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Blocking or weakening the expression of rice circRNA coding site to improve the method of rice seedling growth traits

A technology for growth traits and coding bits, which is applied in the field of plant biology to achieve the effects of easy operation, good quality improvement and concise steps

Active Publication Date: 2022-04-01
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In rice, the CRISPR-Cas system is mainly used to edit coding genes, and there are also some reports on the editing of non-coding miRNAs, and the directional editing (knockout) of non-coding circRNAs and the effective analysis of corresponding biological functions, and then mining has breeding value The work of plant circRNA loci has not been reported yet

Method used

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  • Blocking or weakening the expression of rice circRNA coding site to improve the method of rice seedling growth traits
  • Blocking or weakening the expression of rice circRNA coding site to improve the method of rice seedling growth traits
  • Blocking or weakening the expression of rice circRNA coding site to improve the method of rice seedling growth traits

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of rice Os06circ02797 knockout editing vector pZJP054

[0100] (1) sgRNA design

[0101] From the database website NCBI (https: / / www.ncbi.nlm.nih.gov / ), search and download the rice circRNA coding site Os06circ02797 sequence, and then according to the recognition and cutting rules of the target site by the CRISPR-Cas9 system, Design the sgRNA; then use the CRISPR-P website online (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to predict the mismatch rate and off-target sites of the sgRNA and select the optimal sgRNA3 (SEQ ID No.1 ) and sgRNA4 (SEQ ID No.2) ( figure 1 A). According to the enzyme cleavage site of the knockout vector pZHY988 used in the present invention, the enzyme cleavage site of BsaI was added at the primer end, and two single-stranded nucleotide sequences Os06circ02797-sgRNA-P1F were designed and synthesized (primer sequence such as SEQ ID No.3 ) and Os06circ02797-sgRNA-P1R (primer sequence such as SEQ ID No.4). It was synthesized by ...

Embodiment 2

[0123] Example 2 Verifying the Editing Efficiency of Directed Editing Expression Vectors in Protoplasts

[0124] Protoplast isolation and transformation method references for verification of editing efficiency (Tang X, Ren Q, Yang L, BaoY, Zhong Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretenovic S, Zhang Y, Zheng X , Zhang T, Qi Y, Zhang Y. 2019. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17, 1431-1445.). Rice (Nipponbare) was cultured at 28°C in a dark environment. Cut the rice seedlings into 0.5-1.0 mm long strips with a blade. It was then quickly transferred to a 90 mm Petri dish containing 8-10 ml of enzyme solution (1.5% Cellulase R10, 0.75% Macerozyme R10, 0.6 Mannitol, 10 mM MES at pH 5.7, 10 mM Calcium Chloride and 0.1% BSA) middle. Then infiltrate under vacuum for 30 minutes. Then shake in the dark at 25° C. on a shaker at 60-80 rpm for 5-6 hours. Cell digests were subsequently filtered thro...

Embodiment 3

[0126] Rice genetic transformation mediated by embodiment 3 Agrobacterium

[0127] Agrobacterium-mediated transformation of rice references (Tang X, Ren Q, Yang L, Bao Y, Zhong Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretenovic S, Zhang Y, Zheng X, Zhang T, The experimental method disclosed in Qi Y, ZhangY. 2019. Single transcript unit CRISPR 2.0systems for robust Cas9 and Cas12 amediated plant genome editing. Plant Biotechnol J 17, 1431-1445.).

[0128] The genetic transformation steps of rice specifically include: shelling and disinfecting mature rice (Nipponbare) seeds; inoculating the sterilized seeds on N-6-D solid medium containing 0.4% gellan gum, and culturing under continuous light at 32° C. 5 days; the cultivated seeds were transformed into rice with plasmids pZJP054, pZJP053, pZJP054 and pZJP057 by Agrobacterium-mediated transformation method, and the transformed rice seeds were continuously cultured in the induction selection medium at 32°C under light for 2 weeks; th...

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Abstract

The invention belongs to the field of plant biotechnology, and in particular relates to a method for blocking or weakening the expression of rice circRNA coding sites to improve seedling growth traits. The technical problem to be solved by the invention is to improve the growth traits of rice. In order to solve the technical problem, the technical solution provided is a method for improving the growth rate of rice seedlings. The method is to improve the growth traits such as the growth rate of rice seedlings by blocking or weakening the expression of the circRNA coding site Os06circ02797 in rice. The blocking or weakening of the expression of the circRNA coding site Os06circ02797 in rice is carried out by knocking out the sequence of the rice circRNA coding site Os06circ02797 or interfering with the expression product of the circRNA coding site Os06circ02797. After the method of the present invention knocks out the rice circRNA coding site Os06circ02797, a new material with increased chlorophyll content at the seedling stage and rapid growth of the rice seedling stage is obtained, which has a good prospect.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for blocking or weakening the expression of rice circRNA coding site Os06circ02797 to improve seedling growth traits. Background technique [0002] Circular RNA (circular RNA, circRNA) is a special type of non-coding RNA molecule, which connects the "end" in the 5' upstream sequence through the 3' downstream sequence to form a covalently closed circular molecule without free ends. Due to its special structure, it is not easy to be cut by exoribonuclease, thus maintaining a high stability. Circular RNAs can be composed of exons, introns, exons-introns and intergenic regions of coding genes, respectively. Previous studies have pointed out that circRNA may have the biological function of acting as miRNA sponge, participating in the regulation of transcription process and regulating RNA variable splicing. With the development of high-throughput sequencing tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/113A01H5/00A01H6/46
CPCC12N15/8218C12N15/8261C12N15/113C12N2310/20
Inventor 张勇周建平郑雪莲
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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