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Method for breeding broad-spectrum bacterial blight-resistant rice by editing exon of OsSWEE14 gene

An anti-bacterial blight and exon technology, applied in the field of genetic engineering, can solve the problems of destroying the promoter region, low safety, variation and the like

Inactive Publication Date: 2020-02-14
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the gene editing technology CRISPR / Cas9 system is used to destroy the promoter region of the rice bacterial blight susceptibility gene OsSWEET14, resulting in the inactivation of the susceptibility gene, thereby obtaining rice with bacterial blight resistance
[0003] Rice bacterial blight is a vascular disease, the use of pesticides can only temporarily reduce the damage of bacterial blight, and the cost is high, the pollution is heavy, and the safety is low
Under the pressure of resistance genes for a long time, bacterial blight will inevitably mutate, reducing or losing the resistance of the original resistant varieties

Method used

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  • Method for breeding broad-spectrum bacterial blight-resistant rice by editing exon of OsSWEE14 gene
  • Method for breeding broad-spectrum bacterial blight-resistant rice by editing exon of OsSWEE14 gene
  • Method for breeding broad-spectrum bacterial blight-resistant rice by editing exon of OsSWEE14 gene

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Experimental program
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Embodiment 1

[0058] In this embodiment, a method for cultivating broad-spectrum bacterial blight-resistant rice by editing the exons of the OsSWEE14 gene comprises the following steps:

[0059] (1) Strain activation and plasmid extraction preparation: Strain culture the strain (TOP10F') carrying the pYLCRISPR / Cas9Pubi-H vector on a plate medium containing kanamycin (25 μg / ml) overnight, and the pYLsgRNA1 carrying pYLsgRNA1 - OsU3 / LacZ vector and pYLsgRNA2-OsU6b vector strains (DH10B) were streaked and cultured overnight on a plate medium containing ampicillin (50 μg / ml), and single colonies were picked to culture seed liquid, and then expanded for extraction plasmid;

[0060] (2) Construction of sgRNA1 expression cassette:

[0061] 1) The first round of PCR reaction: take 2-5ng pYLsgRNA1-OsU3 / LacZ plasmid as a template, and do two PCR reactions, one PCR reaction uses primers U-F and OsU3-S14E1, the concentration of primers in the reaction system is 0.2μM, and the product is a; another PC...

Embodiment 2

[0080] This example studies the resistance of CR S14-2, CR S14-6, CR S14-9-I, CR S14-9-II, CR S14-10-I and CRS14-10-II strains to bacterial blight PXO86 .

[0081] CR S14-2, CR S14-6, CR S14-9-Ⅰ, CR S14-9-Ⅱ, CR S14-10-Ⅰ, CRS14-10-Ⅱ strains and Zhonghua 11 rice wild strain was inoculated with PXO86 strain, and the lesion was measured and counted on the 14th day after inoculation.

[0082] The result is as figure 2 shown by figure 2 It can be seen that the lesions of CR S14-2, CR S14-6, CR S14-9-Ⅰ, CR S14-9-Ⅱ, CR S14-10-Ⅰ and CR S14-10-Ⅱ strains inoculated with PXO86 strain were significantly smaller than In the control group, Zhonghua No. 11 rice wild plant (corresponding figure 2 zhonghua 11), indicating that the above-mentioned strains are resistant to bacterial blight PXO86.

Embodiment 3

[0084] This example studies the resistance of the CR S14-6 strain to different bacterial blight bacteria.

[0085] The CR-S14-6 strain and Zhonghua 11 wild rice strain were inoculated with the following bacterial blight strains by leaf-cutting method: PXO86, HB17, AUST2031 / IV24, HLJ72, NX42, AUST2031 / GD14, AUST2031, LC-4, For PX079, 1947, HB21, GD1358, JS49-6, IV-1 and HN1-2, lesion spots were measured and counted on the 14th day after inoculation.

[0086] The result is as image 3 shown by image 3 It can be seen that the lesions of the CR-S14-6 strain inoculated with the above-mentioned bacterial blight strain were significantly smaller than the Zhonghua No. 11 wild rice strain inoculated with the above-mentioned bacterial blight strain (corresponding to image 3 zhonghua 11), indicating that the CR-S14-6 strain is resistant to the above-mentioned bacterial blight strains.

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Abstract

The invention provides a method for breeding broad-spectrum bacterial blight-resistant rice by editing the exon of the OsSWEE14 gene. The method is as follows: editing target sequences of first exon and third exon regions of the OsSWEE14 gene to make the target sequences generate mutations of insertion or deletion of a plurality of bases to obtain the broad-spectrum bacterial blight-resistant rice. The target sequence of the first exon region is as shown in the SEQ ID No.1, and the target sequence of the third exon region is as shown in the SEQ ID No.2. The bacterial blight-resistant rice bredby the method of the invention can resist dozens of types of bacterial blight, and has the advantage of wide resistance spectrum.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for cultivating broad-spectrum bacterial blight-resistant rice by editing OsSWEE14 gene exons. Background technique [0002] Rice bacterial blight is a vascular disease caused by Xanthomonas bacterium. The rice fields infected by Xanthomonas bacterium can reduce the yield by 10%-20% in light cases, and reduce the yield by more than 50% in severe cases. In severe cases, the grains will not be harvested. The current control methods mainly rely on chemical control and agricultural control. Traditional chemical control usually relies on the use of pesticides; agricultural control is mainly to resist bacterial blight by planting disease-resistant varieties. A more cost-effective method. At present, the gene editing technology CRISPR / Cas9 system is also used to destroy the promoter region of the rice bacterial blight susceptibility gene OsSWEET14, res...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00A01H6/46
CPCC12N15/8218C12N15/8281
Inventor 曾璇罗宇芬张明永
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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