WO2018018979A1 - 植物重组载体及无转基因成分的基因编辑植株的筛选方法 - Google Patents

植物重组载体及无转基因成分的基因编辑植株的筛选方法 Download PDF

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WO2018018979A1
WO2018018979A1 PCT/CN2017/082678 CN2017082678W WO2018018979A1 WO 2018018979 A1 WO2018018979 A1 WO 2018018979A1 CN 2017082678 W CN2017082678 W CN 2017082678W WO 2018018979 A1 WO2018018979 A1 WO 2018018979A1
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gene
plant
plants
transgenic
recombinant vector
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French (fr)
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舒庆尧
芦海平
汪庆
黄建中
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浙江大学
浙江之豇种业有限责任公司
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Priority claimed from CN201610597205.1A external-priority patent/CN106222193B/zh
Priority claimed from CN201611247205.5A external-priority patent/CN106636188A/zh
Application filed by 浙江大学, 浙江之豇种业有限责任公司 filed Critical 浙江大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention relates to the field of gene editing technology, in particular to a method for screening a plant recombinant vector and a gene-editing plant without a transgenic component.
  • Gene editing technology can accurately transform the target gene (that is, edit the DNA sequence), which is not only an advanced biological technology, but also an effective means to improve the quality of crops.
  • the technologies that can be applied to gene editing mainly include: ZFNs (Zinc finger nucleases), TALENs (Transcription activator-like effector nucleases), and CRISPR/Cas9 (Clustered regular interspaced short palir) Dromic repeats/CRISPR-associated Cas9, clustered regularly spaced short palindromic repeats/Cas9 nuclease) technique (Bogdanove AJ and Voytas DF, 2011. TAL effectors: customizable proteins for DNA targeting. Science, 333 (6051): 1843 -1846; Carrol D., 2011. Genome engineering with zinc-finger nucleases. Genetics, 188(4): 773-782).
  • CRISPR/Cas9 is a newly developed gene editing technology with great influence, which is favored for its simple operation and wide application.
  • CRISPR/Cpf1 CRISPR from Prevotella and Francisella 1
  • the vector in the CRISPR/Cas9 system consists mainly of two major components: sgRNA (single guide RNA) and Cas9.
  • sgRNA is a non-coding small RNA that is initiated by the U3 or U6 promoter.
  • the Cas9 encoding nuclease protein having a molecular weight greater than 1000 amino acids, cleaves the DNA nucleic acid sequence.
  • the CRISPR/Cas9 system is primarily matched to a specific location (target) of the genome by means of sgRNA, after which the Cas9 nuclease cleaves the DNA here to form a double-stranded incision.
  • DNA damage repair whether it is homologous recombination-based repair (HR) or nonhomologous end-joining (NHEJ)
  • HR homologous recombination-based repair
  • NHEJ nonhomologous end-joining
  • a screening gene is required in the transgenic process to ensure that the recombinant vector can enter the callus to function, such as the hygromycin resistance gene (HygR) and the glyphosate resistance gene (Bar).
  • HygR hygromycin resistance gene
  • Bar glyphosate resistance gene
  • mice Cong L., Ran FA, Cox D., Lin S., Barretto R., Habib N., Hsu PD, Wu X., Jiang). W., Marraffini LA, and Zhang F., 2013.Multiplex genome engineering using CRISPR/Cas system.Science,339(6121):819-823;2013;Wang H.,Yang H.,Shivalila CS,Dawlaty MM,Cheng AW,Zhang F.and Jaenisch R.,2013 .One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas9-mediated genome engineering.
  • Mutant-positive plants were screened from the T 0 generation plant population in the CRISPR/Cas 9 system. This screening process was performed by directly extracting genomic DNA and PCR to obtain target region fragments, which were identified by direct sequencing. Then, the obtained T 0 generation plants are screened, and the seeds (T 1 generation) are planted individually, and each T 1 generation plant is subjected to genome extraction, and a fragment of the recombinant vector (for example, a hygromycin resistance gene is PCR-amplified, HygR) to determine whether to carry the transgenic component, if the amplification result is positive, it means that the transgenic plant.
  • a fragment of the recombinant vector for example, a hygromycin resistance gene is PCR-amplified, HygR
  • the object of the present invention is to provide a vector for cultivating a transgenic plant in a gene editing (such as CRISPR/Cas9 system) experiment, and to provide a corresponding screening method, which provides a cheaper and simpler method for obtaining a transgenic plant. Effective screening methods.
  • a plant recombinant vector comprising a CRISPR/Cas9 vector and an RNAi expression element inserted therein for silencing expression of a plant trait control gene.
  • the CRISPR/Cas9 vector contains an sgRNA gene, a Cas9 gene, and a selection gene.
  • the sgRNA is under the control of the U3 promoter, and the spectinomycin resistance gene is replaced by a Bsa I digestion to a sequence fragment that matches the target gene;
  • the Cas9 nuclease is transcribed by the Ubi promoter and translated into a protein in the host cell.
  • the sgRNA is used to cleave the target, causing the cell to initiate repair work, thereby introducing a mutation;
  • the screening gene is mainly used to ensure that the recombinant vector enters the callus and functions.
  • the screening gene is a hygromycin resistance gene.
  • the present invention inserts RNAi expression elements capable of interfering with plant traits to control gene expression on a CRISPR/Cas9 vector, and RNAi expression elements transcribe to form hairpin RNA, specifically silencing the expression of plant trait control genes, resulting in macroscopic characteristic traits, In the screening of the transgenic offspring, it is judged whether the plant trait control gene is interfered by visually observing whether a characteristic trait is present, and whether the transgenic progeny carries the transgene fragment.
  • the plant trait control gene is a type of gene that does not affect the normal growth of the plant after the RNAi interferes with the characteristic trait of the plant.
  • the characteristic traits mainly include two types, one is a phenotype, such as a leaf type change: curl (Zhao SS et al., 2016; Liu XF et al., 2016), and spots (Lee J et al., 2007; Tamiru). M. et al., 2016); color change (Dong H et al., 2013; Sugimoto H et al., 2007); epidermal hair development (Walker AR et al., 1999) and the like.
  • a leaf type change such as a leaf type change: curl (Zhao SS et al., 2016; Liu XF et al., 2016), and spots (Lee J et al., 2007; Tamiru). M. et al., 2016); color change (Dong H et al., 2013; Sugimoto H et al., 2007); epidermal hair development (Walker AR et al., 1999) and the like.
  • the other type is biochemical properties (the composition of a substance changes, can be detected by a simple chemical reaction), such as low phytic acid: due to high inorganic phosphorus content, ammonium molybdate developer is blue (Zhao HJ et al ., 2016; Shi J et al., R2007), herbicide sensitive (Pan, G. et al., 2006; Saika H. et al., 2014).
  • the characteristic traits are not limited to one gene control, such as the leaf-rolling trait described above, which is controlled by multiple genes in rice, and interference of any one of the genes causes a leaf phenotype to appear.
  • the invention is not limited to the above several phenotypic or biochemical properties.
  • the present invention can select an appropriate trait control gene for RNAi interference according to existing reports. Therefore, the breadth and depth of use of this technology are very large, and have a good use prospect.
  • the plant trait control gene is: an onion-like leaf rolling gene Sll1 (Shallot-like 1, Os09g0395300), a leaf rolling gene Nrl2 (Narrow rolled leaf 3, Os03g0308200), a half-rolled leaf gene in rice.
  • Srl1 (Semi-rolled leaf 1, Os07g0102300), Rice 5 (Rice outmost cell-specific gene 5, Os02g0674800), Drone leaf gene Sle1 (Slender leaf 1, Os12g36890), ligule missing gene OsLg1 (Rice liguleless, Os04g0656500 ), leaf spot gene Spl28 (Spotted leaf 28, Os01g0703600), Sl1 (Sekiguchi lesion, Os12g0268000) or Spl11 (Spotted leaf 1, Os12g05700751), Vyl (Virescent yellow leaf, Os03g0411500), Ygl1 (Yellow green leaf 1, Os05g0349700) or V2 (Virescent 2, Os03g0320900), Elongated uppermost internode (Os05g0482400), dwarf gene d27 (Dwarf 27, Os11g0587000), d18/OsGA3ox2 (Dwarf 18/
  • the RNAi expression element is a BelRNAi expression element for silencing the expression of the bendazone resistance gene Bel, and the base sequence is shown in SEQ ID NO.
