1-aminocyclopropane-1-carboxylate oxidase contained in drug-resistant barnyard grass and coding gene, mutation site and application thereof

An aminocyclopropane, coding gene technology, applied in the field of plant biology, can solve problems such as no report, no clone of ACO gene, few ACO, etc., to achieve the effect of improving drug resistance

Active Publication Date: 2015-05-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few reports on ACO in grasses
So far, no ACO gene has been cloned from barnyardgrass, and there is no report on the impact of ethylene biosynthesis pathway on barnyardgrass resistance from the gene level

Method used

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  • 1-aminocyclopropane-1-carboxylate oxidase contained in drug-resistant barnyard grass and coding gene, mutation site and application thereof
  • 1-aminocyclopropane-1-carboxylate oxidase contained in drug-resistant barnyard grass and coding gene, mutation site and application thereof
  • 1-aminocyclopropane-1-carboxylate oxidase contained in drug-resistant barnyard grass and coding gene, mutation site and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Cloning of the EcACO gene in drug-resistant barnyardgrass and sensitive barnyardgrass and its sequence difference analysis

[0036] The quinclorac-resistant barnyardgrass and sensitive barnyardgrass materials at the 2-3 leaf stage were selected, and the nucleotide and amino acid sequences of their EcACO genes were compared and analyzed.

[0037] 1. Extraction of barnyardgrass total RNA. Using quinclorac-resistant barnyardgrass and sensitive barnyardgrass as research materials, total RNA was extracted from single barnyardgrass seedlings cultured to the 2-3 leaf stage and sensitive barnyardgrass seedlings respectively. Barnyardgrass total RNA was extracted using an ultra-pure total RNA rapid extraction kit (purchased from Yuanping Hao Biotechnology Co., Ltd.), and the operation method was referred to the kit instruction manual. The extracted barnyardgrass total RNA was subjected to 1% agarose electrophoresis to detect the quality and integrity of the RNA, and ...

Embodiment 2

[0042] Example 2. Real-time quantitative PCR analysis of the EcACO gene in drug-resistant barnyardgrass and sensitive barnyardgrass

[0043] Select quinclorac-resistant barnyardgrass and sensitive barnyardgrass materials at the 2-3 leaf stage, and use real-time quantitative PCR (quant itat ive real-time PCR) to analyze the expression level of EcACO gene in these materials, so as to obtain the EcACO gene Relationship between expression levels and quinclorac resistance.

[0044] 1. Extraction of barnyardgrass total RNA. Using quinclorac-resistant barnyardgrass and sensitive barnyardgrass as research materials, total RNA was extracted from single barnyardgrass seedlings cultured to the 2-3 leaf stage and sensitive barnyardgrass seedlings respectively. Barnyardgrass total RNA was extracted using an ultra-pure total RNA rapid extraction kit (purchased from Yuanping Hao Biotechnology Co., Ltd.), and the operation method was referred to the kit instruction manual. The extracted bar...

Embodiment 3

[0047] Example 3. Determination of EcACO Enzyme Activity in Drug-resistant Barnyard Grass and Sensitive Barnyard Grass and Its Difference Comparison

[0048] The quinclorac-resistant barnyardgrass and sensitive barnyardgrass materials at the 2-3 leaf stage were selected, and the enzyme activity of EcACO in these materials was analyzed by the method of Yusuke Kosugi et al. (2014).

[0049] 1. Prokaryotic expression vector construction. The designed primer is EcACO-NdeI-S:5'-GGTATATA CATATG GTGGTTCCAGTGATCG-3' (the underline is the restriction endonuclease NdeI restriction site recognition sequence); EcACO-NdeI-R:5'-GGTATATA CATATG GTGGTCCCTGTGATCG-3' (the underline is the restriction endonuclease NdeI restriction site recognition sequence); EcACO-EcoRV:5'-GGCTCTCA GATATC TCATTAAGCCGCCGGAG-3' (underlined is EcoRV restriction site recognition sequence). The EcACO-R gene and the EcACO-S gene were respectively constructed into the prokaryotic expression vector plasmid pMAL-c5...

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Abstract

The invention discloses a 1-aminocyclopropane-1-carboxylate oxidase contained in drug-resistant barnyard grass and a coding gene, a mutation site and application thereof. The amino acid residue sequence of the 1-aminocyclopropane-1-carboxylate oxidase contained in the drug-resistant barnyard grass is as shown in SEQ ID NO:3, and the nucleotide sequence of the coding gene of the 1-aminocyclopropane-1-carboxylate oxidase is as shown in SEQ ID NO:1. The 1-aminocyclopropane-1-carboxylate oxidase contained in the drug-resistant barnyard grass and the coding gene thereof, which are disclosed by the invention, can be applied to culture a herbicide-resistant transgenic variety.

Description

technical field [0001] The invention relates to barnyardgrass resistance-related genes and applications thereof, in particular to 1-aminocyclopropane-1-carboxylate oxidase in drug-resistant barnyardgrass and its coding gene, mutation site and application, and belongs to the field of plant biotechnology. Background technique [0002] As far as the applicant knows, barnyardgrass (Echinochloa) is a general term for the annual Grass family Echinochloa, and about 35 species of barnyardgrass have been found in the world at present, growing in tropical or subtropical regions, among which 8 species of barnyard grass (Chen&Philips) have been found in my country. ,2006). As one of the ten most malignant and resistant weeds in farmland in the world, barnyardgrass has developed resistance to many types of herbicides (Heap I, 2015). At present, the research on the mechanism of barnyardgrass resistance to herbicides mainly focuses on the mutations of herbicide target protein sites and non...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/82A01H5/00
CPCC12N9/0071C12N15/8274C12Y114/17004
Inventor 杨霞李永丰董明超张自常裴涛
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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