WO2019205919A1 - 一种生物突变体库的构建方法 - Google Patents
一种生物突变体库的构建方法 Download PDFInfo
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Definitions
- the invention relates to a method for constructing a biological mutant library in the field of bioengineering technology.
- Gene editing technology enables precise gene editing in biological cells.
- the technical principle is to form a complex of RNA and protein (referred to as RNP) by binding a guide RNA (sgRNA or gRNA) to a DNA endonuclease (such as Cas9, Cpf1, etc.), which can be searched on the genome and guided RNA.
- sgRNA or gRNA guide RNA
- Cas9, Cpf1, etc. DNA endonuclease
- Complementary target sequences such that the endonuclease precisely cleaves the bound DNA in this region.
- the results of the cleavage are diverse and can be double-stranded DNA cleavage (DSB) at the blunt end or sticky end, or single-stranded DNA cleavage (Nick).
- DSB double-stranded DNA cleavage
- Nick single-stranded DNA cleavage
- plants capable of producing seeds of different editing types are directly obtained by a transgenic process, that is, T1 generation Arabidopsis plants transformed by Agrobacterium tumefax method, or T0 generation plants obtained by tissue culture method.
- the cost of the transgenic process is high and inefficient, making it difficult to obtain large quantities of plants that produce offspring of different editing types.
- the technical problem to be solved by the present invention is how to prepare a mutant library. Numerous studies have shown that the repair of DSBs produced at specific sites results in a large number of different mutation types. In order to enable these new mutant genes to be passed on to the next generation, specifically targeting in germ cells, it is theoretically possible to produce a large number of offspring individuals with different mutant genes. Taking plants as an example, if a large number of seeds with different mutation types can be continuously produced, a mutant seed bank at this site can be established.
- the present invention first provides a method for constructing a biological mutant library, which is X1) or X2):
- the method X1) includes the following X11) and X12):
- X11 introducing a DNA fragment into a target organism to obtain a transgenic organism; the DNA fragment is capable of expressing, in the transgenic organism, a desired element for performing site-directed mutagenesis target DNA;
- the method X2) includes the following X21) and X22):
- X21 introducing a DNA fragment into a target organism to obtain a transgenic organism; the DNA fragment is capable of expressing, in the transgenic organism, a desired element for completing site-directed mutagenesis target DNA;
- X22 cultivating the transgenic organism, selecting an individual (referred to as a mutant) in which the target DNA is mutated from the transgenic organism to obtain a biological mutant library, and selecting the DNA fragment from the transgenic organism and the Individuals whose target DNA has not been mutated (remembered as a mutant stock reserve individual) are propagated to expand the mutant pool.
- the mutant library reserve individual can further generate an individual in which the target DNA is mutated and an individual containing the DNA fragment and the target DNA is not mutated, and the individual whose target DNA is mutated can be classified as a mutant.
- a mutant library wherein the individual containing the DNA fragment and the target DNA is not mutated may be further used as a mutant library stocking individual for breeding mutant and mutant library stock individuals, and will "generate the target DNA to occur This characteristic of the mutated individual and the individual containing the DNA fragment and the target DNA is not mutated is inherited.
- the number of mutants in the mutant pool is expanded by breeding the mutant library stock individuals. The number of breedings can be determined according to specific needs.
- a mutation at a specific site of the target DNA can be produced by cleavage of the target DNA by a DNA endonuclease to repair DSB, or by base deaminase or other base changeable.
- the enzyme is implemented.
- the organism may be a sexual reproductive organism or an asexual reproductive organism such as a plant, an animal, a fungus or a bacterium.
- the organism is a sexual reproductive organism, and the transgenic organism is capable of specifically mutating the target DNA in a germ cell.
- the plant may be a dicot or a monocot.
- the dicotyledonous plant may be a cruciferous plant.
- the germ cells include, but are not limited to, egg cells, oocytes, pollen cells, or pollen mother cells.
- At least one of the elements is activated by a germ cell specific promoter.
- the germ cell-specific promoter may specifically be an egg cell-specific promoter, a pollen cell-specific promoter or a meiosis-specific promoter.
- the egg cell-specific promoter may be a fusion promoter of DD45 (SEQ ID NO: 6), EC 1.1 (SEQ ID NO: 3) or EC 1.1-1.2 (SEQ ID NO: 4).
- the pollen cell-specific promoter may specifically be the rice Os08g0560700 promoter (SEQ ID NO: 7), the tomato LAT52 promoter (SEQ ID NO: 8) or the Arabidopsis AtSPL (SEQ ID NO: 5).
- the meiotic-specific promoter may be a promoter of the Arabidopsis AT4G40020 (SEQ ID NO: 9), AT4G20900 (SEQ ID NO: 10) or AT1G15320 (SEQ ID NO: 11) genes.
- the element may be a component required in the CRISPR/Cas method or the CRISPR/Cpf1 method.