  • the target of the herbicide bentazon is the photosynthetic system, while the normal rice genome contains the benzoic acid resistance gene (CYP81A6/Bel, Os03g0760200), which can release the toxicity, so that the rice treated with the bentazon can still survive. Resistance is shown; however, if the resistance gene is mutated or the RNA level is silenced, it exhibits betadardon sensitivity.
  • a BelRNAi unit i.e., a fragment of the recombinant vector, T-DNA
  • a BelRNAi unit i.e., a BelRNAi expression element
  • sgRNA is required, and Cas9 is present for editing of target genes.
  • the activity of Cas9 is often related to the insertion position.
  • the activity of BelRNAi can be used to measure indirectly. The higher the BelRNAi activity, the more sensitive the plant is. Therefore, in the T 0 generation plants, sensitive plants were screened by applying bentazon.
  • the T 1 generation plant is screened for the bentazone herbicide, if the plant exhibits a sensitive lethal symptom, the plant carrying the transgenic component does not need to be retained; and the surviving is the progeny that does not contain the transgenic fragment. Mutations in the target gene are retained, and it is ensured that the mutant progeny does not contain T-DNA.
  • the promoter in the RNAi expression element is d35S and the terminator is NOS Terminal. These two components are widely used and are maturely used in carrier retrofitting.
  • the CRISPR/Cas9 vector is pHun4c12, pRGE, pCXUN, pDE-cas9 or pKIR, the first three are suitable for monocots, and the latter two are suitable for dicots.
  • the pHun4c12 vector is subjected to HpaI digestion linearization, and a BelRNAi expression element is introduced to obtain a plant recombinant vector, and the base sequence of the plant recombinant vector is shown in SEQ ID NO.
  • the invention also provides a screening method for a genetically modified plant without a transgenic component, comprising:
  • T 0 generation were screened recombinant vector containing the gene of interest has been completed and edited from transgenic plants;
  • T 1 generation plants can exhibit controlling traits to silence gene expression in characters, it is judged that the plants comprising the recombinant vector; the other hand, if the T 1 generation were not exhibit characteristic traits, it is determined that the plants are genetically modified gene is no edit Plant.
  • the screening process includes:
  • step (ii) extracting the DNA of the transgenic plant containing the recombinant vector in the step (i), and sequencing the sequence of the target fragment region of the sgRNA anchored in the transgenic plant, and picking the plant corresponding to the sequence in which the gene mutation has occurred, and obtaining the Recombinant vector of transgenic mutant plants T 0 generation.
  • Another technical solution for screening transgenic plants containing recombinant vectors in step (i) is to select transgenic plants that exhibit characteristic traits.
  • the caller of the plant may be selected from the material to be edited.
  • the plant trait control gene is the bentazone resistance gene Bel of the rice variety.
  • the screening process comprises:
  • the transgenic rice plant is judged to be a transgenic plant containing the recombinant vector; if the leaf coated with the spreader is normally grown, it is judged to be a null plant.
  • the smear further comprises a Tween having a volume ratio of 3 to 6%.
  • the present invention adopts a part of the T 0 generation plants. The leaves were smeared and the reaction to the herbicide bentazon was observed without affecting the growth of the plants.
  • the transgenic rice plants are incubated for one month and then applied with a spreader.
  • the transgenic rice plants obtained in the step (1) are transferred to the outside for growth. After one month, about 4 sheets are grown and one of them is applied. The death of the leaves does not affect the continued growth of the plants.
  • the active ingredient of bentazon is mainly absorbed by the leaves of plants.
  • the smear is applied to the transgenic rice plant A leaf surface at the center of the plant.
  • the leaves at the center are relatively tender, more sensitive to the agent, and the old leaves on the side will have local yellow and other traits, which will interfere with the observation.
  • the present invention performs target gene sequencing on plants which do not exhibit sensitive symptoms after application of bentazon, and no mutation is found, which proves that the method of the present invention can avoid the false negative problem.
  • Bentazon is a contact herbicide that is only damaged at the contact site, which is applied to the surface of a highly hydrophobic plant such as a rice leaf, and therefore must have a certain degree of adhesion. If it does not adhere, the application of the smear into the soil will not only achieve the desired effect, but also endanger the growth of the whole plant, which is not conducive to the survival of the T 0 mutant plants.
  • the research of the invention proves that Tween can not only help the bentazon to stay on the surface of the plant leaves, but also does not affect the function of the bentazon.
  • the ratio of Tween is very critical. When the concentration is too low, the smear cannot adhere to the leaves. When the concentration is greater than 10%, the leaves will show blackness and cannot be distinguished whether it is a positive plant or a normal plant.
  • the spreader contains 1000 mg/L of bentazon and Tween-20 in a volume ratio of 6%.
  • the raw materials are added according to the above ratio, and the foam is stirred by a brush to be foamed, and then applied to the rice leaves.
  • the research experiments show that the adhesion effect of the coating agent of the present invention after coating the foam is better than that of the simple mixing (mixing as above).
  • T 1-generation transgenic rice comprising:
  • the herbicide bentazon is sprayed; if the rice seedlings die, the rice plants containing the recombinant vector insertion; on the contrary, if the seedlings grow normally, the rice plants without the recombinant vector insertion are judged as none.
  • Genetically modified plants of genetically modified components are provided.
  • the present invention determines whether the trait control gene is interfered by visually observing whether a characteristic trait is present in the screening of the transgenic progeny by introducing an RNAi expression element for silencing the plant trait control gene expression in the CRISPR/Cas9 vector, thereby judging the transgenic offspring. whether the fragment containing the transgene, and finally screened to ensure that only the target mutated genes, and not containing the transgene fragment T 1 of the generation of the screening method is cheaper, simpler and more efficient.
  • the screening range can be effectively reduced by observing the characteristic traits, and the screening efficiency is greatly improved.
  • screening for T 1 generation can be large-scale screening.
  • a simple method suitable for high throughput screening of the present invention is more desirable.
  • Figure 1 (a) is a schematic diagram of the recombinant vector of Example 1; wherein the DNA sequence between LB and RB (Fig. 1 (B)) will enter the plant cell;
  • sgRNA is controlled by U3 promoter; spectinomycin resistance gene is replaced by Bsa I to 20bp which matches target gene; Cas9 nuclease is transcribed by Ubi promoter and translated into protein in rice cells, with sgRNA HygR is a hygromycin resistance gene, which is mainly used to screen positive plants in the transgenic process; in the vicinity of RB, the Bel RNAi unit is introduced, and its transcription product forms a hairpin structure, which interferes with Bel in rice. Gene RNA, hindering its translation.
  • FIG. 2 is a schematic diagram showing the construction of pHun4c12-Beli
  • (a) is to link the two fragments of Bel interference (in the opposite direction) into the intermediate vector pBSSK-IN, in order to transcribe the hairpin structure in rice cells; (b) to cut through KpnI and SacI, Beli The element is inserted into the vector pCAMBIA 1300 to obtain the promoter d35S and the Nos terminator, and the entire Beli working element is assembled. (c) was digested with HindIII and EcoRI for d35S-Beli-Nos and blunt-ended by Klenow; on the other hand, HpaI was linearized by the original vector pHun4c12, and the two were ligated to obtain the final vector pHun4c12-Beli .
  • Example 3 is a schematic view showing the working principle of Embodiment 2, Example 3, Example 4, and Example 5;
  • sgRNA will match the target gene (red dot), and the DNA double strand will be cleaved under the action of Cas9 nuclease, and the mutation will be introduced during the repair process.
  • Experimental data analysis showed that the high expression of Cas9 was conducive to the formation of mutations.
  • Beli also interferes with Bel transcripts in rice, so plants with high expression of Cas9 have greater Beli interference intensity and are sensitive to bentazon. Sensitive plants were sequenced on the basis of the first screening of bentazon. T 0 plants undergo meiosis to obtain T 1 generation, and these plants are composed of different gametes.
