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Two oligo DNA groups of sgRNA for fixedpoint knockout of rice OsPLS4 gene

A site-specific knockout, DNA sequence technology, applied in the field of plant genetic engineering, can solve the problems of weakened accumulation, weakened drought resistance and water retention capacity, etc.

Pending Publication Date: 2020-11-03
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been more and more studies on the surface wax of plants, and many research results show that the accumulation of wax on the surface of rice is weakened, which leads to the weakening of its ability to resist drought and water retention.

Method used

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  • Two oligo DNA groups of sgRNA for fixedpoint knockout of rice OsPLS4 gene
  • Two oligo DNA groups of sgRNA for fixedpoint knockout of rice OsPLS4 gene
  • Two oligo DNA groups of sgRNA for fixedpoint knockout of rice OsPLS4 gene

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Experimental program
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Embodiment 1

[0026] Embodiment 1, design and synthesis of rice OsPLS4 gene sgRNA sequence

[0027] The coding region sequence of the rice OsPLS4 gene is shown in SEQ ID NO.9.

[0028] In this embodiment, the CRISPR / Cas9 editing target sequence is 19bp in length, located in the 672-690 and 766-784 nucleic acid sequences of the eighth and ninth exons of the conservative sequence of the OsPLS4 gene coding region, and the edited target sequence is (SEQ ID NO .1): TTACAGTGCTGCCAAGGCT, (SEQ ID NO.2): TGTCAGATGCAATGAAACC, this sequence is specific on the rice genome, and the off-target probability is extremely low.

[0029] Synthesize four oligo DNAs according to the target sequence:

[0030] (SEQ ID NO. 3)PLS4(1)F: GGCATTACAGTGCTGCCAAGGCT

[0031] (SEQ ID NO. 4)PLS4(1)R: AAACAGCCTTGGCAGCACTGTAA

[0032] (SEQ ID NO. 5)PLS4(2)F: GGCATGTCAGATGCAATGAAACC

[0033] (SEQ ID NO. 6)PLS4(2)R: AAACGGTTTCATTGCATCTGACA

Embodiment 2

[0034] Example 2, OsPLS4 editing vector pYL-Hs-PLS4-cas9 construction

[0035] Add ddH to two oligo DNA groups PLS4(1)F and PLS4(1)R, PLS4(2)F and PLS4(2)R respectively 2 Dissolve O to 10 μM, mix according to the reaction system, heat at 95°C for 3 minutes, cool and anneal naturally to form a dimer structure between PLS4(1)F / R and PLS4(2)F / R, and form a dimer structure through annealing reaction , and then respectively connected with the vector pYL-Hs that fused the rice U3 promoter and gRNA backbone sequence. The ligation product was transformed into Escherichia coli by the heat shock method, and a single colony was picked and cultured in LB liquid medium (+kan) for 12 hours, and then sequenced. Select the bacteria with correct sequencing to extract the plasmid DNA, and obtain the editing vector pYL-Hs-PLS4-cas9 containing the target sequence of the rice OsPLS4 gene. For the vector map, see figure 1 .

Embodiment 3

[0036] Embodiment 3, pYL-Hs-PLS4-cas9 carrier Agrobacterium transformation

[0037] Transform pYL-Hs-PLS4-cas9 plasmid into Agrobacterium by freeze-thaw method, add 10 μL of plasmid DNA to 200 μL of Agrobacterium EHA105 competent, ice bath for 30 minutes, freeze in liquid nitrogen for 3 minutes, 37 ° C water bath for 5 minutes, add 1 mL of YEB medium , shake culture at 28°C for 3-4h. Centrifuge at 6000 rpm for 1 min at room temperature, discard the supernatant, add 200 μL of YEB medium to resuspend the bacteria, spread on YEB solid medium (+rifampicin), and incubate at 28°C for 3 days. Single-pick colony identification.

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Abstract

The invention provides sgRNA for knocking out a rice OsPLS4 gene. An sgRNA sequence based on CRISPR / Cas9 is designed for the rice OsPLS4 gene, a DNA fragment containing the coded sgRNA sequence is connected to a carrier carrying the CRISPR / Cas9, rice calluses are transformed with an agrobacterium-mediated method, and the rice OsPLS4 gene is knocked out through screening and identification, whereinthe nucleotide sequence of the sgRNA action site is as shown in SEQ ID NO. 1. The rice endogenous gene OsPLS4 is edited through a CRISPR-CAS9 technology, and an OsPLS4 knockout mutant is obtained. The sgRNA prepared by the invention can efficiently, quickly and accurately target the rice OsPLS4 gene, and has certain significance in fundamental research (rice premature senility molecular mechanism) and production practice (rice premature senility variety improvement and stress-resistant breeding).

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to two oligo DNA groups and applications based on CRISPR-CAS9 technology to knock out the sgRNA of rice OsPLS4 gene. Background technique [0002] The OsPLS4 gene encodes 3-acyl reductase. The function of this enzyme has been studied more in bacteria and animals. It is mainly involved in the second stage of fatty acid de novo synthesis. It belongs to KR protease and is a member of Fatty Acid Synthase (FAS) An important component, and FAS enzyme is one of the important complexes of fatty acid synthesis. The processes of fatty acid synthesis include de novo synthesis of saturated fatty acids, elongation of fatty acid carbon chains, and formation of unsaturated fatty acids. [0003] In plants, epidermal wax is the first barrier for plants to resist external biotic and abiotic stresses. It is mainly composed of ultra-long-chain fatty acids and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113C12N15/82C12Q1/6895A01H5/00A01H6/46
CPCC12N15/1137C12N15/8218C12Q1/6895C12N9/0006C12N2310/20C12Q2600/13
Inventor 周大虎徐杰贺浩华姜志树李婷林小玲范寒雨马莹莹朱昌兰彭小松陈小荣付海辉欧阳林娟
Owner JIANGXI AGRICULTURAL UNIVERSITY
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