CN114369615A - High-throughput molecular identification method for creating gene editing plants based on mixed pool library and application - Google Patents
High-throughput molecular identification method for creating gene editing plants based on mixed pool library and application Download PDFInfo
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Abstract
The invention discloses a high-throughput molecular identification method for creating gene editing plants based on a mixed pool library and application thereof, wherein the method comprises the following steps: detecting the mutation conditions of a plurality of target genes of T0 generation plants; selecting the edited plants from T0 generation plants only transferred into 1 editing carrier for seed collection; after seed harvesting, expanding propagation, and selecting homozygous plants; if no homozygous plant exists, continuously selecting T0 generation for propagation until a homozygous plant is obtained; selecting the edited plants for propagation of T0 generation plants transferred into 2 or n editing carriers; selecting 2 or n plants homozygous for the target genes for seed harvest; selecting plants with most homozygous genes in the T1 generation from plants which are not homozygous for all genes in the T1 generation for propagation to obtain plants of the T2 generation, and selecting plants with homozygous target genes for seed harvest; repeating until obtaining the plant with all genes being homozygously edited. The invention carries out different types of molecular identification aiming at gene editing plants of different generations, and efficiently obtains editing materials of homozygous editing of a plurality of genes.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a high-throughput molecular identification method for creating a gene editing plant based on a mixed pool library and application thereof.
Background
The CRISPR-Cas9 gene editing technology is a 3 rd generation gene editing technology, can quickly realize mutation of a specific gene, and is widely researched and applied to breeding and gene function research at present. When the CRISPR-Cas9 is used for gene function research, the method not only relates to the gene function research of knocking out a single gene, but also relates to the gene function research of knocking out a plurality of genes or one gene family. In the gene editing breeding, gene mutation is realized by a gene editing technology, crops with excellent properties are screened for variety cultivation, and the properties of the crops have characteristics controlled by a single gene and characteristics controlled by multiple genes, so that mutant materials with multiple genes mutated simultaneously need to be created. Therefore, aiming at the mutant material with multiple mutated genes needing to be created simultaneously, a method for creating a gene editing plant based on a mixed pool library is provided, namely, the constructed editing carrier capable of specifically knocking out the specific genes is mixed together to carry out the genetic transformation of the plant, so that the mutant plant with the edited different genes can be quickly obtained, and the research on the gene function and the observation on the character are facilitated.
Conventionally, a first generation sequencing mode is adopted for molecular identification of T0 generation plants created by genetic transformation only by using a single gene editing vector, and because the genetic transformation is carried out by using the single gene editing vector, the gene editing vector which is possibly transferred into the T0 generation plants is determined, and only a target gene specific primer is required to be designed for amplification and identification to judge whether the T0 generation plants are edited or not; and (3) carrying out 23-time expanding propagation on the edited T0 generation plants, planting T1 generation plants, carrying out amplification by using a specific primer, determining the editing mode and the editing type of a target gene, and selecting the plants with the target gene subjected to homozygous editing for research. However, the gene editing vector transferred into the T0 generation plant is uncertain, so that the strategy of genetically transforming the T0 generation plant by adopting a single gene editing vector, namely directly amplifying and identifying by using a target gene specific primer, and judging whether the T0 generation plant is edited or not can not be used for molecular identification of the mixed pool library-based gene editing plant.
The invention aims at the gene editing plant created based on the mixed pool library, has the possibility of transferring 1 or more gene editing carriers, and has the possibility of editing 1 or more genes, and in order to efficiently obtain the homozygous plant edited by the target gene, the invention establishes the molecular identification strategy aiming at the gene editing plant created based on the mixed pool library.
Disclosure of Invention
The invention aims to solve the technical problem of providing a high-throughput molecular identification method for creating gene editing plants based on a mixed pool library and application thereof, and overcoming the defects in the prior art.