- the components required in the CRISPR/Cas method or the CRISPR/Cpf1 method may include a1) or a2):
- A1 DNA endonuclease and sgRNA
- the endonuclease can be Cas9, Cas9n, dCas9 or xCas9.
- the DNA endonuclease used in the CRISPR/Cpf1 method may be Cpf1.
- At least one of the endonuclease and the sgRNA is activated by a germ cell-specific promoter and can be a1), a2) or a3):
- the DNA endonuclease is activated by the germ cell specific promoter, and the sgRNA is activated by a constitutive promoter;
- the sgRNA is activated by the germ cell specific promoter, and the endonuclease is activated by the constitutive promoter;
- the sgRNA targets the target DNA.
- the target DNA may be a coding region of a gene or a regulatory region of a gene, such as a promoter, an enhancer, a 5' UTR, or the like.
- the expression product of the coding region can be a protein, a polypeptide or an RNA.
- the organism is a plant
- the germ cell-specific promoter is an egg cell-specific promoter
- X22 specifically includes: selecting an egg cell containing the DNA fragment and the target DNA is mutated from the transgenic organism An individual formed by fertilization with sperm (the sperm may contain the DNA fragment or may not contain the DNA fragment), the mutant library is selected as a mutant; and the DNA fragment is selected from the transgenic organism without An egg cell in which the target DNA is not mutated and an individual formed by sperm fertilization containing the DNA fragment and the target DNA is not mutated (referred to as individual 1) is stored as a mutant library.
- the method comprises, after X22), the following step X23a): selecting an egg cell and a sperm containing the DNA fragment and the target DNA is mutated from the progeny of the individual 1 (the sperm may also contain the DNA fragment)
- An individual formed by fertilization without the DNA fragment may be selected as a mutant in the mutant library; an egg cell containing no such DNA fragment and the target DNA is not mutated from the progeny of the individual 1 and containing
- the DNA fragment and the subject formed by sperm fertilization in which the target DNA has not been mutated are further used as a mutant library stocking individual for preparing a mutant and an individual having the characteristics of the individual 1 as a mutant stock reserve individual.
- step X23a By repeating step X23a), the number of mutants in the mutant library can be further expanded.
- the number of repetitions may be several times, and the number of repetitions may be determined according to specific needs.
- the organism is a plant
- the germ cell-specific promoter is a sperm cell-specific promoter
- X22 specifically includes: selecting the DNA fragment from the transgenic organism and the target DNA is mutated.
- An individual formed by fertilization of sperm and egg cells (which may or may not contain the DNA fragment), is selected as a mutant in the mutant library; and the genetically modified organism is selected to be free of the DNA fragment and
- the sperm whose target DNA has not been mutated and the individual formed by the fertilization of the egg cell containing the DNA fragment and the target DNA is not mutated are stored as a mutant library.
- the method comprises, after X22), the following step X23b): selecting sperm and egg cells containing the DNA fragment and the target DNA is mutated from the progeny of the individual 2 (the egg cell may also contain the DNA fragment)
- An individual formed by fertilization without the DNA fragment may be selected as a mutant, and a mutant library may be selected as a mutant; and a sperm containing the DNA fragment and the target DNA is not mutated from the progeny of the individual 2 is selected and contained
- the DNA fragment and the individual formed by the fertilization of the egg cell in which the target DNA has not been mutated are further used as a mutant library reserve individual for preparing a mutant and an individual having the characteristics of the individual 2 as a mutant library reserve individual.
- step X23b By repeating step X23b), the number of mutants in the mutant library can be further expanded.
- the number of repetitions may be several times, and the number of repetitions may be determined according to specific needs.
- the DNA fragment may contain b1) or b2):
- the DNA fragment can also express a selection marker that can be used to screen for a transgenic organism.
- the expression cassette refers to a DNA capable of expressing a protein of interest or an RNA of interest in a host, and the DNA may include not only a promoter that activates transcription of a gene encoding a gene of interest or a gene encoding a gene of interest (the promoter is referred to as a promoter S).
- a promoter S A terminator that terminates transcription of a protein encoding gene of interest or a gene encoding a gene of interest may also be included.
- the promoter S can be a constitutive promoter.
- An expression cassette capable of expressing the DNA endonuclease and an expression cassette capable of expressing the sgRNA are designated as Expression cassette 1 and Expression cassette 2, respectively.
- the promoter in at least one of the expression cassette 1 and the expression cassette 2 may be the tissue-specific promoter.
- the DNA fragment further comprises an expression cassette capable of expressing the selection marker (the expression cassette is referred to as expression cassette 3).
- the selection marker is a hygromycin resistance marker.
- the expression cassette 3 contains a hygromycin resistance gene. Further, the expression cassette 3 further comprises a promoter that initiates expression of the hygromycin resistance gene and a terminator that terminates expression of the hygromycin resistance gene.
- the DNA fragment contains only the expression cassette 1 and the expression cassette 2, the order of the upstream and downstream of the expression cassette 1 and the expression cassette 2 is not particularly required as long as the expression of the endonuclease can be expressed.
- the sgRNA can be.
