Application of arabidopsis DNA (Deoxyribonucleic Acid) glycosidase AtOGG1 in aspects of prolonging life of seed and improving germination vigour of seed

A technology of Arabidopsis thaliana and glycosidase, applied in the field of plant genetic engineering, can solve the problems of plant tissue damage, failure to exercise normally, cell rupture, etc., and achieve the effect of improving storage life and vitality, important economic benefits and application prospects

Inactive Publication Date: 2012-07-04
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

ROS can peroxidize DNA, lipids and proteins, making these three types of biomacromolecules unabl

Method used

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  • Application of arabidopsis DNA (Deoxyribonucleic Acid) glycosidase AtOGG1 in aspects of prolonging life of seed and improving germination vigour of seed
  • Application of arabidopsis DNA (Deoxyribonucleic Acid) glycosidase AtOGG1 in aspects of prolonging life of seed and improving germination vigour of seed
  • Application of arabidopsis DNA (Deoxyribonucleic Acid) glycosidase AtOGG1 in aspects of prolonging life of seed and improving germination vigour of seed

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Arabidopsis thaliana DNA glycosidase protein gene AtOGG1 Cloning of full-length gene cDNA

[0027] (1) Preparation of Arabidopsis leaves: wild-type Arabidopsis ( Col-0 ) 2 weeks after the seeds germinated, the leaves were collected.

[0028] (2) Extraction of total RNA: Total RNA was extracted using Trizol product from Invitrogen Company.

[0029] (3) Obtaining cDNA: The RNA in (2) was reverse transcribed using the instructions of Takara's PrimeScript 1st Strand cDNA Synthesis Kit to obtain cDNA.

[0030] (4) get AtOGG1 Full-length cDNA of the gene: the obtained cDNA is used as a template, primers are designed according to the sequence published by GenBank (accession number is NM-102020), and obtained by PCR amplification AtOGG1 Gene, confirmed by sequencing. The PCR primers are as follows:

[0031] Forward primer: SEQ ID NO: 1: 5'- ACGGCGATGAAGAGACCTCGACCT- 3';

[0032] Reverse primer: SEQ ID NO: 2: 5'-AATAACTACGCTCATTTGCCAGGG-3'.

Embodiment 2

[0033] The construction of embodiment 2 plant expression vectors

[0034] (1) Cloning of gene fragments

[0035] Arabidopsis thaliana described in Example 1 AtOGG1 The full-length cDNA of the gene is used as a template, and the restriction site is added by PCR cloning Sma I and Sac PCR fragment of I. The PCR primers are as follows, the underlined part is the restriction site:

[0036] Forward primer: SEQ ID NO:3: 5'-TCC CCCGGG ATGAAGAGACCTCGACCTAC-3';

[0037] Reverse primer: SEQ ID NO:4: 5'-C GAGCTC TCATGGCTTCAACGTATCAC-3'.

[0038] PCR reaction system: 1 μL AtOGG1 Gene, 5 μL dNTP (2.5 mM), 2 μL MgCl 2 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 5 μL 10×PCR buffer, 1 μL KOD plus enzyme (product of TOYOBO), and finally add deionized water to make the total system 50 μL. The reaction conditions of PCR are: 94°C for 5 minutes; then enter the following cycle: 94°C for 30 seconds, 60°C for 45 seconds, 72°C for 70 seconds, a total of 28 cycles; f...

Embodiment 3

[0049] Example 3 Genetic Transformation of Arabidopsis

[0050] 1. Pre-treatment of Arabidopsis transformation

[0051] When the main moss of Arabidopsis thaliana grows to 5-6 cm, cut off the whole inflorescence at the base of the inflorescence, and remove the dominance of its apex. After 1 week, 4-6 new side mosses will grow on the axillary buds, and wait for the side mosses to form flower buds and form. Partial flowering or the formation of 1-2 siliques can be used for transformation, and the grown siliques need to be cut off before transformation. The day before transformation, the plants were well watered and covered with a plastic bag to maintain a high humidity environment.

[0052] 2. Preparation of Infection Medium

[0053] The composition of the dipping medium used for soaking Arabidopsis tidbits is 1 / 2MS medium containing 5% (mass) sucrose, pH=5.8 (adjusted with 1M KOH), autoclaved. When using, add 0.02%~0.05% (mass) surfactant Silwet L-77 and shake well.

[00...

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Abstract

The invention discloses an application of arabidopsis DNA (Deoxyribonucleic Acid) glycosidase AtOGG1 in the aspects of prolonging life of seeds and improving germination vigour of the seeds, belonging to the technical field of plant genetic engineering. In the invention, an AtOGG1 plant expression vector is constructed, a transgenic plant containing the AtOGG1 is prepared, and transgenic arabidopsis seeds have higher artificial aging resistance and higher germination vigour under the stress of methyl viologen, salt, mannitol and high temperature compared with wild type arabidopsis seeds. The AtOGG1 is transferred into important crops such as rice, wheat, corn, soybean and the like, and storage life and vigour of seeds of the important crops also can be improved. The application of the AtOGG1 can greatly reduce global loss caused by deterioration of the seeds and multiple stresses (oxidation, salt, drought and high temperature) every year, has wide application prospect in the fields ofplant variety breeding and agricultural production and has important economic benefit.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of Arabidopsis thaliana DNA glycosidase AtOGG1 in improving seed life span and germination vigor. Background technique [0002] Seeds are the foundation of agricultural production, and their lifespan and vigor play a decisive role in plant reproduction, crop yield, germplasm resource conservation and biodiversity. Mature dry seeds will deteriorate even under suitable storage conditions, the degree of which will increase with the prolongation of storage time, and eventually cause the seeds to lose vigor and fail to germinate. Seed deterioration is usually accompanied by a series of physiological and biochemical changes, such as DNA damage, lipid peroxidation and protein damage. McDonald et al. believed that the lifespan of seeds was jointly affected by internal genetic factors and external environmental factors. Although extensive re...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/82C12N15/66A01H5/10
Inventor 黄上志陈虎辉
Owner SUN YAT SEN UNIV
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