High-efficiency seed labeling method for propagation of plant male sterile line

A male sterility, plant technology, applied in the field of plant genetic breeding and seed production

Active Publication Date: 2013-03-13
北京中农大地科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, the pollen lethal gene and the male fertility restoration gene are constructed in one vector and introduce

Method used

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  • High-efficiency seed labeling method for propagation of plant male sterile line
  • High-efficiency seed labeling method for propagation of plant male sterile line
  • High-efficiency seed labeling method for propagation of plant male sterile line

Examples

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Embodiment 1

[0046] The amplification of embodiment 1Ms45 wild-type allele

[0047] The present invention takes the ms45 male sterile mutant in Table 1 as an example to specifically illustrate the implementation. Firstly, the wild-type allele of ms45 was expanded, which was derived from the inbred line B73, and its sequence is shown in SEQ ID No: 1. We used B73 as a template and referred to the B73 genome sequence (www.maizesequence.org) to design primers to amplify the entire expression element of the gene. The amplification primers are as follows: Ms45F: 5'tgaattcTGCTGAGTTTCTCTTGGGTTATCC 3', Ms45R: 5'tcccgggGGTTGCGCATGAAATAGGGGT 3 '. An EcoRI restriction site was added to the 5' end of the upstream amplification primer, and a SmaI restriction site was added to the 5' end of the downstream amplification primer. The amplification reaction system was: template DNA 2 μL, primer Ms45F 0.5 μL, primer Ms45R 0.5 μL, dNTP 1.6 μL, 10×Buffer 2 μL, high-fidelity taq enzyme 0.3 μL, ddH2O 13.1 μL. ...

Embodiment 2M

[0048] The amplification of embodiment 2Mn1 interfering fragment

[0049] The Mn1 gene encodes a cell wall invertase protein, and the inactivation of this gene will affect the development of the endosperm, resulting in smaller grains ( figure 2shown), but did not affect embryo and plant development. The present invention uses RNAi technology to silence the Mn1 gene. The interference fragment is derived from the inbred line B73, which contains a forward interference fragment, as shown in SEQ ID No: 2, and a reverse interference fragment, such as SEQ ID No: 3, and the intron that forms the hairpin structure in the middle. We have used B73 cDNA as a template, referring to the B73 genome sequence (www.maizesequence.org), and designed primers to amplify the specific conserved sequence of the gene. The amplification primers are as follows: RNAiMn1bF: 5'tggatccggtgaccTAAGTTTCGCTTCGGCGTGC3', RNAiMn1bR 5'gactagtCCACACGATGTTGCCCCATAC 3' . BamHI and BstEII restriction sites were adde...

Embodiment 3

[0050] The cloning of embodiment 3Mn1 gene promoter

[0051] The present invention uses the promoter controlling the grain size gene Mn1 to activate the interference fragment of the gene, and the gene is specially expressed in the endosperm, so the mRNA that is transcribed by the promoter only exists in the endosperm cells. The promoter is derived from the genomic DNA of the maize inbred line B73. The specific sequence is shown in SEQ ID No: 4. We have used the B73 genomic DNA as a template and referred to the B73 genome sequence (www.maizesequence.org) to design primers for the gene The promoter was amplified, and the amplification primers were as follows: Mn1pro bF: 5'atcccggGCTCGCATGAGAGAACAACCA 3', Mn1pro bR: 5'gcaagcttGGGGGTGCTATTTGTACTGTGC 3'. A SmaI restriction site was added to the 5' end of the upstream amplification primer, and a HindIII restriction site was added to the 5' end of the downstream amplification primer. The amplification reaction system was: template DN...

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Abstract

The invention discloses a high-efficiency seed labeling method which can be used for propagation of a plant male sterile line, and brings convenience for hybrid seed production. Furthermore, the invention provides a new method for high-efficiency propagation of the plant male sterile line by utilizing nucleotide sequences that controls plant male fertility and special grain shapes, e.g. size, length, width, thicknesst, etc.; and a transgene technology. According to the invention, the wild-type nucleotide sequence for controlling the male fertility and the nucleotide sequence for controlling the grain shapes are linked and transferred into conventional corns, and then a back cross into a homozygous recessive male sterile line is carried out; and the obtained transgene plant is hybridized with the homozygous recessive male sterile line to simultaneously obtain a lot of seeds of the sterile line and the maintainer line. Due to the effect of the nucleotide sequence for controlling the grain shapes, the seeds can be distinguished into the sterile line and the maintainer line through the grain shapes, wherein the seeds with normal shapes are the sterile line (without transgene sequences), and the seed with abnormal shapes (e.g. small grains) are the maintainer line.

Description

technical field [0001] The invention relates to a new method for propagating male sterile lines of plants. The method utilizes the male sterile gene of the cell nucleus, the gene for controlling the special shape of grains (such as size, length, width, thickness, etc.) and transgenic technology to propagate male sterile plants. The invention belongs to the field of plant genetic breeding and seed production. Background technique [0002] Due to the existence of heterosis, the biomass, resistance to diseases and insect pests, and stress tolerance (drought, high temperature, low temperature, saline-alkali, etc.) of hybrids are considerably improved compared with their parents, such as the yield of hybrid corn and hybrid rice. higher than that of the homozygous parents. The method usually used to produce hybrids is: plant the female parent and the male parent together, remove the tassels of the female parent, and keep the tassels of the male parent, and the seeds harvested by ...

Claims

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Application Information

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IPC IPC(8): A01H1/02A01H5/00C12N15/63
Inventor 赖锦盛赵海铭宋伟彬崔阳
Owner 北京中农大地科技发展有限公司
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