Method for rapid construction of rdna array and use of method in manual regulation of rdna copy number

Abstract

A method for rapid construction of an rDNA array and a use of the method in the manual regulation of an rDNA copy number, relating to molecular biology and molecular technology. The present invention provides a method for rapid construction of an rDNA array, capable of constructing a homogenized and synthetic rDNA array on any selected site in a genome-wide range. On this basis, according to the present invention, a recombinase system, for example, a Cre-loxP system, can be further introduced into rDNA units of a synthetic rDNA array to mediate the rearrangement between the rDNA units, thereby achieving the objective of manually regulating an rDNA copy number.

Description

A rapid method for constructing rDNA arrays and its application in artificially regulating rDNA copy number Technical Field

[0001] The present invention belongs to molecular biology and molecular technology, and in particular relates to a method for rapidly constructing an rDNA array and an application thereof in artificially regulating rDNA copy number. Background Art

[0002] Ribosomal DNA (rDNA) is the DNA sequence within the cellular genome that produces ribosomal RNA (rRNA). Recent studies have shown that rDNA plays a crucial regulatory role in numerous life processes, including environmental stress, DNA damage repair, maintaining genome stability, individual development, and cellular aging. As a major component of the ribosome, rRNA accounts for ~80% of cellular expression, supporting normal cellular life. Consequently, in eukaryotic genomes, rDNA is present in multiple copies, with high sequence similarity between units. Most rDNA sequences are arranged as tandem repeats, forming one or more arrays. In Saccharomyces cerevisiae, approximately 150 rDNA copies are arranged in tandem on chromosome 12, forming a single rDNA array. As a model organism, Saccharomyces cerevisiae boasts a simple and rapid genetic manipulation system, making it widely used in basic research and industrial production. Furthermore, the presence of a single rDNA array makes Saccharomyces cerevisiae an important platform for eukaryotic rDNA research.

[0003] On the one hand, wild-type rDNA units contain multiple functional elements, and the functions of some elements have not yet been accurately elucidated. Furthermore, due to the extremely high copy number of rDNA arrays, even the read length of existing Nanopore sequencing technology cannot cover them, and the sequences between units are highly similar, making correct matching and assembly impossible. The high repetitiveness of rDNA creates difficulties in genetic manipulation and detection. How to rapidly reconstruct an array of synthetic rDNA units on the genome for systematic functional exploration of rDNA is a technical challenge that needs to be solved urgently.

[0004] On the other hand, constructing stable and predictable biological chassis cells is a key area of ​​research in minimal genomes. As one of the most repetitive and unstable regions of the genome, the rDNA array is crucial for constructing minimal eukaryotic genomes. To date, the rDNA copy number required for basic survival in eukaryotic cells remains unknown. Currently, few methods exist for streamlining rDNA regions, and further reductions to approximately 15 rDNA copies are not possible. Another technical challenge remains to be solved: how to construct an rDNA array with artificially controlled copy number to explore the minimum rDNA copy number required for cell survival.

[0005] Currently, rDNA array construction techniques in yeast can be divided into two categories. The first involves integrating a mutant rDNA unit at a target location. This mutation confers hygromycin resistance to the rDNA. Continuously increasing the hygromycin concentration in the culture environment can induce rDNA amplification, ultimately yielding a long rDNA array. In 2000, researchers linked upstream and downstream fragments of the integration site to either side of the mutant rDNA unit. Simultaneously, they inserted an upstream fragment between the rDNA unit and the downstream fragment. The entire fragment was then enzymatically cleaved from the vector and transformed into a yeast strain. Because the rDNA has identical sequences on both sides, amplification of the rDNA unit can be achieved through non-allelic recombination. In 2006, researchers chose a two-step integration method to construct arrays. In the first integrated fragment, an exogenous sequence was added upstream of the mutant rDNA unit. In the second integrated fragment, the exogenous sequence was inserted upstream of the mutant rDNA unit and the NTS2 (non-transcribed spacer 2) sequence in the rDNA unit was connected downstream. The second round of transformation inserted the rDNA between the exogenous sequence and the NTS2 sequence, integrating the second rDNA unit upstream of the first rDNA unit, and achieving rDNA amplification through non-allelic homologous recombination. In 2017, researchers integrated 1, 2, or 2 rDNA units into the genome and then obtained rDNA arrays by continuously increasing the concentration of hygromycin.

[0006] The second approach involves transferring the entire rDNA array, targeting a new integration site after in situ rDNA excision. In 2023, researchers modified CRISPR-Cas9 technology, designing two guide RNAs to target sequences flanking the original site and one guide RNA to target a new site for excision. Three donor DNA strands were also designed: the first strand connects the two ends of the in situ excision, while the second and third strands connect the rDNA array to the ends of the targeted site, respectively. This allows the entire rDNA array to be transferred from the original excision site to the new site.

[0007] However, the above-mentioned technology for constructing rDNA arrays on the genome still has the following technical defects: 1. Dependence on exogenous sequences as homology arms: During the construction process, exogenous sequences are used as homology arms, and by introducing identical sequences on both sides of the rDNA unit, non-allelic homologous recombination is induced to achieve amplification of the rDNA unit. However, the resulting rDNA array contains exogenous sequences between units. In contrast, the present invention directly uses partial sequences of wild-type rDNA as homology arms, and the resulting array sequence is more controllable.

