Separated rice female fertility relevant protein as well as encoding gene and application thereof

A rice and female technology, applied in the field of plant genetic engineering, can solve problems such as unclear molecular mechanisms

Inactive Publication Date: 2010-10-20
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some genes related to plant female fertility have been cloned one after another, the overall molecular mechanism of plant female fertility control is still unclear

Method used

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  • Separated rice female fertility relevant protein as well as encoding gene and application thereof
  • Separated rice female fertility relevant protein as well as encoding gene and application thereof
  • Separated rice female fertility relevant protein as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Cloning of PTB1 gene

[0066] 1. Map-based cloning of the PTB1 gene

[0067] A sterile mutant was found in the rice restorer line 202R, and the mutant showed specific female sterility by cytology and pollen tube observation ( figure 1 A), the root cause of its sterility is the stunted growth of the mutant pollen tubes in the style ( figure 1B). G630 was used as the female parent to cross with the mutant to construct a segregated population, the gene was located, and the gene (PTB1) was cloned by fine mapping and map-based cloning technology.

[0068] Sequence comparison analysis showed that 252 nucleotides were deleted in the mutant ORF, resulting in a premature stop codon, which resulted in premature termination of translation and truncation of the expressed protein, resulting in a sterile phenotype in rice. The normal fertile phenotype can be restored by introducing the normal wild-type gene PTB1 into the mutant by transgenic method.

[0069] The sequenc...

Embodiment 2

[0082] Example 2 Expression pattern of PTB 1 gene

[0083] In this example, pBS-PTB1 and PA7-YFP were digested with Bamh I and Spe I restriction enzymes, and a full-length cDNA fragment of about 1.5K of the PTB1 gene was recovered after the digestion of pBS-PTB1, and ligated into the digested After the same restriction site of PA7-YFP, the recombinant was identified by restriction enzyme digestion and sequencing, and the cell-localized transient expression vector PA7-PT-YFP was constructed. Onion epidermal cells were bombarded with a gene gun, and Arabidopsis protoplasts were transiently transformed, and the fluorescence expression was observed by confocal microscopy. The results show that the PTB1 gene is mainly expressed in the cytoplasm in the cell ( figure 2 ).

[0084] The present inventors also studied the tissue expression and localization of the gene. pBS-P was digested with PstⅠ and BamhⅠ restriction enzymes PTB1 and P1300-GUS, recovery of pBS-P PTB1 The digeste...

Embodiment 3

[0085] Example 3 PTB1 gene rice transgenic test

[0086] In this example, the expression vector PHB (Mao et al., 2005, PNAS 102: 12270-12275, commercially available) was used as the vector for rice transgene. The vector encodes a bacterial origin of replication (ori), a kanamycin resistance gene (Kan r ), hygromycin resistance gene (Hyg r ), herbicide resistance genes (Bar r ), a 2×35S promoter, a NOS gene termination signal sequence, and a multiple cloning site (MCS) for ligating fragments of interest. The PTB1 gene or its fragment can be inserted into the restriction endonuclease site to construct a transgenic plasmid.

[0087] 1. Construction of PTB1 gene complementary expression vector

[0088] In this example, pBS-P was first digested with EcoR I and Bamh I restriction enzymes PTB1 and PHB, recovery of pBS-P PTB1 The promoter fragment of the PTB1 gene of about 1.5K after digestion by enzyme digestion is connected to the vector backbone of about 12K after the digesti...

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Abstract

The invention discloses a separated rice female fertility relevant protein. The primary structure of the separated rice female fertility relevant protein is an animo acid sequence as shown in SEQ ID NO.2, or the animo acid sequence shown in SEQ ID NO.2 forms a derived protein with the same function by substitution, missing or adding of one or a plurality of animo acid residues, or the animo acid sequence shown in SEQ ID NO.2 has at least 70% of homology and has the derived protein with the same function. The invention also provides a gene for encoding the protein and derivatives thereof. The protein and the encoding gene thereof can be used for controlling the fertility of rice female organs, or controlling the growth of pollen tubes of the rice, or identifying the rich female fertility.

Description

technical field [0001] The present invention relates to plant genetic engineering, in particular to an isolated rice female fertility-related protein, its encoding gene and its application. Background technique [0002] Plant female sterility is widespread in nature, and has been reported in plants such as Pinus tabulaeformis, soybean, corn, and rice. Female sterility in plants is usually caused by abnormal development of female organs, including abnormal development of ovules, embryo sacs, and egg cells. According to angiosperm reproductive embryology, female sterility can be divided into three categories, the first category is the absence of female organs or incomplete female organ differentiation; the second category is the lack of normal embryo sac in female organs due to the development of ovules or megaspores Blocked; the third type is the normal development of the embryo sac, but the abnormal development of the embryo after fertilization. As far as rice is concerned...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/68A01H5/00
Inventor 李双成李平王世全邓其明王玲霞郑爱萍刘怀年朱军
Owner SICHUAN AGRI UNIV
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