Vector of rice respiratory burst oxidase gene OsRboh(LOC_Os01g25820) and application thereof

An oxidase and rice technology, applied in the field of genetic engineering, can solve problems such as unclear functions of rice

Active Publication Date: 2016-04-20
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Respiratory Burst Oxidase Homolog (Rboh) gene is directly involved in the formation of ROS, but its function in rice is not clear, it is worthy of further study

Method used

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  • Vector of rice respiratory burst oxidase gene OsRboh(LOC_Os01g25820) and application thereof
  • Vector of rice respiratory burst oxidase gene OsRboh(LOC_Os01g25820) and application thereof
  • Vector of rice respiratory burst oxidase gene OsRboh(LOC_Os01g25820) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of rice PHB-OsRboh (LOC_Os01g25820)-OE vector

[0024] (1) Amplification of OsRboh(LOC_Os01g25820) gene

[0025] According to the OsRboh (LOC_Os01g25820) full-length cDNA (AK065117) sequence published on NCBI, design primers at both ends:

[0026] OsRboh-OE-F: AGATC TATGGCTGACCTGGAAGCAGGCATGG (BamHI)

[0027] OsRboh-OE-R: CACGTG TTAGAAGTTCTCCTTGTGGAAATCA(XbaI)

[0028] The full length of OsRboh (LOC_Os01g25820) was obtained by PCR amplification with the high-fidelity enzyme KOD-Plus using the plasmid AK065117 purchased from the Rice Genome Research Center (RGRC) of Japan as a template. The PCR reaction conditions were: 94°C for 5 min; 94°C for 30 s; 56°C for 30 s; 68°C for 3.0 min, 35 cycles; 68°C for 5 min. The PCR product was detected by electrophoresis with 1.2% agarose, and the size of the amplified fragment was about 2718bp, which was consistent with the expected size ( figure 1 ).

[0029] (2) Gel recovery of OsRboh (LOC_Os01g2582...

Embodiment 2

[0037] Embodiment 2: PHB-OsRboh (LOC_Os01g25820)-OE transfected Agrobacterium EHA105

[0038] (1) Preparation of Agrobacterium Competent Cells

[0039] 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.

[0040] (2) Transformation and identification of Agrobacterium

[0041] 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. Ce...

Embodiment 3

[0042] Embodiment 3: Agrobacterium EHA105 containing PHB-OsRboh (LOC_Os01g25820) transforms rice

[0043] (1) Pretreatment of materials

[0044] The dry seeds are first dehulled, and the dehulled seeds are soaked in 70% ethanol for 1 minute, and then soaked in 50% bleach

[0045] (containing 2% HClO) for 20 minutes, then washed 4 times with sterile water, and transferred the whole seed to MD2 medium

[0046] Plates were cultured in the dark at 26°C for 4 days. When the yellow callus appears on the hypocotyl (usually takes four days, at this

[0047] period, the root is usually 2-5cm long), excised the root and endosperm, and transferred the hypocotyl to a fresh NBD2

[0048] Medium plates, with the blunt side up, cultured in the dark at 26°C for 7-10 days.

[0049] (2) Pick a single colony of Agrobacterium EHA105 and culture it with shaking in 100ml of YEB medium containing corresponding resistance.

[0050] Raise for about 16 hours (200rpm, 28°C), until the OD600 is abou...

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PUM

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Abstract

The invention discloses a rice respiratory burst oxidase OsRboh(LOC_Os01g25820) and an application thereof in leaf color change and fertility reduction, belonging to the field of biotechnology. The vector is a plant expression vector containing double 35S promoters and a rice OsRboh(LOC_Os01g25820) gene. Due to overexpression of OsRboh(LOC_Os01g25820) in rice, the leaf color of the obtained transgenic plant turns yellow, the content of hydrogen peroxide is increased, and the fertility is obviously reduced. The results indicate that the OsRboh(LOC_Os01g25820) plays an important role in the rice development process, and a new gene resource is provided for studying plant development; and the OsRboh(LOC_Os01g25820) also can be used as a potential tool in molecular breeding and genetic improvement so as to improve the plant form.

Description

technical field [0001] The invention relates to a carrier and application of rice respiratory burst oxidase gene OsRboh (LOC_Os01g25820), belonging to the technical field of genetic engineering. Background technique [0002] Reactive oxygen species (reactive oxygen species, ROS) is a by-product of aerobic metabolism of organisms, in addition to harming cells as toxic molecules, but also as an important signal molecule to participate in the growth and development of organisms. [0003] 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. NADPH oxidase uses NADPH located at the edge of the cytoplasm as an electron donor to catalyze O 2 Superoxide anion (O 2 － ), followed by the disproportionation of superoxide anion to generate H 2 o 2 , H 2 o 2 Then generate OH by Fenton reaction. Plant NADPH oxidase is also called respiratory burs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8261C12N15/8287Y02A40/146
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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