[0008] 2. Multiple rounds of transformation are required: First, an rDNA unit with an upstream exogenous sequence is integrated into the genomic locus. In the second round of transformation, the rDNA unit has the exogenous sequence and the upstream rDNA sequence NTS2, respectively, upstream and downstream. The second round of rDNA fragments can be integrated between the exogenous sequence and the NTS2 sequence of the first rDNA fragment. In this invention, we directly utilize rDNA sequences as homology arms. By designing and constructing two rDNA fragments with overlapping regions, we achieve the integration of multiple rDNA units in a single co-transformation, directly forming an rDNA array.

[0009] 3. Hygromycin-induced amplification is required to screen and obtain rDNA arrays: After obtaining a correctly integrated strain, the rDNA units are still amplified by gradually increasing the hygromycin concentration, which is time-consuming and takes about two weeks. The present invention does not require this process.

[0010] 4. Low accuracy (6 / 41): CRISPR can achieve complete array transfer, but subsequent validation results showed that this method has a high off-target rate, with only 6 of the 41 validated clones integrated into the target site. However, this study achieved nearly 100% integration and formed an rDNA array.

[0011] The shortcomings of existing rDNA array streamlining methods: Currently, rDNA streamlining is mainly achieved through top-down deletion, and there is no implementation scheme similar to this scheme. Specifically: in the presence of mutant rDNA plasmids, when cells are faced with hygromycin selection pressure, the number of rDNA copies expressed by the plasmid will increase, and most of the wild-type rDNA on the genome will be induced to recombine and lost. Through this streamlining method, researchers have now reduced the minimum rDNA copy number required for brewer's yeast strains to 15-20, and low-copy strains obtained by similar methods are widely used in rDNA-related research. However, the top-down streamlining method is time-consuming, and after it is reduced to about 15, it is difficult to obtain strains with further streamlined rDNA arrays.

[0012] The Cre-loxP system can mediate recombination at specific sites. It was first discovered in P1 phage and is involved in the circularization of the genome. The Cre protein can recognize and catalyze the recombination of specific DNA sites. The loxP site in the phage genome contains Cre specific recognition sites at both ends. After the Cre enzyme recognizes and binds, it can induce deletion, insertion, transposition and ectopicity in the region. In order to achieve efficient random rearrangement, this study used a bidirectional loxP sequence - loxPsym. In the Saccharomyces cerevisiae synthetic genome project (SC2.0), the loxPsym site was inserted 3 bp downstream of a non-essential gene to induce genome-wide rearrangement and achieve gene deletion or replacement. However, this system has not yet been applied to the transformation of any repetitive sequences. In the present invention, the copy number control of the repetitive region is achieved for the first time by introducing the loxPsym sequence in each rDNA unit. Technical issues

[0013] The present invention proposes a method for rapidly constructing an rDNA array, which can achieve the construction of a uniform synthetic rDNA array across the entire genome. On this basis, the present invention also introduces a sequence that can be recognized by a recombinase (such as the loxPsym sequence in the Cre-loxP system) into a synthetic rDNA array to mediate the rearrangement between rDNA units, thereby achieving the purpose of artificially regulating the rDNA copy number.

[0014] To address the shortcomings of the aforementioned prior art, the present invention aims to provide a technology for constructing uniform synthetic rDNA arrays based on the homologous recombination capabilities of Saccharomyces cerevisiae. This method enables rapid and efficient construction of rDNA arrays at any selected site across the entire genome. Furthermore, recombinase-specific recognition sites are introduced into the rDNA arrays, allowing copy number to be regulated by inducing recombinase expression, thereby achieving deep simplification of the rDNA region. Technical Solutions

[0015] In one aspect, the present invention provides a method for rapidly constructing an rDNA array, comprising the following steps:

[0016] (1) Select a yeast strain of any genotype as the starting strain;

[0017] (2) Construct A and B fragments containing rDNA, where fragment A contains upstream homology arm sequence, selection marker gene, and 1.2 copies of wild-type or synthetic rDNA units in series; fragment B contains one copy of wild-type or synthetic rDNA unit and downstream homology arm sequence, and multiple restriction sites are added at both ends of fragments A and B to release the complete fragments;

[0018] (3) Use restriction endonucleases to completely release fragments A and B from their plasmid vectors;

[0019] (4) The enzyme digestion products containing fragment A and fragment B are co-transformed into the starting strain of step (1) by the lithium acetate transformation method, and spread on the screening plate corresponding to the selection marker gene;

[0020] (5) Screening a yeast strain that has successfully integrated an rDNA array; wherein n of the rDNA array is greater than 3, where n represents the copy number.

[0021] The method is characterized in that when the wild-type rDNA array in the starting strain is not knocked out, the screening method in step (5) includes:

[0022] (a) Randomly select transformants on the screening plate for PCR verification. Use primers upstream and downstream of the integration site to amplify and screen colonies that have correctly integrated the rDNA fragment.

