Virus vector capable of realizing polygene editing of plants as well as construction method and application of virus vector

A construction method, a technology of virus vectors, applied in the direction of plant genetic improvement, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of high cost, high technical requirements, low efficiency, etc.

Active Publication Date: 2020-06-26
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early targeted genome editing technology mainly relied on gene homologous recombination (gene targeting) and somatic cell nuclear transfer technology to complete the transformation of specific genes. The main technical means in this period include: gene recombination technology (Recombineering), Oligonucleotid

Method used

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  • Virus vector capable of realizing polygene editing of plants as well as construction method and application of virus vector
  • Virus vector capable of realizing polygene editing of plants as well as construction method and application of virus vector
  • Virus vector capable of realizing polygene editing of plants as well as construction method and application of virus vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Vector construction:

[0033] 1.1人工合成gRNA-tRNA串联序列GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA,并以TOPO克隆方法连接于载体pCE2,命名为pCE2-GT,在后续步骤中,pCE2-GT作为PCR模板使用,用于合成不同靶标的gRNA-tRNA单元, Targets include:

[0034] SlTCP2-sg1 (Solyc07g062680.1, SEQ ID NO. 2), TACAAGGAGGTCACATTGTG;

[0035] SlOBP3-sg2 (Solyc03g083420.2, SEQ ID NO. 3), TATGTCTCCGTTAGAATCGG;

[0036] SlHSP70-sg3 (Solyc03g082920.2, SEQ ID NO. 4), GTGACAAGATTTCATGGGGA;

[0037] SlHSFA8-sg4 (Solyc09g059520.2, SEQ ID NO. 5), GTGGAGGATGAGTCAACTGA.

[0038] 1.2 Design primers and synthesize polycistronic tRNA-gRNA (PTG) according to Golden Gate assembly.

[0039] Among them, the primer information is as follows:

[0040] SLTCP2-SG1-F (SEQ ID NO. 6): TA GGTCTC CGGTCACATTGTGGTTTTTAGAGCTAGAAATAG

[0041] SLTCP2-SG1-R (SEQ ID NO. 7): CG GGTCTC AGACCTCCTTGTATGCACCAGCCGGGAATCGA;

[0042] SLOBP3-SG2-F (SEQ ID NO. 8): T...

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Abstract

The invention provides a virus vector capable of realizing polygene editing of plants as well as a construction method and application of the virus vector, and relates to the technical field of gene editing of the plants. According to the virus vector, tRNA and sgRNA are sequentially linked to form unit sequences, and the unit sequences are linked with vectors pCE2; a target sequence of a target gene is inserted between the tRNA and sgRNA of each unit sequence by virtue of a ''golden-gate'' cloning method, the multiple unit sequences are linked and arrayed in series to form a series-wound sequence, the series-wound sequence is inserted into a TRV2 virus vector, after plants capable of overexpressing Cas9 proteins are infected by TRV, and a tRNA-sgRNA expression sequence unit assembled withdifferent unit sequences is transcribed, thereby finishing a polygene editing process, so that editing of multiple genes or combined editing of different genes according to targets can be realized. According to the construction method, the expression quantity of mRNA capable of being translated into normal protein sequences in the plants is lower, a gene silencing phenotype is relatively stable,and meanwhile, multiple genes can be precisely knocked out in a targeting manner.

Description

technical field [0001] The invention belongs to the technical field of plant gene editing, and in particular relates to a virus vector capable of multi-gene editing in plants, a construction method and application thereof. Background technique [0002] Targeted genome editing technology refers to the targeted modification of genomic DNA to change specific genes in the genome by targeting exogenous sequences to specific sites on chromosomes, achieving the purpose of site-specific insertion of a gene or DNA element, or knockout of a specific gene sequence , in order to more accurately and deeply understand the pathogenesis of diseases and explore gene functions. Early targeted genome editing technology mainly relied on gene homologous recombination (gene targeting) and somatic cell nuclear transfer technology to complete the transformation of specific genes. The main technical means in this period include: gene recombination technology (Recombineering), Oligonucleotide-mediat...

Claims

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Application Information

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IPC IPC(8): C12N15/83A01H5/00A01H6/82C12Q1/6895
CPCC12N15/8203C12N15/8213C12N15/8218C12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘超超王琼
Owner JIANGSU UNIV OF SCI & TECH
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