An EPSP synthase gene AroA-R1 derived from rhizobium leguminosarum and a preparing method and applications thereof

A kind of technology of EPSP synthase, Rhizobium pea, applied in EPSP synthase gene AroA-R1 and preparation field thereof, can solve problems such as weak protein activity

Inactive Publication Date: 2015-07-01
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some glyphosate-resistant corn contains an altered EPSPS gene from corn. The EPSPS synthetase transcribed and translated from this altered gene is also not sensitive to glyphosate, so it is also resistant to glyphosate, but The proteins expressed by these genes are inactive and must be compensated by overexpression

Method used

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  • An EPSP synthase gene AroA-R1 derived from rhizobium leguminosarum and a preparing method and applications thereof
  • An EPSP synthase gene AroA-R1 derived from rhizobium leguminosarum and a preparing method and applications thereof
  • An EPSP synthase gene AroA-R1 derived from rhizobium leguminosarum and a preparing method and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1 gene synthesis method synthesizes the AroA-R1 gene derived from pea rhizobia

[0093] Utilize PTDS method [Xiong et al., Nucl Acids Res, 2004,32:e98] to synthesize the AroA-R1 gene of pea rhizobia, the primers used are as follows:

[0094] 1. RLAROAI-1:

[0095] GGATCCATGC TGAATGGTTC TGCTTCTAAG CCAGCTACTG CTCGTAAGTC TGCTGGTCTG

[0096] 2. RLAROAI-2:

[0097] GAGAGATAGA CTTGTCACCA GGGATACGAA CAGAACCAGT CAGACCAGCA GACTTACGAG

[0098] 3. RLA ROAI-3:

[0099] TGGTGACAAG TCTATCTCTC ACCGTTCTTT CATGATTGGT GGTCTCGCTT CTGGTGAGAC

[0100] 4. RLA ROAI-4:

[0101] GTTGATAACG TCTTCACCTT CAAGAAGACC AGTGATACGA GTCTCACCAG AAGCGAGACC

[0102] 5. RLA ROAI-5:

[0103] AAGGTGAAGA CGTTATCAAC ACTGGTCGTG CTATGCAAGC TATGGGTGCT CGTATTCGTA

[0104] 6. RLA ROAI-6:

[0105] CCATTGCCAG TGCCTTCAAT GACCCACTGA GCACCTTCCT TACGAATACG AGCACCCATA

[0106] 7. RLA ROAI-7:

[0107] ATTGAAGGCA CTGGCAATGG TGCTCTCCTT GCTCCTGATG CTCCACTCGA CTTCGGCAAT

[0108] 8. RLA ROAI-8:

[0109] TGCCA...

Embodiment 2

[0163] Embodiment 2: AroA-R1 gene prokaryotic expression anti-glyphosate identification

[0164] The AroA-R1 gene clone identified as positive by sequencing was digested with BamHI and SacI, connected to the p251 expression vector, and the concentration was estimated by agarose gel electrophoresis to ensure that the concentration of exogenous DNA in the connection reaction was at least 3- 5 times. The reaction temperature is 16° C., and the connection time is 10-12 hours. Escherichia coli ER2799 is transformed to obtain a prokaryotic expression strain containing the target gene.

[0165] Inoculate the Escherichia coli ER2799 containing the target gene AroA-R1 and the ER2799 bacteria carrying the empty vector p251 into the M9 liquid medium containing 0-100mM glyphosate, culture on a shaker at 37°C for 12h, and measure The growth of the bacteria was compared at OD600, and Escherichia coli ER2799 without inserting the plasmid was used as a supplementary blank control. Result: Und...

Embodiment 3

[0166] Embodiment 3: AroA-R1 gene plant expression vector construction

[0167] First, the chloroplast transit peptide (TSP GenBank M61904) sequence from tobacco was amplified by PCR method, the primers were TSPR (5'-CGCCTGCAGTGGCACAGATTAGCAGCATG-3') and TSPF (5'-CAGAGGATCCTCTGTGCAGTGACCACTGAT-3'), and the amplification conditions were: 94 Preheat at ℃ for 1min; 94℃ for 30s; 50℃ for 30s; 72℃ for 10min, a total of 25 cycles. Add SalI and BamHI restriction sites before the TSP sequence, amplify the AroA-R1 gene, and add BamHI and SacI restriction sites at both ends of the gene, recover the TSP sequence and the PCR fragment of the target gene and connect them to In the pGEM3Z (Promega Madison WI USA) vector, sequence determination was performed to obtain a plasmid containing TSP and AroA-R1 genes. Plasmid DNA was extracted, double-digested with SalI and SacI respectively, and the DNA fragments were recovered. The TSP and AroA-R1 fragments were ligated with the pCAMBIA1301 plasmi...

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Abstract

An EPSP synthase gene AroA-R1 derived from rhizobium leguminosarum and a preparing method and applications thereof are disclosed. The nucleotide sequence of the EPSP synthase gene AroA-R1 is shown as SEQ ID NO1, and an encoded protein sequence is shown as SEQ ID NO2. The EPSP synthase gene AroA-R1 is prepared by utilization of a gene synthesis method. The EPSP synthase encoded by the AroA-R1 gene has resistance to glyphosate, can improve resistance of plants to glyphosate herbicides after the EPSP synthase is transformed into the plants, and can be used for cultivation of glyphosate-resistant transgenic crops.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to an EPSP synthase gene AroA-R1 derived from pea rhizobia and a preparation method and application thereof. Background technique [0002] With the rapid development of chemical herbicide technology, the application of herbicides has become more and more extensive, which has brought great economic benefits to agricultural production. The organophosphorus herbicide glyphosate is a high-efficiency, low-toxicity, and destructive post-emergence herbicide. In recent years, with the cultivation and farming methods tending to be large-scale and intensive, the demand for the pesticide glyphosate has increased significantly. In recent years, the sales volume of glyphosate has increased by 15% every year, occupying the first place in the sales volume of pesticides in the world for many years. [0003] The herbicidal principle of glyphosate is that it can inhibit the synthesis of aromat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/84C12N15/10
Inventor 韩静姚泉洪朱波田永生韩红娟赵伟许晶王波王丽娟
Owner SHANGHAI ACAD OF AGRI SCI
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