- the DNA fragment contains the expression cassette 1, the expression cassette 2, and the expression cassette 3, the order of ligation between the expression cassettes is not particularly required as long as the expression of the DNA endonuclease can be achieved.
- the sgRNA and the resistance marker are sufficient.
- the DNA fragment may also contain a DNA sequence that links each expression cassette.
- the target DNA may be DNA related to biological stress, abiotic stress, yield trait, quality trait or secondary metabolite.
- the target DNA may specifically be a gene associated with bio-adversity, abiotic stress, yield trait, quality trait or secondary metabolite.
- the biological mutant library may be an anti-biotic stress mutant library, an anti-abiotic stress mutant library, a yield trait mutant library, a quality trait mutant library or a secondary metabolite mutant library.
- the invention also protects the use of the method in screening for antibiotic stress, abiotic stress resistance, high yield, good quality or high secondary metabolite mutants.
- the biological stress includes, but is not limited to, diseases, insects, nematodes, viruses, and the like.
- the abiotic stresses include, but are not limited to, herbicides, antibiotics, insecticides, bactericides, drought, cockroaches, cold, heat, salt, heavy metals, and the like.
- the present invention provides a method for constructing a biological mutant library which obtains a transgenic organism by introducing a DNA fragment capable of expressing a desired element for performing site-directed mutagenesis target DNA into a target organism.
- the target organism is a sexual reproductive organism
- the promoter of at least one of the required elements of the site-directed mutant target DNA is a germ cell-specific promoter, and thus the obtained transgenic organism can specifically target the target DNA in the germ cell.
- Site-directed mutagenesis according to the genotype and targeting situation of male and female gametes, to obtain progeny with different genotypes, including progeny with mutation of target gene, non-transgenic progeny with no mutation of target DNA, and transgenic offspring with no mutation of target DNA .
- the progeny of the target gene mutation can be used to screen individuals with the target trait, and the transgenic progeny whose target DNA has not been mutated will be able to continue site-directed mutagenesis of the target DNA in the germ cell and pass it to its offspring, as well as the target DNA. Mutant transgenic offspring. This process can be passed down from generation to generation, and in the process of reproduction, a large number of plants with mutations of target DNA can be produced in each generation, and the mutations are diverse, and thus a mutant library in which a large number of target DNA mutations can be created, and a process of producing a mutant library of plants As shown in Figure 1.
- organisms with the following traits can be screened from the obtained mutant libraries: antibiotic stress, antibiotic abiotic stress, high yield, good quality traits, high secondary metabolite yield, and the like. Therefore, the method of the present invention has great application prospects.
- Figure 1 shows the production process of a library of plant mutants
- Figure 2 shows the results of PCR amplification and sequencing of the target region of ALS by extracting DNA from 13 T1 plants;
- Figure 3 shows the screening of a large number of herbicide resistant mutants
- Figure 4 shows the results of PCR sequencing of DNA from 17 herbicide-tolerant plants.
- Example 1 Construction of a knockout vector for the Arabidopsis ALS gene, the Cas9 gene is specifically expressed in egg cells.
- the target is ttgccgatgatcccgagtgg
- the Arabidopsis ALS gene DNA is shown in SEQ ID NO: 1
- the amino acid sequence thereof is shown in SEQ ID NO: 2.
- Example 2 Transformation of Arabidopsis thaliana ⁇ Methods and materials used in this section, refer to (3) Chen Y, Wang Z, Ni H, Xu Y, Chen Q, Jiang L. 2017. CRISPR/Cas9-mediated base-editing System efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.
- the bacterial solution was applied to YEP solid medium (containing kanamycin and gentamicin) by scribing, in a dark environment at 28 ° C. After 36-48 hours of culture, a single colony was picked and inoculated into 1 ml of liquid YEB medium to which kanamycin and gentamicin had been added, and cultured overnight with shaking (28 ° C, 200 rpm).
- colony PCR was performed, and the positive clones were selected, added to a 50 ml Erlenmeyer flask, and 25 ml of YEP liquid medium (containing kanamycin and gentamicin) was added, and cultured at 28 ° C and 200 rpm. Until the OD600 value rises to within 0.8-1.0. The supernatant was removed by centrifugation, and the cells were resuspended in the same volume of 5% sucrose solution (100 ml sterile deionized water plus 5 g sucrose), and Silwet L-77 (0.02%) was added to the bacterial solution to mix. .
- 5% sucrose solution 100 ml sterile deionized water plus 5 g sucrose
- Silwet L-77 0.02%
- the appropriate Arabidopsis inflorescence was taken in the bacterial liquid for 0.5-1 min, and the black opaque plastic bag was covered on the Arabidopsis seedlings to create dark conditions.
- the cells were placed in a greenhouse at 22 ° C for 24 h and then transferred to light culture. Seeds are harvested after the seeds are ripe.
- the harvested Arabidopsis seeds were sterilized and screened for transgenic plants on MS medium containing hygromycin (25 mg/L). DNA of T1 plants was extracted, and PCR amplification and sequencing were performed on the target region of ALS.