  • Figure 4 is a phenotype of the T 1 generation after treatment with bentazon in Example 2;
  • (a) is the wild type rice variety Jiazhe B, which shows resistance to bentazon; (b) is a sensitive plant of bentazon, which is produced by gamma-ray mutagenesis of Bel mutation; (c) is unmodified The original vector pHun4c12 was subjected to gene knockout to obtain plants, which did not exhibit betadasone sensitivity; (d) single plants of the above three materials; (e) 16 transgenic lines of the target gene OsLCT mutations screened in Example 2 (T) 1st generation plants), growth status before treatment with bentazon; (f) phenotype of 16 transgenic lines after treatment with bentazon.
  • Figure 5 is the number 6 and 30 of the 16 mutant lines in Example 2, and the molecular marker level verification of each individual plant carries a T-DNA fragment;
  • (a), (b) and (c) are the growth status, single-plant comparison and molecular detection results of the sixth mutant strain of bentazon, respectively, and (d)(e)(f) are the 30th mutant line respectively. Growth status, single plant comparison and molecular detection results after treatment with bentazon.
  • Figure 7 is a graph showing the phenotype and expression levels of related genes of T 0 plants after treatment with bentazon in Example 4.
  • (a) is a picture of the sensitivity of the T 0 generation rice plants to bentazon, the left picture shows the whole plant, the right picture shows the comparison of untreated bentazon and the treated leaves of bentazon;
  • (b) The T 0 generation rice plants are insensitive to the performance of bentazon.
  • the left panel shows the whole plant, the right panel shows the comparison of untreated bentazon and the treated leaves of bentazon;
  • (c) the type T 0 rice A comparison chart of the expression levels of Cas9 in plants;
  • (d) is a comparison chart of the expression levels of Bel of various types of T 0 rice plants.
  • Figure 8 is a diagram showing that the T 1 generation plants of Example 4 were treated with bentazon to screen for plants without transgenic components.
  • (a) is a screening of the first generation of T 1 generation rice plants with bentazon; (b) a comparison chart of the bentazon resistant plants and sensitive plants; (c) the above 72 individual plants Molecular marker verification, there are strips indicating that the transgenic plants, and no bands indicate that there are no transgenic plants.
  • Figure 9 is a diagram showing that the T 1 generation plants of Example 5 were treated with bentazon to screen for plants without transgenic components.
  • (a) is a screening of the first generation of T 1 generation rice plants with bentazon; (b) a comparison chart of the bentazon resistant plants and sensitive plants; (c) the above 72 individual plants Molecular marker verification, there are strips indicating that the transgenic plants, and no bands indicate that there are no transgenic plants.
  • RNA interference used here is mainly to interfere with intracellular Bel transcripts by introducing a 300 bp length hairpin structure.
  • the positive sequence was obtained by PCR amplification using the primers Beli-F1 (gagctcAGCTTAGCCATGGATAACGCCTAC, underlined the SacI restriction site) and Beli-R1 (ctgcagAAGGTCACGTCGTGCTCGGTGAAGCACTC, underlined the PstI restriction site).
  • sequence was loaded into PCAMBIA-1301 and assembled with the d35S and NOS terminals on the vector to form the complete functional unit d35S-BelRNAi-NOS terminal.
  • the pHun4c12-Beli carrying the target gene knockout target fragment is mainly transgenic by Agrobacterium-mediated method.
  • the main method is described by Li et al. (2014). (Li WX, Huang JZ, Zhao HJ, Tan YY, Cui HR, Poirier Y., Shu QY, 2014. Production of low phytic acid rice by hairpin RNA-and artificial microRNA- Mediated silencing of OsMIK in seeds. Plant Cell Tiss. Organ Cult., 119:15-25).
  • Example 2 A cadmium (Cd) transport-related gene OsLCT1 (Os06g0579200) was used as a target gene, and screening of a transgenic fragment-free plant was carried out.
  • Cd cadmium transport-related gene
  • the cadmium (Cd) transport-related gene OsLCT1 (Os06g0579200) was used as a target gene, and a 20 bp fragment 5'-TACTATCCCGCGTGCCAATG-3' was introduced as an sgRNA to generate an OsLCT1 mutant.
  • the sgRNA is transcribed at the initiation of the promoter U3 and matched to the gene OsLCT1; the Cas9 gene is also transcribed and translated into nucleases in rice cells. The two synergistically cut the target, and then introduce mutations in the DNA repair process of rice cells.
  • the Bel RNAi hairpin structure is transcribed to form RNA, which matches the Bel RNA present in rice cells, interrupting its fragments and affecting translation.
  • Example 1 By the above procedure of Example 1, 58 rice plants were obtained, and the first 30 strains were selected for subsequent experiments.
  • the DNA of these 30 T 0 plants was extracted, verified by PCR, and screened with HgyR as a marker gene.
  • the primers are as follows:
  • HygR F AGAAGAAGATGTTGGCGACCT
  • HygR R GTCCTGCGGGTAAATAGCT.
  • the PCR system is as follows:
  • the 20 ⁇ l system included: 1 ⁇ l of DNA, 10 ⁇ l of reaction buffer, 0.4 ⁇ l of the upper and lower primers (10 ⁇ M), and double distilled water to make up 20 ⁇ l.
  • the PCR reaction conditions were as follows: 94 ° C for 2 min; followed by 35 three-step cycles of 94 ° C for 10 s, 60 ° C for 30 s, and 72 ° C for 60 s.
  • Reuse primer OsLCT1F CTCGATGTTAAGCATGCTCC
  • the lethality of certain plants was shown in each mutant line, and the proportion of this lethality was not the same, depending on the copy number and location of T-DNA integrated into the rice genome.
  • the highest survival rate is the 16th line, with 33%; and the 17th mutant shows total lethality, which is most likely caused by too many copies inserted. In this case, more plants need to be planted to screen. In order to get the ideal plant. From another perspective, it is precisely because of the need for large groups to screen, so it can better reflect the advantages of this method.
  • the T 1 plant was planted for one month and then treated with bentazon. As shown in Fig. 5 (a) (d), the surviving plants are plants without a transgenic component.
  • the molecular weight level of 84 individual plants of each line was verified to carry a T-DNA fragment.
  • the specific method is as follows:
  • HygR F AGAAGAAGATGTTGGCGACCT
  • HygR R
  • the PCR system was as follows: 20 ⁇ l of the system included: 1 ⁇ l of DNA, 10 ⁇ l of reaction buffer, 0.4 ⁇ l of the upper and lower primers (10 ⁇ M), and double distilled water to make up 20 ⁇ l.
  • the PCR reaction conditions were as follows: 94 ° C for 2 min; followed by 35 three-step cycles of 94 ° C for 10 s, 60 ° C for 30 s, and 72 ° C for 60 s.
  • Example 3 A gene for the control of rice rice abundance, Betaine aldehyde dehydrogenase 2 (OsBADH2), was used as a target gene to screen non-transgenic fragment plants.
  • OsBADH2 Betaine aldehyde dehydrogenase 2
  • the T 1 generation plants were further subjected to a bentazon treatment.
  • Fig. 6 plants of the same line exhibiting sensitivity and resistance to bentazon were shown.
  • the surviving plants are ideal plants in which the gene OsBADH2 has been mutated and without T-DNA insertion, which can be used for production and rice breeding.
  • molecular marker level verification on 84 individuals to carry T-DNA fragments, indicating that the results are consistent with the sensitivity of bentazon.
  • Example 4 Using the Phosphatidylinositol transfer gene as a target gene, screening of T 0 and T 1 mutant plants was performed.
  • the obtained plants of 96 T 0 generation rice plants were hydroponic for one month and then applied to bentazon.
  • Application method Apply one of the top two leaves, hold the blade with one hand, and evenly apply the solution to the blade with a brush.
  • the degree of symptomatic response of the leaves may be inconsistent due to factors such as plant size, and the response in the selected population may be strong.
  • Fig. 7(a)(b) the leaves of sensitive plants showed symptoms of curling and atrophy, while resistant plants had no symptoms.
  • Sensitive plants were taken for subsequent experiments. There are 51 sensitive plants and the remaining 45 are not sensitive. After sequencing 51 strains, 13 strains were found to be mutant aa type (including aa'), 15 strains were Aa type, and 23 strains were AA type (denoted AA-S).
  • strains were selected from each of the four classifications (aa type; Aa type; AA-S type; AA-R type) for subsequent experiments, mainly to determine the expression levels of the two genes Cas9 and Bel.