[0023] (b) Correctly integrated strains were selected for pulsed-field gel electrophoresis and Southern blot analysis. The number of inserted rDNA copies was estimated based on the change in the length of the chromosomal band at the insertion site. Yeast strains that successfully integrated the rDNA array were screened from the corresponding strains; the n of the rDNA array was > 3.

[0024] The method is characterized in that when the wild-type rDNA array on the genome of the starting strain is completely knocked out and a URA3 high-copy plasmid containing a single wild-type rDNA unit is cloned to support cell survival, the screening method in step (5) includes:

[0025] After colonies grow on the screening plates, they are replica-plated onto plates containing 5-FOA for screening, identifying yeast strains that have successfully integrated the rDNA array. Because the expression product of the URA3 gene converts 5-FOA into cytotoxic fluorodeoxyuridine, only strains that have lost the URA3 plasmid can grow on plates containing 5-FOA. To survive, cells require multiple copies of the rDNA unit to express sufficient rRNA. Therefore, 5-FOA plates are used to directly screen yeast strains that have successfully integrated the rDNA array, where n is ≥ 10.

[0026] In the construction method, the upstream homology arm sequence in step (2) is 300 to 1000 bp upstream of the target site; the downstream homology arm sequence is 300 to 1000 bp downstream of the target site; and the selection marker gene includes an auxotrophic gene or a resistance gene. The present invention can construct a new rDNA array in any Saccharomyces cerevisiae strain, and the selection marker gene on rDNA fragment A can be adjusted according to the genotype of the strain, and can be an auxotrophic gene or other resistance gene that can be used in yeast.

[0027] In the method described, the upstream homology arm sequence and the downstream homology arm sequence in step (2) are adjusted to correspond to the position of the selected rDNA insertion, wherein the adjustment method includes molecular cloning or fusion PCR method. Specifically, the upstream and downstream homology arm sequences on rDNA fragments A and B can be adjusted to correspond to the position of the selected rDNA insertion. The adjusted upstream and downstream homology arms are cloned and replaced with the corresponding sequences of the vector containing the original rDNA fragments A and B by molecular cloning, and sequenced for confirmation. The fragments are released by enzyme digestion before use. Fusion PCR can also be used to adjust the upstream and downstream homology arm sequences, and the corresponding array construction can be carried out using linear PCR products.

[0028] In a second aspect, the present invention provides a method for constructing a synthetic rDNA array capable of artificially controlling copy number, comprising the following steps:

[0029] (1) By seamless cloning technology, a specific sequence that can be recognized by the recombinase is inserted into any region of the wild-type rDNA unit to construct a synthetic rDNA unit with a specific sequence. After testing and confirmation that it does not affect the rDNA function, it is used to construct synthetic rDNA fragments and arrays;

[0030] (2) Select a yeast strain of any genotype as the starting strain;

[0031] (3) Construct A and B fragments containing rDNA, where fragment A contains upstream homology arm sequence, selection marker gene, and 1.2 copies of synthetic rDNA unit in series; fragment B contains one copy of synthetic rDNA unit and downstream homology arm sequence, and multiple restriction sites are added at both ends of fragments A and B to release the complete fragment;

[0032] (4) Use restriction endonucleases to completely release fragments A and B from their plasmid vectors;

[0033] (5) The enzyme digestion products containing fragment A and fragment B are co-transformed into the starting strain of step (1) by the lithium acetate transformation method, and spread on the screening plate corresponding to the selection marker gene;

[0034] (6) Screening yeast strains that successfully integrate synthetic rDNA arrays, wherein n of the synthetic rDNA array is greater than 3;

[0035] (7) The recombinase expression plasmid is transferred into the yeast strain that has successfully integrated the synthetic rDNA array. By inducing the expression of the recombinase, the rearrangement between rDNA units is mediated and the rDNA copy number is regulated.

[0036] The construction method described above, wherein the recombinase is an enzyme that can catalyze recombination events at specific DNA sites in different species;

[0037] Preferably, the recombinase is selected from one of Cre and its mutants R32V, R32M, 303GVSdup, and VCre, SCre, Flp, and Dre enzymes;

[0038] Wherein, the specific sequence recognized by Cre recombinase and its mutants is selected from one of loxPsym sequence, loxP sequence, loxP 511 sequence, lox 5171 sequence, lox 2272 sequence, M2 sequence, M3 sequence, M7 sequence, M11 sequence, lox 71 sequence and lox 66 sequence;

[0039] VCre is a recombinase in Vibrio spp., which has 29% similarity with the protein sequence of Cre recombinase. The specific sequence recognized by VCre recombinase is selected from one of the sequences of VloxP, Vlox2272, VloxM2, Vlox43R and Vlox43L.

[0040] SCre recombinase is a recombinase in the genus Shewanella, and the specific sequence recognized by SCre recombinase is selected from one of SloxP, Slox2272, SloxV1R, SloxV1L and SloxM1 sequences;

[0041] The Flp recombinase is derived from the 2μ plasmid in Saccharomyces cerevisiae, and the specific sequence recognized by the Flp recombinase is selected from one of the FRT, F1, F2, F3, F4 and F5 sequences;

[0042] Dre recombinase is also derived from bacteriophage, and the specific sequence recognized by Dre recombinase is selected from one of rox12, rox8, rox34, rox9, rox7, rox61 and rox85 sequences.

