阅读说明：本技术 一种菜薹非转基因突变株的创制方法 (Method for creating non-transgenic mutant strain of flowering Chinese cabbage ) 是由 王桂香 宗梅 刘凡 刘迪 田守卫 韩硕 郭宁 段蒙蒙 缪黎明 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种菜薹非转基因突变株的创制方法。本发明所提供的创制白菜非转基因突变株的方法,是通过将CRISPR/Cas9技术与白菜原位转化技术相结合实现的,具体包括如下步骤：将携带有靶向白菜基因组中目标基因的sgRNA编码序列的CRIPSR/Cas9基因编辑载体通过真空渗入原位转化的方法导入受体白菜。本发明在2032粒种子中获得了两粒PDS等位基因敲除,率先通过不依赖于组织培养的原位转化技术获得非转基因编辑突变株。编辑效率较之前报道的原位转基因效率大大提高,也为转基因安全和无标记基因编辑提供了一个很好的研究思路。(The invention discloses a method for creating a non-transgenic mutant strain of a flowering Chinese cabbage. The method for creating the non-transgenic mutant strain of the Chinese cabbage is realized by combining a CRISPR/Cas9 technology with a Chinese cabbage in-situ transformation technology, and specifically comprises the following steps: the CRIPSR/Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the target Chinese cabbage is introduced into the receptor Chinese cabbage by a vacuum infiltration in-situ transformation method. Two PDS alleles are knocked out from 2032 seeds, and a non-transgenic editing mutant strain is obtained firstly through an in-situ transformation technology independent of tissue culture. The editing efficiency is greatly improved compared with the in-situ transgenic efficiency reported before, and a good research idea is provided for the transgenic safety and the marker-free gene editing.)

1. A method for creating a non-transgenic cabbage mutant is realized by combining a CRISPR/Cas9 technology with a cabbage in-situ transformation technology.

2. The method of claim 1, wherein: the method comprises the following steps: the CRIPSR/Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the target Chinese cabbage is introduced into the receptor Chinese cabbage by a vacuum infiltration in-situ transformation method.

3. The method according to claim 1 or 2, characterized in that: the method comprises the following steps:

(A1) introducing a CRIPSR/Cas9 gene editing vector carrying a sgRNA coding sequence of a target gene in a genome of the targeted Chinese cabbage into agrobacterium to obtain recombinant agrobacterium;

(A2) when the acceptor Chinese cabbage enters the initial flowering phase, carrying out flower soaking treatment according to the following steps: immersing the buds in the resuspension of the recombinant agrobacterium tumefaciens, and carrying out vacuum treatment for 10 minutes under the negative pressure condition of 100-105 Pa;

(A3) and (D) obtaining a non-transgenic mutant strain with the knocked-out target gene from the final seed of the recipient Chinese cabbage subjected to the (A2) flower soaking treatment.

4. The method of claim 3, wherein: before the subject Chinese cabbage is subjected to the flower soaking treatment, the method also comprises the step of watering the subject Chinese cabbage with water the day before.

5. The method according to claim 3 or 4, characterized in that: before the subject Chinese cabbage is subjected to the flower soaking treatment, the method also comprises the step of removing the opened flowers and the pod.

6. The method according to any one of claims 3-5, wherein: the bud is immersed in the suspension of the recombinant agrobacterium and is stopped once during vacuum treatment for 10 minutes under the negative pressure condition of 100-105 Pa.

7. The method according to any one of claims 3-6, wherein: the method also comprises the following steps after the acceptor Chinese cabbage is subjected to the flower soaking treatment: and taking mature pollen of the receptor Chinese cabbage which is not subjected to flower soaking treatment to carry out supplementary pollination on the receptor Chinese cabbage subjected to flower soaking treatment.

8. The method of claim 7, wherein: the method also comprises the following steps after the supplementary pollination is carried out: covering the treated inflorescences with perforated freshness protection bags, covering with silver gray film, removing the freshness protection bags the next day, covering the inflorescences with sodium sulfate paper bags, and taking off the paper bags on the inflorescences in the final flowering period.

9. The method according to any one of claims 1-8, wherein: the CRIPSR/Cas9 gene editing vector is obtained by transforming a marker gene Hyg in a pHSE401 vector into a Bar gene.

10. The method according to any one of claims 1-9, wherein: the Chinese cabbage is non-heading Chinese cabbage;

further, the non-heading Chinese cabbage is a bolting vegetable;

and/or

The target gene is phytoene dehydrogenase gene;

furthermore, a specific recognition sequence in the sgRNA coding sequence of the target gene in the targeted Chinese cabbage genome is shown as SEQ ID No. 1;

furthermore, the CRIPSR/Cas9 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 the pHSE401-Bar vector with a DNA fragment shown in SEQ ID No.1, and the pHSE401-Bar vector is obtained by transforming a marker gene Hyg in the pHSE401 vector into a Bar gene.