The technical problem to be solved by the invention is realized by the following technical scheme:
a high-throughput molecular identification method for creating gene editing plants based on a mixed pool library comprises the following steps:
step (1): selecting a target gene, constructing a gene editing vector, and transferring the gene editing vector into agrobacterium for transformation to obtain a gene editing plant;
step (2): transforming the gene editing vector into a tobacco leaf disc for culture to obtain a T0 generation plant;
and (3): extracting DNA from T0 generation plants, and detecting the mutation condition of a target gene by high-throughput sequencing after PCR amplification;
and (4): for T0 generation plants without sgRNA transfer, false positive plants are transferred, and subsequent detection is not carried out;
and (5): for T0 generation plants only transferred into 1 editing vector, carrying out PCR amplification, and selecting the plants subjected to editing for harvest; carrying out T0 generation seed harvesting and propagation, carrying out PCR amplification, and selecting homozygous plants; if no homozygous editing plant exists, continuously selecting T0 generation for propagation until a homozygous editing plant is obtained;
and (6): carrying out PCR amplification on T0 generation plants transferred with 2 or n knockout carriers, and selecting plants subjected to editing for harvest; after seed collection, carrying out propagation, carrying out PCR amplification on the T1 generation plants, and selecting 2 or n plants with homozygous editing of target genes for propagation;
and (7): for the edited plants which are not homozygous for all genes in the T1 generation in the step (6), continuously selecting the plants with the most homozygous genes in the T1 generation for propagation to obtain the plants of the T2 generation, carrying out PCR amplification, and selecting the plants of which the target genes are homozygous and edited for research;
and (8): and (5) repeating the step (7) until a plant with all genes subjected to homozygous editing is obtained.
Preferably, in the above technical scheme, the target gene in step (1) is a gene in the tobacco starch anabolism pathway.
Preferably, in the above technical scheme, the tobacco plant variety in step (2) is Honghuadajinyuan.
Preferably, in the above technical solution, the step (3) is specifically:
and (3) confirming the transferred editing vector of all created T0 generation plants by adopting a high-throughput sequencing method, designing a specific primer aiming at the gene targeted by the editing vector, and detecting the mutation condition of the target gene by high-throughput sequencing after PCR amplification.
Preferably, in the above technical solution, the step (5) is specifically:
for T0 generation plants only transferred into 1 knockout vector, amplifying by using a specific primer of a target gene, and selecting the plants subjected to editing for seed collection;
carrying out T0 generation seed collection and 23-time propagation, carrying out PCR amplification by using a target gene specific primer for T0 generation detection, determining the editing mode and the editing type of a target gene, and selecting a plant with the target gene subjected to homozygous editing for research;
and if no homozygous editing plant exists, continuously selecting the T0 generation for propagation until a homozygous editing plant is obtained.
Preferably, in the above technical solution, the step (6) is specifically:
for T0 generation plants transferred into 2 or n knockout carriers, amplifying by using specific primers of target genes corresponding to the knockout carriers, and selecting the plants subjected to editing for seed collection;
after seed harvesting, carrying out 46-fold or 23 n-fold amplification, and carrying out PCR amplification on T1 generation plants by using specific primers of target genes to determine the editing mode and the editing type of 2 or n target genes;
and selecting 2 or n plants with homozygous editing of target genes for propagation.
Preferably, in the above technical solution, the step (7) is specifically:
for the edited plant with all homozygous genes which is not obtained in the T1 generation in the step (6), continuously selecting the plant with the most homozygous genes in the T1 generation for 46-fold or 23 n-fold propagation to obtain a T2 generation plant;
and carrying out PCR amplification by using target gene specific primers, and selecting plants with homozygous editing of target genes for research.
An application of a high-throughput molecular identification method for creating a gene editing plant based on a mixed pool library in screening and detecting of progeny individuals of the gene editing plant.
An application of a high-throughput molecular identification method for creating gene editing plants based on a mixed pool library in obtaining homozygous plants with target genes edited.
The technical scheme of the invention has the following beneficial effects:
the efficient molecular identification method of the gene editing plant based on the mixed pool library provided by the invention is used for carrying out different types of molecular identification on the gene editing plants of different generations, and provides technical support for efficiently obtaining the editing material of homozygous editing of a plurality of genes.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.
Example 1
5 genes A-E and 5 genes are key genes in a safflower large golden starch anabolism pathway, 5 sgRNAs aiming at the genes A-E are designed, 1 gene is arranged in each gene, the designed sgRNA sequences are respectively connected to a knockout vector CRISPR-Cas9, 5 gene editing vectors are obtained, the editing vector of the targeted gene A is called A1, and the like.
First, obtaining T0 generation plants
(1) Leaf disc pre-culture: taking 2-month-sized tobacco sterile seedlings, removing leaf edges and veins from mature leaves of the sterile seedlings under sterile conditions (in an ultra-clean workbench), beating into leaf discs with the size of 5mm multiplied by 5mm by a puncher, and pre-culturing for 3 days on an MS culture medium (MS +0.5mg/L NAA +2 mg/L6-BA) solid culture medium.
