Method for editing herbicide-resistant gene of flue-cured tobacco variety

Abstract

The invention relates to a method for editing herbicide-resistant genes of flue-cured tobacco varieties, which comprises the following steps: soaking flue-cured tobacco seeds with EMS (Ethyl Methyl Sulfonate), and sowing the soaked flue-cured tobacco seeds in a culture medium sprayed with a lethal dose of imidazolone herbicide; then, ALS target gene sequencing is carried out on the surviving tobacco plants, and new mutation sites are identified; taking the site as a tobacco target editing area, performing agrobacterium infection on tobacco leaf disc culture tissues, and introducing an editing carrier into flue-cured tobacco cells for targeting editing; then performing mutation screening to obtain a resistance mutation single plant; and carrying out sequencing identification and screening on a target gene mutant type to obtain a single plant and a genotype of which the target site is subjected to directional base substitution, so as to obtain the herbicide-resistant flue-cured tobacco single plant. According to the method, an easily available mutagenic agent EMS is adopted, a CRISPR-iSpyMacCas9 plant single base editing tool is utilized, an editing carrier is introduced into flue-cured tobacco cells, targeting editing is performed, mutation screening is further performed, a herbicide-resistant flue-cured tobacco single plant is finally obtained, and the method is simple to operate and efficient.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a method for editing the herbicide-resistant gene of a flue-cured tobacco variety. Background Art

[0002] With the widespread use of herbicides, farmland weeding has become highly efficient and has gradually become an integral part of modern agricultural production. Although the harm of weeds to crops is not as spectacular as floods, droughts, and pests and diseases, it is the culprit for crop yield reduction, increased labor costs, and reduced income for farmers. Therefore, it is particularly important to cultivate herbicide-resistant plant varieties.

[0003] Patent CN 109392704 A, entitled "A method for cultivating millet germplasm resistant to the herbicide imazethapyr using EMS mutagenesis", discloses a method for cultivating millet germplasm resistant to the herbicide imazethapyr using EMS mutagenesis in the field of crop breeding technology.

[0004] Patent CN 02047844A, named "A highly efficient mutagenesis method for tobacco pollen", which applies EMS chemical mutagenesis to tobacco pollen in a free state in in vitro liquid culture, which can greatly enhance the sensitivity of tobacco genetic material to external mutagenic factors, thereby doubling the mutagenesis frequency and variation range. The mutated pollen is then doubled to maintain and fix the mutant traits in the form of homozygous diploids, overcoming the obstacles of dominant and recessive gene expression, facilitating selection, and greatly shortening the period of mutant trait stabilization, saving breeding time.

[0005] The prior art lacks methods for editing the genes of flue-cured tobacco varieties to resist herbicides. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems in the prior art and provide a method for editing the herbicide-resistant gene of flue-cured tobacco varieties.

[0007] To achieve the above objectives, the present invention is implemented through the following technical scheme: a method for editing the herbicide-resistant gene of a flue-cured tobacco variety, comprising soaking the flue-cured tobacco seeds with EMS and sowing them on a culture substrate sprayed with a lethal dose of imidazoline herbicide; after a small number of tobacco plants germinate and emerge, the surviving tobacco plants are sequenced for the ALS target gene to identify a new mutation site; using the site as a tobacco targeted editing region, the tobacco leaf disc culture tissue is infected with Agrobacterium using the CRISPR-iSpyMacCas9 plant single base editing tool, and the editing vector is introduced into the flue-cured tobacco cells for targeted editing; then the regenerated leaf disc tissue is transferred to a regeneration medium supplemented with herbicides, and mutation screening is performed until tobacco seedlings appear to obtain resistant mutant individual plants; in the mutant offspring, the target gene mutant type is identified and screened by sequencing, and the individual plant and genotype with directed base substitution at the target site are obtained to obtain the herbicide-resistant flue-cured tobacco individual plant.

