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SigRNAs specifically shearing rice xal3 gene promoter using CRISPR/Cas9 system and its application

A promoter and specific technology, applied in the field of sgRNA, can solve the problems of decreased pollen fertility and expression, and achieve the effect of good fertility and no trace of transgenic

Pending Publication Date: 2019-08-06
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

xa13 is a very special rice recessive bacterial blight resistance gene cloned in recent years (Chu et al.2006), there is no difference between xa13 and its dominant allele Xa13 in the coding region of the gene, but the sequence of the xa13 promoter part Mutations lead to a significant decrease in its expression in rice leaves, allowing rice plants to acquire specific resistance to bacterial blight strain PXO99
Constitutive suppression of the xa13 allelic dominant gene Xa13 expression can increase rice resistance to strain PXO99, but at the same time lead to a significant decrease in pollen fertility, indicating that this gene not only controls rice disease resistance but is also related to rice fertility (Bart et al. 2006; Chu et al. 2006)

Method used

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  • SigRNAs specifically shearing rice xal3 gene promoter using CRISPR/Cas9 system and its application
  • SigRNAs specifically shearing rice xal3 gene promoter using CRISPR/Cas9 system and its application
  • SigRNAs specifically shearing rice xal3 gene promoter using CRISPR/Cas9 system and its application

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Experimental program
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Effect test

Embodiment 1

[0043] Selection of Cas9 target and construction of Cas9+P13(g1RNA+g2RNA) expression vector

[0044] According to the literature published by Chu et al. (2006), the sequence number "DQ421395" of the Xa13 gene in Genebank was obtained. The sequence of DQ421395 was obtained in the Genebank of the NCBI website (www.ncbi.nlm.nih.gov) according to this sequence number. Perform blastn on the TIGR Rice website (http: / / rice.plantbiology.msu.edu / LocusNameSearch.shtml) according to the sequence of DQ421395, and obtain its locus identifier in TIGR Rice: Os08g42350.1. 2000 bp forward from the 5' end of the gene was selected as its promoter sequence. Refer to the articles published by Romer (2010) and Yuan (2011) to obtain the sequence of elements in the promoter that are induced and regulated by pathogenic bacteria. A cas9 cleavage target site is selected at the upstream and downstream of the element sequence.

[0045] Referring to the method of Liu Yaoguang’s research group (Ma et al....

Embodiment 2

[0051] (1) Transformation of Cas9+P13(g1RNA+g2RNA) vector

[0052] Referring to the Agrobacterium-mediated indica rice transformation method published by Hiei et al. (1994), the constructed Cas9+P13 (g1RNA+g2RNA) expression vector was transformed into the commonly used japonica rice transformation variety ZH11. The PCR primer Hpt-F / R of hygromycin resistance gene was used for PCR positive screening of transgenic plants, and 40 independent positive transformed plants of T0 generation were obtained; 40 positive transformed plants were detected by PCX-F / R , it was found that 6 strains achieved complete cleavage of the target site.

[0053] (2) PCR sequencing verification of target sites in homozygous sheared transgenic plants

[0054] In order to prove that the deletion of the target genome sequence in this study is indeed caused by the specific cleavage mediated by Cas9 and two sgRNAs, this example uses PCX-F / R to amplify the PCR products obtained from transgenic plants, and cl...

Embodiment 3

[0060] Identification of bacterial blight resistance in transgenic rice with homozygous splicing of the target locus

[0061] In order to detect whether the transgenic plants have obtained the expected disease resistance, all T0 transgenic plants were inoculated with bacterial race PXO99 at the peak tillering stage in this embodiment (using turbidimetric method, the inoculation concentration was controlled at 9- 1.2 billion / ml). Each transgenic plant was inoculated with 5-6 leaves, and the lesion length and lesion area were observed 14 days after inoculation. See Table 2 for the inoculation results. In Table 2: the wild type Zhonghua 11 (ZH11) had obvious disease, the average leaf lesion length was 16.82cm, and the lesion area was 65.82%. The transgenic plants (PD1-PD6) showed significantly improved disease resistance. Calculated according to the standard adopted by Chu Zhaohui (2006) (14 days after inoculation, lesion length <3cm is high resistance level), transgenic T0 ge...

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Abstract

The invention provides a group of sgRNAs specifically shearing a rice xal3 gene promoter by using a CRISPR / Cas9 system and an application thereof, which belong to the technical field of plant geneticengineering. The sgRNA of the present invention comprises g1 RNA and g2 RNA; a nucleotide sequence of the g1 RNA is shown as SEQ ID NO. 1; a nucleotide sequence of the g2RNA is shown as SEQ ID NO. 2.A recombinant expression vector constructed by the sgRNA combination provided is used for transfection of a rice plant, a 149nt promoter region of the xal3 gene can be specifically deleted, so that the xal3 gene in rice loses the ability to be induced and expressed by Xanthomonas oryzae, and the transgenic rice lacking the promoter fragment exhibits resistance to Xanthomonas oryzae. In addition, the method provided by the present invention does not affect other traits of transgenic rice, and the transgenic rice has good fertility.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a group of sgRNAs that use the CRISPR / Cas9 system to specifically cut the rice xal3 gene promoter and applications thereof. Background technique [0002] Breeding for resistance to diseases and insect pests is one of the main goals of breeding in agricultural production. Rice bacterial blight is caused by Xanthomonas oryzae pv. Oryzae (Xoo), which is the most harmful bacterial disease to rice in the world. At present, more than 30 major genes for resistance to bacterial blight have been identified in rice, 5 of which are recessive. xa13 is a very special rice recessive bacterial blight resistance gene cloned in recent years (Chu et al.2006), there is no difference between xa13 and its dominant allele Xa13 in the coding region of the gene, but the sequence of the xa13 promoter part The mutation resulted in a significant decrease in its expression in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00A01H6/46
CPCC12N15/113C12N15/8281C12N2310/10C12N2800/80C12N2810/10
Inventor 李昌焱李威林拥军马伟华
Owner HUAZHONG AGRI UNIV
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