CN117625646A - GhRSL1-2 gene, method for cultivating transgenic plant and application thereof in regulating cotton development - Google Patents
GhRSL1-2 gene, method for cultivating transgenic plant and application thereof in regulating cotton development Download PDFInfo
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Abstract
The invention discloses a GhRSL1-2 gene, a method for cultivating transgenic plants and application thereof in regulating cotton development, and belongs to the field of cotton genetic engineering. GhRSL1-2 was found to localize to the nucleus. Through over-expression and knocking out GhRSL1-2 genes and transferring the cotton, the relative expression quantity of the cotton over-expressed with GhRSL1-2 after the fiber is bloomed is obviously higher than that of the cotton wild. Through detecting the fiber length, the cell wall thickness, the fiber twist number and the seed quality of wild cotton, ghRSL1-2 over-expressed cotton and GhRSL1-2 gene knockout cotton strains, the over-expressed GhRSL1-2 can be found to obviously increase the fiber length, the cell wall thickness, the fiber twist number and the seed weight, and the GhRSL1-2 gene regulation fiber cell fiber length and the cell wall thickness are illustrated. The GhRSL1-2 gene is transferred into cotton to promote the elongation of cotton fiber cells and increase the cell wall thickness of cotton fiber cells, so that the GhRSL1-2 gene has great significance in research and improvement of cotton fiber quality.
Description
Technical Field
The invention belongs to the field of cotton genetic engineering, and particularly relates to a GhRSL1-2 gene, a method for cultivating transgenic plants and application thereof in regulating cotton development.
Background
Cotton fibers are seed trichomes, extremely elongated single cells derived from ovule epidermal cells, whose development undergoes distinct but overlapping stages: initiation, elongation, secondary cell wall synthesis, and maturation. The initial and elongation stages of cotton fibers have a great influence on the number, length and fineness of the fibers, ultimately determining the yield and length of cotton. Biosynthesis of Secondary Cell Walls (SCW) of cotton fibers affects cell wall thickness, which directly affects cotton fiber quality. Each stage of cotton fiber development is crucial to the phenotype of the whole fiber, and the development of the cotton fiber is not regulated by genes, and a plurality of specific genes are involved in the specific stage of cotton fiber development, so that a complex regulation network is formed. However, the known regulation is limited, and all genes involved therein and the regulation mechanism thereof have not been completely clarified, so that a new way of regulating cotton fiber development is required.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a GhRSL1-2 gene, a method for cultivating transgenic plants and application thereof in regulating cotton development.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
in the first aspect of the invention, a GhRSL1-2 gene is disclosed, which can regulate cotton development, and the sequence is shown as SEQ ID NO. 1.
In a second aspect of the invention, the application of GhRSL1-2 gene and related gene thereof in regulating cotton development is disclosed, the GhRSL1-2 gene sequence is shown as SEQ ID NO.1, the related gene has a gene with at least 75% homology with the GhRSL1-2 gene, and the encoding gene has the function of the GhRSL1-2 gene.
Preferably, the cotton develops into a fibroblast development.
Preferably, overexpression of the GhRSL1-2 gene increases cotton fiber length, and knockout of the GhRSL1-2 gene inhibits cotton fiber length.
Preferably, overexpression of the GhRSL1-2 gene can increase the cell wall thickness of the mature cotton fiber, and knockout of the GhRSL1-2 gene can inhibit the cell wall thickness of the mature cotton fiber.
Preferably, overexpression of the GhRSL1-2 gene can increase the weight of mature cotton seeds, and knockout of the GhRSL1-2 gene can inhibit the weight of mature cotton seeds.
Preferably, overexpression of the GhRSL1-2 gene can increase the twisting number of cotton fibers, and knockout of the GhRSL1-2 gene can inhibit the twisting number of cotton fibers.
In a third aspect of the invention, a method for cultivating a transgenic plant is disclosed, wherein a coding gene GhRSL1-2 shown in SEQ ID NO.1 is introduced into a recipient plant to obtain a transgenic plant with different growth and development from the recipient plant.
Preferably, the introduction of the gene GhRSL1-2 into the recipient plant is achieved by introducing a recombinant plasmid containing the GhRSL1-2 gene into the recipient plant.
