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Method for improving citrus canker resistance based on CRISPRCas9 mediated CsWRKY22 site-directed editing

A citrus canker and editing technology, applied in the field of plant genetic engineering, can solve the problems of citrus canker resistance that needs further research, enhance canker susceptibility, etc., and achieve the effect of enhancing citrus canker resistance.

Active Publication Date: 2019-09-27
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]Preliminary studies in our laboratory have shown that the plant immune response PTI (PAMP-triggered immunity) marker gene CsWRKY22 is closely related to the susceptibility of citrus canker. Overexpression of CsWRKY22 in orange significantly enhanced the susceptibility of plants to canker, implying that CsWRKY22 is a susceptibility gene of citrus canker, but whether silencing or knocking out CsWRKY22 can enhance the resistance of citrus canker remains to be further studied

Method used

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  • Method for improving citrus canker resistance based on CRISPRCas9 mediated CsWRKY22 site-directed editing
  • Method for improving citrus canker resistance based on CRISPRCas9 mediated CsWRKY22 site-directed editing
  • Method for improving citrus canker resistance based on CRISPRCas9 mediated CsWRKY22 site-directed editing

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Experimental program
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Effect test

Embodiment 1

[0036] 1. Extraction of DNA

[0037] Select 0.1-1 g of citrus veins, use Aidlab company kit (cat. No. DN15) to extract genomic DNA, and store at -20°C for future use.

[0038] 2. PCR Amplification of Genomic Sequence

[0039]

[0040] The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 1.5min, 35 cycles; 72°C extension, 10min.

[0041] 3. DNA fragment recovery, ligation, transformation into Escherichia coli DH5α

[0042] Under the ultraviolet light, use a clean blade to cut off the agarose gel block containing the fragment of interest. The gel recovery method was carried out according to the instructions of the kit (purchased from Omega Company), and the recovered fragments were quantified by electrophoresis on agarose gel.

[0043] The establishment of the enzyme digestion system and the reaction conditions were carried out with reference to the instructions of the restriction endonuclease kit from TaKaRa Company. The recovered fragments were dig...

Embodiment 2

[0052] Using Wanjincheng genome as a template and WRKY22-f / WRKY22-r as primers (Table 6), the full-length gene of Wanjincheng CsWRKY22 was amplified by PCR, and the electrophoresis band of the target product was about 1.2kb ( figure 1 ), sequencing analysis found that the Wanjincheng CsWRKY22 gene contains three exons and two introns, and there are two alleles in Wanjincheng, and the coding sequence contains abundant single base polymorphism SNP, Based on the first SNP "G / C" difference, the two alleles were named CsWRKY22 respectively G and CsWRKY22 C ( figure 2 ). No insertions or deletions were detected, suggesting that both alleles encode the same functional WRKY22 protein.

[0053] Large-scale (more than 27 clones) sequencing analysis of T clones on the CDS of Wanjincheng Cs WRKY22 coding sequence, three repeated experiments showed that CsWRKY22 G and CsWRKY22 C The heterozygosity in the Wanjincheng genome is 3:1, that is, there are three copies of CsWRKY22 in the Wa...

Embodiment 3

[0055] 1. Design of sgRNA

[0056] Based on the WRKY22 genome ( figure 2 ) and protein sequence ( image 3 ), the sequences that perform the biological functions of WRKY22 are located on the second and third exons, so in order to effectively inactivate the WRKY22 protein function, the present invention uses the first exon sequence of WRKY22 as a target to screen potential Cas9 targets. Four (S1 / S2 / S3 / S4) sgRNAs were selected according to the location and GC content of the target (Table 2). The target sequences are S1: 5'-CCTCTCTTTCGGTTGAGCTGC-3'; S2: 5'-GGCCTCCATCGACTTTGTTA-3'; S3: 5'-GATTTGCAAGCCATAGTAAG-3'; S4: 5'-ACATCTTGTTGCAGACTCGA-3'. The analysis and evaluation results of the four sgRNA targets are shown in Table 2.

[0057] 2. Identification of sgRNA activity

[0058] To verify the in vitro activity of the above sgRNA, the operation steps are as follows:

[0059] (1) sgRNA reverse transcription in vitro.

[0060] The sgRNA in vitro transcription kit (Novoprotein...

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Abstract

The invention discloses a method for improving citrus canker resistance based on CRISPRCas9 mediated CsWRKY22 site-directed editing, and belongs to the field of plant genetic engineering. A CRISPR / Cas9 editing vector guided by S1 and S2 sg RNA is constructed, the epicotyl of wanjicheng orange is transformed with an agrobacterium-mediated method, so that the function of CsWRKY22 protein is lost or reduced, successful editing of CsWRKY22 gene in the citrus genome is realized, CsWRKY22 mutant transgenic plants are obtained, plant resistance to citrus canker is enhanced, and citrus with high canker resistance is obtained.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for improving resistance to citrus canker based on CRISPR Cas9-mediated site-directed editing of CsWRKY22. Background technique [0002] Citrus canker is one of the most devastating diseases of citrus production in the world. It can harm almost all citrus varieties. Citrus canker can cause leaf fall, dead branches and fruit drop by infecting the leaves, branches and fruits of citrus, resulting in a decrease in fruit yield and quality, which seriously affects the economic value of citrus. Citrus canker pathogen Xanthomonas citri subsp.citri (Xcc), one of the most destructive pathogens of commercial citrus in tropical and subtropical countries, is rod-shaped and is a gram Negative bacteria, with a polar flagella, grow in an aerobic environment and colonize the intercellular spaces of host tissues. At present, there is no technical measure to fundamentally solve ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/78
CPCC12N15/8213C12N15/8281C07K14/415C12N2810/10
Inventor 邹修平陈善春王丽娟何永睿彭爱红龙琴
Owner SOUTHWEST UNIVERSITY
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