- the type of editing of the ALS gene was determined by PCR product sequencing, and hygromycin resistance screening, while plants with T-DNA and ALS genes not edited (magic seed plants) were isolated.
- the first genotype contains T-DNA (that is, the ability to express site-directed mutant target DNA in the transgenic organism).
- the DNA fragment of the element due to the specific expression of the egg cell, the target gene on the genome will be edited (ie, mutated), and the second genotype is, without T-DNA, so the target gene on the genome is not Edited, still wild type.
- pollen genotypes the ratio is 1:1.
- the first genotype contains T-DNA. Due to the specific expression of egg cells, the target genes on the genome will not be edited and remain wild type.
- the second genotype is that it does not contain T-DNA, and the target gene on the genome is wild type.
- the harvested seeds had four different genotypes with a ratio of 1:1:1:1.
- the first genotype consists of a copy of the T-DNA and contains the edited target gene;
- the second genotype is a T-DNA containing two copies and contains the edited target gene;
- the genotype is a target gene that does not contain T-DNA and does not contain an edited target gene, that is, a wild type plant;
- the fourth genotype is a T-DNA containing one copy and does not contain an edited target gene.
- the genotypes of the fourth genotype are identical to those of the mother, and the probability of occurrence is 1/4. If the T-DNA is screened (ie, screened by hygromycin), the probability of occurrence is 1/3. .
- These plants can be selected by sequencing the target genes. Of the offspring produced by these plants, 1/2 of the individuals will also have edited target genes, 1/4 of which are wild type and 1/4 of which are maternal genotypes. This process can be looped indefinitely.
- This seed with the fourth genotype produces seeds that are identical to their own genotype and can produce more seeds with independent mutation events, so a mutation that can include an infinite individual can be established from one seed.
- the body library, the seed of this genotype is named "magic seed.”
- plants containing T-DNA and containing an edit and an unedited target gene can continue to generate a large number of independent re-editing events in the offspring.
- T1 plants The DNA of 13 T1 plants was extracted, and the target region of ALS was subjected to PCR amplification and sequencing. The results indicated that approximately 70% of T1 plants produced mutations at the expected location of ALS, as shown in Figure 2.
