Rice female genic male sterility gene engineering expression vector and application thereof

A technology of genetic engineering and expression vectors, applied in the field of agricultural biology, can solve the problems that cannot fully meet the needs of mechanized seed production

Pending Publication Date: 2022-04-12
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although, the present inventor utilizes the dormant bud regeneration or stripping tiller rapid propagation vegetative propagation system of rice (Patent No.: ZL201310043582.7; Li Shichuan, Liu Fugen, Wang Tianjie, etc. Analysis of characteristics of stripping tiller rapid propagation and iterative regeneration of different rice female sterile lines . Molecular Plant Breeding, 2021, 1-16 [2021-12-27]. http: / / kns.cnki.net / kcms / detail / 46.1068.S.20210624.1518.014.html.) solved the problem of the special female nucleus The reproduction problem of the CMS material itself, but it cannot fully meet the requirements of mechanized seed production for mixed sowing and mixed harvesting

Method used

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  • Rice female genic male sterility gene engineering expression vector and application thereof
  • Rice female genic male sterility gene engineering expression vector and application thereof
  • Rice female genic male sterility gene engineering expression vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: Construction of Genetic Engineering Expression Vector for Rice Female Genic Sterility of the Present Invention

[0069] 1.1 Construction of functional RNAi element (p35S::BTZ-RNAi) of rice CYP81A6 gene

[0070] Through NCBI online sequence alignment, a new rice CYP81A6 gene target site was designed, and an efficient and stable functional RNAi element (BTZ-RNAi) was constructed by targeting the conserved domain region of the rice CYP81A6 gene to screen out transgenic components. Progeny plants (such as figure 1 ), the rice CYP81A6 gene coding sequence is shown in SEQ ID NO.3. The T-DNA region of the functional RNAi element (p35S::BTZ-RNAi) expression vector of rice CYP81A6 gene is as follows figure 2 In A, the specific construction process is as follows:

[0071] The leaves of rice Nipponbare seedlings were used as materials, and the total RNA of the leaves was extracted with Trizol Total RNA Extraction Reagent (Tiangen), and the first-strand cDNA was s...

Embodiment 2

[0076] Example 2 Agrobacterium tumefaciens-mediated rice genetic transformation and the acquisition of transgenic rice plants

[0077] 2.1 Induction of rice anther culture to obtain callus

[0078] At the booting stage of the rice GMS line "Yunling 319FS" (i.e. female GMMS rice plants), cut the rice bracts with a leaf-to-pillow distance of 1-5 cm, cut off the excess leaves and stem nodes, and keep the whole rice bracts After the surface was sprayed with 70% v / v alcohol for surface disinfection, it was wrapped in tin foil to avoid light, placed in an ice box and brought back to the laboratory, and placed in an incubator at 10°C for 10 days in the dark. After the low-temperature pretreatment is completed, wipe the surface of the rice buds with 70% v / v alcohol cotton balls, cut open the rice buds with a scalpel in an ultra-clean workbench, and carefully take out the young ears with tweezers and place them in a sterile petri dish. After the spikelet was cut off from the bottom, t...

Embodiment 3

[0081] Embodiment 3 genetically engineered female nuclear sterile line (FST T / fst) filter

[0082] 3.1 Transgenic T 0 Identification of the female fertility characteristics of the strains and identification of bentazone susceptibility

[0083] transgenic T 0 Whether the strains can develop normally and obtain seeds at the mature stage was used as the standard, and 11 transgenic candidate strains whose female fertility returned to normal were obtained through screening.

[0084] Using wild-type rice female male sterile line "Yunling 319FS" plants as controls, control plants and 11 transgenic T plants were stripped by tiller stripping. 0 The new tillers of the plants are carried out in the greenhouse culture of normal seedlings. When the plants survive and grow new leaves to the 3-leaf stage (seedling stage), spray bentazone solution on the leaves with a small hand-held sprayer. The spray concentration is 1200mg / L. Till water droplets can be seen attached to the leaves. Th...

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Abstract

The invention relates to a rice female genic male sterility gene engineering expression vector and application thereof, and belongs to the technical field of agricultural biology. In order to realize the breeding of seeds and seedlings of the rice female genic male sterile line, the invention provides a technical means of transgene transfer deletion to realize the breeding of a non-transgene homozygous female genic male sterile line. Specifically, after selfing and fruiting of a genetic engineering female genic male sterile line, namely a hybrid type transgenic T0 strain, a transgenic T1 segregation population is obtained, in the seedling stage of the transgenic T1 segregation population, transgenosis female fertility recovery plants are screened out by spraying bentazone herbicide, and survival T1 plants are non-transgenosis female genic male sterile (fst/fst) plants. The non-transgenic female genic male sterile line can meet the mechanical seed production operation of a hybrid rice seed production technical system of 'FS + MS (FM-line)', namely 'female sterile line + male sterile line (FM line method)'.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a genetically engineered rice expression vector for breeding female male sterile lines of rice constructed on the basis of rice FST and CYP81A6 genes and a method applied to the breeding of rice female genetic male sterile lines. [0002] technical background [0003] As the first country in the world to develop and popularize hybrid rice, China began research on male sterility in rice in 1964 (Yuan Longping. Male Sterility of Rice. Science Bulletin, 1966,17:185–188), to 1973 In 1975, the "three-line matching" of rice male sterile line, maintainer line and restorer line was successfully established, and a relatively complete hybrid rice seed production technology system was established in 1975 and began to be popularized and applied (Ren Guangjun, Yan Longan, Xie Huaan . Review and Prospect of Three-line Hybrid Rice Breeding Research. Science Bulletin, 2016, 6...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29A01H5/00A01H6/46A01H1/02
Inventor 李东宣陈丽娟李伟朱骞郭效琼吴文斌余卫霖李仕川罗迪王天杰王春丽
Owner YUNNAN AGRICULTURAL UNIVERSITY
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