[0043] The above five recombinases can trigger sequence rearrangement in Saccharomyces cerevisiae by recognizing specific sequences, thereby achieving editing of the target gene.

[0044] In the construction method, the recombinase is induced by one of galactose, β-estradiol or light control.

[0045] In a third aspect, the present invention provides application of any of the methods described above to rapid reconstruction of rDNA arrays.

[0046] In a fourth aspect, the present invention provides the application of any one of the construction methods described above to the rapid reconstruction of a synthetic rDNA array with artificially regulated copy number;

[0047] Preferably, the application can achieve deep pruning of the rDNA region, reducing the copy number in Saccharomyces cerevisiae to less than 8.

[0048] Specifically, the present invention achieves rearrangement of units in an rDNA array containing a loxPsym sequence by inducing the expression of the Cre enzyme, obtaining a series of yeast strains with different rDNA copy numbers, demonstrating that the present invention can achieve regulation of the rDNA array copy number; by combining the copy number with the strain phenotype, deep streamlining of the rDNA region is achieved, reducing the copy number in brewer's yeast to ~8.

[0049] The present invention not only has advantages in constructing wild-type arrays, but also can delete and reprogram rDNA sequences as needed, which can be used to study the functions and regulatory mechanisms of rDNA sequence elements. It can also introduce exogenous sequences, such as other genes, between each rDNA unit, as in other inventions, thereby achieving multi-copy integration of the corresponding gene.

[0050] The design principle of the present invention takes Cre enzyme as an example:

[0051] 1. One-step integration method to construct rDNA array

[0052] This paper proposes an efficient method for constructing rDNA arrays based on homologous recombination in Saccharomyces cerevisiae. This method can rapidly construct rDNA arrays at any selected site throughout the entire genome of Saccharomyces cerevisiae, enabling the study of rDNA-related functions and regulatory mechanisms. Site-specific integration of the rDNA array requires two fragments containing rDNA units: A and B. These fragments can be released from their respective vectors by restriction endonuclease digestion. Fragment A contains upstream homology arm sequences (300–1000 bp upstream of the target site), a selection marker gene, and 1.2 tandem copies of the rDNA unit (9 Kb + 2 Kb). Fragment B contains a complete rDNA unit (9 Kb) and downstream homology arm sequences (300–1000 bp downstream of the target site).

[0053] The rDNA units within segments A and B can be sequenced according to different experimental objectives. The released linear segments A and B are transferred together into the yeast strain to be transformed, where they are integrated into the target site through homologous recombination within the yeast. Following transformation, the corresponding auxotrophic plates are plated. To obtain strains with correctly integrated rDNA arrays, the present invention provides two screening strategies based on the genotypic differences of the starting strains.

[0054] The first approach is universal. PCR (Polymerase Chain Reaction) is first used to identify whether rDNA has been integrated into the target location. Pulsed-field gel electrophoresis and Southern Blot are then used to determine the number of integrated rDNA units, thereby obtaining strains that have successfully integrated the rDNA array (n>3).

[0055] The second approach targets a specific type of yeast strain: wild-type rDNA is deleted from its genome, and a high-copy URA3 plasmid containing one rDNA unit is used to express rRNA to maintain basic cell survival. The approach employed involves directly replicating colonies from the transformed plate onto plates containing 5-FOA to screen for colonies that have lost the URA3 plasmid, indicating that the cell genome has integrated sufficient rDNA units to maintain cell survival. Several colonies on the 5-FOA plate can then be randomly selected for subsequent rDNA copy number analysis, resulting in a nearly 100% success rate in rDNA array construction.

[0056] The present invention has been applied to the construction of various rDNA arrays. Examples have demonstrated the successful construction of rDNA arrays on different chromosomes of the genome (PCR, pulsed-field gel electrophoresis, Southern Blot, and third-generation sequencing technologies confirmed the correct integration of the rDNA array at the target location) and characterized the copy number of the corresponding rDNA array.

[0057] 2. Artificially regulating rDNA copy number

[0058] In the present invention, loxPsym sites are inserted into the sequence of rDNA units, and a synthetic rDNA array is constructed on the genome through the one-step integration method described above. Subsequently, expression of the Cre enzyme is induced in cells to induce rearrangements between rDNA units, thereby causing changes in rDNA copy number, thereby achieving artificial regulation of rDNA copy number. Beneficial effects

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] 1. The present invention can achieve efficient and rapid construction of any synthetic rDNA array at any position in the genome through the design of two rDNA fragments and a one-step co-transformation strategy.

[0061] 2. This method uses the rDNA unit sequence itself as the homology arm, eliminating the need for exogenous artificial sequences. It can integrate varying numbers of rDNA units into target sites, achieving the construction of uniform rDNA arrays. Similar to seamless cloning technology, this approach avoids the introduction of scar sequences between rDNA units, making the resulting array sequence more controllable. This is particularly important when testing the function of sequence elements within rDNA units, as it avoids the introduction of interfering factors.

