisense oligonucleotide is modified with 2'-O-methyl. At the same time, BLAST software was used to analyze and determine the miRNA antisense oligonucleotide random control sequence. miRNA antisense oligonucleotides were synthesized by Shanghai Gemma Company.

[0022]2. Plant material and cultivation conditions

[0023] Arabidopsis ( Arabidopsis thaliana ) seeds were rinsed with 70% ethanol for 1 minute. After absorbing the ethanol solution, add 5% sodium hypochlorite solution to rinse for 15 minutes. During this period, shake the seeds from time to time to suspend the seeds and increase the contact area between the...

Embodiment 2

[0039] Example 2 Silencing efficiency of high-purity water-antisense oligonucleotides on Arabidopsis endogenous miRNA

[0040] As in Example 1, the antisense oligonucleotide solution in Step 3 is 2.5 μM anti-ath-miR169h-n (SEQ ID NO: 1: 5'-CAGGCAAGUCAUCCUUGGCUA-3') and high-purity water, sucrose solution (5mM , 10mM), sodium chloride solution (5mM, 10mM) five combination solutions. anti-ath-miR169h-n was used to silence ath-miR169h-n (SEQ ID NO:3:5'-UAGCCAAGGAUGACUUGCCUG-3'). The control group was treated with anti-mock (SEQ ID NO: 2: 5'-GGCGCCCUAGGUAGGACAACC -3'), and the treatment conditions were the same as above.

[0041] See attached for processing results figure 1 . figure 1 It shows that at the concentration of 2.5 μM antisense oligonucleotide, the expression of ath-miR169h-n in the high-purity water-antisense oligonucleotide solution treatment group decreased to about 1% of the control group; The expression level of ath-miR169h-n in the sense oligonucleotide solu...

Embodiment 3

[0042] Example 3 Sustained time-dependent effect of high-purity water-antisense oligonucleotides on endogenous miRNA silencing in Arabidopsis

[0043] As in Example 1, but the Arabidopsis seedlings in step 3 were treated with 2.5 μM anti-ath-miR169h-n or anti-mock, and the materials were collected at 0 hours, 24 hours, 3 days and 5 days to extract RNA. Other steps are identical with embodiment 1.

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Abstract

The invention discloses a method for silencing Arabidopis thaliana endogenous miRNA through utilizing antisense oligonucleotides, and belongs to the technical field of the application of the antisense oligonucleotides. According to the invention, the target miRNA is specifically silenced in plants through introducing the miRNA antisense oligonucleotides into plant cells by immersing Arabidopis thaliana roots in an aqueous solution of the miRNA antisense oligonucleotides. The method has the advantages of simple and fast operation, substantial interference effect and lasting stability, avoids the disadvantages comprising long experiment period, time consumption and labor consumption because of the dependence on a transgenic technology in present methods, breaks through the limitation of the utilization of a sugar solution medium to realize the antisense oligonucleotide transmembrane transport, and provides a new technical means for researches on the functions of the Arabidopis thaliana miRNA.

Description

technical field [0001] The invention relates to a method for silencing Arabidopsis endogenous miRNA by using antisense nucleotides. Background technique [0002] MicroRNA (miRNA) is a kind of endogenous non-coding small molecule RNA of about 22nt. In eukaryotes, miRNA plays a role in post-transcriptional negative regulation by complementary pairing with target mRNA to inhibit gene translation or shear mRNA. Studies on model plants such as Arabidopsis and rice have shown that plant miRNAs not only regulate various growth and development processes such as root system development and flowering timing, but also play a role in stress responses such as oxygen free radicals, drought, salinity, severe cold, and inorganic nutrient deficiency. important role. As widely distributed post-transcriptional regulators, miRNA has increasingly become an indispensable component in functional genomics research, but the functions of most miRNAs are still unclear. [0003] At present, there ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87A01H5/00
Inventor 唐恬谢睦南施苏华吴仲义张添元
Owner SUN YAT SEN UNIV