Parthenogenetic haploid induced gene DMP and application thereof
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A haploid induction line, haploid technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as inability to combine
Active Publication Date: 2020-11-27
CHINA AGRI UNIV
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Problems solved by technology
However, due to the lack of biologically induced haploids in dicotyledonous plants, it cannot be combined with gene editing technology
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Embodiment 1
[0090] Embodiment 1, the preparation and application of the Arabidopsis mutant of AtDMP8 and / or AtDMP9 gene knockout
[0091] 1. Using the CRISPR / Cas9 system to knock out AtDMP8 and / or AtDMP9 genes
[0092] The AtDMP8 and / or AtDMP9 gene in Arabidopsis was knocked out by CRISPR / Cas9 system, and the AtDMP8 and / or AtDMP9 gene knockout Arabidopsis mutants were obtained. Specific steps are as follows:
[0093] 1. Selection of sgRNA sequence
[0094] The target site sequences were designed on the AtDMP8 and AtDMP9 genes respectively, with a length of 20bp.
[0095] Target site 1 is located at positions 98-117 of sequence 1 and at positions 144-163 of sequence 3, and the sequence of sgRNA target site 1 is GAGAAAACAGAGGAAAGCGT.
[0096] Target site 2 is located at positions 290-309 of sequence 3, and the sequence of sgRNA target site 2 is AAGAGGTCGAAAACGTCGCA.
[0097] Target site 3 is located at positions 368-387 of sequence 3, and the sequence of sgRNA target site 3 is TCAAGAGTG...
Embodiment 2
[0165] Embodiment 2, the preparation and application of the tomato mutant of SlDMP gene knockout
[0166] 1. Using the CRISPR / Cas9 system to knock out the SlDMP gene
[0167] The SlDMP gene in tomato was knocked out by CRISPR / Cas9 system, and the tomato mutant with SlDMP gene knockout was obtained. Specific steps are as follows:
[0168] 1. Selection of sgRNA sequence
[0169] Design the target site sequence on the SlDMP gene, the length is 20bp.
[0170] Target site 1 is located at positions 76-95 of sequence 5, and the sequence of sgRNA target site 1 is TATCCTACTAATTTACCACA.
[0171] Target site 2 is located at positions 247-266 of sequence 5, and the sequence of sgRNA target site 2 is TTCTCCTTTACCAAATGA.
[0172] 2. Construction of CRISPR / Cas9 vector
[0173] The CRISPR / Cas9 vector is the DNA molecule (sgRNA expression element) shown in sequence 8, the DNA molecule (Cas9 expression element) shown in sequence 11, the DNA molecule (fluorescent protein expression element)...
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Abstract
The invention discloses a parthenogenesis haploid induced gene DMP and application thereof. According to the invention, parthenogenesis haploid induced genes AtDMP8 and AtDMP9 are cloned from arabidopsis thaliana. Experiments prove that mutation of the AtDMP8 and the AtDMP9 can generate parthenogenesis haploid inducibility, so that the application of parthenogenesis induced haploid extends to dicotyledonous crops. Verification is further carried out in tomatoes, and the invention also finds that mutation of SlDMP can generate parthenogenesis haploid inducibility in tomatoes. The invention laysan important foundation for widening the application of haploid breeding technology in dicotyledons and revealing the biological mechanism of parthenogenetic haploid production. In view of the universality of haploid breeding technology utilization in the current breeding industry, the DMP has very wide application space and market prospect.
Description
technical field [0001] The present invention relates to the field of agricultural biotechnology and the field of crop genetics and breeding based on genome editing technology, in particular to a method for preparing a plant maternal haploid induction line and its application, and in particular to an orphan obtained by gene editing technology. The application of the female germline haploid-inducing gene DMP mutant as a plant haploid-inducing line in inducing plants to produce female parental haploid. Background technique [0002] Breeding of superior inbred lines is the basis and key for crops to utilize heterosis and breed superior hybrids. However, the traditional breeding method needs 7 to 8 generations to obtain a relatively stable inbred line, while the haploid breeding technology only needs 2 generations (Weber D F, 2014), which greatly shortens the breeding cycle. At present, the production of haploids in dicotyledonous crops mainly depends on the in vitro culture of ...
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