Vector sgRNA CRISPR/Cas9 relevant with character of rice reddish brown glume, vector construction and application

A carrier and rice technology, applied in the field of genetic engineering, can solve problems such as low efficiency and difficulty in obtaining homozygous mutants, and achieve high efficiency

Inactive Publication Date: 2018-05-15
SAAS BIOTECH & NUCLEAR TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that both alleles of OsCHI need to be mutated to produce brown rice, and the efficiency of mutating two genes at the same time is low, and it is difficult to obtain homozygous mutants of two gene mutations

Method used

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  • Vector sgRNA CRISPR/Cas9 relevant with character of rice reddish brown glume, vector construction and application
  • Vector sgRNA CRISPR/Cas9 relevant with character of rice reddish brown glume, vector construction and application
  • Vector sgRNA CRISPR/Cas9 relevant with character of rice reddish brown glume, vector construction and application

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Such as figure 1 As shown in a, the sgRNA fragment design of the present invention.

[0033] Genetic material: rice cinnamyl alcohol dehydrogenase (CAD) gene, the full name of which is Oryza sativa (japonicacultivar-group) cinnamyl alcohol dehydrogenase (CAD) gene, GenBank: BK003969.1, see http: / / www.ncbi.nlm .nih.gov / nuccore / 64519465. The base sequence of the coding region of the gene is shown in SEQ ID NO.7.

[0034] Select three sgRNA targeting sites on the rice cinnamyl alcohol dehydrogenase (CAD) gene, refer to http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR target site prediction and design, use http: / / www.rgenome.net / cas-offinder / Analysis of off-target effects. Finally, three sgRNAs were selected and named as T1, T2, T3 ( figure 1a). The base sequences of T1, T2, and T3 are shown in SEQ ID NO.1, 2, and 3, respectively.

[0035] The gRNAs contained in the three sgRNA fragments all specifically target the fifth exon (SEQ ID NO.6) of the rice cinnamyl alcohol dehydrog...

Embodiment 2

[0037] Such as figure 1 As shown, the CRISPR / Cas9 gene editing vector comprising the sgRNA fragment of the present invention was constructed.

[0038] The three sgRNA fragments obtained in Example 1 were respectively ligated into rice OsU6a (position 1-485 in SEQ ID NO.8), OsU6b (position 1-371 in SEQ ID NO.13), OsU6c (position 1 to 780) downstream of the three promoters, followed by the Ploy-T terminator (positions 505 to 628 in SEQ ID NO.8, positions 391 to 515 in SEQ ID NO.13, and position 800 in SEQ ID NO.18 ~923 bits). The specific operation takes T1 as an example. First, use the primers of the primers SEQ ID NO.10 and SEQ ID NO.9 containing the T1 sequence and the primers of SEQ ID NO.11 and SEQ ID NO.12 to carry out the first round of PCR to obtain two PCR products, and then Using these two PCR products as templates and using SEQ ID NO.9 and SEQ ID NO.12 as primers, a second round of PCR was performed to obtain OsU6a::T1 (SEQ ID NO.8). Similarly, OsU6b::T2 (SEQ ID NO...

Embodiment 3

[0042] The application of the CRISPR / Cas9 gene editing vector of the present invention in gene editing specifically targeting the rice cinnamyl alcohol dehydrogenase gene.

[0043] Plant material: Rice Nipponbare (Oryza.Sativa L.spp.japonica)

[0044] The final vector obtained in Example 2 was used to transform Escherichia coli DH5α, single clones were picked and sequenced, and the correctly sequenced strains were amplified, and the extracted plasmids were transferred into Agrobacterium strain EHA105 to obtain bacterial liquid for dipping.

[0045] Mature Nipponbare seeds were used as materials. The seeds were sterilized with 1‰ mercury liter, washed five times with sterile water, and cultured on MS medium to induce callus. The mature embryo callus of rice Nipponbare was obtained by culturing under light at 30°C for two weeks. Cut the callus into 2mm-3mm pellets and culture them on fresh MS medium.

[0046] After 4 days, the EHA105 bacterial solution was used to impregnate t...

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Abstract

The invention discloses a vector sgRNA CRISPR / Cas9 relevant with character of rice reddish brown glume, vector construction and application, for overcoming the defect that rice with reddish brown glume can be acquired only on the condition of mutation of two alleles OsCHi. The invention provides an sgRNA segment which comprises a gRNA of which specificity is targeted to the fifth exon of cinnamylalcohol dehydrogenase(CAD) gene of rice; the sgRNA refers to T1, T2 and T3 specifically. The invention further provides a CRISPR / Cas9 gene editing vector comprising the sgRNA and a construction methodof the vector. The vector can edit cinnamyl alcohol dehydrogenase(CAD) gene of rice. Besides, application of the segments and the vector in acquiring rice having characters of reddish brown glume andstem earing, and primer sequence for detecting CRISPR / Cas9-CAD gene reconstruction are also provided. According to the invention, conventional breeding cost can be reduced, mechanization level of hybrid rice seed production can be improved, and seeding cycle can be shortened.

Description

technical field [0001] The present invention relates to a sgRNA fragment, a CRISPR / Cas9 editing vector, a vector construction method, and an application related to rice chaff traits of reddish-brown chaff. It belongs to the field of genetic engineering. Background technique [0002] Using natural mutation and artificial mutation, a small amount of breeding materials with different glume colors can be obtained from rice germplasm resources. The different colors of glumes can be used to accurately separate the hybrids on the sterile line from the restorer seeds harvested with a harvester in rice mechanized seed production. Therefore, different glume colors have a high effect in hybrid rice seed production. technical value. However, if the traditional hybridization and backcrossing methods are used to transfer the shell color traits to restorer lines and maintainer lines without changing the characteristics of the original materials, it will take nearly ten years to achieve t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11C12Q1/6895A01H5/00A01H4/00A01H6/46
CPCA01H4/00C12N15/113C12N15/8213C12N2310/10C12Q1/6895
Inventor 蒲志刚向小利王平
Owner SAAS BIOTECH & NUCLEAR TECH RES INST
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