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A kind of method and its application of regulation and control plant pollen fertility

A pollen-specific, dicotyledonous plant technology, applied in botany equipment and methods, angiosperms/flowering plants, biochemical equipment and methods, etc., can solve the problems of Barnase protein temperature sensitivity, insufficient specificity, toxicity leakage, etc. , to avoid abnormal tissue and organ development and low temperature sensitivity

Active Publication Date: 2015-11-25
SHENZHEN INST OF MOLECULAR CROP DESIGN +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A series of problems were also found in the process of using Barnase to create plant male sterile lines, such as the temperature sensitivity of Barnase protein, the specificity of the pollen-specific promoter driving its expression, or the strong promoters such as CaMV35S upstream Toxic leakage problems caused by

Method used

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  • A kind of method and its application of regulation and control plant pollen fertility
  • A kind of method and its application of regulation and control plant pollen fertility
  • A kind of method and its application of regulation and control plant pollen fertility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1. synthetic BarnaseW nucleotide fragment

[0033] According to the high-level structure of Barnase, the codon preference of Bacillus amyloliquefaciens, and the codon preference of dicotyledonous plants, the nucleotide sequence of Barnase was modified, and the important amino acids that determine the temperature sensitivity of Barnase were also point-mutated ( Q16I, T17R, K20R, G65S, K66A and K108R). The modified and point-mutated nucleotide sequence, such as SEQ ID NO: 1, was named BarnaseW, and the sequence was synthesized by Bao Biological Engineering (Dalian) Co., Ltd.

Embodiment 2

[0034] Embodiment 2. Construction of plant expression vector pBnSK

[0035] Specific primers designed to amplify Pat2g38500:

[0036] Primer 1: 5'-gCCCTCgAgACgATTTgTCCTggTTCAgTgCA-3' (SEQ ID NO: 9)

[0037] Primer 2: 5'-CCgagatctCATTggAgAgAgCg-3' (SEQ ID NO: 10)

[0038] Genomic DNA of Arabidopsis thaliana was used as a template to amplify pAt2g38500 with the above primers. The PCR product was detected and recovered by 1% agarose gel electrophoresis, and the product was ligated into pMD18-T. Positive clones were screened and verified by sequencing. The augmented sequence is the expected promoter sequence of PAt2g38500, as shown in SEQ ID NO: 3 in the sequence listing.

[0039] Specific primers designed to amplify BarW-N:

[0040] Primer 3: 5'-CCGagatctATGGCTCAAGTG-3' (SEQ ID NO: 11)

[0041] Primer 4: 5'-gatggtgaccttaCCATCCAAGAGCCTGAGCC-3' (SEQ ID NO: 12)

[0042] Using the artificially synthesized BarnaseW gene as a template, the above primers were used to amplify BarW-N...

Embodiment 3

[0061] Example 3. Agrobacterium-mediated genetic transformation of Arabidopsis

[0062] The plant expression vector pBnSK was transformed into Agrobacterium EHA105 strain by electric shock method.

[0063] Infect Arabidopsis thaliana with the method of dipping flowers with Agrobacterium, co-cultivate in the dark for 2-3 days, and then cultivate normally until the seeds are harvested. Seeds with red fluorescent markers were picked under a fluorescence microscope, and molecular detection was performed to obtain pBnSK-transformed Arabidopsis plants.

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Abstract

The invention belongs to the field of plant gene engineering and plant breeding technology, concretely relates to a method, which comprises the following steps: dividing Barnase into an N terminal and C terminal, respectively using two pollen-specific promoters with overlapped expression periods to drive expression thereof in plant, thereby enhancing tissue specificity of Barnase expression, and overcoming toxicity leakage problem during establishment of plant male sterility line by using Barnase gene.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering and plant breeding, and specifically relates to a method of dividing Barnase into N-terminal and C-terminal fragments, respectively utilizing pollen-specific promoters with overlapping expression periods to drive its expression in plants, Therefore, the method for strengthening the tissue specificity of Barnase expression and overcoming the problem of toxicity leakage when using the Barnase gene to create male sterile lines of plants. Background technique [0002] Heterosis is a common phenomenon in the biological world, and the use of heterosis can significantly improve crop yield, quality and resistance. Hybrid breeding has become the main way to breed new varieties of many crops, and the selection of crop male sterile lines is a key link in the utilization of heterosis. Due to the problems of long cycle, slow effect, and sensitivity to environmental factors, the conventional ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/55C12N15/82A01H5/00
Inventor 刘东风常振仪卢启清唐晓艳邓兴旺
Owner SHENZHEN INST OF MOLECULAR CROP DESIGN
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