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 Chinese cabbage.

Background

Transgene safety has been a concern due to problems with food safety and gene drift. And the CRISPR/Cas9 mediated gene editing technology can replace the traditional transgenic technology to obtain non-transgenic improved materials. Safe non-transgenic improved material can be obtained by methods of sexual propagation and marker selection of transgenic offspring to remove exogenous vectors containing Cas9/sgRNA and retain editing mutations. At the same time, the CRISPR/Cas9 technology also provides the possibility of directly obtaining non-transgenic mutants without progeny separation. Including Agrobacterium-mediated transient expression of CRISPR/Cas9 (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), etc., but all rely on tissue culture and ex vivo regeneration.

Chinese cabbage as a main vegetable crop has slow development of transformation technology research, and is mainly due to difficult germination of explants and low transformation efficiency. The agrobacterium-mediated floral dip (floral-dip) transformation method is a simpler and more convenient transgenic method independent of tissue culture. The method has the advantages that tissue culture and in vitro regeneration can be avoided, transgenic seeds can be directly obtained, the method is simple, and time and labor are saved. In recent years, many scholars have achieved some favorable progress in transgenic research on Chinese cabbage by using in situ transformation. Cao Chung Qing (2000) successfully transforms non-heading Chinese cabbage by vacuum infiltration; the Chinese cabbage is transformed by adopting a microinjection method in the severe brave (2003); in addition, the Chinese cabbage is successfully transformed by a vacuum infiltration method in the east of China (2007) in Zhang Guang Hui (1998) and Yan Yong (2004), but the transformation efficiency is low, which is about 0.01 percent mostly. A flowering Chinese cabbage (Brassica campestris l.ssp. chinensis) is a conventional cultivar of non-heading Chinese cabbage type with tender flower stems as the main edible organs, and the period from sowing to harvest of the product is about 40-60 days, so that the method is a good material for researching the in-situ conversion technology of the white vegetables.

At present, although related reports are established on genetic transformation of Chinese cabbage and a CRISPR/Cas9 gene editing system, no related report exists on creating a non-transgenic mutant strain of the Chinese cabbage by combining in-situ transformation and a CRISPR/Cas9 technology.

Disclosure of Invention

The invention aims to provide a method for creating a non-transgenic mutant strain of a flowering Chinese cabbage.

The method for creating the non-transgenic mutant strain of the Chinese cabbage is realized by combining a CRISPR/Cas9 technology with a Chinese cabbage in-situ transformation technology.

Further, the method may comprise the steps of: the CRIPSR/Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the target Chinese cabbage is introduced into the receptor Chinese cabbage by a vacuum infiltration in-situ transformation method.

Still further, the method may comprise the steps of:

(A1) and (3) introducing the CRIPSR/Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the targeted Chinese cabbage into the agrobacterium to obtain the recombinant agrobacterium.

(A2) When the acceptor Chinese cabbage enters the initial flowering phase, carrying out flower soaking treatment according to the following steps: immersing the bud in the recombinant Agrobacterium resuspended solution, and vacuum treating for 10 minutes under the negative pressure condition of 100-105Pa (such as 104 Pa).

Wherein, the bud is immersed in the resuspension of the recombinant agrobacterium, which can be specifically carried out as follows: and (3) poking the flower buds to be opened by using a pair of tweezers, and bending branches during flower soaking so that the whole inflorescence is soaked in the resuspension of the recombinant agrobacterium tumefaciens.

(A3) And (D) obtaining the non-transgenic mutant strain with the knocked-out target gene from the final seed of the receptor Chinese cabbage subjected to flower soaking treatment (A2).

Before the subject Chinese cabbage is subjected to the flower soaking treatment, the method also comprises the step of watering the subject Chinese cabbage with water the day before.

The method also comprises the step of removing the opened flowers and carob fruits before the subject Chinese cabbage is subjected to the flower soaking treatment.

The OD600 of the resuspension of the recombinant Agrobacterium is 0.05. The heavy suspension of the recombinant agrobacterium may specifically be: the recombinant agrobacterium was suspended in 1/2MS (containing 5% (i.e. 5g/100mL) sucrose, 0.05% by volume Silwet L-77) to an OD600 of 0.05.

In a specific embodiment of the invention, the agrobacterium is agrobacterium tumefaciens, in particular agrobacterium tumefaciens C58.

