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Function and application of miR164 genes in controlling development and fertility of root system of rice

A technology of mir164 and sexual function, applied in the application field of rice genetic improvement, can solve the problems such as not found

Inactive Publication Date: 2009-09-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, miRNA transcripts do not contain protein coding regions, and several small ORFs exist on pre-miRNA molecules, but no pre-miRNA molecules that can be translated into proteins have been found so far

Method used

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  • Function and application of miR164 genes in controlling development and fertility of root system of rice
  • Function and application of miR164 genes in controlling development and fertility of root system of rice
  • Function and application of miR164 genes in controlling development and fertility of root system of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Isolation of the precursor pre-miR164 of clone miR164

[0024] Adopt TRIZOL reagent (purchased from Invitrogen Company) to extract leaf total RNA (extraction method according to above-mentioned TRIZOL reagent manual) from rice variety " Nipponbare " (a kind of public report), utilize reverse transcriptase SSII (purchased from Invitrogen Company) to extract cDNA was synthesized by reverse transcription, and the reaction conditions were: 65°C for 5 minutes, 42°C for 120 minutes, and 70°C for 10 minutes. Amplify with primers GPF (5'-GGTACCAATGGTACCTGGCGACACAGAGAGAGA-3', sequence-specific primer plus adapter KpnI site) and GPR (5'-GGATCCAATGGATCCCGAACTGAGCTGGAGAGACA-3', sequence-specific primer plus adapter BamHI) with restriction site adapter The cDNA (899bp) of pre-miR164 was obtained. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; 35 cycles of 30 sec at 94°C, 30 sec at 53°C, and 1 min at 72°C; extension at 72°C for 10 min. The amplifie...

Embodiment 2

[0025] Example 2: Construction and genetic transformation of pre-miR164 overexpression vector

[0026] In order to better analyze the function of miR164, the applicant overexpressed its precursor pre-miR164 in rice. The function of the gene was studied from the phenotype of transgenic plants.

[0027] The overexpression vector construction method is as follows: first, the positive clone pGEM-pre-miR164 plasmid obtained in Example 1 (obtained in Example 1) is double-digested with BamHI and KpnI, and the exogenous fragment is reclaimed; meanwhile, the same method is used to Restriction digestion of the genetic transformation vector pCAMBIA1301U carrying the Ubiquitin promoter was completed, extracted with chloroform:isoamyl alcohol (volume ratio 24:1), and the restriction product was purified. The enzyme-digested fragment containing pre-miR164 and the digested vector were used for ligation reaction to transform Escherichia coli DH10β (the Escherichia coli DH10β strain was purch...

Embodiment 3

[0166] Example 3: Detection of the expression level of rice endogenous miR164

[0167] The rice variety "Minghui 63" (Oryza sativa L.ssp.Indica, a rice variety publicly promoted in China) was used as the material, and the RNA of the following 13 different tissues and organs at different growth stages was extracted and the expression level of mature miR164 was detected by northern blot. The 13 selected tissues are: 1, root at tillering stage; 2, stem at jointing stage; 3, leaf sheath at 4 leaf stage; 5, ear (less than 0.5cm); 6, ear (0.5-1cm); 7, Spike (3-5cm); 8, spike (greater than 10cm); 9, stamen; 10, pistil; 11, seedling germinated for 1 day; 12, seedling germinated for 3 weeks; 13, seedling for 3-week yellow flower. Total RNA was extracted using TRIZOL reagent (purchased from Invitrogen Company) (the extraction method was according to the above-mentioned TRIZOL reagent instruction manual). According to the experimental operation method of Xie et al. (Xie K, Wu C, Xiong L...

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Abstract

The invention relates to the technical field of rice gene engineering. Application of miR164 small RNA of rice for controlling development and fertility of a root system of a plant to genetic improvement of the rice is obtained through separation, cloning and functional verification, and the gene is one of the following nucleotide sequences: a DNA sequence shown in a sequence table SEQ NO:1 and an RNA sequence shown in SEQ NO:2. After connecting with an exogenous promoter, a nucleotide sequence containing miR164 precursor is transferred into the rice, the root system of the transgenic rice is developed and sterile, and the fertility can be recovered by externally applying hormone.

Description

technical field [0001] The invention relates to the technical field of rice genetic engineering. It specifically involves the isolation, cloning and functional verification of a microRNA (miRNA) that can affect plant root development and fertility and the application of its precursor in rice genetic improvement. The miRNA regulates the root development and growth of plants and the fertility of floral organs. The precursor of the miRNA is combined with the ubiquitin promoter (Ubiquitin promoter) and directly transferred to rice, the root development of the transgenic rice plants is significantly enhanced, and the plants become sterile; and the external application of hormones can increase the fertility of the transgenic plants. get partially restored. This technology can be used to cultivate sterile lines or maintainer lines with well-developed root systems, which can be conveniently used in the cultivation of hybrid rice. Background technique [0002] miRNA is a small RNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/11
CPCC12N15/8287C12N15/8262A01H5/0255A01H6/1424
Inventor 熊立仲谢卡斌侯昕
Owner HUAZHONG AGRI UNIV
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