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Acetolactate synthase inhibitor resistance-related protein uvals and its coding gene and application

A technology of acetolactate and synthetase, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of plant individual death, inability to complete mitosis, interference with DNA synthesis, etc., and achieve the effect of high application value

Active Publication Date: 2019-09-06
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Acetolactate synthase inhibitor herbicides inhibit the activity of acetolactate synthase in plants, thereby preventing the synthesis of branched-chain amino acids, resulting in the destruction of protein synthesis, hindering DNA synthesis during cell division, thereby stopping the mitosis of plant cells In the S phase of the G1 phase (DNA synthesis phase) and the M phase of the G2 phase, DNA synthesis is interfered, so the cells cannot complete mitosis, and the plant stops growing, eventually leading to the death of individual plants

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, discovery of UVALS protein and UVALS gene

[0039] 1. Determination of the optimal mutagenesis time

[0040] 1. Transfer Aspergillus niger TR-H to PDA slant medium and culture at 28°C for 3-5 days.

[0041] 2. Wash the Aspergillus niger spores on the inclined surface with sterile physiological saline, pour them into a sterilized triangular flask containing 20-30 glass beads with a diameter of 4-5mm, and shake and cultivate at 220rpm for 1 hour to fully disperse the spores. Then use 4 layers of sterile lens paper to filter and remove mycelia to make a concentration of 10 5 / ml of spore suspension.

[0042] 3. Take 5ml of spore suspension in a plate with a diameter of 9cm, put a sterile magnetic stirrer and place it on the magnetic stirrer, open the lid of the dish, and place it 30cm vertically away from the ultraviolet lamp (power 30W), and irradiate it under stirring respectively. 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 or 600s, turn off the UV la...

Embodiment 2

[0057] Example 2, functional verification of UVALS protein

[0058] 1. Construction of recombinant plasmids

[0059] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0060] 2. Using the DNA molecule synthesized in step 1 as a template, PCR amplification is performed using a primer pair composed of AncDF1 and AncDF1 to obtain a PCR amplification product.

[0061] AncDF1: 5'-GA GGATCC GTATGATGCCTATGAGACCTTC-3';

[0062] AncDF1: 5'-TC GTC GAC TTAGAAACCGGGAACTTTCC-3'.

[0063] 3. Take the PCR amplification product obtained in step 2, perform double digestion with restriction endonucleases BamH I and Sal I, and recover the digested product.

[0064] 4. Take the pGBKT7 vector (purchased from Clontech, catalog number: 630443), perform double digestion with restriction endonucleases BamH I and Sal I, and recover the vector skeleton of about 7.3 kb.

[0065] 5. Ligate the digested product obtained in step 3 with the vector backbone o...

Embodiment 3

[0088] Embodiment 3, the acquisition and identification of transgenic plants

[0089] 1. Construction of recombinant plasmids

[0090] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0091] 2. Using the double-stranded DNA molecule obtained in step 1 as a template, a primer pair composed of Primer1 and Primer2 is used for PCR amplification to obtain a PCR amplification product, and a DNA fragment of about 2000 bp is recovered.

[0092] Primer1: 5'-GC TCTAGA ATGATGCCTATGAGACCTTC-3';

[0093]Primer2: 5'-TC GAGCTC TTAGAAACCGGGAACTTTCC-3'.

[0094] 3. Digest the DNA fragment obtained in step 2 with restriction endonucleases XbaI and SacI, and recover the digested product.

[0095] 4. Digest the Super1300 vector with restriction endonucleases XbaI and SacI, and recover the vector backbone of about 10,000 bp. Super1300 carrier: purchased from Shanghai Beinuo Biotechnology Co., Ltd., the article number is addgene 0595.

[0096] 5...

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Abstract

The invention discloses an acetolactate synthetase inhibitor resistance related protein UVALS and an encoding gene and application thereof. The UVALS protein is the following (a) or (b): (a) protein composed of amino acid sequence shown as sequence 1 in a sequence table, and (b) protein substituted and / or deleted and / or added by one or more amino acid residues of amino acid sequence of the sequence 1, related to the resistance of organism to acetolactate synthetase inhibitor and derived from the sequence 1. The invention further discloses applications of the UVALS protein, including (c1) regulating the resistance of plants to the acetolactate synthetase inhibitor; (c2) promoting the increase of resistance of plants to the acetolactate synthetase inhibitor; (c3) regulating the resistance of microorganism to the acetolactate synthetase inhibitor; and (c4) promoting the increase of resistance of microorganism to the acetolactate synthetase inhibitor. The protein UVALS has high application values to breeding the new variety of plant with high resistance to the acetolactate synthetase inhibitor.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an acetolactate synthase inhibitor resistance-related protein UVALS and its coding gene and application. Background technique [0002] Modern biotechnology has caused major changes in several fields of agriculture, the most prominent of which is the expression of foreign genes introduced into plants, which has brought revolutionary changes to crop breeding and freed people from using traditional hybridization techniques to breed new varieties. The situation has entered a new era of free construction of genetic traits. Breeding transgenic herbicide-resistant crop varieties through genetic engineering is the fastest-growing, deepest-studied, and most accepted case by farmers. The most critical link is the acquisition of herbicide-resistant genes, isolation from mutant herbicide-resistant plants and microorganisms Resistance genes are the most important pathway. [0003] Acetolactate sy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/19A01H5/00A01H6/20
Inventor 陶波乔禹欣马诚义邵百惠张洪岩宋秀丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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