Cytoplasmic male sterility restorer gene in rice and application thereof

A technology for male sterility and gene recovery, which is applied in the field of genetic engineering to achieve the effects of improving selection efficiency, speeding up the breeding process, and eliminating the need for test crosses.

Active Publication Date: 2014-06-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rf3 and Rf4 Has been located in specific intervals of chromosome 1 and 10 (Zhang et al., 199

Method used

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  • Cytoplasmic male sterility restorer gene in rice and application thereof
  • Cytoplasmic male sterility restorer gene in rice and application thereof
  • Cytoplasmic male sterility restorer gene in rice and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0044] Example 1 Rice restorer gene Rf4 Location and cloning

[0045] The invention uses map-based cloning to clone Rf4 gene.

[0046] First, cross the wild abortive cytoplasmic male sterile line Zhenshan 97A with the restorer line Minghui 63 to establish a F 2 Separate the population and select about 2000 sterile plants for Rf4 Positioning. According to the reported preliminary location on rice chromosome 10 Rf4 The approximate location of the site (Zhang et al., 1997; Zhang Qunyu et al., 2002; Ahmadikhah and Karlov, 2006), in which multiple polymorphic molecular markers as shown in Table 1 were created. Use these marks and F 2 Linkage analysis of sterile populations constructed a Rf4 The linkage map of the locus region, namely figure 1 . There are 2 marks in the picture, namely M19391 and M19256 and Rf4 There is only 1 recombination event between the sites, and there are 4 markers and Rf4 Complete co-separation, that is, zero recombination. which is Rf4 It is located i...

Example Embodiment

[0051] Example 2 Rf4 Candidate gene transformation and functional testing

[0052] Design gene-specific PCR amplification oligonucleotide primers according to the upstream and downstream sequences of Os10g0495200. The primer sequence is as SEQ ID NO: 11 (the underline is restriction endonuclease Kpn I cut point) and SEQ ID NO: 12 (underlined Xba I tangent point) as shown.

[0053] SEQ ID NO:11: 5'-ACTTAT ggtacc TAGCAACTGCGAACACACCCTCAAT-3’

[0054] SEQ ID NO: 12: 5'-CGGTCA tctaga ACCAGCAACCTCCCAACCATGACTAT-3’

[0055] The 7.8 kb genome fragment containing the promoter, coding region, and downstream termination sequence was amplified from the restorer line Minghui 63 by PCR technology. The PCR reaction system and reaction conditions are as follows: a 50 μl reaction contains 1 x reaction buffer, 200 μM dNTPs, 100 ng Minghui 63 genomic DNA, 0.3 μM each of the primers, and 1.0 U KOD FX polymerase. 30 cycles: 97 ℃ 15S, 68 ℃ 6 min. Restriction endonuclease Kpn I and Xba I cut a...

Example Embodiment

[0057] Example 3 Rf4 Gene sequence analysis

[0058] Using PCR amplification and sequencing, the functional types of 5 wild-failed restorer lines, Minghui 63, IR24, HNF-W1, HNF-W2, and HNF-W8, were analyzed. Rf4 Allele ( Rf4-M, Rf4-I, Rf4-W1, Rf4-W2, Rf4-W8 ) And the non-functional type of two wild abortive sterile lines Zhenshan 97A and Jin 23A rf4 Allele (rf4-Z, rf4-J ). Among them, HNF-W1, HNF-W2, HNF-W8 Rf4 Allele ( Rf4-W1, Rf4-W2, Rf4-W8 ) Is derived from common wild rice, obtained by crossing and backcrossing indica rice lines with different common wild rice lines. The sequences shown in SEQ ID NO:1 and SEQ ID NO:2 are Minghui 63 and IR24, respectively Rf4-M with Rf4-I Allelic genomic DNA fragments (including the promoter region, CDS, and 3'untranslated region sequences); the sequences shown in SEQ ID NO: 3 to SEQ ID NO: 5 are HNF-W1, HNF-W2, HNF-W8 Rf4-W1, Rf4-W2, Rf4-W8 Allele CDS sequence. These functional types Rf4 The open reading frame of the allele has 234...

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Abstract

The invention relates to the field of plant genetic engineering, and specifically discloses a cytoplasmic male sterility restorer gene in rice and an application thereof. The invention provides a nucleotide sequence of a wild abortive cytoplasmic male sterility restorer gene (i) Rf4 (/i) isolated and cloned from rice and an amino acid polypeptide sequence encoded by the restorer gene. The gene belongs to a member of PPR (pentatricopeptide repeats) gene family and is a constitutively expressed gene. The fertility is restored by negatively regulating the expression levels of sterility gene (i) WA352 (/i) in mitochondrial genome. A new restorer line can be cultivated by transforming (i) Rf4 (/i) into the original rice varieties containing no restorer gene. Transgenic restorer lines are hybridized with a wild abortive type sterile line to produce hybrids having normal sterility for production.

Description

technical field [0001] The invention relates to the field of genetic engineering, and more specifically relates to rice cytoplasmic male sterility restoration gene and application thereof. Background technique [0002] Plants can produce hybrids through cross-pollination, and hybrids have advantages in growth vigor, adaptability, yield, etc., called heterosis. Harnessing heterosis in crops can improve yield, quality and resistance. The phenomenon that the male reproductive organs of plants cannot produce normal fertile male gametes, that is, pollen, is called male sterility. Crop varieties or lines with the genetic trait of male sterility are called sterile lines. On the other hand, hybrids produced by crossing some crop varieties (strains) with sterile lines have restored normal fertility, and these crop varieties (strains) are called restorer lines. By using the sterile line as the female parent and the restorer line as the male parent, the operation of detasseling can ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C12N15/11C07K14/415C12Q1/68A01H5/00
Inventor 刘耀光唐辉武罗荡平陈乐天张群宇
Owner SOUTH CHINA AGRI UNIV
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