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Plant glyphosate-resistant gene and applications thereof

A glyphosate-resistant gene technology, applied in the fields of plant molecular biology and plant genetic engineering, to increase diversity and reduce ecological risks

Inactive Publication Date: 2012-12-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although glyphosate can inhibit 5-enolpyruvate-3-phosphoshikimate synthase (EPSPS) in the synthesis of aromatic amino acids in most plants and bacteria, some bacterial EPSPS have been found to be insensitive to glyphosate , was isolated, and some plants were also found to be resistant to glyphosate in production practice

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Glyphosate resistance experiments of Ophiopogon japonicus, Ophiopogon japonicus and broad-leaved Ophiopogon japonicus

[0030] Select Ophiopogon japonicus with uniform growth, soil Ophiopogon japonicus and broad-leaved soil Ophiopogon japonicus for spraying treatment. The concentration of glyphosate was set at 0, 375, 750, 1500, 3000, 6000, 12000, 24000ai.g / ha, and the test was performed according to the designed dosage, and each treatment was repeated four times. Observe the degree of plant damage after spraying, investigate and record the level of phytotoxicity every week, draw the inhibition rate curve and perform Logistic regression analysis to calculate the ED 50 value. repeat three times. The results showed that the glyphosate resistance of Ophiopogon japonicus, Ophiopogon japonicus and broad-leaved Ophiopogon japonicus were 4.73, 5.06, and 5.8 times that of common plants, respectively.

Embodiment 2

[0031] Example 2. Cloning of EPSPS gene

[0032] Using the reverse transcription product of Ophiopogon japonicus total RNA as a template, primers were designed using the conserved sequence of the EPSPS gene, and RT-PCR was performed. The amplified fragment was cloned into the pMD19-T vector for sequencing and BLAST.

[0033] After obtaining the sequence of the conserved region, the full-length clone of the EPSP synthetase gene was carried out by RACE (rapid-amplification of cDNA ends) technology. The reverse transcription of Ophiopogon japonicus, Ophiopogon japonicus, and Ophiopogon japonicus broad-leaved RNA was carried out with the adapter primer AP, and gene-specific primers were designed using the sequence of the conserved region of the gene, and RT-PCR was performed with the specific primer at the 3' end.

[0034] According to the obtained target gene fragment, the 5' end was cloned by TdT terminal transferase method. Design gene-specific primers for reverse transcriptio...

Embodiment 3

[0037] Example 3. Analysis and comparison of amino acid sequences encoded by Ophiopogon japonicus, Ophiopogon japonicus and broad-leaved Ophiopogon japonicus EPSPS genes

[0038] By MAGA software analysis and comparison of about 260 EPSPS synthase amino acid sequences with higher sexness of the EPSP synthase system of Ophiopogon japonicus, Ophiopogon japonicus and Ophiopogon japonicus broad-leaved, it was found that the Compared with other plants, there are four specific amino acids in the amino acid sequence of EPSP synthase, which are 144m, 184i, 232a, and 273m (equivalent to positions 70, 107, 153, and 192 of EPSP synthase in Escherichia coli. 146m, 186i, 234a and 275m in Ophiopogon japonicus), the amino acids at these positions are conserved in Ophiopogon japonicus, Ophiopogon japonicus and Ophiopogon japonicus broad-leaved, but they do not exist in the same positions in other plants. It is the existence of these amino acids that lead to the resistance of Ophiopogon japoni...

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PUM

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Abstract

The invention discloses a plant glyphosate-resistant gene and applications thereof. The nucleotide sequence of the glyphosate-resistant gene is SEQIDNO:1, SEQIDNO:3 or SEQIDNO:5 and encoded amino acid sequences are SEQIDNO:2, SEQIDNO:4 or SEQIDNO:6 respectively. A glyphosate-resistant plant prepared throuhg the plant glyphosate-resistant gene can apparently display glyphosate-resistant herbicides, novel choices are provided for cultivating glyphosate-resistant herbicides, and the diversity of the transgenic technology of the glyphosate-resistant herbicides is added, accordingly, ecological risks of the transgenic technology of the glyphosate-resistant herbicides are reduced.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and plant genetic engineering, in particular, the invention relates to three plant-derived glyphosate-resistant / tolerant genes and proteins encoded by the genes. Through the method of genetic transformation, the gene is expressed in the plant so that the plant acquires resistance to glyphosate, so that glyphosate can be used to selectively control weeds in crop fields. The present invention can also be used in the screening of crop breeding and plant cell culture. Background technique [0002] The molecular formula of glyphosate is C 3 h 8 NO 5 P, is a high-efficiency, broad-spectrum, low-toxicity, low-residue herbicide that does not damage the soil environment and kills most plants. It has the characteristics of non-toxic to humans and animals, low frequency of resistance to weeds and crops under natural conditions, low soil residue, etc., and has huge market potential. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/10C12N15/63C12N5/10A01H5/00C12N15/82C12Q1/68
CPCC12N15/8275C12Q1/68C12N2770/32351C12N15/10C12N2770/32322C12N2800/22C12N15/63C12N2770/32323C07K14/415C12N9/1092C12Q1/6895C12Q2600/13C12Q2600/158
Inventor 强胜毛婵娟陈世国戴伟民宋小玲
Owner NANJING AGRICULTURAL UNIVERSITY
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