  • the mutant plant aa has the highest Cas9 expression, while the Aa plant also has a higher level of Cas9 (there is no difference between the two), probably because Cas9 has a higher Mutations are likely to result in mutations.
  • AA-S also exhibits betadarsone sensitivity, its low expression level is not sufficient to induce mutations.
  • Insensitive AA plants (AA-R) have lower expression of Cas9 and are less likely to elicit mutants.
  • the above experiment proves that the amount of Cas9 is negatively correlated with the amount of Bel, and we can narrow the screening range by the principle that the more sensitive it is, the more likely it is to be a mutant.
  • the screening probability can be increased from the original 28/96 (28 mutants selected from 96 strains) to 28/51 (51 sensitive plants were directly identified by sequencing to identify 28 mutants).
  • the aa and aa 'T 1 generation seed planting type plants wait until the seedlings to grow (grow without strict time limits) of 1000mg / L bentazon treatment.
  • the resistant plants are rice that has reached the target gene Phosphatidylinositol transfer mutation but does not carry T-DNA plants.
  • the stripless plants were all resistant plants.
  • Example 5 14-3-3C (Os08g0430500) gene as the target gene, substituting for T 0 and T 1 plants generation mutants screened.
  • Example 4 The method of Example 4 with reference to the embodiment will be T 0 transgenic plants screened, 48 T 0 generation of plants, there are 21 exhibits bentazon sensitive, after 21 Sequencing revealed 16 is mutants (including Aa, aa type). The remaining 27 plants were also sequenced and no mutants were found, ie there was no problem of false negatives. You can increase the screening range from 16/48 to 16/21 with a 2x increase in probability.
  • the aa and aa 'type plants T 1 of the generation seed planting, seedlings were grown to wait until 1000mg / L bentazon treatment.
  • some plants in one line showed resistance to bentazon, while some were resistant plants.
  • the resistant plants are rice that has reached the 14-3-3C mutation of the target gene but does not carry the T-DNA plant.
  • Fig. 9(c) further, by molecular marker verification, consistent with the above phenotype, the stripless plants were all resistant plants.

Abstract

提供了一种植物重组载体及无转基因成分的基因编辑植株的筛选方法，该重组载体包括CRISPR/Cas9载体和插入其中的RNAi表达元件，所述RNAi表达元件用于沉默植物性状控制基因的表达。

Description

植物重组载体及无转基因成分的基因编辑植株的筛选方法 技术领域
本发明涉及基因编辑技术领域，尤其涉及一种植物重组载体及无转基因成分的基因编辑植株的筛选方法。
背景技术
基因编辑技术可以准确地对目标基因进行改造(即对DNA序列进行编辑)，不仅是一项先进的生物学技术，同时也是改良农作物品质的有效手段。
目前，可应用于基因编辑的技术主要包括：ZFNs(Zinc finger nucleases，锌指核酸酶)，TALENs(Transcription activator-like effector nucleases，转录起始因子核酸酶)以及CRISPR/Cas9(Clustered regularly interspaced short palir dromic repeats/CRISPR-associated Cas9,成簇规律间隔的短回文重复序列/Cas9核酸酶)技术(Bogdanove A.J.and Voytas D.F.,2011.TAL effectors:customizable proteins for DNA targeting.Science,333(6051):1843-1846；Carrol D.,2011.Genome engineering with zinc-finger nucleases.Genetics,188(4):773-782)。其中，CRISPR/Cas9是新发展起来的一项有着巨大影响力的基因编辑技术，其简单的操作以及广泛的应用而受到青睐。在此基础上建立的又一项基因编辑技术CRISPR/Cpf1(CRISPR from Prevotella and Francisella 1)将基因编辑的重点从引入突变到高效地对基因进行“替换”，“定向编辑”等精准的方式(Zetsche B.,Gootenberg J.S.,Abudayyeh O.,Slaymaker,I.M.,Makarova,K.S.,Essletzbichler,P.,Volz,S.E.,Joung,J.,Oost J.,Regev,A.,Koonin,E.V.,Zhang F.,2015.Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.Cell,163:759-771)。由此可见，基因编辑技术的应用前景是十分广阔的。
CRISPR/Cas9体系中的载体主要由两大元件构成：sgRNA(single guide RNA)以及Cas9。sgRNA是一种非编码的小RNA，由U3或者U6启动子启动。Cas9编码核酸酶蛋白，分子量大于1000个氨基酸，可以切割DNA核酸序列。CRISPR/Cas9体系主要借助sgRNA来匹配到基因组的特定位置(靶点)，之后Cas9核酸酶将此处DNA切断，形成双链切口。在DNA损伤修复过程中，无论是同源重组修复(Homologous recombination-based repair,HR)还是非同源末端连接修复(Nonhomologous end-joining,NHEJ)，都会在切口处引入突变。此外，在转基因过程中还需要一个筛选基因来保证重组载体能够进入愈伤组织发挥功能，例如潮霉素抗性基因(HygR)，草甘膦抗性基因(Bar)。
