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Dwarf male-sterile rice cultivation method and DNA (Deoxyribonucleic Acid) used thereby

A technology of rice and rice, which is applied in the direction of recombinant DNA technology, DNA / RNA fragments, botanical equipment and methods, etc., can solve the problem of difficult to obtain rice plants, and achieve the effect of convenient cultivation

Inactive Publication Date: 2011-09-07
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Practice has proved that it is difficult to obtain rice plants that exhibit both male sterility and dwarf traits through conventional hybridization techniques in rice

Method used

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  • Dwarf male-sterile rice cultivation method and DNA (Deoxyribonucleic Acid) used thereby
  • Dwarf male-sterile rice cultivation method and DNA (Deoxyribonucleic Acid) used thereby
  • Dwarf male-sterile rice cultivation method and DNA (Deoxyribonucleic Acid) used thereby

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the construction of hairpin RNAi expression vector IRSACK

[0043] 1. Materials

[0044] 1. Rice varieties for testing: Japonica rice commercially available varieties: QX1, Nongyuan 238, and Jijing 88.

[0045] 2. Bacterial strains: Escherichia coli (Escheriehia coli) DH5a and DH10B.

[0046] 3. Vector: pGEM-Teasy vector was purchased from Promega Company; RNAi vector pTCK303, its construction method can be found in ZHENWANG et al.A Practical Vector for Efficient Knockdown of Gene Expression in Rice (Oryzasativa L.).Plant Molecular Biology Reporter 22:409 -417. December 2004 2004 International Society for Plant Molecular Biology. Printed in Canada. The Crop Research Institute of the Chinese Academy of Agricultural Sciences is also preserved.

[0047] 2. Method

[0048] 1. Primer design

[0049] (1) According to the RTS gene sequence (see GenBank accession number U12171.1), a pair of primers RTSF and RTSR capable of amplifying the entire rts fragment...

Embodiment 2

[0194] Embodiment two, hairpin RNAi expression vector IRSACK transforms rice

[0195] 1. Materials

[0196] (1) Receptor materials for rice transformation: rice varieties Nongyuan 238 and Jijing 88.

[0197] (2) Strains: Escherichia coli DH5a and DH10B and Agrobacterium tumefacieus strain EHA105.

[0198] 2. Method

[0199] 1. Preparation of Agrobacterium Competent Cells

[0200] (1) Plate Agrobacterium tumefaciens EHA105 on LB (containing rifampicin 40mg / L form f) solid medium, culture at 28°C for 2-3 days, and pick a single colony.

[0201] (2) Inoculate a single colony of EHA105 in 2ml of SOB culture solution, take the seed solution after 12 hours, inoculate it in 400ml of SOB culture solution, cultivate to OD at 28°C, 180rmp 550 . = 0.5-0.6. The following operations were performed on ice.

[0202] (3) Collect Agrobacterium by centrifugation at 2200 rpm, 4° C. for 10 minutes, and suspend the bacteria with 10% glycerol.

[0203] (4) 2500rpm, 4°C, centrifuge for 10 mi...

Embodiment 3

[0258] Dwarfing degree and fertility of embodiment three transgenic plants

[0259] In order to verify that the transgenic plants exhibiting both dwarf and pollen abortion traits are indeed caused by the RNAi technology adopted in the present invention, the transgenic plants are selected for PCR amplification to verify whether there is a transformed gene sequence. RTSF-B was used for the upstream primer, and the primer In-cla (5'->3'GAGGCGGTACAATGATCA ACCATGA) designed for the downstream primer according to the intron (Intron) sequence in the RNAi vector pTCK303 was used to select the transgenic plants prepared in Example 2 for PCR amplification. increase. Among them, the PCR amplification parameters are as follows:

[0260] The reaction system is:

[0261]

[0262] Reaction parameters: Denaturation at 94°C for 5 minutes, then PCT cycle, namely 94°C for 1 min, 56°C for 1 min, 72°C for 90 seconds, a total of 37 cycles, and finally 72°C for 10 minutes.

[0263] The PCR amp...

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Abstract

The invention discloses a cultivation method for obtaining rice plants, which simultaneously show male-sterile and dwarf properties, through genetic engineering. Dwarf male-sterile rice with simultaneous incomplete expression of male-fertile genes and plant height genes is cultivated with an RNAi (Ribonucleic Acid interfere) technology by using a GA20-oxidase gene (OsGA20o*2), which has an effect of stretching rice stems and is used for gibberellin synthesis, and an RTS (Ricetapetum-Specific) gene which plays a key role in development of rice anther tapetum; the male-sterile and dwarf properties of the dwarf male-sterile rice simultaneously occur; and the rice can be normally fertilized to bear fruits after receiving wild type plant pollen. The dwarf male-sterile rice can be widely applied to rice recurrent selection breeding and has great significances to rice genetic breeding and production.

Description

technical field [0001] The present invention relates to a method for cultivating rice plants through genetic engineering technology, in particular to a method for cultivating rice plants with simultaneous expression of male sterility and dwarf traits, and more specifically relates to the use of RNAi technology to control genes related to rice anther development and stem elongation. The expression of long-acting genes is interfered and down-regulated or silenced at the same time, so that the rice plants show dwarf traits and male sterility at the same time, and present complete linkage dominant inheritance. Background technique [0002] Recurrent selection is a periodic population improvement method. Based on a population with rich genetic background, through periodic repeated outcrossing and selection, beneficial genes are assembled, so that the frequency of good or synergistic genes in the population gradually increases, and the frequency of bad or synergistic genes graduall...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00C12R1/91
Inventor 赵开军王坚王春连高英刘丕庆
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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