Preferably, the recombinant plasmid is pBI121-GhRSL1-2, pCAMBIA2300-GhRSL1-2 or pHellsgate4-GhRSL1-2.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of GhRSL1-2 gene in regulating cotton development, firstly cloning the coding sequence of GhRSL1-2 to pCAMBIA2300-GFP vector, detecting the localization of the gene in tobacco as nucleus; by exploring the evolutionary relationship between the GhRSL1-2 protein and the model plant Arabidopsis thaliana protein, the affinity between the GhRSL1-2 protein and the AtRHD6 protein is shown to be recent. Secondly, respectively transferring the over-expressed and knocked out GhRSL1-2 genes into cotton, screening to obtain GhRSL1-2 transgenic cotton, and detecting the relative expression level of the over-expressed GhRSL1-2 cotton and wild cotton at 20d (the development stage of the secondary cell wall) after fiber flowering by using qRT-PCR technology, wherein the relative expression amount of the over-expressed GhRSL1-2 cotton at 20d after fiber flowering is found to be significantly higher than that of the wild cotton, and the GhRSL1-2 is positioned at the cell nucleus. Finally, by detecting the fiber length, the cell wall thickness, the fiber twist number and the seed quality of wild cotton, ghRSL1-2 over-expressed cotton and GhRSL1-2 gene knockout cotton lines, the over-expressed GhRSL1-2 is found to obviously increase the fiber length, the cell wall thickness, the fiber twist number and the seed quality, and the GhRSL1-2 gene knockout can obviously inhibit the fiber length, the cell wall thickness, the fiber twist number and the seed quality, which indicates that the GhRSL1-2 gene regulates the fiber length and the cell wall thickness of the fiber cell. Therefore, the GhRSL1-2 gene is transferred into cotton, so that the elongation of cotton fiber cells can be promoted, the cell wall thickness of the cotton fiber cells can be increased, and the GhRSL1-2 gene has great significance for research and improvement of cotton fiber quality.
Drawings
FIG. 1 is a graph of subcellular localization of GhRSL1-2 in tobacco leaves;
FIG. 2 is a graph showing the evolutionary relationship between the GhRSL1-2 protein and a model plant Arabidopsis protein;
FIG. 3 is a graph showing the molecular identification result of GhRSL1-2 over-expressed transgenic cotton plants;
FIG. 4 is a graph of relative expression levels of GhRSL1-2 over-expressed cotton fibers in 20DPA fibers;
FIG. 5 is a graph showing the molecular identification results of GhRSL1-2-Crispr-Cas9 transgenic cotton plants;
FIG. 6 is a graph of the phenotype identification results of transgenic cotton; wherein A: wild cotton, ghRSL1-2 overexpression and GhRSL1-2 knockout of mature fiber phenotype of cotton, B: fiber length statistics of wild cotton, ghRSL1-2 overexpression and GhRSL1-2 knockout cotton, C: wild cotton, ghRSL1-2 overexpression and GhRSL1-2 gene knockout cotton mature fiber twist phenotype, D: wild cotton, ghRSL1-2 overexpression and GhRSL1-2 gene knockout cotton 1cm fiber cell twist count, E: wild cotton, ghRSL1-2 overexpression and GhRSL1-2 gene knockout cotton mature fiber cross section was stained with calcium fluowhite, F: based on E-made wild cotton, ghRSL1-2 overexpression and GhRSL1-2 knockout cotton cell wall thickness statistics, G: wild-type cotton, ghRSL1-2 overexpression and GhRSL1-2 Gene knockout mature fiber sections stained with Pontamine Fast Scarlet B, H: statistics of cell wall thickness of wild cotton, ghRSL1-2 over-expression and GhRSL1-2 knockout cotton based on G, I: ghRSL1-2 high expression phenotype of mature seed of cotton plant, J: ghRSL1-2 gene knockout phenotype of mature seed of cotton plant, K: statistical analysis of 100 mature grain weights of wild cotton, ghRSL1-2 over-expression and GhRSL1-2 knockout cotton.
Detailed Description
The following further details of the invention in connection with specific embodiments thereof are set forth merely to illustrate the invention and are not intended to limit the scope of the invention.