目前，CRISPR/Cas9系统已经广泛运用于动植物中，包括转基因小鼠(Cong L.,Ran F.A.,Cox D.,Lin S.,Barretto R.,Habib N.,Hsu P.D.,Wu X.,Jiang W.,Marraffini L.A.,and Zhang F., 2013.Multiplex genome engineering using CRISPR/Cas system.Science,339(6121):819-823；2013；Wang H.,Yang H.,Shivalila C.S.,Dawlaty M.M.,Cheng A.W.,Zhang F.and Jaenisch R.,2013.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas9-mediated genome engineering.Cell,153(4):910-918.)，斑马鱼(Hwang W.Y.,Fu Y.,Reyon D.,Maeder M.L.,Tsai S.Q.,Sander J.D.,Peterson R.T.,Yeh J.R.J.and Joung J.K.,2013.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat.Biotechnol.,31(3):227-229)等动物；同时在植物中，已经在水稻，拟南芥，烟草等作物之中取得成功(Feng Z.Y.,Mao Y.F.,Xu N.F.,Zhang B.T.,Wei P.L.,Wang Z.,Zhang Z.L.,Yang D.L.,Yang L.,Zeng L.,Liu X.D.,and Zhu J.K.,2014.Muti-generation analysis reveals the inheritance,specificity and patterns of CRISPR/Cas induced gene modifications in Arabidopsis.Proc.Natl.Acad.Sci.USA,111(12):4632-4637；Miao J.,Guo D.,Zhang J.,Huang Q.,Qin G.,Zhang X.,Wan J.,Gu H.,Qu L.J.,2013.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Res.,23(10):1233；Nekrasov V.,Staskawicz B.,Weigel D.,Jones J.D.G.and Kamoun S.,2013.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.Nat.Biotechnol.,31:691-693；Shan Q.,Wang Y.,Li J.,Zhang Y.,Chen K.,Liang Z.,Zhang K.,Liu J.,Xi J.J.and Qiu J.L.,2013.Targeted genome modification of crop plants using a CRISPR/Cas-mediated genome engineering.Cell,153:910-918)。
以往转基因的经典材料，例如抗除草剂和抗虫的转基因作物，都必须使后代植株一直保留有转基因成分才能表现出一定的生物学功能。相反，由于CRISPR/Cas9系统在T0代植株中就可以实现靶基因的编辑，而在T1代植株中不再需要外源进入的CRISPR/Cas9等T-DNA遗传成分。因此，只要通过自交或者回交可以使已完成编辑的靶标基因与T-DNA元件得到分离，获得无转基因成分的基因编辑植株。通过CRISPR/Cas9这样的系统来培育无重组载体片段植株的优点是显而易见的：第一，从无转基因食品安全的角度出发，是可以被大众广泛接受的。第二，由于T-DNA整合进基因组的随意性，很有可能会破坏植株别的功能基因，造成副作用。因此，无转基因的植株运用于实验或者生产都会显得更加准确与安全。
目前，想要获得无转基因成分植株还是依靠传统的方法。从CRISPR/Cas 9体系中的T0代植株群体中筛选获得突变阳性植株，这个筛选过程通过直接提取基因组DNA，PCR获得靶点区域片段，通过直接测序的方法来鉴定。之后，筛选得到的T0代植株，将其种子(T1代)单株种植，对每株T1代植株进行基因组提取，PCR扩增重组载体中的片段(例如潮霉素抗性基因,HygR)来判断是否携带转基因成分，如果扩增结果是阳性就表示是转基因植株。上述方法不仅需要提取DNA、进行PCR和凝胶电泳等繁琐的实验步骤，而且若上述步骤中任何一个环节出现错误，都会造成特异性条带无法扩增出来，而误以为某植株是无转基因片段的植株，进而造成假阴性结论。
因此，有必要针对CRISPR/Cas 9体系工作原理的特殊性及优势所在，探究一种更为简便的鉴定方法，以解决上述传统方法带来的缺陷。
发明内容
本发明的目的是提供一种应用于基因编辑(如CRISPR/Cas9系统)实验中培育无转基因植株的载体，并给出相应的筛选方法，为获得无转基因植株提供了一种更为廉价、简单、有效的筛选手段。
一种植物重组载体，包括CRISPR/Cas9载体和插入其中的RNAi表达元件，所述RNAi表达元件用于沉默植物性状控制基因的表达。
所述CRISPR/Cas9载体含有sgRNA基因、Cas9基因和筛选基因。其中，sgRNA受U3启动子控制，壮观霉素抗性基因通过Bsa I酶切来替换成与目标靶基因匹配的序列片段；Cas9核酸酶由Ubi启动子启动转录并在宿主细胞中翻译成蛋白，与sgRNA配合切开靶点，导致细胞启动修复工作，从而引入突变；筛选基因主要用于保证重组载体进入愈伤组织，并发挥功能。作为优选，筛选基因为潮霉素抗性基因。
本发明通过在CRISPR/Cas9载体上插入能够干扰植物性状控制基因表达的RNAi表达元件，RNAi表达元件转录形成发夹RNA，特异地沉默植物性状控制基因的表达，导致发生肉眼可见的特征性状，在转基因后代的筛选中，通过肉眼观察是否出现特征性状来判断该植物性状控制基因是否被干扰，进而判断该转基因后代中是否携带转基因片段。
所述植物性状控制基因为RNAi干扰后植物表现明显的特征性状，且不影响植物正常生长的一类基因。
所述特征性状主要包括两类，一类是表型，例如叶型变化：卷曲(Zhao S.S et al.,2016；Liu X.F et al.,2016)、斑点(Lee J et al.,2007；Tamiru M.et al.,2016)；颜色变化(Dong H et al.,2013；Sugimoto H et al.,2007)；表皮毛发育(Walker A.R et al.,1999)等。另一类是生化特性(某个物质的成分发生改变，可以通过简单的化学反应进行检测)，例如低植酸：因无机磷含量高，钼酸铵显色剂呈蓝色(Zhao H.J et al.,2016；Shi J et al.,R2007)，除草剂敏感(Pan,G.et al.,2006；Saika H.et al.,2014)等。
所述特征性状不限于一个基因控制，例如上述的卷叶性状，在水稻中是由多个基因控制，任何一个基因的干扰都会导致卷叶表型出现。
本发明并不局限于上述几种表型或生化特性。本发明可以根据现有报道选择合适的性状控制基因进行RNAi干扰。因此，本技术的使用广度和深度都是十分巨大的，有着很好地利用前景。
作为优选，所述植物性状控制基因为：水稻中的葱状卷叶基因Sll1(Shallot-like 1，Os09g0395300)、卷叶基因Nrl2(Narrow rolled leaf 3，Os03g0308200)、半卷叶基因 Srl1(Semi-rolled leaf 1，Os07g0102300)、外卷叶片基因Roc 5(Rice outmost cell-specific gene5，Os02g0674800)、下垂叶片基因Sle1(Slender leaf 1，Os12g36890)、叶舌缺失基因OsLg1(Rice liguleless，Os04g0656500)、叶片斑点基因Spl28(Spotted leaf 28，Os01g0703600)、Sl1(Sekiguchi lesion，Os12g0268000)或Spl11(Spotted leaf 1，Os12g05700751)、叶色基因Vyl(Virescent yellow leaf，Os03g0411500)、Ygl1(Yellow green leaf 1，Os05g0349700)或V2(Virescent 2，Os03g0320900)、株高基因Eui(Elongated uppermost internode，Os05g0482400)、矮杆基因d27(Dwarf 27，Os11g0587000)、d18/OsGA3ox2(Dwarf 18/gibberellins 3β-hydroxylase gene，Os01g0177400)、d1(Dwarf 1，Os05g0333200)或d53(Dwarf 53，Os11g0104300)、谷粒长宽基因Pgl1(Positive regulator of grain length 1，Os03g0171300)、Pgl2(Positive regulator of grain length 2，Os02g0747900)或Gw7(grain width 7，Os07g0603300)、苯达松抗性基因Bel(Bentazon lethality，Os03g0760200)；玉米中的卷叶基因Rld1(Roll leaf 1，Zm0001d048527)或Mwp1(Milkweed pod 1，Zm0001d020384)、叶舌缺失基因Lgn 1(Zm0001d045945)或Lgn 2(Zm0001d042777)、叶片斑点基因Les22(Lesion mimic 22，Zm00001d029074)或Lls 1(Lethal leaf spot 1，Zm00001d027656)、叶色基因ClpP5(Chloroplast caseinolytic protease 5，Zm00001d028895)。
