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Nucleotide sequence for detecting maize plant NAZ-4 and detection method of nucleotide sequence

A sequence, technology of corn, applied in the field of plant biology

Pending Publication Date: 2020-05-29
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event

Method used

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  • Nucleotide sequence for detecting maize plant NAZ-4 and detection method of nucleotide sequence
  • Nucleotide sequence for detecting maize plant NAZ-4 and detection method of nucleotide sequence
  • Nucleotide sequence for detecting maize plant NAZ-4 and detection method of nucleotide sequence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] The construction of embodiment 1 transformation vector

[0112] A forward primer containing a HindIII anchor site was designed at the 5' end of the ABP9 promoter Pabp9 sequence, and a reverse primer containing a HindIII and SpeI anchor site (from outside to inside) was designed at the 3' end, and the maize genomic DNA was used as a template for PCR amplification. Pabp9 was increased; the PCR product was cloned into the pCAMBIA3301 vector by HindIII digestion to obtain the pCAMBIA3301-Pabp9 intermediate vector. The ABP9 cDNA cloning vector T-easy-ABP9 was digested with XhoI to make up, and then digested with XbaI. The recovered small fragment was ligated with the pBI121 vector digested by XbaI and Ecl136II to obtain the intermediate vector pBI121-35S-ABP9. pCAMBIA3301-Pabp9 was digested with SpeI and then digested with AflII to recover a large fragment; pBI121-35S-ABP9 was digested with XbaI and then digested with AflII to recover a small fragment; the two recovered frag...

Embodiment 2

[0114] Embodiment 2 Maize genetic transformation

[0115] The method used for transforming corn is the Agrobacterium-mediated method, and its operation procedure is as follows:

[0116] (1) select the corn ears of pollination 10-13 days, and peel off the immature embryos (1.5-2.0mm) of moderate size therefrom;

[0117] (2) Use an inoculation loop to pick a ring full of Agrobacterium LBA4404 (containing the expression vector) from the YEP plate grown at 19°C for three days, suspend it in the infection culture medium, room temperature, 75rpm, 2-4h, to OD 550 = 0.3-0.5, infecting detached immature embryos;

[0118] (3) The immature embryos that have been infected are transferred to the co-culture medium, and cultured in the dark at 20°C for 3 days;

[0119] (4) Three days later, the immature embryos were transferred to the recovery medium, and cultured in the dark at 28°C for 7 days;

[0120] (5) transfer the immature embryos to the selection medium after the recovery culture,...

Embodiment 3

[0123] The screening of embodiment 3 transformants

[0124] (1) Molecular detection of bar and ABP9 target gene was carried out on the transformed seedlings obtained by transformation. According to gene sequence design PCR primer pair, primer sequence is respectively SEQ ID NO:16 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ IDNO:19. Genomic DNA was extracted from transformed seedling leaves and amplified according to the following PCR parameters:

[0125] reaction system:

[0126]

[0127] Reaction procedure:

[0128]

[0129]

[0130] Positive transformed seedlings were screened according to the expected amplified fragment size of 672 bp of the bar gene and 582 bp of the expected amplified fragment size of the ABP9 gene.

[0131] (2) Cross the positive plants with Zheng 58 and backcross once to harvest BC 1 f 1 seed. to BC 1 f 1 The plants were identified for glufosinate-ammonium herbicide, and the transformants NAZ-1~NAZ-6 with excellent resistance were selecte...

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Abstract

The invention relates to a nucleotide sequence for detecting maize plant NAZ-4 and a detection method of the nucleotide sequence. The nucleotide sequence of the maize plant comprises a sequence shownin SEQ ID NO:1 or a complementary sequence of the sequence, or a sequence shown in SEQ ID NO:2 or a complementary sequence of the sequence. The maize plant NAZ-4 provided by the invention has good resistance to drought and glufosinate-ammonium herbicides, and by adopting the detection method, whether a biological sample contains DNA (deoxyribonucleic acid) molecules of a transgenic maize event NAZ-4 or not can be accurately and rapidly identified.

Description

technical field [0001] The invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid sequence for detecting corn plant NAZ-4 and its detection method, especially relates to a transgenic corn event NAZ-4 which is resistant to drought and glufosinate herbicide application and is used for detecting biological Whether the sample contains the nucleic acid sequence of the specific transgenic maize event NAZ-4 and its detection method. Background technique [0002] Field weeds compete with crops for water, fertilizer, light and growth space, directly affecting crop yield and quality. At the same time, many weeds are intermediate hosts of crop pathogenic bacteria and pests, and are one of the important biological limiting factors for crop production. According to statistics from the Food and Agriculture Organization of the United Nations, the annual loss of food production due to weeds in the world is as high as 95 billion US dollars, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82C12N5/10C12Q1/6895A01H1/02A01H4/00A01H1/08A01H5/00A01H5/10A01H6/46A01G22/20
CPCC12Q1/6895C12N15/8273C12N15/8277A01H1/02A01H4/008A01H4/005A01H1/08A01G22/20C12Q2600/13
Inventor 赵军冷鹏飞王磊范云六
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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