- T1 plants Using the characteristics of egg-specific targeting, these T1 plants re-edit the unedited ALS locus when seed (T2 generation), independently generating new mutation types.
- the technical principle of re-targeting also known as the "magic seed” system is shown in Figure 1.
- T2 generation seeds showed herbicide-resistant mutants.
- T2 generation seeds harvested from the above T1 plants were sterilized and spread on MS medium containing 0.24 mg/L of methimazole nicotinic acid, and the herbicide-resistant mutants were screened, as shown in Fig. 3, and a large number of successfully screened. Herbicide resistant plants.
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Abstract
本发明公开了一种生物突变体库的构建方法。本发明公开的生物突变体库的构建方法包括:1)向靶标生物中导入DNA片段,得到转基因生物;该DNA片段能在转基因生物中表达完成定点突变靶标DNA的所需的元件;2)培养转基因生物,得到生物突变体库;该生物为有性生殖生物,转基因生物能特异在生殖细胞中定点突变靶标DNA。利用本发明的方法得到的突变体库可筛选到具有以下性状的生物:抗生物逆境,抗非生物逆境,高产量,好品质性状,高次生代谢物产量等。因此,本发明的方法具有巨大的应用前景。
Description
本发明涉及生物工程技术领域中,一种生物突变体库的构建方法。
基因编辑技术,特别是CRISPR/Cas9技术,在生物细胞中实现了精准的基因编辑。其技术原理是通过一段引导RNA(sgRNA或gRNA)与DNA内切酶(如Cas9、Cpf1等)结合形成RNA和蛋白的复合物(简称RNP),该复合物可以在基因组上搜索与引导RNA上互补的目标序列,从而使得DNA内切酶精准地在该区域对结合的DNA进行剪切。根据不同的DNA内切酶特性,剪切的结果多种多样,可以是平末端或粘性末端的双链DNA断裂(DSB),也可以是单链DNA断裂(Nick)。生物体对DSB或Nick的修复会导致***或删除(Indel),从而实现了对靶标基因的精准编辑。
目前具有能够产生不同编辑类型种子的植株均是通过转基因过程直接获得,即农杆菌蘸花法转化得到的T1代拟南芥植株,或者通过组织培养的方法获得的T0代植株。转基因过程的成本高、效率低,很难大量获取这种能够产生不同编辑类型后代的植株。
发明内容
本发明所要解决的技术问题是如何制备突变体库。大量的研究表明,针对特定位点产生的DSB的修复结果是产生了大量不同的突变类型。为了能够使这些新的突变基因能够传递到下一代,在生殖细胞中特异地启动打靶,理论上会产生大量含有不同突变基因的后代个体。以植物为例,如果能够持续不断的产生大量的具有不同突变类型的种子,就能够建立一个在该位点的突变体种子库。
为解决上述技术问题,本发明首先提供了一种生物突变体库的构建方法,其为X1)或X2):
所述方法X1)包括下述X11)和X12):
X11)向靶标生物中导入DNA片段,得到转基因生物;所述DNA片段能在所述转基因生物中表达完成定点突变靶标DNA的所需的元件;
X12)培养所述转基因生物,得到生物突变体库;
所述方法X2)包括下述X21)和X22):
X21)向靶标生物中导入DNA片段,得到转基因生物;所述DNA片段能在所述转基因生物中表达完成定点突变靶标DNA的所需的元件;
X22)培养所述转基因生物,从所述转基因生物中选择所述靶标DNA发生突变的个体(记为突变体)得到生物突变体库,从所述转基因生物中选择含有所述DNA片段且所述靶标DNA 未发生突变的个体(将其记为突变体库储备个体)进行繁殖,以扩大所述突变体库。
所述突变体库储备个体能进一步产生所述靶标DNA发生突变的个体和含有所述DNA片段且所述靶标DNA未发生突变的个体,所述靶标DNA发生突变的个体可作为突变体归入生物突变体库,所述含有所述DNA片段且所述靶标DNA未发生突变的个体可作为突变体库储备个体进一步用于繁殖突变体和突变体库储备个体,并将“产生所述靶标DNA发生突变的个体和含有所述DNA片段且所述靶标DNA未发生突变的个体”的这种特征遗传下去。通过繁殖所述突变体库储备个体,扩大所述突变体库中突变体的数量。所述繁殖的次数可根据具体需要确定。
具体的,所述靶标DNA的特定位点产生突变(即定点突变)可通过DNA内切酶对所述靶标DNA切割后修复DSB产生,也可以是通过碱基脱氨酶或其它可改变碱基的酶来实现。
上述方法中,所述生物可为有性生殖生物或无性生殖生物,如植物、动物、真菌或细菌。
上述方法中,所述生物为有性生殖生物,所述转基因生物能特异在生殖细胞中定点突变所述靶标DNA。
