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CN-111647620-B - Method for creating non-transgenic mutant strain of flowering cabbage

CN111647620BCN 111647620 BCN111647620 BCN 111647620BCN-111647620-B

Abstract

The invention discloses a method for creating a non-transgenic mutant strain of a flowering cabbage. The method for creating the non-transgenic mutant of the cabbage is realized by combining a CRISPR/Cas9 technology with a cabbage in-situ transformation technology, and specifically comprises the following steps of introducing a CRISPSR/Cas 9 gene editing vector carrying an sgRNA coding sequence of a target gene in a genome of the cabbage into a recipient cabbage by a vacuum infiltration in-situ transformation method. In the invention, two PDS alleles are knocked out from 2032 seeds, and a non-transgenic editing mutant is obtained by an in-situ transformation technology independent of tissue culture. Compared with the in-situ transgene efficiency reported before, the editing efficiency is greatly improved, and a good research idea is provided for transgene safety and marker-free gene editing.

Inventors

  • WANG GUIXIANG
  • ZONG MEI
  • LIU FAN
  • LIU DI
  • TIAN SHOUWEI
  • HAN SHUO
  • GUO NING
  • DUAN MENGMENG
  • MIAO LIMING

Assignees

  • 北京市农林科学院

Dates

Publication Date
20260505
Application Date
20200610

Claims (6)

  1. 1. A method for creating non-transgenic mutant of Chinese cabbage is realized by combining CRISPR/Cas9 technology with Chinese cabbage in-situ conversion technology; the method comprises the following steps: (A1) Introducing a CRISPSR/Cas 9 gene editing vector carrying an sgRNA coding sequence of a target gene in a cabbage genome into agrobacterium to obtain recombinant agrobacterium; (A2) When the recipient Chinese cabbage enters the initial flowering stage, the flower dipping treatment is carried out according to the following steps that flower buds are immersed in the heavy suspension of the recombinant agrobacterium and are subjected to vacuum treatment for 10 minutes under the condition of negative pressure of 104 Pa; Immersing the flower buds in the suspension of the recombinant agrobacterium, and performing vacuum treatment for 10 minutes under the condition of negative pressure of 104Pa, wherein the intermediate pause is that the flower buds are stopped for 5 minutes, and pressurizing again after the air pressure is slowly reduced to normal pressure; (A3) Obtaining a non-transgenic mutant strain from which the target gene is knocked out from the final seed of the recipient cabbage subjected to the flower dipping treatment of (A2); The Chinese cabbage is non-heading Chinese cabbage; the non-heading Chinese cabbage is a cabbage stalk; The vegetable bolts are vegetable bolt varieties of 'four nine vegetable cores'; the CRISPSR/Cas 9 gene editing vector is obtained by modifying a marker gene Hyg in a pHSE401,401 vector into a Bar gene; the target gene is a phytoene dehydrogenase gene; The specific recognition sequence in the sgRNA coding sequence of the target gene in the target cabbage genome is shown as SEQ ID No. 1.
  2. 2. The method of claim 1, further comprising the step of watering said subject cabbage with water one day before said flower dipping treatment.
  3. 3. The method of claim 1, further comprising the step of removing flowers and fruits that have been opened before said flower dipping treatment is performed on said recipient cabbage.
  4. 4. The method according to claim 1, wherein said flower dipping treatment is carried out on said subject Chinese cabbage, further comprising the step of taking mature pollen of said subject Chinese cabbage which is not flower dipping treated and carrying out auxiliary pollination on said subject Chinese cabbage which is flower dipping treated.
  5. 5. The method of claim 4, wherein the supplementary pollination is performed by covering the treated inflorescences with a perforated fresh-keeping bag, covering the treated inflorescences with silver gray film, tearing off the fresh-keeping bag the next day, covering the inflorescences with a sodium sulfate paper bag, and removing the paper bag on the inflorescences in the final flowering stage.
  6. 6. The method of claim 1, wherein the CRISPSR/Cas 9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the Chinese cabbage is a recombinant vector obtained by replacing a small fragment between two BsaI sites of pHSE401-Bar vector with a DNA fragment shown in SEQ ID No.1, and the pHSE401-Bar vector is a vector obtained by modifying a marker gene Hyg in the pHSE401 vector into a Bar gene.