[0062] 3. The present invention can directly screen on the screening plate corresponding to the marker gene to obtain the correctly integrated rDNA array strain without the need to use hygromycin to induce the amplification of the rDNA unit.

[0063] 4. For special strain backgrounds, the present invention proposes the use of 5-FOA to counter-screen the URA3 plasmid with high copy number wild-type rDNA, effectively enriching strains integrating the rDNA array with n>10, without the need for manual screening steps relying on PCR verification and copy number analysis.

[0064] 5. The present invention successfully constructed a series of rDNA with n>10 by deleting the intrinsic wild-type rDNA array and reducing the interference of the endogenous copy number dynamic regulation system (fob1 knockout). loxp The arrayed strains, through recombinase systems such as Cre-loxP, mediated the rearrangement between rDNA units, and obtained a series of strains with different copy numbers, realizing rapid and efficient artificial regulation of rDNA copy number, completing deep streamlining of the rDNA region, and obtaining strains with fewer copy numbers than the known minimum, expanding existing cognition.

[0065] 6. The present invention inserts specific sequences recognized by recombinases or their mutants into the non-coding regions of rDNA units, inducing rearrangements between rDNA units and achieving artificial regulation of rDNA copy number. For example, inserting the loxPsym sequence into the NTS2 region of the wild-type yeast rDNA sequence yields a synthetic rDNA unit bearing the loxPsym sequence that can independently support yeast survival. Based on the Cre-loxP system, inducing rearrangements between rDNA units allows for artificial regulation of rDNA copy number. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 is a schematic diagram of the one-step integration method for constructing rDNA arrays and a schematic diagram of the principle of artificially regulating rDNA copy number; wherein A is a schematic diagram of the one-step integration method for constructing rDNA arrays in Saccharomyces cerevisiae; B is a schematic diagram of the principle of artificially regulating rDNA copy number;

[0067] FIG2 is a schematic diagram of the restriction sites on both sides of the target fragment in fragment A and fragment B;

[0068] Figure 3 is the electrophoresis result of the enzyme digestion product;

[0069] FIG4 shows the results of colony PCR identification of three randomly selected transformants;

[0070] Figure 5 shows the pulsed field electrophoresis and Southern Blot detection results of the strains;

[0071] Figure 6 is the electrophoresis result of the enzyme digestion product;

[0072] FIG7 shows the pulsed-field gel results and rDNA-specific hybridization Southern Blot results of the strains;

[0073] Figure 8 shows that the correct size of the colony PCR band indicates that the rDNA unit has been integrated into the ChrIII target site;

[0074] Figure 9 shows rDNA in LEU2-rdnp loxp Unit schematic diagram;

[0075] Figure 10 shows rDNA loxp Unit functional testing;

[0076] Figure 11 shows ectopic integration of rDNA loxp Pulsed-field and Southern Blot results of the strains;

[0077] Figure 12 shows the results of Nanopore third-generation sequencing of the IIIsp strain, where A is the colinearity plot of the third-generation sequencing of the strain, showing that the rDNA in the IIIsp strain is correctly integrated on ChrIII; B is the rDNA copy number calculated by the relative depth of sequencing, which is approximately 14;

[0078] Figure 13 shows the changes in rDNA copy number of strains after induced rearrangement;

[0079] FIG14 shows that the rDNA copy number in the strain was determined to be 6-8 using ddPCR technology. Modes for Carrying Out the Invention

[0080] Example 1: Construction of multi-rDNA array strains

[0081] As shown in Figure 1, A is a schematic diagram of the one-step integration method for constructing rDNA arrays in Saccharomyces cerevisiae; B is a schematic diagram of the principle of artificially regulating rDNA copy number.

[0082] (1) The starting strain was the wild-type strain BY4742, which has ~150 copies of the rDNA array on ChrXII.

[0083] (2) Construct a plasmid vector containing rDNA fragments A and B. In this case, fragment A includes the 812 bp homology arm upstream of the ChrXV integration site (SEQ ID NO.5), the auxotrophic marker LEU2, and 1.2 rDNA units (SEQ ID NO.2) in series; fragment B includes one rDNA unit (SEQ ID NO.1) and the 787 bp homology arm downstream of the ChrXV integration site (SEQ ID NO.6). At the same time, multiple restriction sites are designed at both ends of the rDNA fragment, and multiple groups of restriction enzymes can be used to release the complete rDNA fragment (Figure 2). The constructed fragments are cloned into the Escherichia coli vector for sequencing and amplification.

[0084] (3) In this case, the target fragments were released from their plasmids by enzyme digestion (fragment A: NotI, fragment B: NotI, as shown in Figure 3), and the digestion products were co-transformed into the Saccharomyces cerevisiae strain BY4742 by lithium acetate transformation.

[0085] (4) After transformation, the cells were plated on SC-Leu plates and cultured at 30°C for 48 h. Three transformants were randomly selected for yeast colony PCR verification. 20 μL of 20 mM sodium hydroxide was added to the colonies to be verified until the solution became turbid. The cell walls were broken using the PCR program shown in Table 1. The left and right primers in Table 3 (SEQ ID NO. 9.12.13.14) and 1 μL of digested yeast cells were added to the PCR system (Table 2) as templates. The program was run as shown in Table 4. The positions of the left and right verification primers were designed on both sides of the two homologous arms and within the rDNA unit, respectively (Figure 1). The results showed that the integration of the rDNA unit was detected at the designed site in all three colonies.