In the following examples, the test methods, unless otherwise specified, are all conventional; materials, reagents, and the like used, unless otherwise specified, are commercially available; the quantitative experiment is carried out by setting three repeated experiments, and the result is averaged; the material is selected from cotton fiber tissue 20 days after flowering, hereinafter abbreviated as 20DPA, etc., and so on. The light and dark alternate culture is that light culture and dark culture alternate, and the specific culture period can be specifically: light culture for 14 hours/dark culture for 10 hours.
In the examples below, the cotton variety is Xuzhou cotton 142, which is commonly available to the public.
In the following examples, agrobacterium tumefaciens GV3101 is described in the following literature: the effect of Agrobacterium tumefaciens GV3101 on transcription was injured by Arabidopsis thaliana and inoculated by Sho Wei Min, zhao Mingchen, min, su Chenggang, du Xiaobing, report on agricultural biotechnology, 2013,21 (5): 537-545.
In the examples below, plasmid pCAMBIA2300 is described in the following literature: yuanyong, feng Yongkun, ni Mochao, shu Gongmei, guo Shuqiao, liu Laihua.
In the examples below, the plant expression vector pCAMBIA2300-35S-GUS-CaMVterm was constructed and verified as described in J.Biotechnology in China 2013,33 (3): 86-91.
The nucleotide sequence of the gene GhRSL1-2 of the present invention may be mutated by a known method by a person of ordinary skill in the art, and those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the gene GhRSL1-2 of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the gene GhRSL1-2 and have the function of the gene GhRSL1-2.
Example 1 GhRSL1-2 Gene analysis
1. Localization of GhRSL1-2 in tobacco
1. Two single enzyme cutting sites on pCAMBIA2300-GFP vector are selected, primers are designed according to a target gene GhRSL1-2 (the sequences are shown as SEQ ID NO.1 in table 1), and the primers need to be added with enzyme cutting sites and homology arms (the primer sequences are shown as SEQ ID NO.2 and SEQ ID NO.3 in table 1), wherein the enzyme cutting sites are KpnI and XbaI, and target gene fragments containing the enzyme cutting sites are obtained by amplification in a PCR instrument according to the front primer and the rear primer.
Table 1 sequence listing
2. Double enzyme digestion is carried out on the pCAMBIA2300-GFP vector, enzyme digestion sites are KpnI and XbaI, the enzyme digestion sites are consistent with that added before a gene primer, a target gene fragment containing the enzyme digestion sites is connected to the pCAMBIA2300-GFP vector, and then E.coli (DH 5 alpha) competence is transferred.
3. And (3) selecting a monoclonal, carrying out PCR reaction, selecting a positive strain, sequencing, and extracting plasmids of the corresponding strain after sequencing is successful. Meanwhile, a plasmid without the target gene GhRSL1-2 is used as a control.
4. Each set of plasmids was introduced into GV3101 Agrobacterium tumefaciens strain, transiently co-expressed in tobacco leaves, nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI), and a confocal laser scanning microscope was used to detect Green Fluorescent Protein (GFP) fluorescent signals.
As shown in FIG. 1, it was revealed that the cell state was good, and that the localization of GhRSL1-2 in tobacco was nuclear by fluorescence.
2. Evolutionary relationship between GhRSL1-2 protein and model organism Arabidopsis protein
The amino acid sequence of GhRSL1-2 protein (shown as sequence SEQ ID NO. 4) is compared with the amino acid sequence of model organism Arabidopsis protein obtained from database, and phylogenetic tree is constructed, cotton RHD is named according to phylogenetic relation with AtRHD, and the result is shown as figure 2, which shows that the affinity of GhRSL1-2 protein and AtRHD6 is nearest.
Example 2 acquisition and phenotypic analysis of GhRSL1-2 transgenic Cotton plants
1. Construction of recombinant plasmid PC2300S-GhRSL1-2 overexpression
1. Designing a primer according to the CDS sequence (the sequence is shown as SEQ ID NO. 1) of the GhRSL1-2, wherein the primer sequence is shown as SEQ ID NO.2 and SEQ ID NO.3 in the table 1, taking cDNA of tissues with relatively high expression of the GhRSL1-2 in cotton as a PCR template, and amplifying the CDS sequence of the GhRSL1-2 by using a KOD-Plus-Neo PCR amplification system.
