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A screening method for recombinant vectors and non-transgenic gene editing plants

A recombinant vector and gene editing technology, applied in the field of plant gene editing, can solve the problems of inability to amplify specific bands, side effects, false negatives, etc., and achieve the effect of cheap screening methods

Active Publication Date: 2019-09-20
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, due to the randomness of T-DNA integration into the genome, it is likely to destroy other functional genes of the plant and cause side effects. Therefore, non-transgenic plants are also required for experiments or production.
The above method not only requires tedious experimental steps such as DNA extraction, PCR, and gel electrophoresis, but also if any of the above steps is wrong, the specific band will not be amplified, and it will be mistaken for a plant without transgenic fragments. plants, and then the false negative conclusion of the primers

Method used

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  • A screening method for recombinant vectors and non-transgenic gene editing plants
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  • A screening method for recombinant vectors and non-transgenic gene editing plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Preparation of BelRNAi gene transcription unit

[0041] The RNA interference used here mainly interferes with Bel transcripts in cells by introducing a 300bp long hairpin (Hairpin) structure.

[0042] Using primers Beli-F1 (gagctcAGCTTAGCCATGGATAACGCCTAC, the lowercase underline is the SacⅠ restriction site) and Beli-R1 (ctgcagAAGGTCACGTCGTGCTCGGTGAAGCACTC, the lowercase underline is the PstⅠ restriction site), and the forward sequence was obtained by PCR amplification.

[0043] On the other hand, primers Beli-F2 (ggtaccAGCTTAGCCATGGATAACGCCTAC, the lowercase underline is the KpnI restriction site) and Beli-R2 (ctcgagAAGGTCACGTCGTGCTCGGTGAAGCACTC, the lowercase underline is the XhoI restriction site) were used. Sequencing was performed after connecting the two sequences to the T vector, and the sequencing result was SEQ ID NO.1.

[0044] After the sequencing is correct, the sequence is loaded into PCAMBIA-1301, assembled with the d35S and NOS terminal on the carrier...

Embodiment 2

[0049] Example 2 Using the gene OsLCT1 (Os06g0579200) related to cadmium (Cd) transport as the target gene, screening of plants without transgenic fragments

[0050] The cadmium (Cd) transport-related gene OsLCT1 (Os06g0579200) was used as the target gene, and the 20bp fragment 5'-TACTATCCCGCGTGCCAATG-3' was introduced as sgRNA to generate the OsLCT1 mutant.

[0051] Such as figure 2 shown, at T 0 In the generation, on the one hand, the sgRNA will be transcribed under the promoter U3 and matched to the gene OsLCT1; the Cas9 gene is also transcribed and translated into a nuclease in rice cells. The two work together to cleave the target and then introduce mutations during DNA repair in rice cells. On the other hand, the Bel RNAi hairpin structure is transcribed to form RNA, which matches the Bel RNA existing in rice cells, interrupts its fragments, and affects translation.

[0052] Through the above steps of Example 1, 58 rice plants were obtained, and the first 30 plants w...

Embodiment 3

[0070] Example 3 Using the gene Betaine aldehyde dehydrogenase 2 (OsBADH2) that controls the aroma of rice as the target gene, screening of plants without transgenic fragments

[0071] In order to further verify the applicability of the newly constructed vector pHun4c12-Beli. At the same time, the gene Betaine aldehyde dehydrogenase 2 (OsBADH2) was used as the target gene for experiments, and the OsBADH2 mutant plants will show rice aroma and improve rice quality.

[0072] Experimental process and steps are consistent with embodiment 2.

[0073] Select two of the mutant lines, for T 1 Substitute plants were treated with bentazone.

[0074] The result is as Figure 6 As shown, plants showing bendazone sensitivity and resistance in the same line. Among them, the surviving plants are the ideal plants with mutation of gene OsBADH2 and no T-DNA insertion, which can be used for production and rice breeding.

[0075]

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Abstract

The invention discloses a recombinant vector and a non-GMO gene editing plant screening method. An initial carrier in the recombinant vector is a CRISPR / Cas9 carrier which contains sgRNA gene, Cas9 gene and screening label gene and is used for plant gene editing, the recombinant vector carries a BelRNAi expression element, and a hairpin RNA interference fragment interfering with bentazone resistance gene is generated through transcription of the BelRNAi expression element. By introducing the BelRNAi expression element to the initial carrier and conducting bentazone herbicide screening on future generation plants, offspring with target gene mutated are reserved, and it is also ensured that the mutated offspring do not contain transgenic fragments; screening method is cheaper, simpler and more effective.

Description

technical field [0001] The invention relates to the technical field of plant gene editing, in particular to a screening method for recombinant vectors and non-transgenic gene editing plants. Background technique [0002] Gene editing technology can accurately modify the target gene (that is, edit the DNA sequence). It is not only an advanced biological technology, but also an effective means to improve the quality of crops. [0003] At present, the technologies that can be applied to gene editing mainly include: ZFNs (Zinc finger nucleases, zinc finger nucleases), TALENs (Transcription activator-like effector nucleases, transcription initiation factor nucleases) and CRISPR / Cas9 (Clustered regularly interspaced short palir dromicrepeats / CRISPR-associated Cas9, clustered regularly interspaced short palindromic repeats / Cas9 nuclease) technology (Bogdanove A.J.and Voytas D.F., 2011. TAL effectors: customizable proteins for DNA targeting. Science, 333(6051): 1843-1846 ; Carrol D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/65A01H5/00A01H6/46
Inventor 舒庆尧芦海平邓丽刘松梅
Owner ZHEJIANG UNIV
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