Rice anther epidermis and pollen wall fat deposition gene OsABCG15, recombinant expression vector and application

An expression vector and pollen wall technology, which is applied in the field of genetic engineering, can solve the problems of limited parents, influence, and infertility of cytoplasmic-nucleus interaction sterile lines, and achieves the effect of wide application prospects.

Inactive Publication Date: 2012-07-18
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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  • Rice anther epidermis and pollen wall fat deposition gene OsABCG15, recombinant expression vector and application
  • Rice anther epidermis and pollen wall fat deposition gene OsABCG15, recombinant expression vector and application
  • Rice anther epidermis and pollen wall fat deposition gene OsABCG15, recombinant expression vector and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, osabcg15 Obtaining Mutant Plants and Morphological Observation

[0027] Indica rice Jinhui No. 1 mutant named osabcg15 Mutants were preserved by our laboratory, using II-32B as recurrent parent, and osabcg15 The mutants were crossed and alternated backcrossed, and the mutants were finally preserved. The sterile material and II-32B were a pair of near-isogenic lines. osabcg15Mutants were crossed with II-32B, and the F1 generation was all fertile, and segregation occurred in the self-crossed F2 generation, of which 567 were normal plants and 187 were mutants, and the ratio was 3:1 (χ2 = 0.0071 osabcg15 Morphological observation of mutant plants: II-32B rice ears droop after fruiting ( figure 1 A left), while osabcg15 The mutant spikelets were not fruiting and were erect ( figure 1 A right); II-32B anther contains mature pollen and appears yellow ( figure 1 on B), osabcg15 The mutant anthers are white ( figure 1 Lower B); II-32B pollen...

Embodiment 2

[0028] Embodiment 2, OsABCG15 Gene mapping and cloning

[0029] 1. Positioning groups

[0030] Will osabcg15 The mutant was crossed with the japonica rice Nipponbare, the F2 generation was obtained by selfing, and the male sterile plants were selected as the positioning population.

[0031] 2. Extraction of rice DNA

[0032] 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...

Embodiment 3

[0039] Embodiment 3, OsABCG15 Functional Analysis of Genes

[0040] In order to further determine OsABCG15 The gene is the gene that causes the phenotype of the male sterile mutant, from containing OsABCG15 Gene cloned from rice BAC (bacterial artificial chromosome) AP005446 OsABCG15 Gene and expression control region (comprising exons and introns of 5887bp, 1596bp upstream promoter and 1060bp downstream terminator), the nucleotide sequence is as shown in SEQ ID NO.31, digested with EcoR I and Sbf I , connected into the pCAMBIA1301 vector that was digested with the same restriction enzymes to obtain a 8.5kb pCAMBIA1301- OsABCG15 Recombinant expression vector, transform the obtained recombinant expression vector into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and obtain the recombinant expression vector pCAMBIA1301- OsABCG15 Agrobacterium tumefaciens LBA4404, named LBA4404- OsABCG15 .

[0041] The resulting LBA4404- OsABCG15 Transformed rice osabcg15 ...

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Abstract

The invention discloses a rice anther epidermis and pollen wall fat deposition gene OsABCG15 and protein coded by the gene. The protein comprises the amino acid sequence shown as the SEQ ID NO.1 or the amino acid sequence shown as the SEQ ID NO.1 subjected to substitution, deletion or addition of at least one amino acid and has the function as same as that of the rice anther epidermis and pollen wall fat deposition protein. The invention further discloses a recombinant expression vector of the OsABCG15 gene. The protein coded by the OsABCG15 gene has a function of transferring lipid to form specific ornamentation of the anther and the pollen wall. The gene is necessary for microspore to be developed into fertile pollen. Due to deletion of the gene function, the protective layer of the anther and the pollen wall is not formed and rice male sterility is generated.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to genes for lipid deposition in rice anther epidermis and pollen wall OsABCG15 , also involved in rice anther epidermis and pollen wall lipid deposition genes OsABCG15 Recombinant expression vectors and applications. 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. In my country, the sown area of ​​rice accounts for 30% of the sown area of ​​grain crops in the country, but the total output accounts for more than 42% of the grain crop output. The important reason is the successful application of rice heterosis. [0003] Jones discovered the phenomenon of heterosis in rice as early as 1926. However, rice is a self-fertilizing crop, and it is difficult to obtain a large number of high-purity hybrids, which hinders the process of rice heterosis utilization. It was not until 19...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C07K14/415A01H5/00
Inventor 张毅黄远新吕俊杨昆赵勇王忠伟武丽娜管玉圣
Owner SOUTHWEST UNIVERSITY
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