In the process of immersing the flower buds in the suspension of the recombinant agrobacterium tumefaciens and carrying out vacuum treatment for 10 minutes under the negative pressure condition of 100-105Pa (such as 104Pa), the process is stopped once (the process can be stopped for 5 minutes, and the pressure is pressurized again after the air pressure is slowly reduced to the normal pressure, so that the plant is more favorable for being in a state of being easily invaded by bacterial liquid). Then the vacuum pump is closed, the air valve is opened slowly, and the plant is taken out after the normal pressure is recovered.

The method also comprises the following steps after the acceptor Chinese cabbage is subjected to the flower soaking treatment: and taking mature pollen of the receptor Chinese cabbage which is not subjected to flower soaking treatment to carry out supplementary pollination on the receptor Chinese cabbage subjected to flower soaking treatment.

The supplementary pollination can also comprise the following steps: covering the treated inflorescences with perforated freshness protection bags, covering with silver gray film, removing the freshness protection bags the next day, covering the inflorescences with sodium sulfate paper bags, and taking off the paper bags on the inflorescences in the final flowering period.

In a specific embodiment of the invention, the CRIPSR/Cas9 gene editing vector is a pHSE401-Bar vector. The pHSE401-Bar vector is obtained by transforming a marker gene Hyg in the pHSE401 vector into a Bar gene.

In the method, the cabbage may be non-heading cabbage.

Further, the non-heading Chinese cabbage may be a bolt.

In a specific embodiment of the invention, the Chinese cabbage is a Chinese cabbage of a bolting variety 'four-nine heart'.

In a specific embodiment of the present invention, the target gene is a phytoene dehydrogenase gene.

Correspondingly, a specific recognition sequence (corresponding to spacer) in the sgRNA coding sequence of the target gene in the genome of the targeted Chinese cabbage is shown as SEQ ID No. 1. The CRIPSR/Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the genome of the targeted Chinese cabbage is a recombinant vector obtained by replacing a small segment between two BsaI sites of a pHSE401-Bar vector with a DNA segment shown in SEQ ID No. 1. The pHSE401-Bar vector is obtained by transforming a marker gene Hyg in the pHSE401 vector into a Bar gene.

The invention refers to and improves the transformation technology on the basis of the vacuum-mediated Chinese cabbage in-situ transformation technology. 2032 seeds of a flowering Chinese cabbage 'four-nine heart' with lycopene dehydrogenase gene PDS (phytoene desaturase) encoding gene knocked out by a CRISPR/Cas9 system (Bar gene is carried on an editing vector) are obtained. Seeds of T0 generation were sown and phenotypical observation, Basta protein test strip detection and PDS gene sequencing were performed, respectively. The result shows that 2 plants exhibiting dwarfing and albino (PDS gene knockout phenotype) are negative in Basta protein test strip detection, no exogenous vector sequence is detected by PCR amplification, but PDS gene sequencing verifies that base deletion mutation occurs in a target region to cause premature termination and knockout of PDS genes, and wild PDS genes are not reserved. The successful gene editing and knockout in the bolting through in-situ transformation in the experiment shows that: the transformation vector is not integrated into the plant genomic DNA, but is transiently expressed in the host cell, resulting in gene editing. Two PDS allele knockouts were obtained in 2032 seeds, and non-transgenic editing mutants were obtained first by in situ transformation techniques independent of tissue culture. The editing efficiency is greatly improved compared with the in-situ transgenic efficiency reported before, and a good research idea is provided for the editing of the marker-free gene.

Drawings

FIG. 1 shows construction of a bolting PDS gene editing knockout vector and identification of in-situ transformed plants. a, the first four exon sequences of the 'four nine-cabbage heart' PDS gene, different color backgrounds represent different exons, gRNA is marked at the tail end of the first exon by underlining, and three bases in red font are PAM regions. Schematic representation of the left and right boundaries of the pHSE401-Bar-PDS transformation vector. gRNA is transcribed by U6-26 promoter, and contains Bar plant selection marker gene. c, two seedlings of 2032 are always weakly albino. And d, the albino seedling PDS gene target section PCR sequencing result shows that sequence diversity variation occurs in the gRNA target area. And e, performing monoclonal sequencing on the PCR product, wherein the two albino seedlings generate different mutation types in the PDS target region.

FIG. 2 shows the result of Basta protein test paper. a, detecting the sensitivity of a Basta protein test paper, wherein CK1 is an untransformed normal strain and shows a negative reaction; CK2 is an identified transformed seedling, and shows positive reaction; 20,40 and 80 are mixed liquid of 1 positive seedling and 19, 39 and 79 non-transformed seedlings respectively, and all show positive reactions. b, converting the detection result of part of test paper in 2032 seedlings in situ.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The early-maturing variety of the flowering Chinese cabbage referred to in the following examples is 'four-nine heart': products of Yuanzhongye, Ltd.

The pHSE401 vector referred to in the following examples: addgene, Inc., cat # 62201.