[0086] Two strains with correct integration were randomly selected and analyzed for the number of integrated rDNA units using pulsed-field electrophoresis and Southern blot analysis. The results in Figure 4 show that in strain b, in addition to rDNA signals detected at chrXII, rDNA unit signals were also detected at ChrXV. ChrXV was significantly shifted compared to the wild-type strain, and based on length estimation, approximately 15 rDNA units were integrated. Figure 5 shows the pulsed-field electrophoresis and Southern blot results for the strains. These results demonstrate that we have successfully constructed an additional rDNA array within the chrXV region of the wild-type strain.

[0087] Table 1 Saccharomyces cerevisiae cell wall disruption procedure

[0088]

[0089] Table 2 Yeast colony PCR reaction system

[0090]

[0091] Table 3 Primer combinations for colony PCR verification of rDNA integration in ChrXV

[0092]

[0093] Table 4 Yeast colony PCR operation program

[0094]

[0095] Example 2: One-step integration method to achieve ectopic construction of rDNA array to support strain survival

[0096] (1) The wild-type rDNA array on ChrXII in the starting strain was completely deleted, and the strain was supported by a high-copy plasmid (URA3-RDN) carrying the wild-type rDNA unit and the URA3 gene. At the same time, to reduce the fluctuation of the copy number in the rDNA array, the FOB1 gene in the starting strain was knocked out by the HygMX4 resistance marker gene.

[0097] (2) Construct a plasmid containing rDNA fragments A and B. In this case, fragment A includes the 450 bp homology arm upstream of the ChrIII integration site (SEQ ID NO. 3), the auxotrophic marker LEU2, and 1.2 rDNA units in series; fragment B includes one rDNA unit and the 387 bp homology arm downstream of the ChrIII integration site (SEQ ID NO. 4). At the same time, multiple restriction sites were designed at both ends of the rDNA fragment, and multiple groups of restriction enzymes were used to release the complete rDNA fragment (Figure 1B). The constructed fragments were cloned into an E. coli vector for sequencing and amplification.

[0098] (3) In this case, the target fragments were released from their plasmids by enzyme digestion (fragment A: NotI, fragment B: NotI, Figure 6), and the digestion products were co-transformed into the starting Saccharomyces cerevisiae strain in step (1) by lithium acetate transformation.

[0099] (4) After transformation, the cells were plated on SC-Leu plates and cultured at 30°C for 24 hours. The cells were then transferred to SC-Leu+5-FOA plates. After culture at 30°C for 24 hours, four cells were randomly selected from the 5-FOA plates for pulsed-field electrophoresis and Southern Blot analysis. The experimental results in Figure 7 show that the size of the ChrIII chromosomes of the four monoclonal clones increased significantly, and the signals overlapped with chrIX. Based on the size differences between chromosomes, it was estimated that at least 10 rDNA units were inserted into each of these strains. These results indicate that we have successfully introduced an rDNA array into chrIII.

[0100] (5) Although not necessary for screening, we also performed colony PCR on the four strains selected above to verify the upstream and downstream of the integration site. First, use a pipette tip to add 20 μL of 20 mM sodium hydroxide to each of the colony cells to be verified until the solution becomes turbid. The template digestion procedure is shown in Table 1. Then, 1 μL of the digested template and the two pairs of primers in Table 5 were added to the PCR verification system (SEQ ID NO. 8, 9, 10, 11). The results showed that the rDNA in all four strains was correctly integrated at the target site (Figure 8).

[0101] Table 5 Primer combinations for colony PCR verification of rDNA integration on ChrIII

[0102]

[0103] Example 3: rDNA loxp Array construction

[0104] (1) The wild-type rDNA array on ChrXII in the starting strain was knocked out, and the strain contained a high-copy plasmid containing a wild-type rDNA unit and the URA3 gene (URA3-RDN) to support survival. At the same time, to reduce the fluctuation of the copy number in the rDNA array, the FOB1 gene in the starting strain was knocked out by the HygMX4 resistance marker gene.

[0105] (2) Construction of rDNA units with loxPsym sequences and their functional testing

[0106] The wild-type rDNA array fragment was cloned into the high-copy plasmid vector pRS425 to facilitate subsequent amplification and gene manipulation, resulting in the LEU2-RDN plasmid. Based on the wild-type rDNA unit sequence, the loxPsym sequence (SEQ ID NO. 7) was inserted into the NTS2R (-206) region upstream of the 35S gene promoter, as shown in Figure 9, to obtain the LEU2-rdnp plasmid.