2. And (3) connecting the GhRSL1-2 target gene fragment obtained in the step (1) into a vector pCAMBIA2300 by using a homologous recombination method to obtain a recombinant plasmid pCAMBIA2300-GhRSL1-2.
3. And (3) introducing the recombinant plasmid PC2300S-GhRSL1-2 obtained in the step (2) into agrobacterium tumefaciens LB4404 to obtain the GhRSL1-2 over-expression recombinant agrobacterium.
2. Construction of recombinant plasmid Crispr-Cas9-GhRSL1-2 gene knockout vector
1. The specific nucleotide sequence of about 20bp in GhRSL1-2 is found out as two targets to be knocked out, and the targets are shown as SEQ ID NO.9 and SEQ ID NO.10 in the table 1. Target design is carried out on cotton gene knockout sgRNA (the sequence of which is shown as SEQ ID NO.7 in Table 1), and single gene double targets are adopted.
2. Primers were designed according to the target spots, and sgRNA and tRNA fragments were amplified on the basis of pGTR4 vectors, the sequences of which are shown in SEQ ID NO.8 in Table 1. The first PCR amplification takes the vector as a template to obtain two fragments of tRNA+target 1 (the primers are shown as SEQ ID NO.11 and SEQ ID NO. 12) and target 1+sgRNA1+tRNA+target 2 (shown as SEQ ID NO.13 and SEQ ID NO. 14). And amplifying by using overlap extension PCR for the second time, and obtaining tRNA+target 1+sgRNA1+tRNA+target 2 containing homologous arms of the expression vector by using the two fragments obtained by the first amplification as templates and the primer sequences as shown in SEQ ID NO.15 and SEQ ID NO. 16. The expression vector is a PRGEB32 vector, the vector contains a U6 promoter and gRNA2, and has two BsaI enzyme cutting sites, and BsaI enzyme is used for carrying out double enzyme cutting on the PRGEB32 vector.
3. The tRNA+target 1+sgRNA1+tRNA+target 2 obtained by homologous recombination and the digested carrier PRGEB32 are connected and amplified, the connection product is used for converting the competent cells of the Trans1-T1, the competent cells are coated on a culture medium with kana resistance for culture, positive colonies are picked for PCR detection, and plasmids are extracted from the positive colonies which are identified.
4. The recombinant plasmid PRGEB32-GhRSL1-2-gRNA is introduced into agrobacterium tumefaciens GV3101 to obtain GhRSL1-2 knockout recombinant agrobacterium.
In the GhRSL1-2 knockout experiment, the mechanism of GhRSL1-2 silencing is as follows: cas9 protein naturally exists in plant body, when agrobacterium is transfected into plant, recombinant plasmid PRGEB32-GhRSL1-2-gRNA can bind to GhRSL1-2 in plant body under the positioning of sgRNA and transfer action of tRNA, and Cas9 binds to autonomously recognized PAM site (Cas 9/gRNA can target any DNA site containing 5, -N20-NGG-3' (n=a, T, G, C) or 5' -CCN-N20-3 '), wherein NGG is Cas9 to recognize required PAM and can precisely cleave target DNA duplex at about 3bp before PAM), and when Cas9 is bound to a part of sequence of Cas9, cas9 protein can knock out the part of target point bound to sgRNA, after target is cleaved by CRISPR system, biological individual can initiate own repair mechanism, and mutation is generated after repair, thus realizing gene silencing.
3. Obtaining transgenic plants
The GhRSL1-2 over-expression recombinant agrobacterium obtained in the first step and the GhRSL1-2 knock-out recombinant agrobacterium obtained in the second step are respectively used for infecting the hypocotyl sections of Xu Zhoumian seedlings to obtain embryogenic callus and sterile seedlings, and positive seedlings are identified after grafting to obtain positive plants; and carrying out generation propagation to obtain T3 generation homozygous transgenic plants, namely a GhRSL1-2 over-expression cotton plant line and a GhRSL1-2 knockout cotton plant line.
