Application of nicotinamide in improving wheat gene editing efficiency
阅读说明：本技术 尼克酰胺在提高小麦基因编辑效率中的应用 (Application of nicotinamide in improving wheat gene editing efficiency ) 是由 王轲 代文双 叶兴国 邹枨 刘会云 杜丽璞 于 2020-06-29 设计创作，主要内容包括：本发明公开了尼克酰胺在提高小麦基因编辑效率中的应用。尼克酰胺提高小麦基因编辑效率是通过尼克酰胺处理小麦幼胚或成熟胚实现的,小麦为含有Cas9蛋白和针对靶基因的sgRNA且靶基因未发生或者部分发生编辑的小麦。实验证明,2.5mM尼克酰胺处理14天获得的外源基因GUS和内源基因TaWaxy编辑效率最高,与对照组(未经尼克酰胺处理)相比编辑效率分别提高了36.0％和22.5％。本发明可以提高小麦基因编辑效率,具有重要的应用价值。(The invention discloses application of nicotinamide to improvement of wheat gene editing efficiency. The nicotinamide-enhanced wheat gene editing efficiency is realized by treating wheat immature embryos or mature embryos with nicotinamide, wherein the wheat contains Cas9 protein and sgRNA aiming at a target gene, and the target gene is not edited or is partially edited. Experiments prove that the editing efficiency of the exogenous gene GUS and the endogenous gene TaWaxy obtained by treating for 14 days with 2.5mM nicotinamide is the highest, and the editing efficiency is respectively improved by 36.0 percent and 22.5 percent compared with that of a control group (without the nicotinamide treatment). The invention can improve the wheat gene editing efficiency and has important application value.)
1. Application of nicotinamide in improving wheat gene editing efficiency.
2. The use of claim 1, wherein: the nicotinamide-enhanced wheat gene editing efficiency is realized by treating a wheat explant with nicotinamide.
3. Use according to claim 2, characterized in that: the nicotinamide treatment of the wheat explant is to culture the wheat explant on a culture medium containing 2.0-5.0mM nicotinamide for more than 7 days.
4. Use according to any one of claims 1 to 3, wherein: the wheat contains a Cas9 protein and sgrnas for the target genes.
5. Use according to any one of claims 1 to 3, wherein: the wheat contains a Cas9 protein and sgRNA aiming at a target gene, and the target gene is not edited or is partially edited.
6. Use according to claim 2 or 3, characterized in that: the explant is a immature embryo or a mature embryo.
7. A method for improving wheat gene editing efficiency comprises the following steps: wheat explants containing Cas9 protein and sgRNA against the target gene were treated with nicotinamide.
8. The method of claim 7, wherein: the "treatment of wheat explants containing Cas9 protein and sgRNA directed to a target gene with nicotinamide" is to culture wheat explants containing Cas9 protein and sgRNA directed to a target gene on a medium containing 2.0-5.0mM nicotinamide for more than 7 days.
9. The method of claim 7 or 8, wherein: the wheat containing the Cas9 protein and the sgRNA aiming at the target gene is wheat which contains the Cas9 protein and the sgRNA aiming at the target gene and does not edit or partially edits the target gene.
10. The method of claim 7 or 8, wherein: the explant is a immature embryo or a mature embryo.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of nicotinamide in improving the gene editing efficiency of wheat.
Background
The editing efficiency of the CRISPR/Cas9 system is greatly different among different species, for example, the mutation efficiency of the diploid plant rice CRISPR/Cas9 technology can reach over 80 percent. The construction of effective gene editing systems for polyploid plants, especially wheat, potato, etc., remains a difficult point in current research. Wheat is a hexaploid composed of A, B and D genomes, the genome is about 17Gb, which is about 40 times of that of rice, and the repetitive sequence is as high as 85% to 90%. Simultaneous editing of multiple copies in the wheat genome using CRISPR/Cas9 technology remains very difficult, which is a major reason limiting the application of wheat gene editing. Although the gene editing efficiency in wheat can reach 80% at most, the efficiency of simultaneously editing three homologous genes is only about 30% at most. Therefore, the research work for improving the editing efficiency of wheat, especially three genomes, is far from being done.
Histone acetylation plays a major role in nucleosome remodeling, which is the earliest studied and most characteristic post-translational modification. Histone acetylation can alter chromatin structure, regulate gene transcription, DNA replication and repair. Nicotinamide (Nicotinamide) is a common histone deacetylase inhibitor (HDACi) used to study the level of histone acetylation modification. Nicotinamide is a derivative of vitamin B3, a histone deacetylase inhibitor belonging to SIR2 class, which significantly inhibits histone deacetylase in mammals and yeast when added in excess. Currently, no application of nicotinamide is reported in wheat.
Disclosure of Invention
The invention aims to improve the wheat gene editing efficiency.
The invention firstly protects the application of nicotinamide in improving the gene editing efficiency of wheat.
In the application, the nicol amide is used for improving the gene editing efficiency of the wheat by treating the wheat explant.
In the above application, the nicotinamide treatment of the wheat explant can be carried out by culturing the wheat explant on a culture medium containing 2.0-5.0mM (such as 2.0-2.5mM, 2.5-5.0mM, 2.0mM, 2.5mM or 5.0mM) nicotinamide for more than 7 days (such as 7-14 days, 7 days or 14 days).
The culture medium may specifically be 1/2MS culture medium. 1/2 the MS culture medium is prepared by dissolving 2.215g MS powder, 20g sucrose and 0.5g MES in appropriate amount of distilled water, and adding distilled water to desired volume of 1L; adding 3g of plant gel, and adjusting the pH value to 5.8; sterilizing at 121 deg.C for 15 min.