[0008] The above-mentioned method for editing the herbicide-resistant gene of flue-cured tobacco varieties comprises the following steps: S1: Soak the dried flue-cured tobacco seeds in 0.05-0.15% EMS solution for 1-3 hours, rinse the seeds and sow them in a plug tray with a matrix at a sowing density of 8-15 g / m 2 During the seed germination period, water with imidazoline until green seedlings emerge; S2: Transplant the green seedlings into flower pots and take leaves for whole-genome DNA sequencing, focusing on comparing and analyzing base mutations in the acetolactate synthase gene, i.e., the ALS coding region; S3: Identify the screened base mutation haplotypes, exclude unintentional mutations, verify the frameshift mutation and base substitution mutation haplotypes, clone the haplotypes and introduce them into wild-type flue-cured tobacco materials to confirm the resistance function of the mutant genotype; S4: Using the genotype with confirmed resistance function as a template and gene editing tools, the bases of the target gene functional sites of wild-type tobacco are edited into mutant types to obtain new herbicide-resistant flue-cured tobacco materials.

[0009] In the above-mentioned method for editing the herbicide-resistant gene of flue-cured tobacco varieties, the concentration of the EMS solution in S1 is 0.1-0.15%.

[0010] The above-mentioned method for editing the herbicide-resistant gene of flue-cured tobacco varieties, wherein the seed density in S1 is 10-13 g / m 2 .

[0011] In the above-mentioned method for editing the gene of flue-cured tobacco varieties to resist herbicides, the concentration of imazethapyr in S1 is 95-110 mg / L.

[0012] In the above-mentioned method for editing the gene of herbicide resistance in flue-cured tobacco varieties, the wild-type flue-cured tobacco material in S4 is K326.

[0013] The present invention has positive effects: the present invention adopts the easily available mutagen EMS and utilizes the CRISPR-iSpyMacCas9 plant single base editing tool to introduce the editing vector into the flue-cured tobacco cells, perform targeted editing, and then perform mutation screening, and finally obtain herbicide-resistant flue-cured tobacco plants, with simple operation and high efficiency. DETAILED DESCRIPTION

[0014] The technical scheme of the present invention is clearly and completely described below through embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] (Example 1) Soak the dried flue-cured tobacco seeds in 0.1% EMS solution for 1 hour, rinse the seeds three times with distilled water, and sow them in a plug tray with a substrate at a sowing density of 10 g / m 2 During the seed germination period, water with 100 mg / L imidazopyrine until green seedlings emerge. Transplant the green seedlings into pots and take leaves for DNA whole genome sequencing, focusing on comparison and analysis of base mutations in the coding region of the acetolactate synthase gene (ALS).

[0016] The screened base mutation haplotypes were identified, unintentional mutations were excluded, frameshift mutation and base substitution mutation haplotypes were verified, and the haplotypes were cloned and introduced into the wild-type flue-cured tobacco material K326 to confirm the resistance function of the mutant genotype.

[0017] Using the genotype with confirmed resistance function as a template and gene editing tools, the bases of the target gene functional sites of wild-type tobacco are edited into mutant types to obtain new herbicide-resistant flue-cured tobacco materials.

[0018] (Example 2) Soak the dried flue-cured tobacco seeds in 0.08% EMS solution for 1.5 hours, rinse the seeds three times with distilled water, and sow them in a plug tray with a matrix at a sowing density of 12 g / m 2 During the seed germination period, water with 105 mg / L of imidazole acetylcholine until green seedlings emerge. Transplant the green seedlings into flower pots, take leaves for DNA whole genome sequencing, and focus on comparing and analyzing base mutations in the coding region of the acetolactate synthase gene (ALS).

[0019] The screened base mutation haplotypes were identified, unintentional mutations were excluded, frameshift mutation and base substitution mutation haplotypes were verified, and the haplotypes were cloned and introduced into the wild-type flue-cured tobacco material K326 to confirm the resistance function of the mutant genotype.