进一步优选，所述RNAi表达元件为用于沉默苯达松抗性基因Bel表达的BelRNAi表达元件，碱基序列如SEQ ID NO.1所示。
除草剂苯达松的作用靶点是光合系统，而正常水稻的基因组中含有可以解除毒性的苯达松抗性基因(CYP81A6/Bel，Os03g0760200)，使得苯达松处理后的水稻依旧能够存活，表现出抗性；但是，若该抗性基因突变或者RNA水平被沉默，则会表现出苯达松敏感。
上述特点在本发明中被巧妙地运用，通过在原始载体中引入BelRNAi单元(即BelRNAi表达元件)，使得含有转基因片段(即该重组载体的片段，T-DNA)与BelRNAi绑定。在T0代植株中，需要sgRNA，Cas9存在来进行靶基因的编辑，Cas9的活性往往会与插入位置有关，此时可以利用BelRNAi的活性来间接衡量，BelRNAi活性越高则植株越敏感。因此，在T0代植株中，通过涂抹苯达松来筛选出敏感的植株。在T1代植株进行苯达松除草剂筛选时，若植株表现出敏感致死症状，则为携带转基因成分的植株，是不需要保留的；而存活下来的则为不含有转基因片段的后代，既保留了目标基因的突变，又确保了该突变后代中不含有T-DNA。
作为优选，所述RNAi表达元件中启动子为d35S，终止子为NOS Terminal。这两个元件应用广泛而且被成熟地运用于载体改造中。
在重组载体构建时，目前有多种多样的载体，只要保证基本元件(sgRNA，Cas9)存在，都能达到相同的目的。不同物种的载体相差较大，根据待基因编辑植株选择相应的载体。作为优选，所述CRISPR/Cas9载体为pHun4c12、pRGE、pCXUN、pDE-cas9或pKIR，前三种适用于单子叶植物，后两种适用于双子叶植物。
进一步优选，对pHun4c12载体进行HpaⅠ酶切线性化，引入BelRNAi表达元件，获得植物重组载体，所述植物重组载体的碱基序列如SEQ ID NO.2所示。
本发明还提供了一种无转基因成分的基因编辑植株的筛选方法，包括：
(1)将所述的植物重组载体转入待基因编辑材料，培养获得转基因植株；
(2)从转基因植株中筛选含重组载体且目标基因已完成编辑的T0代植株；
(3)继续培养T0代植株直至获得其种子为T1代；
(4)若T1代植株表现出性状控制基因表达沉默的特征性状，则判断该植株含有重组载体；反之，若T1代植株不表现特征性状，则判断该植株为无转基因成分的基因编辑植株。
步骤(2)中，所述筛选的过程包括：
(ⅰ)选取存在重组载体筛选基因的植株，得到含重组载体的转基因植株；
(ⅱ)提取步骤(ⅰ)中含重组载体的转基因植株DNA，经测序，确定转基因植株中sgRNA锚定的靶片段区域的序列，从中挑取已发生基因突变的序列所对应的植株，获得含重组载体的转基因突变植株T0代。
步骤(ⅰ)中筛选含重组载体的转基因植株的另一种技术方案：选取表现出特征性状的转基因植株。
所述待基因编辑材料可选用植物的愈伤组织。
所述植物性状控制基因为水稻品种的苯达松抗性基因Bel。作为优选，步骤(2)中，所述筛选的过程包括：
(a)将转基因水稻植株培育至长出多张叶片，将含有苯达松的涂抹剂涂抹在其中一张叶片上，继续培育3～4天之后观察；
如果涂有涂抹剂的叶片出现卷曲、萎缩的敏感症状，则判断该转基因水稻植株为含有重组载体的转基因植株；如果涂有涂抹剂的叶片正常生长，则判断为无效植株。
(b)提取含有重组载体的转基因植株DNA，对靶点区域进行PCR扩增后，经测序，确定转基因植株中sgRNA锚定的靶点区域是否突变，从中挑取已发生突变序列所对应的植株，获得所述T0代植株；
所述涂抹剂还包含体积占比3～6％的吐温。
由于敏感植株对苯达松表现出致死，在筛选T0代植株时，如果使用整株喷洒方法，那么目标植株就死亡，则无法获得有用植株，因此，本发明采用对T0代植株的部分叶片进行涂抹，在不影响植株生长的前提下，又能观察到对除草剂苯达松的反应。
作为优选，步骤(a)中，将转基因水稻植株培育一个月后涂抹涂抹剂。将步骤(1)中获得的转基因水稻植株转移到外界生长，一个月后，长出大概4张，涂抹其中一张，这张叶片致死的情况下不会影响植株继续生长。
苯达松的有效成分主要通过植株叶片吸收。作为优选，所述涂抹剂涂抹在转基因水稻植 株中心的一张叶片表面。中心处的叶片比较嫩，对药剂更加敏感，边上的老叶本身会有局部黄色等性状，会干扰观察。
本发明在研究中发现，涂抹苯达松的叶片表现的敏感程度越高，该植株为突变体的可能性越高。机理分析：Cas9基因和BelRNAi是共线的，在转录水平上会具有一致性。Cas9转录越多，可能越有利于突变体的产生。这个Cas9的转录可以间接通过BelRNAi的量反应出来，BelRNAi的高丰度表达会导致水稻细胞中Bel的转录本受到干扰，表现出除草剂苯达松的敏感。因此，敏感症状越明显也就意味着突变体可能性越大，以此为原则可以进一步地缩小筛选范围。
本发明对使用苯达松涂抹后没有表现敏感症状的植株进行靶基因测序，确实没有发现突变，证明本发明方法可以避免假阴性问题。
苯达松为接触性除草剂，只有接触部位才会受伤，所述涂抹剂针对像水稻叶片这样的疏水性很强的植株表面上使用，因此必须要求具有一定的粘附性。如粘附不住，涂抹剂流入土壤中，不仅达不到预期的效果，同时会危害整个植株的生长，不利于T0代突变植株的存活。
本发明研究证明吐温不仅能够帮助苯达松停留在植株叶片表面，而且不影响苯达松发挥功能。吐温的配比非常关键，当浓度过低，涂抹剂无法粘附在叶片上，当浓度大于10％，无论是阳性植株还是正常植株，叶片都会出现发黑症状，无法区分。作为优选，所述涂抹剂中包含1000mg/L的苯达松和体积比为6％的吐温-20。
制备涂抹剂时，按上述配比添加原料后，利用毛笔进行搅拌打出泡沫后，再涂刷到水稻叶片上。研究实验表明，本发明的涂抹剂打出泡沫后进行涂覆的粘附效果比简单混合(如上下混匀)的方法要好。
以苯达松抗性基因作为植物性状控制基因，T1代无转基因水稻的筛选过程包括：
在T1代幼苗期间，喷施除草剂苯达松；若水稻幼苗死亡，则为含有重组载体插入的水稻植株；反之，若幼苗正常生长，则为无重组载体插入的水稻植株，判定为无转基因成分的基因编辑植株。
本发明具备的有益效果：
本发明通过在CRISPR/Cas9载体中引入沉默植物性状控制基因表达的RNAi表达元件，在转基因后代的筛选中，通过肉眼观察是否出现特征性状来判断该性状控制基因是否被干扰，进而判断该转基因后代中是否包含转基因片段，最后筛选到既保证目标基因发生突变，又不含转基因片段的T1代，筛选方法更为廉价，简单和有效。
在T0代的筛选中，相较于传统PCR方法，通过观察特征性状可以有效缩小筛选范围，大大提高筛选效率。
在针对T1代进行筛选时，可以进行大规模筛选。尤其是遇到多个插入拷贝的时候(获得无转基因植株的概率变小)，更加需要本发明这种适合高通量筛选的简便方法。
附图说明
图1(a)为实施例1中重组载体的图谱示意图；其中LB和RB之间的DNA序列(图1(B))都将进入植物细胞内；
其中，sgRNA受U3启动子控制；壮观霉素抗性基因通过Bsa I酶切来替换成与靶基因匹配的20bp；Cas9核酸酶由Ubi启动子启动转录并在水稻细胞中翻译成蛋白，与sgRNA配合切开靶点；HygR是潮霉素抗性基因，主要是在转基因过程中用于筛选阳性植株；在RB附近，引入Bel RNAi单元，其转录产物会形成发夹结构，干扰水稻中的Bel基因RNA，阻碍其翻译。
图2为构建pHun4c12-Beli的示意图；
其中，(a)为将Bel干扰的两个片段(方向相反)连接进中间载体pBSSK-IN，以期在水稻细胞中可以转录形成发夹结构；(b)为通过KpnⅠ和SacⅠ酶切，将Beli元件插入载体pCAMBIA1300，以便获得启动子d35S和Nos终止子，整个Beli工作元件组装完成。(c)为d35S-Beli-Nos通过HindⅢ和EcoRⅠ酶切获得，并通过Klenow进行平末端化；另一面，对原始载体pHun4c12进行HpaⅠ酶切线性化，两者相连，获得最终的载体pHun4c12-Beli。
图3为实施例2，例3，例4，例5的工作原理示意图；
