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RNA (Ribonucleic Acid) interference vector and application thereof

A technology of RNA interference and interference carrier, which is applied in the field of RNA interference carrier, can solve problems such as linkage burden, prolong breeding cycle, increase workload, etc., and achieve the effects of reducing errors, improving work efficiency, and flexible operation

Active Publication Date: 2011-09-21
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Improving the plant height of crops through conventional breeding has achieved great success in crops such as rice and wheat, but this method also has some shortcomings: one is the phenomenon of linkage drag in conventional breeding, that is, favorable genes may be associated with other The unfavorable genes are closely linked, so it is difficult to eliminate these unfavorable genes by backcrossing; the second is to introduce some other unfavorable traits of the donor parent into the recipient parent while introducing the favorable traits of the donor parent into the donor parent Therefore, it is necessary to eliminate these undesirable traits through several backcrosses, which increases the workload and prolongs the breeding cycle

Method used

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  • RNA (Ribonucleic Acid) interference vector and application thereof
  • RNA (Ribonucleic Acid) interference vector and application thereof
  • RNA (Ribonucleic Acid) interference vector and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1, the construction of RNA interference vector pLHRNAi

[0068] The RNA interference vector pLHRNAi uses the method of homologous recombination directional cloning. The intron of the Arabidopsis FAD2 gene is forward cloned into the plant binary vector pCUbi1390 with a hygromycin selection marker. The structure diagram of the RNA interference vector pLHRNAi is shown in figure 1 As shown, the specific method is as follows:

[0069] 1. Obtaining the intron fragment of the Arabidopsis FAD2 gene

[0070] Arabidopsis thaliana (L.) was extracted by CTAB method (Kraft E, Stone SL, Ma LG, Su N, Gao Y, Lau OS, Deng XW, Callis J(2005) Genome analysis and functional characterization of the E2 and RING-Type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 139:1597-1611, publicly available from Institute of Crop Science, Chinese Academy of Agricultural Sciences.) Genomic DNA.

[0071] The primer sequences for the amplified introns were designed as follows (...

Embodiment 2

[0101] Embodiment 2, the application of RNA interference vector pLHRNAi

[0102] 1. Construction of D2 Gene RNA Interference Vector pLHRNAi-D2

[0103] 1. Obtaining the D2 gene interference fragment

[0104] Brassinosteroid (BR) is an important plant hormone controlling plant height, which is synthesized by a series of brassinosteroid synthases. In rice, the CYP90D2 / D2 gene encodes a key synthetase in the brassinosteroid synthesis pathway.

[0105] 1) Primer design

[0106] (1) Primers used to amplify the sense interference fragment of the D2 gene

[0107] The underlined letters indicate the homologous recombination sequences on both sides of the Sac I restriction site on the upstream multiple cloning site of the pLHRNAi vector (which can be cloned into the vector through homology and same group orientation), and the left slash indicates Kpn I and Sac I restriction endonuclease cleavage site (can be cloned onto the vector by double-enzyme digestion connection), the primer ...

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Abstract

The invention discloses an RNA (Ribonucleic Acid) interference vector and application thereof. The RNA interference vector provided by the invention comprises FAD2 (fatty acid desaturase 2) gene intron and an upstream polyclone site and a downstream polyclone site on two sides of the FAD2 gene intron, wherein the FAD2 gene intron is the 60th-1190th site nucleotide from the 5' terminal of the sequence 1 in a sequence table. Proven by experiments of the invention, the invention provides an RNA interference vector suitable for plants; shRNA (short hairpin RNA) formed by expression of the RNA interference vector in a receptor is diced by Dicer into siRNA (small interfering RNA); and the siRNA can specifically inhibit the expression of a target gene, thereby achieving the aims of controlling the plant height of paddy and studying the CYP90D2 / D2 gene function.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to an RNA interference carrier and its application. Background technique [0002] Plant height is an important agronomic trait in crop breeding. Appropriate plant height is conducive to increasing the biomass of crops, enhancing lodging resistance and increasing yield. In the conventional breeding of plant height, the dwarf source parent is usually used as the donor parent, and the high-quality variety with a high plant height is used as the recipient parent. First, the dwarf source gene of the donor parent is introduced into the recipient parent by hybridization. Then through continuous backcrossing, excellent lines with appropriate plant height were selected from the backcrossed progeny. Improving the plant height of crops through conventional breeding has achieved great success in crops such as rice and wheat, but this method also has some shortcomings: one is the phenomenon ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113A01H5/00
Inventor 万建民李辉程治军金田蕴江玲郭秀萍王久林张欣雷财林王洁吴赴清
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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