所述植物可为双子叶植物或单子叶植物。所述双子叶植物可为十字花科植物。
所述生殖细胞包括但不仅限于***、***、花粉细胞或花粉母细胞。
上述方法中,所述元件中至少有一个由生殖细胞特异启动子启动表达。
上述方法中,所述生殖细胞特异启动子具体可为***特异启动子、花粉细胞特异启动子或减数***特异启动子。所述***特异启动子可为DD45(SEQ ID NO:6)、EC1.1(SEQ ID NO:3)或EC1.1-1.2的融合启动子(SEQ ID NO:4)。所述花粉细胞特异启动子具体可为水稻Os08g0560700启动子(SEQ ID NO:7)、番茄LAT52启动子(SEQ ID NO:8)或拟南芥AtSPL(SEQ ID NO:5)。所述减数***特异启动子可为拟南芥AT4G40020(SEQ ID NO:9)、AT4G20900(SEQ ID NO:10)或AT1G15320(SEQ ID NO:11)基因的启动子。
上述方法中,所述元件可为CRISPR/Cas方法或CRISPR/Cpf1方法中所需的元件。
上述方法中,所述CRISPR/Cas方法或CRISPR/Cpf1方法中所需的元件均可包括a1)或a2):
a1)DNA内切酶和sgRNA;
a2)所述DNA内切酶、crRNA和tracrRNA。
所述DNA内切酶可为Cas9、Cas9n、dCas9或xCas9。所述CRISPR/Cpf1方法中所用的DNA内切酶可为Cpf1。
具体的,所述DNA内切酶和所述sgRNA中至少有一种由生殖细胞特异启动子启动表达可为下述a1)、a2)或a3):
a1)所述DNA内切酶由所述生殖细胞特异启动子启动表达,所述sgRNA由组成型启动子启动表达;
a2)所述sgRNA由所述生殖细胞特异启动子启动表达,所述DNA内切酶由所述组成型启动子启动表达;
a3)所述DNA内切酶和所述sgRNA均由所述生殖细胞特异启动子启动表达。
上述方法中,所述sgRNA靶向所述靶标DNA。所述靶标DNA可以为基因的编码区,也可以为基因的调控区,例如启动子、增强子、5’UTR等。所述编码区的表达产物可以为蛋白质、多肽或RNA。
上述方法中,所述生物为植物,所述生殖细胞特异启动子为***特异启动子,X22)具体可包括:从所述转基因生物中选择含有所述DNA片段且所述靶标DNA发生突变的***与***(该***可含有所述DNA片段也可不含所述DNA片段)受精所形成的个体,作为突变体入选所述突变体库;从所述转基因生物中选择不含有所述DNA片段且所述靶标DNA未发生突变的***和含有所述DNA片段且所述靶标DNA未发生突变的***受精所形成的个体(将其记为个体1),作为突变体库储备个体。
进一步,所述方法在X22)后包括如下步骤X23a):从所述个体1的后代中选择含有所述DNA片段且所述靶标DNA发生突变的***与***(该***可含有所述DNA片段也可不含所述DNA片段)受精所形成的个体,作为突变体入选所述突变体库;从所述个体1的后代中选择不含有所述DNA片段且所述靶标DNA未发生突变的***和含有所述DNA片段且所述靶标DNA未发生突变的***受精所形成的个体,进一步作为突变体库储备个体用于制备突变体和具有所述个体1特征的可作为突变体库储备个体的个体。
重复步骤X23a),可进一步扩大所述突变体库中突变体的个数。所述重复的次数可为若干次,重复次数可根据具体需要进行确定。
上述方法中,所述生物为植物,所述生殖细胞特异启动子为***特异启动子,X22)具体可包括:从所述转基因生物中选择含有所述DNA片段且所述靶标DNA发生突变的***与***(该***可含有所述DNA片段也可不含所述DNA片段)受精所形成的个体,作为突变体入选所述突变体库;从所述转基因生物中选择不含有所述DNA片段且所述靶标DNA未发生突变的***和含有所述DNA片段且所述靶标DNA未发生突变的***受精所形成的个体(将其记为个体2),作为突变体库储备个体。
进一步,所述方法在X22)后包括如下步骤X23b):从所述个体2的后代中选择含有所述DNA片段且所述靶标DNA发生突变的***与***(该***可含有所述DNA片段也可不含所述DNA片段)受精所形成的个体,作为突变体入选所述突变体库;从所述个体2的后代中选择不含有所述DNA片段且所述靶标DNA未发生突变的***和含有所述DNA片段且所述靶标DNA未发生突变的***受精所形成的个体,进一步作为突变体库储备个体用于制备突变 体和具有所述个体2特征的可作为突变体库储备个体的个体。
重复步骤X23b),可进一步扩大所述突变体库中突变体的个数。所述重复的次数可为若干次,重复次数可根据具体需要进行确定。
上述方法中,所述DNA片段可含有b1)或b2):
b1)能表达所述DNA内切酶的表达盒和能表达所述sgRNA的表达盒;
b2)能表达所述DNA内切酶的表达盒、能表达所述crRNA的表达盒和能表达所述tracrRNA的表达盒。
所述DNA片段还能表达能用于筛选转基因生物的筛选标记。
所述表达盒是指能够在宿主中表达目的蛋白或目的RNA的DNA,该DNA不但可包括启动目的蛋白编码基因或目的RNA编码基因转录的启动子(将该启动子记为启动子S),还可包括终止目的蛋白编码基因或目的RNA编码基因转录的终止子。所述启动子S可为组成型启动子。
将能表达所述DNA内切酶的表达盒和能表达所述sgRNA的表达盒分别记为表达盒1和表达盒2。在所述生物为所述植物或所述动物时,所述表达盒1和所述表达盒2中至少有一个表达盒中的启动子可为所述组织特异性启动子。
具体的,所述DNA片段还含有能表达所述筛选标记的表达盒(将该表达盒记为表达盒3)。
在本发明的一个实施例中,所述筛选标记为潮霉素抗性标记。进一步,所述表达盒3含有潮霉素抗性基因。更进一步,所述表达盒3还含有启动所述潮霉素抗性基因表达的启动子以及终止所述潮霉素抗性基因表达的终止子。
