Anther tapetum specific expression promoter POsABCG15, recombinant expression vector thereof and application thereof

An anther tapetum and expression vector technology, which is applied in the field of genetic engineering, can solve the problems of limited parents, unfree combination 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: 2012-07-11
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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  • Anther tapetum specific expression promoter POsABCG15, recombinant expression vector thereof and application thereof
  • Anther tapetum specific expression promoter POsABCG15, recombinant expression vector thereof and application thereof
  • Anther tapetum specific expression promoter POsABCG15, recombinant expression vector thereof and application thereof

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, osabcg15 Obtaining Mutant Plants and Morphological Observation

[0026] 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. osabcg15 Mutants 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

[0027] Embodiment 2, OsABCG15 Gene mapping and cloning

[0028] 1. Positioning groups

[0029] 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.

[0030] 2. Extraction of rice DNA

[0031] 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 wel...

Embodiment 3

[0038] Embodiment 3, anther tapetum-specific expression promoter POsABCG15 analyze

[0039] The analysis found that the finely positioned OsABCG15 The upstream 2034bp of the gene has an RNA polymerase recognition site, and the nucleotide sequence is shown in SEQ ID NO.31, named as anther tapetum-specific expression promoter, referred to as POsABCG15 . Primers are designed according to the sequence shown in SEQ ID NO.31, the upstream primer is P OsABCG15F:5'-gac aagctt cgaaatgctcggttatatg-3' (SEQ ID NO.32), the underline is the Hind III restriction site; the downstream primer is P OsABCG15R:5'-gc tctaga ctcctccaagagacacag-3' (SEQ ID NO.33), the underline is the Xba I restriction site, amplified by PCR, the PCR reaction conditions are: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 45 seconds, annealing at 61°C for 45 seconds, Extend at 72°C for 2 minutes, 28 cycles, and finally extend at 72°C for 10 minutes. The PCR product was identified by agaros...

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Abstract

The invention discloses an anther tapetum specific expression promoter POsABCG15. The promoter has a nucleotide sequence shown as SEQ ID NO. 31. The promoter can regulate and control a target gene, so that the target gene is expressed to anther tapetum specifically; and the POsABCG15 in rice regulates and the controls the expression of a lipid deposition gene OsABCG15 on anther epidermis and pollen wall of the rice and influences the accumulation of lipids on surfaces of anther and pollen, so that the anther tapetum specific expression promoter POsABCG15 can be used for generating a new rice male sterile line or regulating and controlling the expression of other genes in the tapetum, and has an important effect in agricultural production.

Description

[0001] technical field [0002] The invention belongs to the field of genetic engineering, in particular to an anther tapetum-specific expression promoter POsABCG15 , also involving the anther tapetum-specific expression promoter POsABCG15 Recombinant expression vectors and applications. Background technique [0003] 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. 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 1970 that Libihu in my country discov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/02
Inventor 吕俊张毅王忠伟武丽娜管玉圣黄远新杨昆赵勇
Owner SOUTHWEST UNIVERSITY
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