其中，sgRNA会匹配到靶基因上(红色圆点)，在Cas9核酸酶的作用下切开DNA双链，修复过程中引入突变。实验数据分析得到Cas9表达高有利于突变的形成。同时，Beli也会干扰水稻中的Bel转录本，因此Cas9高表达的植株，其Beli干扰强度也越大，表现出苯达松敏感。在苯达松初筛的基础上，对敏感植株进行测序确认。T0植株经过减数分裂获得T1代，这些植株是由不同的配子组合而来。在T1代中，CRISPR/Cas9元件已经履行完生物学功能，目的基因(红色圆点)都将保持突变。若植株是带有T-DNA，那么Bel hpRNAi单元会干扰水稻细胞中的Bel转录本，表现致死。相反，若T1植株不带有T-DNA，那么在苯达松处理下依旧能够存活。因此，此方法可以高效地从T0代中选出突变植株，从T1代中选出无T-DNA成分的植株。
图4为实施例2中T1代在苯达松处理之后的表型；
其中，(a)为野生型水稻品种嘉浙B，表现出苯达松抗性；(b)为苯达松敏感植株，通过γ射线诱变Bel突变产生的；(c)为未改造过的原始载体pHun4c12进行基因敲除获得植株，并不表现出苯达松敏感；(d)上述三种材料的单株；(e)实施例2筛选出来的目的基因OsLCT突变的16个转基因系(T1代植株)，苯达松处理前的生长状况；(f)16个转基因系经苯达松处理之后的表型。
图5为实施例2中针对16个突变系中的第6和第30号，对每个单株进行分子标记水平验证是否携带T-DNA片段；
其中(a)(b)(c)分别为第6号突变系苯达松处理后的生长状况、单株比较和分子检测结果，(d)(e)(f)分别为第30号突变系苯达松处理后的生长状况、单株比较和分子检测结果。
图6(a)为实施例3中T1代植株其中一个突变系经苯达松处理之后的表型，(b)为(a)中单株比较。
图7为实施例4中T0代植株经苯达松处理之后的表型及相关基因的表达量；
其中，(a)为T0代水稻植株对苯达松表现敏感症状的图片，左边图为整个植株，右边图为未处理苯达松和处理苯达松后叶片的对比图；(b)为T0代水稻植株对苯达松表现不敏感的图片，左边图为整个植株，右边图为未处理苯达松和处理苯达松后叶片的对比图；(c)为各类型T0代水稻植株Cas9表达量的比较图；(d)为各类型T0代水稻植株Bel表达量的比较图。
图8为实施例4中T1代植株经苯达松处理来筛选无转基因成分植株
其中，(a)为对其中一个系的T1代水稻植株进行苯达松筛选；(b)为苯达松抗性植株和敏感植株的对比图；(c)对上述的72个单株进行分子标记验证，有条带说明是转基因植株，无条带则说明是无转基因成分植株。
图9为实施例5中T1代植株经苯达松处理来筛选无转基因成分植株
其中，(a)为对其中一个系的T1代水稻植株进行苯达松筛选；(b)为苯达松抗性植株和敏感植株的对比图；(c)对上述的72个单株进行分子标记验证，有条带说明是转基因植株，无条带则说明是无转基因成分植株。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
1、BelRNAi基因转录单元的制备
这里用到的RNA干扰主要是通过引入300bp长度的发夹(Hairpin)结构来干扰细胞内的Bel转录本。
用引物Beli-F1(gagctcAGCTTAGCCATGGATAACGCCTAC,小写下划线为SacⅠ酶切位点)和Beli-R1(ctgcagAAGGTCACGTCGTGCTCGGTGAAGCACTC,小写下划线为PstⅠ酶切位点)，经PCR扩增获得正向序列。
另一方面，用引物Beli-F2(ggtaccAGCTTAGCCATGGATAACGCCTAC,小写下划线为KpnⅠ酶切位点)和Beli-R2(ctcgagAAGGTCACGTCGTGCTCGGTGAAGCACTC,小写下划线为XhoⅠ酶切位点)。将这两段序列连接T载体之后进行测序，测序结果为SEQ ID NO.1。
测序正确之后，将序列装进PCAMBIA-1301，与其载体上的d35S和NOS terminal拼装，最终形成完整功能单元d35S-BelRNAi-NOS terminal。
2、重组载体的构建
将这个完整单元用EcoRI和HindⅢ酶切之后，装进已经被HpaI切开的原始载体pHun4c12，形成最终的载体。整个载体序列为SEQ ID NO.2。
3、重组载体的转入和植株细胞的培养
携带有目的基因敲除靶片段的pHun4c12-Beli主要是通过农杆菌介导的方法进行转基因。主要方法参照Li et al.(2014).(Li W.X.,Huang J.Z.,Zhao H.J.,Tan Y.Y.,Cui H.R.,Poirier Y.,Shu Q.Y.,2014.Production of low phytic acid rice by hairpin RNA-and artificial microRNA-mediated silencing of OsMIK in seeds.Plant Cell Tiss.Organ Cult.,119:15-25)。
实施例2  以镉(Cd)转运相关的基因OsLCT1(Os06g0579200)作为靶基因，进行无转基因片段植株的筛选。
以镉(Cd)转运相关的基因OsLCT1(Os06g0579200)作为靶基因，引入20bp的片段5'-TACTATCCCGCGTGCCAATG-3'作为sgRNA来产生OsLCT1突变体。
在T0代中，一方面，sgRNA在启动子U3的启动下会转录出来，并匹配到基因OsLCT1上；Cas9基因也在水稻细胞中转录翻译出来核酸酶。两者协同作用，切割靶点，之后水稻细胞DNA修复过程中引入突变。另一方面，Bel RNAi发夹结构转录出来形成RNA，与水稻细胞内存在的Bel RNA匹配，打断其片段，影响翻译。
通过实施例1的上述步骤，获得58株水稻，选取前30株进行后续实验。
提取这30株T0代植株的DNA，对其进行PCR验证，以HgyR作为标记基因进行筛选。引物如下：
HygR F:AGAAGAAGATGTTGGCGACCT；
HygR R:GTCCTGCGGGTAAATAGCT。
PCR体系如下：
20μl体系包括：1μl DNA,10μl反应缓冲液,0.4μl上下引物(10μM),双蒸水补足20μl。
PCR反应条件如下：94℃2min；之后进行35个三步循环94℃10s,60℃30s,72℃60s。
再利用引物OsLCT1F:CTCGATGTTAAGCATGCTCC，
和Os LCT1 R:AGAGTCAGGAACGCGGCTAC进行扩增，方法及程序如上所述。再通过测序找出突变体以及确定突变位点(如表1所示)。
表1  T0代30株植株靶点测序
Figure PCTCN2017082678-appb-000001
Figure PCTCN2017082678-appb-000002
Figure PCTCN2017082678-appb-000003
注：加下环线表示插入，*表示缺失。
将aa和aa'类型植株的T1代种子进行种植，共16个品系，待其长至幼苗进行1000mg/L的苯达松处理。
如表2所示，在每个突变系中都表现出一定植株的致死，这个致死的比例并不相同，取决于T-DNA整合进水稻基因组的拷贝数及位置。存活率最大的是第16个系，有33％；而第17号突变系表现出全致死，这很有可能是插入的拷贝数过多引起的，这样的情况需要种植更多的植株来筛选，以便获得理想的植株。另外一个角度，正是因为需要大群体来筛选，因此更能体现出此方法的优势所在。
表2  16个突变系经苯达松处理之后的存活植株统计结果
Figure PCTCN2017082678-appb-000004
我们选取第6和30号转基因系进行具体分析。T1代植株种植一个月后进行苯达松处理。如图5(a)(d)所示，存活的植株，即为无转基因成分的植株。
对每个系的84个单株进行分子标记水平验证是否携带T-DNA片段。具体方法如下：
提取这84株T1代植株的DNA，对其进行PCR验证，以HgyR作为标记基因进行筛选。引物如下：HygR F:AGAAGAAGATGTTGGCGACCT；和HygR R:
GTCCTGCGGGTAAATAGCT。PCR体系如下：20μl体系包括：1μl DNA,10μl反应缓冲液,0.4μl上下引物(10μM),双蒸水补足20μl。PCR反应条件如下：94℃2min；之后进行35个三步循环94℃10s,60℃30s,72℃60s。
结果如图5(c)(f)所示，该结果与苯达松处理(见表2)结果一致。
实施例3  以控制水稻稻米香味的基因甜菜碱醛脱氢酶2(Betaine aldehyde dehydrogenase 2，OsBADH2)作为靶基因，进行无转基因片段植株的筛选。
为了进一步验证新构建载体pHun4c12-Beli的适用普遍性。同时，以基因甜菜碱醛脱氢酶2(Betaine aldehyde dehydrogenase 2，OsBADH2)作为靶基因进行实验，OsBADH2突变的植株会表现出稻米芳香，提高稻米品质。
实验过程及步骤同实施例2一致。
之后，进一步再对其中T1代植株进行苯达松处理。结果如图6所示，同一个系中表现出苯达松敏感和抗性的植株。其中，存活的植株正是基因OsBADH2已经突变，同时不带有T-DNA插入的理想植株，可以用于生产及水稻育种。同样，我们对84个单株进行分子标记水平验证是否携带T-DNA片段，表明其结果与苯达松敏感与否是一致的。通过这个例子，我们进一步验证了此载体和方法在CRISPR体系中筛选无转基因植株的有效性和高效性。
