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Preparation method of rice miRNA homozygous lethal mutant

A lethal mutation, rice technology, applied in the field of plant biology, can solve the problem of inability to germinate the lethal mutant seeds of rice

Pending Publication Date: 2021-05-28
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to use the CRISPR-Cas12a system to knock out rice OsmiR390 to obtain rice lethal mutant seeds that cannot germinate

Method used

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  • Preparation method of rice miRNA homozygous lethal mutant
  • Preparation method of rice miRNA homozygous lethal mutant
  • Preparation method of rice miRNA homozygous lethal mutant

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preparation example Construction

[0031] Furthermore, the present invention provides a method for preparing rice miRNA homozygous lethal mutants. The method includes the following steps:

[0032] a, preparing the expression vector according to any one of claims 2 to 6;

[0033] b, using the expression vector obtained in step a to transform rice, and using the CRISPR-Cas12a gene editing system to obtain transformed plants;

[0034] c. Collecting the seeds of the transformed plants, and screening out the homozygous seeds for miR390 knockout to obtain the rice miRNA homozygous lethal mutant.

[0035] Based on the content of the present invention disclosed above, those skilled in the art can easily use existing materials and plant molecular biology methods to implement the above method and obtain rice miRNA homozygous lethal mutants.

[0036] Specifically, the preparation method of the rice miRNA homozygous lethal mutant in the technical solution of the present invention can be carried out according to the follo...

Embodiment 1

[0044] Example 1 Construction of rice miR390 knockout vector CRISPR-Cas12a

[0045] (1) crRNA design

[0046] The crRNA was designed according to the recognition and cleavage rules of the target site by CRISPR-Cas12a. According to the rice OsmiR390 precursor genome sequence (SEQ ID No.4), design single-stranded nucleotide sequence OsmiR390-crRNA1-F (sequence as shown in SEQ ID No.5) and OsmiR390-crRNA1-R (sequence as shown in SEQ ID No.6) Show).

[0047] (2) Annealing of single-stranded nucleotide sequences

[0048] Dilute the upstream and downstream OsmiR390-crRNA1-F and OsmiR390-crRNA1-R single-stranded nucleotide sequences of the target site by 10 times, take 10 μL each, denature at 98°C for 5 minutes, cool naturally, and dilute the annealed product 20 times for use.

[0049] (3) Digestion, gel recovery, connection

[0050] The backbone vector used in the experiment was pTX377, which was constructed by our laboratory based on the vector pYPQ230 shown in the literature (...

Embodiment 2

[0059] Rice genetic transformation mediated by embodiment 2 Agrobacterium

[0060] The method disclosed in Agrobacterium-mediated transformation of rice reference (Toki et al., Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. The Plant Journal, 2006, 47(6):969-976.). The genetic transformation steps of rice are as follows:

[0061] The mature seeds of rice (Nihonbare) are dehulled and sterilized; the sterilized seeds are inoculated on the N-6-D solid medium containing 0.4% gellan gum, and cultivated under continuous light at 32° C. for 1 to 5 days; the cultivated seeds are passed through the agricultural The bacillus-mediated transformation method transfers the plasmid pMIR390-1 into rice, and the transformed rice seeds are continuously cultured in the induction selection medium at 32°C under light for 2 weeks; the callus produced by proliferation is transferred into the RE-III medium; Young plants produced from callus were tran...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a preparation method of a rice miR390 homozygous lethal mutant. The technical problem to be solved by the invention is to obtain rice lethal mutant seeds which cannot germinate. The technical scheme of the invention is to provide crRNA for directional knockout of a rice OsmiR390 gene in a CRISPR-Cas12a gene editing method, and a method for preparing a rice miR390 homozygous lethal mutant. According to the method disclosed by the invention, the rice OsmiR390 gene can be effectively knocked out, and a functional deletion mutant with a homozygous lethal phenotype is obtained, so that the method has a good prospect in the aspects of genome function research of rice and research and an application of miRNAs.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for preparing rice miR390 homozygous lethal mutants. Background technique [0002] MicroRNAs (miRNAs) are a class of non-coding endogenous small molecule RNAs (generally 19-24nt) in eukaryotes, which regulate gene expression by splicing target mRNA or inhibiting translation, thereby performing post-transcriptional level regulation. It plays an important role in the process of plant organ formation, growth and development, signal transduction and abiotic stress response. The MicroRNA390 (miR390) family is an ancient and highly conserved family. Its main target gene, AGO7, is an important component of the RNA silencing complex, and is widely involved in the splicing of target miRNAs. It plays a role in sexual formation, floral organ formation and stress. However, the exact function of miR390 in rice is still poorly understood, mainly due to the lack of mut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC12N15/113C12N15/8218C07K14/415C12N2310/20
Inventor 周建平张勇郑雪莲全泉祁彩燕唐旭
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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