在所述DNA片段仅含有所述表达盒1和所述表达盒2时,所述表达盒1和所述表达盒2的上下游顺序无特殊要求,只要能实现能表达所述DNA内切酶和所述sgRNA即可。在所述DNA片段含有所述表达盒1、所述表达盒2以及所述表达盒3时,各表达盒间的连接顺序无特殊要求,只要能实现能表达所述DNA内切酶、所述sgRNA以及所述抗性标记即可。所述DNA片段还可含有连接各表达盒的DNA序列。
上述方法中,所述靶标DNA可为与生物逆境、非生物逆境、产量性状、品质性状或次生代谢物相关DNA。
所述靶标DNA具体可为与生物逆境、非生物逆境、产量性状、品质性状或次生代谢物相关的基因。
进一步,所述生物突变体库可为抗生物逆境突变体库、抗非生物逆境突变体库、产量性状突变体库、品质性状突变体库或次生代谢物突变体库。
本发明还保护所述方法在筛选抗生物逆境、抗非生物逆境、高产量、好品质或高次生代 谢物突变体中的应用。
所述生物逆境包括但不仅限于病、虫、线虫、病毒等。所述非生物逆境包括但不仅限于除草剂、抗生素、杀虫剂、杀菌剂、旱、涝、寒、热、盐、重金属等。
本发明提供了一种生物突变体库的构建方法,该方法通过将能表达完成定点突变靶标DNA的所需元件的DNA片段导入靶标生物中,得到了一种转基因生物。当靶标生物为有性生殖生物时,定点突变靶标DNA的所需元件中至少有一个元件的启动子为生殖细胞特异启动子,因此,得到的转基因生物能够特异地在生殖细胞中对靶标DNA进行定点突变,根据雌雄配子的基因型及打靶情形,进而得到具有不同基因型的后代,其中包括靶标基因发生突变的后代、靶标DNA未发生突变的非转基因后代、以及靶标DNA未发生突变的转基因后代。靶标基因发生突变的后代可用于筛选含有目标性状的个体,而靶标DNA未发生突变的转基因后代将能够在生殖细胞中对靶标DNA继续进行定点突变并传至其后代,同时也会产生靶标DNA未发生突变的转基因后代。此过程可以世代相传,并且在繁殖的过程中,每代均可产生大量靶标DNA发生突变的植株,并且突变多样,进而可以创制大量靶标DNA发生突变的突变体库,植物突变体库的产生过程如图1所示。根据靶标DNA的不同,可以从得到的突变体库中筛选具有以下性状的生物:抗生物逆境,抗非生物逆境,高产量,好品质性状,高次生代谢物产量等。因此,本发明的方法具有巨大的应用前景。
图1显示了植物突变体库的产生过程;
图2显示了提取13株T1代植株的DNA,针对ALS的靶点区域进行PCR扩增并测序的结果;
图3显示了筛选到大量抗除草剂突变体;
图4显示了17株抗除草剂植株的DNA,进行PCR测序的结果。
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂、仪器等,如无特殊说明,均可从商业途径得到。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1.构建针对拟南芥ALS基因的敲除载体,其Cas9基因在***中特异表达。
其中,靶点为
ttgccgatgatcccgagtgg,拟南芥ALS基因DNA示于SEQ ID NO:1中,其氨基酸序列示于SEQ ID NO:2中。
将DNA片段5’
ttgccgatgatcccgagtgg3’克隆至pHEE401E载体{参考(1)Xing H L,Dong L,Wang Z P,et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327.(2)Wang Z P,Xing H L,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J].Genome Biology,2015,16(1):144.}中的sgRNA表达盒中,转入农杆菌GV3101中,以供通过蘸花法转化拟南芥。
实施例2.对拟南芥进行转化{该部分所用方法和材料,参考(3)Chen Y,Wang Z,Ni H,Xu Y,Chen Q,Jiang L.2017.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}
将构建好的载体通过冻融法转化到农杆菌GV3101菌株中之后,将菌液通过划线法涂于YEP固体培养基中(含有卡那霉素和庆大霉素),在28℃黑暗环境下培养36-48h后,挑取单菌落接种于1ml的已加入卡那霉素和庆大霉素的液体YEB培养基中,振荡培养过夜(28℃200rpm)。此时进行菌落PCR鉴定,并选取其中阳性克隆,加入到50ml锥形瓶中,加入25ml YEP液体培养基(含有卡那霉素和庆大霉素),在28℃200rpm的条件下震荡培养,直到OD600值升高到0.8-1.0内。取菌液离心沉淀去除上清液,并用相同体积的5%的蔗糖溶液(100ml无菌去离子水加5g蔗糖)重悬菌体,并在菌液中加入SilwetL-77(0.02%)混匀。将适宜的拟南芥花序在菌液中蘸取0.5-1min后,在拟南芥苗上面遮盖黑色不透光塑料袋,创造黑暗条件,放置于22℃温室培养24h后转入光照培养,待种子成熟后收获种子。收获的拟南芥种子经过消毒后,在含有潮霉素(25mg/L)的MS培养基上筛选转基因植株。提取T1代植株的DNA,针对ALS的靶点区域进行PCR扩增并测序。
实施例3.鉴定T1代植株的基因型
通过PCR产物测序,以及潮霉素抗性筛选确定对ALS基因的编辑类型,同时分离具有T-DNA且ALS基因未被编辑的植株(魔法种子植株)。