(2) And (3) culturing agrobacterium: 5 single colonies, which were successful in transforming the A1-E1 knock-out vector, were picked up and cultured overnight at 200rpm in 2mL YEB medium (containing 50. mu.g/mL rifampicin, 50. mu.g/mL streptomycin, and 50. mu.g/mL kanamycin) at 28 ℃; 1mL of the cell suspension was added to 50mL of YEB medium containing the same resistance, cultured at 28 ℃ and 200rpm until OD600 became 0.5, centrifuged at 4 ℃ and 3,000rpm for 15min to collect the cells, and the cells were suspended in 20mL of MS medium.
(3) Knockout vector mixing: 4mL of each of the bacterial solutions containing the knockout vectors A1, B1, C1, D1 and E1 were collected and mixed.
(4) Infection of agrobacterium: soaking the pre-cultured leaf disc in agrobacterium LBA4404 which is used for transformation and contains a mixed knockout vector and is resuspended in an MS liquid culture medium, infecting for 10min, taking out the leaf disc, absorbing bacterial liquid on sterilized filter paper, contacting the upper surface of the leaf disc with the culture medium, flatly paving the leaf disc on a solid co-culture medium (MS +0.5mg/L NAA +2 mg/L6-BA), and co-culturing for 3 days under the dark condition at the temperature of 28 ℃.
(5) Selecting and culturing: washing the co-cultured leaf disc with sterile water containing 250mg/L of carbenicillin (Cb), sucking excess water on the leaf surface with sterile filter paper, contacting the upper surface of the leaf with culture medium, culturing on selective culture medium (MS +0.5mg/L NAA +2 mg/L6-BA +250mg/LCb +50mg/L Kan) for inducing callus differentiation, changing the culture medium every 2 weeks, and observing white or green enlarged callus at the edge of the leaf disc after about 1 month.
(6) Rooting culture: when the callus grows green buds (after a leaf disc is placed in a callus differentiation culture medium for about 2 months), cutting the green buds (with the height of 1-2 cm), transferring the green buds into an MS solid culture medium (without hormone) added with corresponding antibiotics (250mg/L of carboxin (inhibiting agrobacterium) +100mg/L of kanamycin) for rooting, enabling the green buds to grow roots after about 10 days, and transplanting the green buds into soil after 1 month in the MS culture medium for rooting to obtain a T0 generation plant.
Second, plant DNA extraction
The genome of the Plant was extracted using DNeasy Plant Mini Kit from QIAGEN.
Third, detection of transfer-in knock-out vector
Primers are designed at two ends of the target site, the amplified fragment contains a target site sequence, and the two ends of the primers contain barcode with 6 basic groups for distinguishing different transgenic plants. And (3) amplifying the sgRNA-F and sgRNA-R primers by taking the extracted transgenic plant leaf DNA as a template, recovering a PCR product, building a library and sequencing.
Fourth, detection of target gene mutation
Designing a specific primer according to the genome position of a target site, wherein the target site needs to be within 10-100bp from a left primer or a right primer, and amplifying a DNA fragment containing a target site sequence by utilizing the designed specific primer in a first round of PCR reaction; the second round of PCR reaction is to add barcode sequence to the product of the first round of PCR to distinguish each sample. And performing mixed sequencing on the amplified second round PCR product, wherein the sequencing platform is illumina Hiseq X, the sequencing strategy is PE150, and the sequencing data volume is 1G.
And comparing the effective data to a reference sequence through BWA software, and detecting variation information.