4. Identification of transgenic plants
1. Identification of GhRSL1-2 overexpressed cotton
Cotton leaf DNA of each group was extracted by using GhRSL1-2 overexpressed cotton (GhRSL 1-2-OE) as an experimental group and wild cotton Xu Zhoumian (WT) as a control group, and PCR identification was performed using gene primers (sequences shown as SEQ ID NO.5 and SEQ ID NO.6 of Table 1).
As a result, referring to FIG. 3, the extracted DNA band showed that GhRSL1-2 was overexpressed in GhRSL1-2 overexpressed plants compared to Xuzhou cotton 142, indicating that the GhRSL1-2 overexpressing vector had been transferred into cotton.
Wild cotton Xu Zhoumian (WT) was used as a control group and GhRSL1-2 overexpressed cotton (GhRSL 1-2-OE) was used as an experimental group. Genetic analysis was performed on GhRSL1-2 overexpressed cotton and wild-type Xuzhou cotton 142, respectively. And (3) taking the fiber of 20d after the fiber is bloomed, grinding the sample by liquid nitrogen, extracting each group of RNA, detecting the concentration of the RNA, carrying out reverse transcription by using a reverse transcription kit to obtain cDNA of the fiber of 20d, and detecting the relative expression level of GhRSL1-2 by qRT-PCR.
As a result, referring to FIG. 4, the relative expression level of GhRSL1-2 gene of Xuzhou cotton 142 of wild type cotton was 0.3, the average relative expression level of GhRSL1-2 over-expressed cotton was 21, and the relative expression level of GhRSL1-2 over-expressed cotton after 20d of fiber flowering was significantly higher than that of wild type cotton, further indicating that GhRSL1-2 over-expression vector had been transferred into cotton.
2. Identification of GhRSL1-2 knockout cotton
Wild cotton Xu Zhoumian (WT) was used as a control group and GhRSL1-2 knockout cotton was used as an experimental group. Selecting the GhRSL1-2 knockdown recombinant agrobacterium obtained in the second step, designing a primer on a genome sequence according to the vicinity of a designed target point, then performing PCR, purifying a product, sequencing the product with a T vector, comparing the product with the genome sequence, and identifying a knockdown transgene.
As a result of the identification, referring to FIG. 5, the GhRSL1-2 knockdown recombinant sgRNA1 and sgRNA2 were each inserted with one base compared to the wild type sequence, and were confirmed to have been knocked down.
5. Phenotypic analysis of GhRSL1-2 transgenic cotton
Randomly selecting 10 mature fibers of the GhRSL1-2 over-expression cotton strain obtained in the third step, the GhRSL1-2 gene knockout cotton strain and the wild Xuzhou cotton 142 strain, randomly taking 15 cotton bolls from each strain, pulling the fibers for several times, enabling the fibers to be parallel, and removing the attached fibers. Placing the fiber bundles on a black fluff plate, measuring the lengths by using a ruler, recording the phenotype of each group of cotton mature fibers, counting the fiber lengths, identifying the twisting phenotype of the mature fibers and counting the twisting number; then, the cross section is dyed by calcium fluoride white, and the thickness of each group of cell walls is counted; performing Pontamine Fast Scarlet B mature fiber section staining, and counting cotton cell wall thickness; determining the phenotype of mature seeds of each group of cotton plants, and counting the mature grain weight.
The results in FIGS. 6A and B show that the average length of wild-type plant fibers is 27mm, the length of GhRSL1-2 overexpressed plant fibers is 29mm, the length of GhRSL1-2 gene knockout plant fibers is 23mm, and the length of overexpressed plant fibers is increased by 7% relative to the length of wild-type fibers; compared with the wild type fiber length, the GhRSL1-2 gene knockout strain is shortened by 15%, which indicates that the over-expression of GhRSL1-2-OE increases the fiber length, and the knockout of the Crispr-Cas9-GhRSL1-2 gene significantly inhibits the fiber length, which indicates that the GhRSL1-2 gene regulates and controls the development of fiber cells.
