Application of LEPTO1 and encoded protein thereof in rice fertility control

A protein activity, protein technology, applied in the application, the use of vectors to introduce foreign genetic material, angiosperms/flowering plants, etc., can solve the problems of less identification of male sterile plants, difficulties, etc., to improve rice quality and increase rice yield. Effect

Active Publication Date: 2019-02-19
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, male sterile plants in natu

Method used

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  • Application of LEPTO1 and encoded protein thereof in rice fertility control
  • Application of LEPTO1 and encoded protein thereof in rice fertility control
  • Application of LEPTO1 and encoded protein thereof in rice fertility control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Cloning of LEPTO1 gene

[0058] 1. Phenotype analysis of lepto1

[0059] Indica rice variety Guangluai 4 60 Co-γ radiation mutagenesis, a series of mutants were obtained.

[0060] Comparison of lepto1 mutant and wild type Guangluai 4, the results are as follows figure 1 , (A) wild type (left) and lepto1 (right) plant comparison; (B) wild type (left) and lepto1 (right) ear comparison; (C) wild type (left) and lepto1 (right) small flower morphology Comparison; (D) I of wild type anther 2 / KI staining; (E) I of lepto1 anther 2 / KI staining. Ruler, 10μm; among them, lepto1 is not significantly different from the wild type in the vegetative growth stage, but it is not firm ( figure 1 A and 1B). However, compared with the wild type, the anthers of lepto1 are small and white ( figure 1 C), I 2 / KI staining shows that lepto1 presents a pollen-free phenotype ( figure 1 D and 1E).

[0061] 2. Map-based cloning of mutant genes

[0062] The plant that can produce shape-separate...

Embodiment 2

[0067] Example 2. Expression pattern of LEPTO1 gene

[0068] 1. Real-time quantitative PCR

[0069] Real-time quantitative PCR was used to detect the expression of LEPTO1 gene in the roots, internodes, leaves and ears of Guangluai 4, and the expression of LEPTO1 gene in lepto1 mutant young ears (LEPTO1). Ubiquitin gene is used as an internal control.

[0070] The result is figure 2 A, R, root; In, internode; L, leaf; P1, ear of 1cm; P2, ear of 2cm; P3, ear of 3cm; P4, ear of 4cm; P10, ear of 10cm. Ubiquitin is used as an internal control, and the error bar represents the standard error of three biological replicates; it shows that LEPTO1 is expressed in different tissues, and the highest expression level is in 1cm ears.

[0071] 2. In situ hybridization

[0072] The pollen of Guanglu'ai No. 4 was used for RNA in situ hybridization to analyze the precise temporal and spatial expression pattern of LEPTO1 gene.

[0073] The result is figure 2 B-2G (2B, two periods; 2C, three times; 2D a...

Embodiment 3

[0074] Example 3, Lepto1 cytological phenotype analysis

[0075] In order to determine the reason why the mutant lepto1 has no pollen, the chromosomal behaviors in the pollen mother cells of Guangluai 4 (wild type) and lepto1 mutants were observed by DAPI staining of knock-off florets and semi-thin resin sections of anthers. .

[0076] The result is image 3 As shown, (A) chromosome morphology in the pollen mother cells of knocked-off florets in wild-type and lepto1; (B) DAPI staining observation of wild-type anther semi-thin resin sections; (C) DAPI staining observation of lepto1 anther semi-thin resin sections ; Ruler, 10μm; It can be seen that in the wild type, the first period after the initiation of meiosis is the pre-thin-line stage, at this time, the chromosomes are scattered and partially agglomerated; in the early-thin-line stage, The chromosomes stretched out. In the thin line stage, the chromosomes are processed to grow long lines; while in lepto1, the florets of diffe...

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Abstract

The invention discloses an application of LEPTO1 protein and the encoded gene thereof in regulating and controlling male fertility of plants. The present invention provides the use of a substance which inhibits the activity and/or level of LEPTO1 protein in the cultivation of male sterile plants, or a substance for inhibiting or reducing the expression of the LEPTO1 protein coding gene in the cultivation of male sterile plants. The inventors of the present invention apply a genetic editing technology to knock out the CRISPR-Cas9 gene in wild-type rice, which causes the development of pollen mother cells to stagnate in the pre-filament line stage and lead to chromosome degradation and final pollen abortion. The site-specific mutation LEPTO1 protein coding gene can be used for rice fertilitycontrol and hybrid seed production in combination with a tissue-specific gene knock-out technology, so that the method has important significance in rice breeding and can provide important biologicalresources for increasing rice yield and improving rice quality.

Description

Technical field [0001] The invention belongs to the field of biotechnology, in particular the application of LEPTO1 and its encoded protein in rice fertility control. Background technique [0002] Rice is one of the most important food crops in the world. Nearly half of the world's population feed on rice. Rice is my country's largest food crop, so how to increase the output of rice is a key issue in my country's agricultural production. With the increase of the world's population, the decrease of arable land and the deterioration of climatic conditions, the demand for the increase of rice output has become increasingly prominent. In the two "green revolutions", rice yields have achieved breakthrough growth. The first "green revolution" took place in the middle of the 20th century. The main feature was to turn the high stalks of rice into short stalks. With the promotion of short stalk varieties, rice yields have been greatly improved, solving problems in many countries. Self-...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/113A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8289C12N2310/10C12N2310/20
Inventor 程祝宽赵婷婷李亚非唐丁沈懿杜桂杰
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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