Example 2
The molecular identification method for the edited plants is as follows:
M01-M04 was selected from 100T 0-generation tobacco plants produced in example 1. Taking leaves in the big cross stage for molecular identification; based on the used CRISPR-Cas9 vector, universal primers for detecting the sgRNA are designed on two sides of the position where the sgRNA is connected to the vector, a high-throughput sequencing method is adopted for sequencing amplification, and the editing vector of a T0-generation tobacco plant is confirmed to be transferred, and the table 1 shows. Then, specific primer design for detecting target genes is carried out on genes targeted by the editing vector contained in M1-M04, specific primers a1 are designed according to the position of the editing vector editing A gene of the A gene, and after amplification, the mutation conditions of a plurality of target genes are detected by a high-throughput method, wherein the detection conditions are as follows:
TABLE 1 molecular identification method and results of T0 generation plant M01-M04 transformed based on pooled library
For T0 generation plants without sgRNA transfer: m01 transferred without the sgRNA is transferred into a false positive plant, subsequent detection is not carried out, and the research is abandoned;
for T0 plants transformed with 1 sgRNA: amplifying and sequencing by a common primer of the sgRNA, and confirming that an editing vector of a T0-generation tobacco plant is A1, so that a1 specific primer is used for amplifying and sequencing to detect target gene mutation, and M02 with an edited A gene is selected; carrying out 23-time propagation on M02 seeds, sampling T1 generation, carrying out amplification sequencing again by using a specific primer a1, and selecting a plant subjected to homozygous editing of A gene for research; if no homozygous editing plant exists, M02 continues to propagate until a homozygous editing plant is obtained;
for T0 plants transformed with 2 sgrnas: (1) amplifying and sequencing by a common primer of the sgRNA, and confirming that an editing vector transferred into a T0 tobacco substitute plant is A1 and B1, so that PCR amplification and sequencing are carried out by using a1 specific primer and a B1 specific primer, and 2M 03 plants with target genes A and B edited are selected; (2) after M03 is harvested, carrying out amplification propagation according to 46 times, sampling T1 generation, carrying out amplification sequencing again by using specific primers a1 and B1, and selecting a plant in which the A gene and the B gene are homozygously edited for amplification propagation; (3) if the homozygous edited plant of the A and B genes is not obtained in the T1 generation, continuously selecting the plant in which the A or B gene is homozygous edited in the T1 generation for 46-fold propagation to obtain a T2 generation plant, carrying out PCR amplification by using target gene specific primers a1 and B1, and selecting the plant in which the A and B genes are homozygous edited for research; (4) repeating the step (3) until obtaining a plant with homozygous editing of both A and B genes;
for T0 plants transformed with 3 sgrnas: (1) amplifying and sequencing by a universal primer of the sgRNA, and confirming that an editing vector transferred into a T0-generation tobacco plant is A1, B1 and C1, so that PCR amplification and sequencing are carried out by using a1, B1 and C1 specific primers, and selecting 3M 04 plants with edited target genes of A1, B1 and C1; (2) after M04 is harvested, carrying out amplification propagation according to 46 times, sampling T1 generation, carrying out amplification sequencing again by using specific primers a1, B1 and C1, and selecting a plant in which the A gene, the B gene and the C gene are homozygously edited for amplification propagation; (3) if no plant with both A, B and C genes homozygous edited is obtained in the T1 generation, continuing to select plants with both A and B genes or both A and C genes or both B and C genes homozygous edited in the T1 generation for 46-fold propagation to obtain plants of the T2 generation, performing PCR amplification by using target gene specific primers a1, B1 and C1, and selecting plants with both A, B and C genes homozygous edited for research; (4) repeating (3) until obtaining a plant with homozygous editing of both A, B and C genes;
for T0 generation plants with n sgRNAs (n is less than or equal to 5): (1) amplifying and sequencing by using a common primer of the sgRNA, and confirming that an editing vector transferred into a T0-generation tobacco plant is A1, B1 … … and N1, so that PCR (polymerase chain reaction) amplification and sequencing are performed by using a1, B1 … … and N1 specific primers, and an M0N plant with N target genes A, B … … and N edited is selected; (2) after M0N is harvested, carrying out amplification propagation according to 23N times, sampling T1 generation, carrying out amplification sequencing again by using specific primers a1, b1 … … and N1, and selecting plants of which A, B … … and N genes are homozygously edited for amplification propagation; (3) if no plant with homozygous editing of both A, B … … and N genes is obtained in the T1 generation, continuously selecting a plant with homozygous editing of (N-1) genes in the T1 generation for 23N times of propagation to obtain a T2 generation plant, performing PCR amplification by using target gene specific primers a1, b1 … … and N1, and selecting a plant with homozygous editing of both A, B … … and N genes for research; (4) repeat (3) until plants are obtained in which both A, B … … and the N gene are homozygously edited.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.