The results of FIGS. 6C and D show that the average twist number of wild-type cotton per 1cm of mature fiber is about 57, the twist number of GhRSL1-2 overexpressed cotton per cm of mature fiber is about 66, the twist number of GhRSL1-2 knockout cotton per cm of mature fiber is about 18, and the twist number of overexpressed plant fiber is increased by 16% relative to the twist number of wild-type fiber; compared with the length of wild fiber, the GhRSL1-2 gene knockout strain is shortened by 68%, which indicates that over-expression of GhRSL1-2-OE increases the twisting number of fiber, while knockout of Crispr-Cas9-GhRSL1-2 gene significantly inhibits the twisting number of fiber, which indicates that the GhRSL1-2 gene regulates the twisting degree of fiber.
The cotton cross sections of E and F in FIG. 6 were stained with calcium fluowhite, showing that the cell wall thickness of the wild type cotton mature fiber was about 5.5 μm, the average cell wall thickness of the GhRSL1-2 overexpressed cotton mature fiber was about 7.1 μm, the cell wall thickness of the GhRSL1-2 knock-out cotton mature fiber was about 4.3 μm, and the cell wall thickness of the overexpressed plant mature fiber was increased by 29% relative to the cell wall thickness of the wild type mature fiber; the GhRSL1-2 gene knockout line is shortened by 22% relative to the length of wild type fibers, and graphs G and H are mature fiber sections dyed with Pontamine Fast Scarlet B, and the fiber cell wall thickness shown by statistics is the same as the data of E and F, so that the reliability of the data is ensured, and the over-expression of GhRSL1-2-OE is shown to increase the cell wall thickness of cotton mature fibers, and the knockout of Crispr-Cas9-GhRSL1-2 gene remarkably inhibits the cell wall thickness of mature fibers, and the GhRSL1-2 gene is shown to regulate the cell wall development of fibers.
The mature seed weight statistics of I, J and K in FIG. 6 show that the wild type plant mature seed weight average is about 7.5g, the GhRSL1-2 overexpressing plant mature seed weight average is about 9.3g, the GhRSL1-2 knockout line mature seed weight average is about 7.2g, the overexpressing plant mature seed weight is increased by 24% relative to the wild type and knockout mature seed weight; it is shown that overexpression of GhRSL1-2-OE increases mature seed weight, and that GhRSL1-2 gene regulates seed development.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
- The GhRSL1-2 gene is characterized by being capable of regulating cotton development, and the sequence is shown as SEQ ID NO. 1.
- The application of GhRSL1-2 gene and related gene in regulating cotton development is characterized in that the sequence of GhRSL1-2 gene is shown in SEQ ID NO.1, the related gene has at least 75% of homology with GhRSL1-2 gene, and the encoding gene has the function of GhRSL1-2 gene.
- 3. The use according to claim 2, wherein the cotton develops into a fibroblast development.
- 4. The use according to claim 2, wherein overexpression of the GhRSL1-2 gene increases cotton fiber length and knockout of the GhRSL1-2 gene inhibits cotton fiber length.
- 5. The use according to claim 2, wherein overexpression of the GhRSL1-2 gene increases the cell wall thickness of the mature cotton fiber, and knocking out the GhRSL1-2 gene suppresses the cell wall thickness of the mature cotton fiber.
- 6. The use according to claim 2, wherein overexpression of the GhRSL1-2 gene increases the weight of mature cotton seed and knocking out the GhRSL1-2 gene suppresses the weight of mature cotton seed.
- 7. The use according to claim 2, wherein overexpression of the GhRSL1-2 gene increases the twist count of cotton fibers, and knocking out the GhRSL1-2 gene suppresses the twist count of cotton fibers.
- 8. A method for breeding transgenic plants, characterized in that a transgenic plant having a growth different from that of a recipient plant is obtained by introducing into the recipient plant a coding gene GhRSL1-2 as shown in SEQ ID No. 1.
- 9. The method according to claim 8, wherein the introduction of the gene GhRSL1-2 into the recipient plant is effected by introducing a recombinant plasmid containing the GhRSL1-2 gene into the recipient plant.
- 10. The method of claim 8, wherein the recombinant plasmid is pBI121-GhRSL1-2, pCAMBIA2300-GhRSL1-2, or pHellgate 4-GhRSL1-2.
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