amiRNA for inhibiting rice gene OsRboh(LOC_Os01g25820)

A gene and rice technology, applied in the field of genetic engineering, can solve difficult problems such as specific silencing of target genes, off-target effects, etc.

Active Publication Date: 2016-04-06
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the common use of RNAi technology has the advantages of high efficiency and directness, its main disadvantage is that it can silence other genes with high homology in the target gene family at the same time, resulting in off-target effects, and it is difficult to specifically silence the target gene.

Method used

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  • amiRNA for inhibiting rice gene OsRboh(LOC_Os01g25820)
  • amiRNA for inhibiting rice gene OsRboh(LOC_Os01g25820)
  • amiRNA for inhibiting rice gene OsRboh(LOC_Os01g25820)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Obtaining the sequence of rice OSBROH gene amiRNA

[0023] Use the tigr online database to find the rice OsRboh (LOC_Os01g25820) gene and download it. According to the online software http: / / wmd3.weigelworld.org to search for possible amiRNA sequences, and according to the reaction thermodynamic parameters, effective free hybridization energy (ΔG) and hybridization energy (ΔGint) and other parameters, finally select “TTAAACAACCGAAGGATACGC” as the target amiRNA sequence. After the candidate amiRNA is obtained, it is reverse-complemented, the T at the 6th position is changed to A, and the T at the 10th position is changed to A to obtain amiRNA*.

Embodiment 2

[0024] Embodiment 2: Construction of rice PHB-OSBROH-amiRNA vector

[0025] The amiRNA and amiRNA* sequences of OsRboh (LOC_Os01g25820) were respectively replaced with the amiRNA and amiRNA* sequences in the backbone OsmiR528 to obtain the OsRboh (LOC_Os01g25820)-amiRNA sequence. According to the principle that the target sequence and the carrier PHB do not repeat restriction sites, the restriction sites BamHI and XbaI were added to the two ends of the OsRboh(LOC_Os01g25820)-amiRNA sequence fragment respectively. Then it was handed over to Shanghai Jierui Bioengineering Co., Ltd. for gene synthesis, and the target sequence was connected to the cloning vector pMD18T.

[0026] BamHI and XbaI digest pMD18T-OsRboh(LOC_Os01g25820)-amiRNA, and the size of the target band can be clearly cut out, see figure 1 . Recover the digested product from agarose gel, connect it with the expression vector PHB digested product with T4DNA ligase, transform Escherichia coli DH5α competent cells, ...

Embodiment 3

[0027] Example 3: PHB-OsRboh(LOC_Os01g25820)-amiRNA transfected Agrobacterium EHA105

[0028] (1) Preparation of Agrobacterium Competent Cells

[0029] Pick a single colony of Agrobacterium EHA105 and inoculate it in 5ml of YEB medium, shake it overnight at 28°C, inoculate it in 50ml of YEB medium at a ratio of 1:100, and continue culturing at 28°C for about 6-7h until OD600=0.4-0.6. Put the bacterial solution on ice for 30min; centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, and suspend the bacteria in 10ml of 0.15M NaCl; 2 , 4°C) to suspend gently, aliquot 200 μl per tube, or add sterile glycerol with a final concentration of 20%, and store at -70°C.

[0030] (2) Transformation and identification of Agrobacterium

[0031] Add 10 μl of plasmid DNA to 200 μl of Agrobacterium competent, mix evenly, ice-bath for 30 minutes, freeze in liquid nitrogen for 3-5 minutes, bathe in water at 37°C for 5 minutes, add 1ml of YEB medium, and shake at 28°C for 3-4 hours. Cent...

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Abstract

The invention relates to amiRNA for inhibiting the rice gene OsRboh(LOC_Os01g25820) and discloses an amiRNA sequence of silence rice respiratory burst oxidase OsRboh, and the sequence is TTAAACAACCGAAGGATACGC. According to the amiRNA for inhibiting the rice gene OsRboh(LOC_Os01g25820), the OsRboh(LOC_Os01g25820)-amiRNA sequence is artificially synthesized and connected to an expression carrier to be converted into rice, obtained transgenic rice plant leaves turn into white, and the fertility is lowered. By means of the prepared rice gene OsRboh(LOC_Os01g25820) amiRNA, the rice target gene can be targeted to efficiently, and certain significance is achieved on the aspects of basic research and production practice.

Description

technical field [0001] The invention relates to an amiRNA inhibiting OsRboh (LOC_Os01g25820) gene expression and its application, belonging to the technical field of genetic engineering. Background technique [0002] Reactive oxygen species (reactive oxygen species, ROS) are a by-product of aerobic metabolism of organisms. On the one hand, they can act as toxic secondary metabolites to damage normal molecules in the body; on the other hand, they participate in the growth of organisms as important signaling molecules. and hair. Studies have found that the sources of ROS include plasma membrane NADPH oxidase, peroxidase and amine oxidase, among which the ROS mediated by NADPH oxidase has attracted the most attention. [0003] Plant NADPH oxidase, also known as respiratory burst oxidase (Respiratory burst oxidase homologue, Rboh), is the homologue of mammalian macrophage NADPH oxidase catalytic subunit gp91phox. Current research shows that the functions of plant Rboh genes ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC12N15/1137C12N15/8218C12N2310/141
Inventor 胡丽芳贺浩华刘世强朱昌兰欧阳林娟彭小松陈小荣贺晓鹏傅军如边建民徐杰孙晓棠周大虎
Owner JIANGXI AGRICULTURAL UNIVERSITY
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