Claims (9)
1. A high-throughput molecular identification method for creating gene editing plants based on a mixed pool library is characterized by comprising the following steps:
step (1): selecting a target gene, constructing a gene editing vector, and transferring the gene editing vector into agrobacterium for transformation to obtain a gene editing plant;
step (2): transforming the gene editing vector into a tobacco leaf disc for culture to obtain a T0 generation plant;
and (3): extracting DNA from T0 generation plants, and detecting the mutation condition of a target gene by high-throughput sequencing after PCR amplification;
and (4): for T0 generation plants without sgRNA transfer, false positive plants are transferred, and subsequent detection is not carried out;
and (5): for T0 generation plants only transferred into 1 editing vector, carrying out PCR amplification, and selecting the plants subjected to editing for harvest; carrying out T0 generation seed harvesting and propagation, carrying out PCR amplification, and selecting homozygous plants; if no homozygous editing plant exists, continuously selecting T0 generation for propagation until a homozygous editing plant is obtained;
and (6): carrying out PCR amplification on T0 generation plants transferred with 2 or n knockout carriers, and selecting plants subjected to editing for harvest; after seed collection, carrying out propagation, carrying out PCR amplification on the T1 generation plants, and selecting 2 or n plants with homozygous editing of target genes for propagation;
and (7): for the edited plants which are not homozygous for all genes in the T1 generation in the step (6), continuously selecting the plants with the most homozygous genes in the T1 generation for propagation to obtain the plants of the T2 generation, carrying out PCR amplification, and selecting the plants of which the target genes are homozygous and edited for research;
and (8): and (5) repeating the step (7) until a plant with all genes subjected to homozygous editing is obtained.
2. The method for high-throughput molecular identification of plants based on mixed pool library gene editing creation according to claim 1, wherein the target gene in step (1) is a gene in the tobacco starch anabolic pathway.
3. The method for high-throughput molecular identification of plants with gene editing created based on mixed pool library according to claim 1, wherein the tobacco plant variety in step (2) is Honghuadajinyuan.
4. The method for high-throughput molecular identification of plant based on mixed pool library gene editing creation according to claim 1, wherein the step (3) is specifically:
and (3) confirming the transferred editing vector of all created T0 generation plants by adopting a high-throughput sequencing method, designing a specific primer aiming at the gene targeted by the editing vector, and detecting the mutation condition of the target gene by high-throughput sequencing after PCR amplification.
5. The method for high-throughput molecular identification of plant based on mixed pool library gene editing creation according to claim 1, wherein the step (5) is specifically:
for T0 generation plants only transferred into 1 knockout vector, carrying out PCR amplification by using a specific primer of a target gene, and selecting the plants subjected to editing for seed collection;
carrying out T0 generation seed collection and 23-time propagation, carrying out PCR amplification by using a target gene specific primer for T0 generation detection, determining the editing mode and the editing type of a target gene, and selecting a plant with the target gene subjected to homozygous editing for research;
and if no homozygous editing plant exists, continuously selecting the T0 generation for propagation until a homozygous editing plant is obtained.
6. The method for high-throughput molecular identification of plant based on mixed pool library gene editing creation according to claim 1, wherein the step (6) is specifically:
for T0 generation plants transferred into 2 or n knockout carriers, amplifying by using a specific primer group of a target gene corresponding to the knockout carrier, and selecting the plants which are edited for seed collection;
after seed harvesting, carrying out 46-fold or 23-fold amplification, and carrying out PCR amplification on T1 generation plants by using a specific primer group of target genes to determine the editing mode and the editing type of 2 or n target genes;
and selecting 2 or n plants with homozygous editing of target genes for propagation.
7. The method for high-throughput molecular identification of plant based on mixed pool library gene editing creation according to claim 1, wherein the step (7) is specifically:
for the edited plant with all homozygous genes which is not obtained in the T1 generation in the step (6), continuously selecting the plant with the most homozygous genes in the T1 generation for 46-fold or 23 n-fold propagation to obtain a T2 generation plant;
and carrying out PCR amplification by using target gene specific primers, and selecting plants with homozygous editing of target genes for research.
8. The use of the method for high-throughput molecular identification of gene-edited plants based on the mixed pool library of any of claims 1-7 in screening and detecting the progeny individuals of the gene-edited plants.
9. Use of the method for high-throughput molecular identification of plants with gene editing created based on mixed pool library according to any of claims 1-7 for obtaining homozygous plants with edited target genes.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200385747A1 (en) * | 2017-11-27 | 2020-12-10 | Riken | Genome-edited plant production method |
| CN109234310A (en) * | 2018-11-13 | 2019-01-18 | 云南省烟草农业科学研究院 | It is quickly obtained the recombinant vector and application method of no transgene gene editor plant |
| CN112609019A (en) * | 2020-12-21 | 2021-04-06 | 华智生物技术有限公司 | Screening method of gene editing site homozygote without transgene |
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