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Construct for regulating fertility of plant pollens and usage thereof

A technology for pollen development and introduction into plants, applied in botany equipment and methods, angiosperms/flowering plants, biochemical equipment and methods, etc., can solve problems such as easy leakage, easy to affect plant growth and development, Barnase genotoxicity, etc., to achieve The effect of low temperature sensitivity and avoiding abnormal development of tissues and organs

Active Publication Date: 2015-06-10
BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In the present invention, in order to solve the problem that Barnase genotoxicity is too large and easy to leak, and it is easy to affect the normal growth and development of plants, the present invention divides the Barnase protein into N-terminal (amino acids 1-36) and C-terminal (37-110 Amino acid) Two small peptides, N-terminal contains 2 α-helices, C-terminal contains 5 β-sheets

Method used

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  • Construct for regulating fertility of plant pollens and usage thereof
  • Construct for regulating fertility of plant pollens and usage thereof
  • Construct for regulating fertility of plant pollens and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1. synthetic BarnaseW nucleotide fragment

[0035] According to the high-level structure of Barnase, the codon preference of Bacillus amyloliquefaciens, and the codon preference of monocotyledonous plants, the nucleotide sequence of Barnase was modified, and the important amino acids that determine the temperature sensitivity of Barnase were also point-mutated ( Q15I, T16R, K19R, 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 Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0036] Embodiment 2. Construction of plant expression vector pTa95v2

[0037] Specific primers designed to amplify PTaPSG719:

[0038] Primer 1: 5'-aagcttCGCATTCGCAAGGTTCACT-3'

[0039] Primer 2: 5'-ctgcagGCATTTCTATATGATATGACCGGCCAA-3'

[0040] Using the genomic DNA of wheat as a template, PTaPSG719 was amplified with the above primers. The PCR product was detected and recovered by 1% agarose gel electrophoresis, and the product was connected into pEASY-T3. Positive clones were screened and verified by sequencing. is the expected PTaPSG719 promoter sequence, as shown in SEQ ID NO:3 in the sequence listing.

[0041] Specific primers designed to amplify BarW-N:

[0042] Primer 3: 5'-ctgcag ATGGCGCAAGTTATTAATACGTTC-3'

[0043] Primer 4: 5'-gtcgac TCACCAGCCTAGAGCCTGC-3'

[0044] Using the artificially synthesized BarnaseW gene as a template, the above primers were used to amplify BarW-N. The PCR product was detected and recovered by 2.5% agarose gel electrophoresis. The produ...

Embodiment 3

[0063] Example 3. Agrobacterium-mediated genetic transformation of rice and wheat

[0064] The plant expression vector pTa95v2 was transformed into Agrobacterium AGLO strain by heat shock method.

[0065] Infect rice embryogenic calli with Agrobacterium, co-cultivate in the dark for 2-3 days, and then go through two steps of resistance selection, pre-differentiation, differentiation and rooting culture, and finally obtain the transgenic rice with kanamycin resistance ( Transplant pTa95v2 rice) T 0 Substitute plants.

[0066] Callus was induced secretly with mature wheat embryos. Wheat calli were infected with Agrobacterium and co-cultured in the dark for 3 days. Put the calli co-cultivated with Agrobacterium on the induction medium added with cefotaxime to resume culture in the dark for 1 week, then transfer to the selection medium for 4-6 weeks, and transfer the resistant callus to differentiation The medium induces the differentiation of shoots, and then the differentiat...

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Abstract

Disclosed is a method for creating a male sterility line of a plant, comprising: using two different specific expression promoters of the late development stage of plant pollens to drive two nucleotide fragments, namely the N terminus and C terminus of the ribonuclease gene Barnase of Bacillus, to express in the plant, respectively. As both the small peptides encoded by the N terminus and C terminus of Barnase have no ribonuclease activity, when driven to express in the same cell by the two pollen-specific promoters of which the expression periods overlap, the peptides can recover partially natural ribonuclease activity, leading to male sterility of the plant.

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): C12N15/55C12N15/82C12N15/84A01H5/00A01H5/10C12N5/10
CPCC12N15/8289
Inventor 马力耕李健邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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