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Application of ath-eTM160 in inhibiting functions of microRNA160

A technology with the same function and DNA molecules, applied in the direction of DNA / RNA fragments, applications, recombinant DNA technology, etc.

Active Publication Date: 2013-01-09
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the endogenous mimic target genes of other miRNAs have not been reported

Method used

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  • Application of ath-eTM160 in inhibiting functions of microRNA160
  • Application of ath-eTM160 in inhibiting functions of microRNA160
  • Application of ath-eTM160 in inhibiting functions of microRNA160

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, the acquisition of ath-eTM160 gene

[0030] 1. Extraction of Arabidopsis total RNA

[0031] 1) Extraction by Trizol method:

[0032] (1) Take an appropriate amount of wild-type Arabidopsis thaliana (Arabidopsis thaliana, purchased from ABRC, the United States) material (0.1g) and add liquid nitrogen to grind for 15 minutes until it becomes powdery, and pour the powder into a 2mL centrifuge tube (about 1 / 2 tube) , then immediately add 1 mL of Trizol reagent. Mix the powder with Trizol reagent and incubate at room temperature for 5 minutes to completely dissociate the nucleic acid-protein complex.

[0033] (2) Centrifuge the centrifuge tube at 12000 rpm at 4°C for 15 min.

[0034] (3) Transfer the supernatant to a new 2mL centrifuge tube, and add an equal volume of 0.5ml chloroform to it, shake the tube vigorously by hand for 15s, and let it stand at room temperature for 3min.

[0035] (4) Centrifuge the centrifuge tube at 12000 rpm at 4°C for 15 min.

...

Embodiment 2

[0073] Example 2, the acquisition and functional identification of ath-eTM160 Arabidopsis

[0074] 1. Obtaining ath-eTM160 Arabidopsis

[0075] 1. Construction of recombinant vector containing ath-eTM160 overexpression

[0076] The plasmid ath-eTM160-EZ-T obtained in Example 1 was digested with Xba I and Sac I restriction endonucleases ath-eTM160-EZ-T, and the target fragment of about 194bp was recovered, and Xba I and Sac I were used to restrict the endonuclease The expression vector pCAMBIA1300 (purchased from CAMBIA, Australia) was digested with Dicer, the vector fragment was recovered, and then the target fragment was connected with the vector fragment to obtain the recombinant expression vector ath-eTM160-1300. The recombinant expression vector was sent for sequencing, and the result was The vector obtained by inserting the sequence 1 in the sequence listing from the 8th to 187th nucleotides of the 5' end between the XbaI and SacI restriction sites of pCAMBIA1300.

[00...

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Abstract

The invention discloses application of ath-eTM160 in inhibiting functions of microRNA160. The DNA (deoxyribonucleic acid) disclosed by the invention is any one of the following (1)-(3): (1) DNA molecules disclosed as 8th-187th nucleotides from the 5' tail end in Sequence 1 in the sequence table; (2) DNA molecules with identical functions, which can be hybridized with the DNA sequences defined in (1) under strict conditions; and (3) DNA molecules with identical functions, which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequences defined in (1). The experiment proves that the gene segment ath-eTM160, which is obtained from Arabidopsis thaliana, can inhibit the expression of microRNA160 in the Arabidopsis thaliana after being transferred into Arabidopsis thaliana for overexpression.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an ath-eTM160 and its application in inhibiting the function of microRNA160. Background technique [0002] MicroRNA (miRNA) is an important class of small molecule RNA, which is involved in the regulation of plant growth and development, stress response and other pathways. In plants, miRNAs mainly function by degrading target genes. First, the mature miRNA binds to the target gene through base complementary matching, and then causes the cleavage of the target gene mRNA at the binding position (the cleavage mainly occurs between the 10 and 11 positions of the complementary region), resulting in the degradation of the target gene. Recently, some researchers reported an IPS1 gene. Although the gene can partially match the miR399 sequence, IPS1 forms a 3-base bulge at the cutting position, which prevents IPS1 from being cleaved by miR399, that is, Cannot be degraded. When IPS1 is high...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/21C12N15/11C12N15/84A01H5/00
Inventor 王秀杰王猛吴华君王志敏
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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