实施例4  以Phosphatidylinositol transfer基因作为靶基因，进行T0代和T1代突变植株的筛选。
1、获取T0代植株
以Phosphatidylinositol transfer基因(Os05g0545000)作为靶基因，在pHun4c12-Beli载体上插入20bp的片段：AAATGCAAGTGCGAGGCATT，以期获得该基因突变的植株。最终，我们选取了96株T0代水稻植株进行后续实验。
2、T0代转基因植株筛选
制备涂抹剂：以水为溶剂，苯达松浓度设置为1000mg/L，加入6％(V/V)吐温-20提高粘稠度，利用毛笔搅拌打出泡沫。
将获得的96株T0代水稻植株水培一个月后的植株进行苯达松涂抹。涂抹方法：对最上两张叶片的一张进行涂抹，一手托住叶片，用毛笔蘸取溶液均匀涂布在叶片上。
三至四天之后进行观察，叶片的症状反应程度可能会因植株大小等因素不一致，选取群体中反应强烈的即可。
如图7(a)(b)所示，敏感植株叶片呈现卷曲，萎缩的症状，而抗性植株没有症状。我们选 取敏感的植株进行后续实验。敏感的植株有51株，剩下45株不敏感。我们对51株进行测序之后，发现13株是突变体aa型(含aa')，15株是Aa型，23株是AA型(记为AA-S)。
为了防止有突变体遗漏在不敏感的植株中造成假阴性，我们进一步对45株不敏感的植株(记为AA-R)都进行了靶基因测序，确实没有发现突变。因此，此方法在假阴性问题上还是可以控制的。
之后，进一步从四个分类中(aa型；Aa型；AA-S型；AA-R型)各选出10株进行后续实验，主要是测定两个基因Cas9和Bel的表达水平。
从图7(c)中可见，突变体植株aa型有最高的Cas9表达量，而Aa型植株中Cas9也有着较高水平(两者之间并没有差异)，可能正是因为Cas9有着较高的突变容易导致突变产生。尽管AA-S也表现出苯达松敏感，但其表达量较低还是不足以诱发突变。不敏感的AA型植株(AA-R)中Cas9表达更低，更加不可能引发突变体。
从图7(d)中可见，以Bel基因出发，由于受到RNA干扰，突变体植株aa型中的Bel表达量最低，Aa型的植株中Bel的表达也很低。两者相近，这与两者中Cas9表达相近一致。敏感AA-S中，Bel基因也一定程度上被干扰。在AA-S群体中，Bel的表达量和Cas9的表达量一样，都是处于一个中间状态。AA-R群体中，Bel表达更不受影响，因此也没有敏感症状。
上述实验证明了，Cas9的量与Bel的量是负相关的，我们可以通过越是敏感越有可能是突变体的原则来缩小筛选范围。可以将筛选概率从原来的28/96(96株中选出28株突变体)提高到28/51(51株敏感的植株直接通过测序发现28株突变体)。
3、T1代无转基因片段植株筛选
将aa和aa'类型植株的T1代种子进行种植，共13个品系，待其长至幼苗(没有严格的生长时间限制)进行1000mg/L的苯达松处理。
如图8所示，其中一个系中有些植株表现出苯达松敏感，而有些是抗性的植株。其中，抗性植株就是已经达到目的基因Phosphatidylinositol transfer突变，但不携带T-DNA植株的水稻。如图8(c)所示，进一步通过分子标记验证，与上述表型一致，无条带植株均为抗性植株。
实施例5  以14-3-3C(Os08g0430500)基因作为靶基因，进行T0代和T1代突变植株的筛选。
用pHun4c12-Beli对基因14-3-3 C(Os08g0430500)进行定点突变诱导。sgRNA靶点碱基序列为：ACTCTGATCAAGGAGTACC G。
通过实施例1的方法获得了48株T0代水稻植株。
参照实施例4的方法进行T0代转基因植株筛选，48株T0代植株中，有21株表现出苯达松敏感，21株测序后发现16株是突变体(含Aa，aa型)。剩下的27株植株也全部测序并没有发现突变体，也即不存在假阴性的问题。可以将筛选范围从16/48提高到16/21，概率提高2倍。
将aa和aa'类型植株的T1代种子进行种植，待其长至幼苗进行1000mg/L的苯达松处理。如图9所示，一个系中有些植株表现出苯达松敏感，而有些是抗性的植株。其中，抗性植株就是已经达到目的基因14-3-3C突变，但不携带T-DNA植株的水稻。如图9(c)所示，进一步通过分子标记验证，与上述表型一致，无条带植株均为抗性植株。
Figure PCTCN2017082678-appb-000005
Figure PCTCN2017082678-appb-000006
Figure PCTCN2017082678-appb-000007
Figure PCTCN2017082678-appb-000008
Figure PCTCN2017082678-appb-000009
Figure PCTCN2017082678-appb-000010
Figure PCTCN2017082678-appb-000011
Figure PCTCN2017082678-appb-000012
Figure PCTCN2017082678-appb-000013
Figure PCTCN2017082678-appb-000014

Claims (12)

  1. 一种植物重组载体，其特征在于，包括CRISPR/Cas9载体和插入其中的RNAi表达元件，所述RNAi表达元件用于沉默植物性状控制基因的表达。
  2. 如权利要求1所述的植物重组载体，其特征在于，所述植物性状控制基因为：水稻中的葱状卷叶基因Sll1、卷叶基因Nrl2、半卷叶基因Srl1、外卷叶片基因Roc 5、下垂叶片基因Sle1、叶舌缺失基因OsLg1、叶片斑点基因Spl28、Sl1或Spl11、叶色基因Vyl、Ygl1或V2、株高基因Eui、矮杆基因d27、d18/OsGA3ox2、d1或d53、谷粒长宽基因Pgl1、Pgl2或Gw7、苯达松抗性基因Bel；玉米中的卷叶基因Rld1或Mwp1、叶舌缺失基因Lgn 1或Lgn 2、叶片斑点基因Les22或Lls 1、叶色基因ClpP5。
  3. 如权利要求2所述的植物重组载体，其特征在于，所述RNAi表达元件为用于沉默苯达松抗性基因Bel表达的BelRNAi表达元件，碱基序列如SEQ ID NO.1所示。
  4. 如权利要求1所述的植物重组载体，其特征在于，所述RNAi表达元件中启动子为d35S，终止子为NOS Terminal。
  5. 如权利要求1所述的植物重组载体，其特征在于，所述CRISPR/Cas9载体为pHun4c12、pRGE、pCXUN、pDE-cas9或pKIR。
  6. 如权利要求3或5所述的植物重组载体，其特征在于，所述植物重组载体的碱基序列如SEQ ID NO.2所示。
  7. 一种无转基因成分的基因编辑植株的筛选方法，包括：
    (1)将权利要求1-6任一项所述的植物重组载体转入待基因编辑材料，培养获得转基因植株；
    (2)从转基因植株中筛选含重组载体且目标基因已完成编辑的T0代植株；
    (3)继续培养T0代植株直至获得其种子为T1代；
    (4)若T1代植株表现出性状控制基因表达沉默的特征性状，则判断该植株含有重组载体；反之，若T1代植株不表现特征性状，则判断该植株为无转基因成分的基因编辑植株。
  8. 如权利要求7所述的筛选方法，其特征在于，步骤(2)中，所述筛选的过程包括：
    选择表现出特征性状的转基因植株，提取其基因组DNA，对目标基因靶点区域进行PCR扩增，经测序确定该转基因植株中sgRNA锚定的靶点区域是否突变，从中挑取已发生突变序列所对应的植株，获得所述T0代植株。
  9. 如权利要求8所述的筛选方法，其特征在于，所述植物性状控制基因为水稻品种的苯达松抗性基因Bel。
  10. 如权利要求9所述的筛选方法，其特征在于，步骤(2)中，所述筛选的过程包括：
    (a)将转基因水稻植株培育至长出多张叶片，将含有苯达松的涂抹剂涂抹在其中一张叶片上，继续培育3～4天之后观察；
    如果涂有涂抹剂的叶片出现卷曲、萎缩的敏感症状，则判断该转基因水稻植株为含有重 组载体的转基因植株；如果涂有涂抹剂的叶片正常生长，则判断为无效植株。
    (b)提取含有重组载体的转基因植株DNA，对靶点区域进行PCR扩增后，经测序，确定转基因植株中sgRNA锚定的靶点区域是否突变，从中挑取已发生突变序列所对应的植株，获得所述T0代植株；
    所述涂抹剂还包含体积占比3～6％的吐温。
  11. 如权利要求10所述的筛选方法，其特征在于，所述涂抹剂中包含1000mg/L的苯达松和体积比为6％的吐温-20。
  12. 如权利要求9所述的筛选方法，其特征在于，步骤(4)中，在T1代幼苗期间，喷施除草剂苯达松；若水稻幼苗死亡，则为含有重组载体插入的水稻植株；反之，若幼苗正常生长，则为无重组载体插入的水稻植株，判定为无转基因成分的基因编辑植株。
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