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Oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, mutant gene, and recombinant expression vector and application thereof

A technology for pollen germination and fertility genes, which is applied in the field of genetic engineering, can solve the problems of limited parents, infertility of cytoplasmic-nucleus interaction sterile lines, high risk of large-scale seed production, etc., and achieves the effect of wide application prospect.

Inactive Publication Date: 2017-06-13
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of free mating of the plasmogenetic-nuclear interaction male sterile line, the available parents are limited, and the seed production is more complicated than the two-line method. The fertility of the temperature-photosensitive male sterile line is affected by natural temperature and light conditions, and the risk of large-scale seed production is high. and other reasons, the heterosis of rice cannot be fully reflected through these two male sterile lines

Method used

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  • Oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, mutant gene, and recombinant expression vector and application thereof
  • Oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, mutant gene, and recombinant expression vector and application thereof
  • Oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, mutant gene, and recombinant expression vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Obtaining and morphological observation of embodiment 1, osaom mutant plant

[0029] The osaom mutant is derived from the mutation of the indica rice Minghui 63. The normal Minghui 63 is used as the male parent to cross with the osaom mutant, and the mutant gene is preserved in the hybrid. Mutant osaom can be isolated. osaom mutant crossed with Minghui 63, F l All generations are fertile, inbred F 2 Segregation occurred in the generation, among which 293 were normal plants and 120 were mutant plants, and the ratio was consistent with It indicated that the phenotype of the male sterile mutant was caused by a nuclear gene mutation. Morphological observation of osaom mutant plants: Minghui 63 rice ears droop after fruiting ( figure 1 , A), while the spikelets of the osaom mutant were not fruiting and were erect ( figure 1 , B); Minghui 63 anthers were thick and plump ( figure 1 , C), the osaom mutant had thinner anthers ( figure 1 , D); Minghui 63 pollen iodine ...

Embodiment 2

[0030] Embodiment 2, location and cloning of OsAOM gene

[0031] 1. Positioning groups

[0032] F 2 In the next generation, the male sterile plants were selected as the positioning group.

[0033] 2. Extraction of rice DNA

[0034] Parents were extracted using the improved CTAB method, including the following steps: Take 0.1-0.2 grams (about half a piece) of leaves and put them in a small mortar, add an appropriate amount of liquid nitrogen, grind them to powder immediately, put them into a 2m1 centrifuge tube, and add 700μL Put the 1.5×CTAB solution preheated at 100°C in a centrifuge tube, mix it carefully, put it in a 65°C water bath, take out the centrifuge tube after 20 minutes, add an equal volume of chloroform / isoamyl alcohol, mix vigorously, centrifuge at 13000rpm for 10 minutes, and take Put the supernatant in a new tube, add 900 μL of absolute ethanol and mix well, then put it at -20°C for more than half an hour. The precipitated DNA was centrifuged at 14000 rpm f...

Embodiment 3

[0041] Example 3, Functional analysis of OsAOM gene

[0042] In order to further confirm that the OsAOM gene is the gene that causes the phenotype of the male sterile mutant, the OsAOM gene and its expression regulatory region (comprising 5603 bp of exons and introns) were expanded from the rice BAC (bacterial artificial chromosome) OSJNBb0062H16r containing the OsAOM gene. promoter, 1254bp upstream promoter and 1589bp downstream terminator), the nucleotide sequence is shown in SEQ ID NO.25. The specific operation is segmented amplification and connection into the pCAMBIA1301 vector: using the single restriction site XbaI in the middle of the coding region, a restriction site KpnI is added to the end of the first half of the upstream primer CF1, and the first half of the downstream primer CR1 is located at the XbaI position After the point, the second half of the upstream primer CF2 is located in front of the XbaI site, and the end of the second half of the downstream primer C...

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Abstract

The invention relates to an oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, a mutant gene, and a recombinant expression vector and application thereof. The gene encoded protein is protein with the amino acid sequence shown as SEQ ID NO.1, or protein which is obtained by substituting, missing or adding at least one amino acid and has the same function as the OsAOM; the protein formed by the amino acid sequence shown as SEQ ID NO.1 has the nucleotide sequence shown as SEQ ID NO.2. The invention also discloses the recombinant expression vector of the OsAOM gene. The OsAOM gene disclosed by the invention encodes one piece of GDSL lipase; the functions of regulating and controlling the pollen germination aperture growth, the microspore cell membrane expansion and the pollen inner wall and cytoplasm development are realized; the gene is a necessary gene for developing microspores into fertile pollen; the gene function deficiency can cause germination aperture structure disorder, microspore cell membrane and cytoplasm degradation, pollen inner wall formation incapability and oryza sativa male sterility.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to rice pollen germination pore development and pollen fertility gene OsAOM, a mutant gene of rice pollen germination pore development and pollen fertility gene OsAOM, and also relates to rice pollen germination pore development and pollen fertility gene OsAOM Applications in the regulation of pollen fertility. Background technique [0002] Rice (Oryza sativa) is one of the main food crops, and about half of the world's population uses rice as a staple food. The successful application of heterosis has played an important role in the improvement of rice varieties in my country. Rice is a self-fertile crop, and the utilization of its heterosis depends on the selection of sterile lines. [0003] At present, the heterosis of rice is mainly realized through the cytoplasmic-nuclear interaction sterile line (three-line method) and the temperature-photosensitive sterile line ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/20C12N15/82A01H5/00
CPCC12N9/20C12N15/8289C12Y301/01003
Inventor 张毅吕俊杨昆黄远新管玉圣武丽娜
Owner SOUTHWEST UNIVERSITY
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