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Brassica napus Polima cytoplasmic male sterility restoring gene Rfp and application thereof

A technology of male sterility in Brassica napus, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as large labor costs and long breeding cycle

Inactive Publication Date: 2017-09-05
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Utilizing the Polyma CMS system by conventional breeding methods requires the cultivation of sterile lines, maintainer lines and restorer lines to achieve three-line matching, and a large number of crosses and multi-generation backcrosses in the process of three-line selection require a lot of labor costs and long breeding cycles

Method used

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  • Brassica napus Polima cytoplasmic male sterility restoring gene Rfp and application thereof
  • Brassica napus Polima cytoplasmic male sterility restoring gene Rfp and application thereof
  • Brassica napus Polima cytoplasmic male sterility restoring gene Rfp and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of a physical map containing the Rfp segment of the cytoplasmic male sterility restorer gene in Brassica napus and fine mapping of the sterility restorer gene Rfp

[0025] 1. Experimental materials

[0026] The raw material used in this example is rapeseed polima cytoplasmic male sterile line 6330A (Zeng, F.Q., et al., Identification of AFLP and SCAR markers linked to the male fertility restorer gene of pol CMS (Brassica napus L.). Euphytica, 2009.165(2): p.363-369) and another rapeseed polima cytoplasmic male sterility restorer line Bing409 (document above: Zeng, F.Q., et al., 2009), the restorer line Bing409 was selected as the donor parent , and using 6330A as the recurrent parent, a near-isogenic line was obtained by multi-generation backcrossing and sibling crossing (Zeng, F.Q., et al., 2009).

[0027] The near-isogenic line constructed after the sister crossing of sterile and fertile strains (see the literature for the construction method ...

Embodiment 2

[0055] Example 2: Isolation and functional verification of the dominant gene Rfp

[0056] 1. Determination of Rfp candidate genes

[0057] The model organism used in this example is Blast from the report of the Arabidopsis website. 10 (http: / / www.arabidopsis.org / blast) software analyzes the sequence of Brassica napus containing Rfp between the molecular markers SSR48 and SSR53, showing that there may be 10 genes in this segment, one of which is PPR (pentatricopeptide repeat, accession number IPR002885) family members. The transcription initiation site of this gene was determined by analyzing the reference genome sequence, and high-fidelity PCR polymerase (Phusion TM High-Fidelity DNA Polymerse (purchased from New England Biolads Company) was used to isolate a fragment of about 1.5 kb in the upstream untranslated region of the gene (that is, including the promoter region of the gene), and the amplified fragment was cloned into pMD18-T (purchased from Bao Bioengineering Dalia...

Embodiment 3

[0068] Example 3: Cytological slice analysis

[0069] In order to further determine the cytological characteristics of Porima cytoplasmic male sterility and abortion, and clarify the function of the Rfp gene, the present invention carried out semi-thin section observations on the anthers, focusing on observing the microspores and Tapetum development, the differences in the development of tapetum and microspores in fertile and sterile anthers at different stages were compared. The result of semi-thin section showed that the stage of abortion occurred in the stage of differentiation of protosporium ( Figure 7 Figure C in and Figure 7 Figure I in the sterile single plant anther, the inner wall, middle layer, tapetum and microspore mother cells that the L2 layer cells in the sterile single anther cannot normally differentiate; the sterile single plant anther develops to the pollen mother cell period ( Figure 7 Figure D in and Figure 7 Figure J in ) has been unable to form f...

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Abstract

The invention belongs to the technical field of rapeseed breeding. It specifically relates to the Brassica napus polima cytoplasmic male sterility restorer gene Rfp and its application. The dominant gene Rfp involved in the present invention is the homologous gene of the function gain variation of the recessive gene rfp. The invention also discloses the cloning, verification and application of the restorer gene Rfp and the breeding of cytoplasmic male sterile lines, maintainer lines and restorer lines of crops such as rapeseed. The nucleotide sequence of the Brassica napus polima cytoplasmic male sterility restorer gene Rfp of the present invention is shown in SEQ ID NO:1, and its protein sequence is shown in SEQ ID NO:2. The cloned gene of the present invention can also be directly used in the cultivation of the restorer line of Brassica napus through transgenic technology.

Description

technical field [0001] The invention belongs to the technical field of rapeseed breeding. It specifically relates to the Brassica napus polima cytoplasmic male sterility restorer gene Rfp and its application. The dominant gene Rfp involved in the present invention is the homologous gene of the function gain variation of the recessive gene rfp. The cloning, verification and application of the restorer gene Rfp can speed up the breeding of cytoplasmic male sterile lines, maintainer lines and restorer lines of rapeseed and other crops, thereby greatly shortening the breeding cycle. Background technique [0002] Plant male sterility refers to the phenomenon that the male gametes develop abnormally, while the female gametes develop normally and can accept foreign normal pollen to set fruit (Hanson, M.R. and S.Bentolila, Interactions of mitochondria and nuclear genes that affect male gametophyte development. Plant Cell, 2004.16 :p.S154-S169). This phenomenon was first discovere...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415A01H5/00
CPCC07K14/415C12N15/8289
Inventor 易斌洪登峰杨宗辉刘智傅廷栋涂金星杨光圣马朝芝沈金雄文静
Owner HUAZHONG AGRI UNIV
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