Method for creating rehmannia heterozygous mutant and application of rehmannia heterozygous mutant

Abstract

The invention relates to a method for creating a rehmannia heterozygous mutant and application of the rehmannia heterozygous mutant, and belongs to the technical field of molecular biology. A CRISPR/Cas9 system targets a rehmannia RgPDS1 gene, a nucleotide sequence of a coding region of the RgPDS1 gene is as shown in SEQ ID NO.1, and a target site sequence is as shown in SEQ ID NO.2. A pair of complementary upstream and downstream primers is synthesized according to the target sequence; after the primers are annealed to form double strands, the double strands are inserted into a CRISPR/Cas9 plant expression vector pKSE401 through an enzyme digestion connection method, and successfully constructed recombinant plasmids are further screened out; and the recombinant plasmid is transformed into agrobacterium tumefaciens, and a rehmannia explant is infected by adopting a leaf disc method genetic transformation technology to obtain the heterozygous mutant plant. The CRISPR/Cas9 gene editing system is used for creating the heterozygous mutant in the rehmannia for the first time, a new research means is provided for accelerating the rehmannia molecular breeding process, improving varieties and innovating germplasm resources, and the functions of researching some key regulatory genes can be further achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method and application for creating a heterozygous mutant of Rehmannia glutinosa. Background technique [0002] The CRISPR / Cas9 system is one of the gene editing technologies widely used in the modification of target DNA in organisms. It consists of gRNA and Cas9. CRISPR / Cas9 technology has the advantages of simple construction method, wide application range, high editing efficiency and low cost. CRISPR / Cas9 gene editing technology has been widely used in crops such as Arabidopsis thaliana, rice, soybean, tomato, etc., but it is less used in medicinal plants. The silencing or deletion of phytoene dehydrogenase (PDS) will inhibit the normal synthesis of carotenoids, thereby affecting the protection of chlorophyll by carotenoids, causing the green plants rich in chlorophyll to fade and appear albino. Therefore, the PDS gene is often used as a marker gene t...

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Problems solved by technology

However, due to the long-term asexual reproduction of Rehmannia glutinosa, there are many types of pests and diseases, and the species degenerates seriously.

Moreover, the genome of Rehmannia glutinosa is highly heterozygous, the genetic basis is narrow, and self-incompatibility makes it impossible to obtain self-seeds, so it is difficult to obtain pure lines

Therefore, the breeding process of Rehmannia glutinosa is slow and there are few new varieties, which restrict the standardized and industrialized production of Rehmannia glutinosa

However, there is no research on the application of CRISPR / Cas9 gene editing system in Rehmannia glutinosa

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