[0107] rDNA verification by Plasmid Shuffling method loxp Whether the unit is functional: pRS425, pLEU2-RND and LEU2-rdnp plasmids were transformed into a strain with wild-type rDNA array knockout and URA3-RDN as the only rDNA source. Since the expression product of the URA3 gene can convert 5-FOA into cytotoxic fluorodeoxyuridine, only the strain that has lost the URA3 plasmid can grow on the plate containing 5-FOA. Therefore, the cells of the transformed single colony were cultured in SC-Leu liquid medium at 30℃ overnight and diluted to the same cell concentration (OD 600=1), and then a 10-fold gradient dilution was performed. The cells of each dilution gradient were spotted on SC-Leu and SC-Leu+5-FOA plates for survival analysis. The results showed that on 5-FOA plates, LEU2-rdnp could support cell survival just as the wild-type rDNA plasmid LEU2-RDN did, indicating that after the strain lost the wild-type rDNA URA3-RDN plasmid, rDNA loxp The unit can function and support the survival of the strain (as shown in Figure 10).

[0108] (3) Construct a plasmid containing rDNA fragments A and B. In this case, fragment A includes the 450 bp homology arm upstream of the ChrIII integration site, the auxotrophic marker LEU2, and 1.2 rDNA fragments in series. loxp Unit; segment B includes an rDNA loxp The rDNA unit and the 387 bp homology arm downstream of the ChrIII integration site were designed. Multiple restriction sites were designed at both ends of the rDNA fragment, and multiple enzyme digestion combinations were used to release the intact rDNA fragment (Figure 1B). The corresponding loxPsym sequences were added to the corresponding positions of the rDNA unit in fragments A and B to construct the modified fragments, which were cloned into an E. coli vector for sequencing and amplification.

[0109] (4) In this case, the target fragments were released from their plasmids by enzyme digestion (fragment A: NotI, fragment B: NotI), and the digestion products were co-transformed into the starting Saccharomyces cerevisiae strain in step (1) by lithium acetate transformation.

[0110] (5) After transformation, the cells were plated on SC-Leu plates and cultured at 30°C for 24 hours. The cells were then transferred to SC-Leu+5-FOA plates. After cultured at 30°C for 24 hours, 6 colonies were randomly selected from the 5-FOA plates for pulsed field electrophoresis and Southern Blot experiments. The experimental results in Figure 11 showed that the 6 colonies had rDNA signals on the ChrIII chromosome only, and the size of the ChrIII chromosome increased significantly. The signal overlapped with chrIX. Based on the size differences between chromosomes, it was estimated that at least 10 rDNA units were inserted into each of these strains. These results indicate that we have successfully introduced an rDNA on chrIII. loxp One strain was randomly selected and named IIIsp, and Nanopore third-generation sequencing was performed. The sequence assembly results showed that rDNA loxp The array was correctly integrated at the target location (Fig. 12A). By dividing the average sequencing depth of the rDNA region in the sequencing data by the average depth of other chromosomal regions (Fig. 12B), it was calculated that ∼14 copies of the rDNA array were successfully obtained in this strain.

[0111] Example 4: Streamlining of rDNA regions based on Cre-loxP recognition

[0112] (1) The starting strain was the IIIsp in Example 3, in which the wild-type rDNA array on the genome was knocked out and a short rDNA was constructed on ChrIII. loxp array, the FOB1 gene was knocked out by the HygMX4 resistance marker gene.

[0113] (2) The pGal-Cre plasmid was transferred into the cells by lithium acetate transformation method, and the transformed cells were resuspended in SC-His liquid medium and cultured at 30°C for 48 hours. The cell culture was then diluted 1:100 into fresh SC-His liquid medium and cultured at 30°C for 48 hours. The cell mixture was then diluted to OD 600 = 0.1. After culturing at 30°C for 4 hours, an appropriate volume of 20% galactose was added to a final concentration of 2%. Induction culture was continued at 30°C for 2 hours. The induced cell mixture was serially diluted 10-fold. The diluted cells were plated onto YPD plates (~200 colonies per plate). Subsequently, colonies from the YPD plates were replicated onto SC-His and YPD plates to obtain a large number of his- colonies. Forty-eight his- colonies were randomly selected and serially diluted 10-fold. Cells from different dilutions were then spotted onto YPD plates to test the growth rate of the strain, using the IIIsp strain as a control.

[0114] (3) Six colonies (S1-S6) that grew slower than IIIsp were randomly selected from the his- colonies for pulsed-field electrophoresis and Southern Blot analysis (Figure 13). The results showed that the rDNA copy number in these strains fluctuated significantly, with a significant downward shift in the signal detected, indicating a decrease in rDNA copy number. Some strains also had more than one length of chrIII. Flow cytometry revealed that the genomes of strains 1 and 5 had doubled after induced rearrangement.

[0115] (4) Digital PCR was used to accurately quantify the rDNA copy number in the S2 and S3 strains, which had not undergone genome diploidization. The results showed that the copy number of each of the three randomly selected monoclonal clones was between 6 and 8 (Figure 14). These results demonstrate that the present invention can achieve artificial regulation of rDNA copy number, thereby achieving the purpose of streamlining the rDNA region.