The culture can be carried out by light-dark alternate culture at 23-27 deg.C (such as 23-25 deg.C, 25-27 deg.C, 23 deg.C, 25 deg.C or 27 deg.C). The period of the light-dark alternate culture can be specifically 16h of light and 8h of darkness; the light intensity during light culture is specifically 300 μmol/m2/s。
In any of the above applications, the wheat may contain a Cas9 protein and a sgRNA for a target gene.
In any of the above applications, the wheat may be wheat that contains the Cas9 protein and sgRNA for the target gene and the target gene is unedited or partially edited.
In any of the above applications, the explant may be a immature embryo or a mature embryo.
The invention also provides a method for improving the wheat gene editing efficiency, which comprises the following steps: wheat explants containing Cas9 protein and sgRNA against the target gene were treated with nicotinamide.
In the above method, the treating of the explant of wheat containing Cas9 protein and sgRNA directed to the target gene with nicotinamide can be culturing the explant of wheat containing Cas9 protein and sgRNA directed to the target gene on a medium containing 2.0-5.0mM (e.g., 2.0-2.5mM, 2.5-5.0mM, 2.0mM, 2.5mM, or 5.0mM) nicotinamide for more than 7 days (e.g., 7-14 days, 7 days, 14 days).
The culture medium may specifically be 1/2MS culture medium. 1/2 the MS culture medium is prepared by dissolving 2.215g MS powder, 20g sucrose and 0.5g MES in appropriate amount of distilled water, and adding distilled water to desired volume of 1L; adding 3g of plant gel, and adjusting the pH value to 5.8; sterilizing at 121 deg.C for 15 min.
The culture can be carried out by light-dark alternate culture at 23-27 deg.C (such as 23-25 deg.C, 25-27 deg.C, 23 deg.C, 25 deg.C or 27 deg.C). The period of the light-dark alternate culture can be specifically 16h of light and 8h of darkness; the light intensity during light culture is specifically 300 μmol/m2/s。
In any of the above methods, the wheat containing the Cas9 protein and the sgRNA directed to the target gene can be wheat containing the Cas9 protein and the sgRNA directed to the target gene with no or partial editing of the target gene.
In any of the methods described above, the explant can be a immature embryo or a mature embryo.
Any one of the genes can be a wheat exogenous gene or an endogenous gene.
Any one of the Cas9 proteins described above may specifically be a SpCas9 protein.
Any of the sgrnas described above that contain a Cas9 protein and are directed against a target gene may specifically be a vector containing gene editing.
Any of the above wheat may be a transgenic plant containing a gene editing vector but having no edited target gene.
Any one of the target genes may be a GUS gene (exogenous gene) or a Tawax gene (endogenous gene).
Any one of the gene editing vectors can be specifically a vector pMWB110-SpCas9-TaU3-Gus (a vector containing Ubi and TaU3 promoters for respectively starting SpCas9 and sgRNA aiming at exogenous gene GUS) or a vector pMWB110-SpCas9-TaU3-WAXY (a vector containing Ubi and TaU3 promoters for respectively starting SpCas9 and sgRNA aiming at endogenous gene TaWaxy).
Any one of the above wheat may be specifically a transgenic plant not subjected to GUS gene editing, a transgenic plant not subjected to tawax gene editing or a transgenic plant subjected to single genome editing of tawax gene as mentioned in the examples.
The invention utilizes nicotinamide to treat young embryo or mature embryo of transgenic wheat containing gene editing vector but with target gene not edited or partially edited to obtain mutant plant with target gene edited, thereby improving wheat gene editing efficiency. Among them, the exogenous GUS gene and the endogenous TaWaxy gene obtained by treating for 14 days with 2.5mM nicotinamide have the highest editing efficiency, and the editing efficiency is respectively improved by 36.0% and 22.5% compared with that of a control group (without the treatment of nicotinamide). The invention has important application value.
Drawings
FIG. 1 shows the results of the detection of mutation types in the partially edited plant of step two in example 1. Wherein, A is the detection result without nicotinamide treatment, M is DNA marker, lanes 1-7 are 2-CK-G,
FIG. 2 is the partial results of the detection of the type of post-juvenile mutation in transgenic plants with no editing of the Nicotinamide-treated Tawax gene in step two of example 2. Wherein M is DNA marker, lanes 1-5 are 14-CK-W1, lanes 6-8 are 14-2.5-W1, lanes 9-10 are 14-5-W1,
FIG. 3 is the partial results of the post-embryonic mutation type of 5 in step two of example 2 in transgenic plants with single genome editing of the Nicotinamide-treated Tawax gene. Where M is a 2kb DNA marker, lanes 1-2 are edited in the transgenic plant only 7D, lanes 3-4 are edited in the transgenic plant only 4A, lanes 5-6 are edited in the transgenic plant only 7A,
FIG. 4 is a partial examination of mutation types after mature embryos of 5 step two nicotinamide-treated unedited plants of example 3. Wherein M is a 2kb DNA marker, lanes 1-5 are 14-2.5-W3, lanes 6-10 are 14-5-W3, lanes 11-15 are 14-CK-W3,
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.
1/2MS culture medium: 2.215g of MS powder, 20g of cane sugar and 0.5g of MES are dissolved in a proper amount of distilled water, and then the volume is determined to be 1L by using the distilled water; adding 3g of plant gel, and adjusting the pH value to 5.8; sterilizing at 121 deg.C for 15 min.
2 × Taq PCR StarMix is a product of Novowed Biotechnology Ltd, catalog No. P213-03. The plant genome DNA extraction kit is a product of biological science and technology limited company of century, and the product catalog number is CW 0531M.