如图1所示,转基因植株产生的***有两种类型,比例为1:1,第一种基因型是含有T-DNA(即能在所述转基因生物中表达完成定点突变靶标DNA的所需的元件的DNA片段),由于***特异表达的特性,基因组上的靶点基因将被编辑(即发生突变),第二种基因型是,不含有T-DNA,因此基因组上的靶点基因没有被编辑,仍然是野生型。花粉的基因型也同样有两种,比例为1:1,第一种基因型是含有T-DNA,由于***特异表达的特性,基因组上的靶点基因不会被编辑,仍为野生型,第二种基因型是,不含有T-DNA,基因组上的靶点基因为野生型。该植株自交后,收获的种子具有四种不同的基因型,比例为1:1:1:1。第一种基因型为含有一个拷贝的T-DNA且含有被编辑过的靶点基因;第二种基因型为含有两个拷贝的T-DNA且含有被编辑过的靶点基因;第三种基因型为不含有T-DNA且不含有被编辑过的靶点基因,即野生型植株;第四种基因型为含有一个拷贝的T-DNA且不含有被编辑过的靶点基因。第四种基因型的植株与母代的基因型完全相同,出现的概 率为1/4,如果对T-DNA进行筛选的话(即通过潮霉素筛选),则其出现的概率为1/3。这些植株可以通过对靶点基因的测序挑选出来。这些植株产生的后代中也将有1/2的个体含有被编辑过的靶点基因,1/4为野生型,1/4为母代基因型。该过程可以无限循环下去。这种具有第四种基因型的种子能够产生与自己基因型完全相同的种子,还能够产生更多的具有独立突变事件的种子,因此从一粒种子开始即可建立一个可包括无限个体的突变体库,将这种基因型的种子命名为“魔法种子”。此外,根据“魔法种子”的技术原理,含有T-DNA且含有一个编辑和一个未编辑靶点基因的植株,在后代中也可以继续产生大量独立的重新编辑事件。
提取13株T1代植株的DNA,针对ALS的靶点区域进行PCR扩增并测序。结果表明约70%的T1植株在ALS的预期位置上产生了突变,如图2所示。
利用***特异打靶的特性可知,这些T1植株在产生种子(T2代)的时候,会对未编辑的ALS位点重新进行编辑,独立产生新的突变类型。重新打靶的技术原理(又称“魔法种子”体系)如图1所示。
实施例4.筛选抗除草剂突变体
利用“魔法种子”的技术路线,T2代种子出现了抗除草剂的突变体。
将从上述T1代植株上收获到的T2代种子,消毒后铺到含有0.24mg/L甲咪唑烟酸的MS培养基上,筛选抗除草剂突变体,如图3所示,成功筛选到大量抗除草剂植株。
随机提取其中17株抗除草剂植株的DNA,PCR测序表明这些植株的ALS基因发生了如图4所示的基因突变。
说明书中提及的所有出版物和专利申请均通过引用并入本文,如同每篇出版物或专利申请被单独、特别地通过引用并入本文一样。
尽管为清楚理解起见,前述发明已通过举例说明和实施例的方式较为详细地进行了描述,但显而易见的是,可以在所附权利要求书的范围内实施某些改变和修改,这样的改变和修改均在本发明的范围之内。
Claims (10)
- 一种生物突变体库的构建方法,其特征在于:所述方法为X1)或X2):所述方法X1)包括下述X11)和X12):X11)向靶标生物中导入DNA片段,得到转基因生物;所述DNA片段能在所述转基因生物中表达完成定点突变靶标DNA的所需的元件;X12)培养所述转基因生物,得到生物突变体库;所述方法X2)包括下述X21)和X22):X21)向靶标生物中导入DNA片段,得到转基因生物;所述DNA片段能在所述转基因生物中表达完成定点突变靶标DNA的所需的元件;X22)培养所述转基因生物,从所述转基因生物中选择所述靶标DNA发生突变的个体得到生物突变体库,从所述转基因生物中选择含有所述DNA片段且所述靶标DNA未发生突变的个体进行繁殖,以扩大所述突变体库。
- 根据权利要求1所述的方法,其特征在于:所述生物为有性生殖生物或无性生殖生物。
- 根据权利要求1所述的方法,其特征在于:所述生物为有性生殖生物,所述转基因生物能特异在生殖细胞中定点突变所述靶标DNA。
- 根据权利要求1-3中任一所述的方法,其特征在于:所述元件中至少有一个由生殖细胞特异启动子启动表达。
- 根据权利要求4所述的方法,其特征在于:所述生殖细胞特异启动子为***特异启动子、花粉细胞特异启动子或减数***特异启动子。
- 根据权利要求1-5中任一所述的方法,其特征在于:所述元件为CRISPR/Cas方法或CRISPR/Cpf1方法中所需的元件。
- 根据权利要求6所述的方法,其特征在于:所述CRISPR/Cas方法或CRISPR/Cpf1方法中所需的元件均包括a1)或a2):a1)DNA内切酶和sgRNA;a2)所述DNA内切酶、crRNA和tracrRNA。
- 根据权利要求1-7中任一所述的方法,其特征在于:所述DNA片段含有b1)或b2):b1)能表达所述DNA内切酶的表达盒和能表达所述sgRNA的表达盒;b2)能表达所述DNA内切酶的表达盒、能表达所述crRNA的表达盒和能表达所述tracrRNA的表达盒;进一步,所述DNA片段还能表达能用于筛选转基因生物的筛选标记。
- 根据权利要求1-8任一所述的方法,其特征在于:所述靶标DNA为与生物逆境、非生物逆境、产量性状、品质性状或次生代谢物相关DNA。
- 权利要求1-9中任一所述方法在筛选抗生物逆境、抗非生物逆境、高产量、好品质或高次生代谢物突变体中的应用。
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WANG, ZHIPING ET AL.: "Egg Cell-Specific Promoter-Controlled CRISPR/Cas9 Efficiently Generates Homozygous Mutants for Multiple Target Genes in Arabidopsis in a Single Generation", GENOME BIOLOGY, vol. 16, 31 December 2015 (2015-12-31), pages 1 - 12, XP021228694 * |
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