Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice

A rice and carrier technology, applied in the field of plant genetic engineering, can solve the problems of large blindness, difficulty in obtaining mutants, and difficult realization of miRNA, and achieve the effect of simple operation and high knockout rate

Active Publication Date: 2017-02-01
成都极谷基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are time-consuming, labor-intensive, blind, and difficult to implement in the study of miRNA functions, mainly because miRNAs are very small (about 21bp), and mutants are difficult to obtain

Method used

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  • Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice
  • Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice
  • Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Using the method of the present invention to create a single miRNA mutant (taking rice OsmiR528 as an example)

[0040] 1. Vector construction of CRISPR-Cas9-miRNA mutants

[0041] (1) Primer design

[0042] Primers were designed according to the recognition and cutting rules of the target site by CRISPR / Cas9. For the precursor genome sequence of rice OsamiR528 (GenBank number: GQ419957.2), the primers designed were OsmiR528-sgRNA1-F: 5'-gtgtGAAGGGGCATGCAGAGGAGC-3'; OsmiR528-sgRNA1-R: 5'-aaacGCTCCTCTGCA TGCCCCTTC-3'.

[0043] (2) Primer annealing

[0044] Dilute the upstream and downstream primers of each target site 10 times, take 10 μL each, denature at 98°C for 5 minutes, cool naturally, and dilute the annealed product 20 times for use.

[0045] (3) Digestion, gel recovery, connection

[0046] The backbone vector used in the experiment was pZHY988 ( figure 1 ), constructed by our laboratory, the construction process is as follows: first synthesize (syn...

Embodiment 2

[0090] Example 2 Using the method of the present invention to create multiple miRNA mutants (taking rice OsmiR397a and OsmiR97b as examples)

[0091] 1. Vector construction of CRISPR-Cas9-miRNAs mutants

[0092] (1) OsmiR397a and OsmiR397b double-site knockout vector construction method

[0093]The OsmiR397 family has two members, OsmiR397a (GenBank number: AP014962:28489785...28489898) and OsmiR97b (GenBank number: XM_015769221). In order to obtain the OsmiR397 knockout mutant, a two-site knockout mutation vector in which OsmiR397a and OsmiR97b were simultaneously knocked out was constructed. According to the precursor sequence of OsmiR397a and OsmiR97b, design and synthesize OsmiR397a-sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, OsmiR397b-sgRNA1 sequence OsmiR397a-gRNA1-OsmiR397b-gRNA1-F and OsmiR397a-gRNA1-OsmiR397b-gRNA1- R (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.). OsmiR397a-gRNA1-OsmiR397b-gRNA1-F:

[0094] gtgtGAGTGCAGCGTTGATGAACAAGTTTTAGAGCTAG...

Embodiment 3

[0105] Example 3 Using the method of the present invention to create miRNA large fragment deletion mutants (taking rice OsmiR408 as an example)

[0106] 1. Vector construction of CRISPR-Cas9-miRNAs mutants

[0107] (1) OsmiR408-sgRNA1 and OsmiR408-sgRNA2 double-site knockout vector construction method

[0108] When considering knocking out all or most of the precursor miRNAs or when it is difficult to find a suitable PAM site near some mature miRNA precursors, it is necessary to consider the strategy of using double-site knockout to create large fragment deletions.

[0109] According to the precursor sequence of OsmiR408, the sequences OsmiR408-gRNA1-gRNA2-F and OsmiR408-gRNA1-gRNA2-R containing sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, and sgRNA2 were designed and synthesized (submitted by Shanghai Yingjun Biotechnology Co., Ltd. Ltd Synthetics).

[0110] OsmiR408-gRNA1-gRNA2-F:

[0111] gtgtGATGAGGCAGAGCATGGGATGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT...

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Abstract

The invention belongs to the technical field of plant genetic engineering and specifically relates to a method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice. Aiming at solving the technical problems, a method and a detection means are provided for directionally modifying miRNA sites of plants. The invention discloses a method for obtaining miRNA mutants of the paddy rice; specific operation of the method comprises: knocking out single miRNA, knocking out a plurality of miRNAs or knocking out large fragments of the miRNA by adopting a CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- associated 9) method. The method provided by the invention is suitable for creation and screening of directionally knocking out the miRNA of the paddy rice.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for directional knockout of rice miRNA. Background technique [0002] miRNA (microRNA) is a kind of small non-coding RNA with a length of about 19-24 nucleotides that is ubiquitous in eukaryotes. It is encoded by the endogenous miRNA gene and transcribed by RNA polymerase II. It mainly regulates the expression level of its target gene mRNA through shear degradation, inhibition of translation, and chromosomal remodeling (methylation). miRNA plays an important role in plant growth and development, metabolism, epigenetics, and abiotic stress response. [0003] The traditional methods to study the function of miRNA mainly include: (1) Indirect method to study the function of miRNA by transgenic expression of miRNA to inhibit the function of its target gene. However, since miRNAs often regulate multiple target genes with the same or different f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/113C12N15/8216C12N2310/141C12N2800/80C12N2810/10
Inventor 周建平张勇郑雪莲唐旭程燕邓科君
Owner 成都极谷基因科技有限公司
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