Description

Method for creating non-transgenic mutant strain of flowering cabbage Technical Field The invention relates to the technical field of biology, in particular to a method for creating a non-transgenic mutant strain of a flowering cabbage. Background Transgenic safety has been a concern due to food safety and gene drift issues. And CRISPR/Cas9 mediated gene editing technology can replace traditional transgenic technology to obtain non-transgenic improved material. Safe non-transgenic improved materials can be obtained by removing the exogenous vector containing Cas9/sgRNA and retaining the editing mutations, typically by sexual propagation and marker selection of transgenic offspring. At the same time the CRISPR/Cas9 technology also offers the possibility to obtain non-transgenic mutants directly without the need for progeny isolation. Including agrobacterium-mediated CRISPR/Cas9 transient expression (Chen et al, 2018;Iaffaldano et al, 2016), cas9/gRNA complex (RNP) transfection (Woo et al, 2015;Murovec et al, 2018; park et al, 2019), and the like, but all rely on tissue culture and regeneration ex vivo. Cabbage is used as a main vegetable crop, the research of transformation technology of the cabbage is slow, and the transformation efficiency is low mainly because of the difficulty in sprouting of explants of the cabbage. The agrobacterium-mediated flower dipping (flower-dip) transformation method is a simpler and more convenient transgenic method independent of tissue culture. The method has the advantages of avoiding tissue culture and in-vitro regeneration, directly obtaining the transgenic seeds, along with simple method, time saving and labor saving. In recent years, many students have made some preferential progress in transgenic research on Chinese cabbage by using an in-situ transformation method. Cao Mingqing (2000) successfully converts non-heading Chinese cabbage by a vacuum infiltration method, yan Jiyong (2003) converts Chinese cabbage by a microinjection method, zhang Anhui (1998), yan Jiyong (2004) successfully converts Chinese cabbage by a vacuum infiltration method in Zhaodong (2007), but the conversion efficiency is low, which is about 0.01 percent at most. The flowering cabbage (Brassica campestris L. Ssp. Chinensis) is a conventional cultivar taking tender flower stems as main edible organs in the type of non-heading cabbage, and is a good material for researching the in-situ conversion technology of the cabbage vegetables, and about 40-60 days are required for sowing to harvest the product. At present, although related reports are established on genetic transformation of Chinese cabbage and CRISPR/Cas9 gene editing systems, related reports on creating non-transgenic mutant strains of Chinese cabbage by combining in-situ transformation and CRISPR/Cas9 technology are not yet available. Disclosure of Invention The invention aims to provide a method for creating a non-transgenic mutant strain of a flowering cabbage. The method for creating the non-transgenic mutant of the cabbage is realized by combining a CRISPR/Cas9 technology with a cabbage in-situ conversion technology. Further, the method can comprise the step of introducing a CRISPSR/Cas 9 gene editing vector carrying an sgRNA coding sequence of a target gene in a cabbage genome into a recipient cabbage by a vacuum infiltration in situ transformation method. Still further, the method may comprise the steps of: (A1) And introducing a CRISPSR/Cas 9 gene editing vector carrying an sgRNA coding sequence of a target gene in a cabbage genome into agrobacterium to obtain recombinant agrobacterium. (A2) When the recipient Chinese cabbage enters the initial flowering stage, the flower dipping treatment is carried out by immersing the flower buds in the heavy suspension of the recombinant agrobacterium and carrying out vacuum treatment for 10 minutes under the condition of negative pressure of 100-105Pa (104 Pa, for example). The flower bud is immersed in the heavy suspension of the recombinant agrobacterium, and the flower bud to be opened is pulled out by forceps, and the branch is bent during flower dipping, so that the whole inflorescence is soaked in the heavy suspension of the recombinant agrobacterium. (A3) Obtaining the non-transgenic mutant strain from which the target gene is knocked out from the final seed of the receptor cabbage subjected to the flower dipping treatment in the step (A2). The method may further comprise the step of watering the recipient cabbage a day before the flower dipping treatment is performed on the recipient cabbage. The method may further comprise the step of removing the already opened flowers and fruits before the flower dipping treatment of the recipient cabbage. The OD600 of the recombinant Agrobacterium suspension was 0.05. The recombinant agrobacterium may be suspended in 1/2MS (5% (i.e., 5g/100 mL) sucrose, 0.05% volume percentage Silwet L-77) to od600=0.05. In a specific embodiment of the invention, th