Claims

1. A method for rapidly constructing an rDNA array, characterized in that: The following steps are involved: (1) Select a yeast strain of any genotype as the starting strain; (2) Constructing A and B fragments containing rDNA, wherein fragment A sequentially contains an upstream homology arm sequence, a screening marker gene, and 1.2 copies of a wild-type or synthetic rDNA unit in series; fragment B sequentially contains one copy of a wild-type or synthetic rDNA unit and a downstream homology arm sequence, and multiple restriction sites are added at both ends of fragments A and B to release the complete fragments; (3) Use restriction endonucleases to completely release fragment A and fragment B from the plasmid vector in which they are located; (4) co-transforming the enzyme digestion products containing fragment A and fragment B into the starting strain of step (1) by lithium acetate transformation method, and coating the resultant products on a screening plate corresponding to the screening marker gene; (5) Screening yeast strains that successfully integrate rDNA arrays.

2. The method according to claim 1, characterized in that When the wild-type rDNA array in the starting strain is not knocked out, the screening method in step (5) includes: (a) Randomly select transformants on the screening plate for PCR verification, use primers corresponding to the upstream and downstream of the integration site to amplify, and screen the colonies with correctly integrated rDNA fragments; (b) The correctly integrated strains were selected for pulsed-field gel electrophoresis and Southern Blot analysis. The number of inserted rDNA copies was estimated based on the change in the length of the chromosome band where the insertion site was located, and the yeast strains that successfully integrated the rDNA array were screened from the corresponding strains.

3. The method according to claim 1, characterized in that When the wild-type rDNA array on the genome of the starting strain is completely knocked out, and the URA3 high-copy plasmid cloned with a single wild-type rDNA unit supports cell survival, the screening method described in step (5) includes: After the colonies on the screening plate grow out, they are copied onto a plate containing 5-FOA for screening to obtain yeast strains that have successfully integrated the rDNA array.

4. The method according to claim 1, characterized in that In step (2), the upstream homology arm sequence is 300 to 1000 bp upstream of the target site; the downstream homology arm sequence is 300 to 1000 bp downstream of the target site; and the selection marker gene includes an auxotrophic gene or a resistance gene.

5. The method according to claim 1, characterized in that In step (2), the upstream homology arm sequence and the downstream homology arm sequence are adjusted to correspond to each other according to the selected rDNA insertion position, wherein the adjustment method includes molecular cloning or fusion PCR method.

6. A method for constructing a synthetic rDNA array capable of artificially regulating the copy number, characterized in that: The following steps are involved: (1) Through seamless cloning technology, a specific sequence that can be recognized by the recombinase is inserted into any region of the wild-type rDNA unit to construct a synthetic rDNA unit with a specific sequence. After being tested and confirmed to have no effect on the rDNA function, it is used to construct synthetic rDNA fragments and arrays; (2) Select a yeast strain of any genotype as the starting strain; (3) Constructing A and B fragments containing rDNA, where fragment A contains the upstream homology arm sequence, the selection marker gene, and 1.2 copies of the synthetic rDNA unit in series; fragment B contains one copy of the synthetic rDNA unit and the downstream homology arm sequence, and multiple restriction sites are added at both ends of the A and B fragments to release the complete fragments; (4) Use restriction endonucleases to completely release fragment A and fragment B from the plasmid vector in which they are located; (5) co-transforming the enzyme digestion products containing fragment A and fragment B into the starting strain of step (2) by lithium acetate transformation method, and spreading them on the screening plate corresponding to the screening marker gene; (6) Screening yeast strains that successfully integrate synthetic rDNA arrays; (7) The recombinase expression plasmid is transferred into the yeast strain that has successfully integrated the synthetic rDNA array. By inducing the expression of the recombinase, the rearrangement between rDNA units is mediated and the rDNA copy number is regulated.

7. The construction method according to claim 6, characterized in that: The recombinase is an enzyme that can catalyze recombination events at specific DNA sites in different species; Preferably, the recombinase is selected from Cre and its mutants R32V, R32M, 303GVSdup, and one of VCre, SCre, Flp, and Dre enzymes; Wherein, the specific sequence recognized by Cre recombinase and its mutants is selected from one of loxPsym sequence, loxP sequence, loxP 511 sequence, lox 5171 sequence, lox 2272 sequence, M2 sequence, M3 sequence, M7 sequence, M11 sequence and lox 66 sequence; The specific sequence recognized by VCre recombinase is selected from one of VloxP, Vlox2272, VloxM2, Vlox43R and Vlox43L sequences; The specific sequence that can be recognized by the SCre recombinase is selected from one of SloxP, Slox2272, SloxV1R, SloxV1L and SloxM1 sequences; The specific sequence recognized by the Flp recombinase is selected from one of the FRT, F1, F2, F3, F4 and F5 sequences; The specific sequence that can be recognized by the Dre recombinase is selected from one of the sequences of rox12, rox8, rox34, rox9, rox7, rox61 and rox85.

8. The construction method according to claim 6, characterized in that: The induction mode of the recombinase is one of galactose, β-estradiol or light control.

9. Use of the method according to any one of claims 1 to 5 for rapid reconstruction of rDNA arrays.

10. Application of the construction method according to any one of claims 6 to 8 to rapid reconstruction of a synthetic rDNA array with artificially controllable copy number; Preferably, the application can achieve deep simplification of the rDNA region and reduce the copy number in Saccharomyces cerevisiae to less than 8.

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