[0020] Using the genotype with confirmed resistance function as a template and gene editing tools, the bases of the target gene functional sites of wild-type tobacco are edited into mutant types to obtain new herbicide-resistant flue-cured tobacco materials.

[0021] (Example 3) Soak the dried flue-cured tobacco seeds in 0.12% EMS solution for 2 hours, rinse the seeds three times with distilled water, and sow them in a plug tray with a matrix at a sowing density of 9 g / m 2 During the seed germination period, water with 98 mg / L of imidazole acetylcholine until green seedlings emerge. Transplant the green seedlings into flower pots, take leaves for DNA whole genome sequencing, and focus on comparing and analyzing base mutations in the coding region of the acetolactate synthase gene (ALS).

[0022] The screened base mutation haplotypes were identified, unintentional mutations were excluded, frameshift mutation and base substitution mutation haplotypes were verified, and the haplotypes were cloned and introduced into the wild-type flue-cured tobacco material K326 to confirm the resistance function of the mutant genotype.

[0023] Using the genotype with confirmed resistance function as a template and gene editing tools, the bases of the target gene functional sites of wild-type tobacco are edited into mutant types to obtain new herbicide-resistant flue-cured tobacco materials.

[0024] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for editing the herbicide-resistant gene of a flue-cured tobacco variety, characterized in that: After the flue-cured tobacco seeds were soaked in EMS, they were sown in a culture medium sprayed with a lethal dose of imidazoline herbicide; after a few tobacco plants germinated and emerged, the ALS target gene was sequenced for the surviving tobacco plants to identify new mutation sites; Using this site as the tobacco targeted editing region, the CRISPR-iSpyMacCas9 plant single base editing tool was used to infect tobacco leaf disc culture tissue with Agrobacterium, and the editing vector was introduced into flue-cured tobacco cells for targeted editing; the regenerated leaf disc tissue was then transferred to a regeneration medium supplemented with herbicide for mutation screening until tobacco seedlings appeared, and resistant mutant plants were obtained; in the mutant offspring, the target gene mutant type was identified and screened by sequencing, and the plants and genotypes with directed base substitutions at the target site were obtained, thereby obtaining herbicide-resistant flue-cured tobacco plants.

2. The method for editing the herbicide-resistant gene of flue-cured tobacco varieties according to claim 1, characterized in that: The steps include: S1: Soak the dried flue-cured tobacco seeds in 0.05-0.15% EMS solution for 1-3 hours, rinse the seeds and sow them in a plug tray with a matrix at a sowing density of 8-15 g / m 2 During the seed germination period, water with imidazoline until green seedlings emerge; S2: Transplant the green seedlings into flower pots and take leaves for whole-genome DNA sequencing, focusing on comparing and analyzing base mutations in the acetolactate synthase gene, i.e., the ALS coding region; S3: Identify the screened base mutation haplotypes, exclude unintentional mutations, verify the frameshift mutation and base substitution mutation haplotypes, clone the haplotypes and introduce them into wild-type flue-cured tobacco materials to confirm the resistance function of the mutant genotype; S4: Using the genotype with confirmed resistance function as a template and gene editing tools, the bases of the target gene functional sites of wild-type tobacco are edited into mutant types to obtain new herbicide-resistant flue-cured tobacco materials.

3. The method for editing the herbicide-resistant gene of flue-cured tobacco varieties according to claim 1, characterized in that: The concentration of the EMS solution in S1 is 0.1-0.15%.

4. The method for editing the herbicide-resistant gene of flue-cured tobacco varieties according to claim 1, characterized in that: The planting density in S1 is 10-13 g / m 2 .

5. The method for editing the herbicide-resistant gene of flue-cured tobacco varieties according to claim 1, characterized in that: The concentration of imidacloprid in the S1 is 95-110 mg / L.

6. The method for editing the herbicide-resistant gene of flue-cured tobacco varieties according to claim 1, characterized in that: The wild type flue-cured tobacco material in S4 is K326.