Replication start site fragment of fragia parviflora as well as identification method and application of replication start site fragment

Abstract

The invention relates to the technical field of gene engineering, in particular to a replication start site fragment of erenchium leptosphaereum as well as an identification method and application of the replication start site fragment. The invention provides a replication starting site fragment of the fragia parviflora, the replication starting site fragment of the fragia parviflora is an ori-1 sequence, and the ori-1 sequence is as shown in Seq. No.1. The invention also provides a construction method of the replication starting site fragment of the fragia parviflora, and the construction method of the replication starting site fragment of the fragia parviflora. On the basis of the determined replication starting site fragment of the Amphyperenchum parvum, a new idea and an important basis are provided for research on a replication mechanism of the Amphyperenchum parvum, and a replication mechanism of other eukaryotes is explored.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and in particular relates to a fragment of a replication initiation site of Physcomitrella patens and an identification method and application thereof. Background Art

[0002] Conservative replication of genetic material is essential to maintain the integrity of the genome. DNA replication in eukaryotic cells initiates at multiple sites, called replication origins, which are dispersed throughout the genome.

[0003] In yeast, there is an autonomously replicating sequence (ARS), which is a sequence that can independently initiate DNA replication. Due to the simplicity of the yeast system and the short culture cycle, it is widely used to identify the replication start site. The main function of plant extracellular circular DNA (eccDNA) is gene identification and transmission, indicating that eccDNA may replicate through a similar mechanism. Studies have found that the self-replicating sequence in the replicon of Amaranth eccDNA is similar in function to yeast ARS. However, no clear conserved sequence of the replication start site has been found so far.

[0004] Physcomitrium patens is an important model plant with simple structure, short growth cycle and strong homologous recombination ability. The study of the self-replication ability of eccDNA in Physcomitrium patens helps to understand plant stress resistance and yield, and provide new solutions for agricultural production. However, the replication initiation site sequence (ori) associated with eccDNA in Physcomitrium patens has not yet been identified. Summary of the invention

[0005] The purpose of the present application is to provide a fragment of the replication initiation site of Physcomitrella patens and an identification method and application, aiming to solve the problem that the replication initiation site sequence related to eccDNA in Physcomitrella patens has not been identified in the prior art.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a fragment of a replication initiation site of Physcomitrella patens, wherein the fragment of a replication initiation site of Physcomitrella patens is an ori-1 sequence, wherein the ori-1 sequence is shown in Seq. No.1.

[0008] In a second aspect, the present application provides a primer pair for identifying a fragment of a replication start site of Physcomitrella patens, the primer pair comprising a first primer and a second primer, wherein the sequence of the first primer is shown in Seq. No. 2, and the sequence of the second primer is shown in Seq. No. 3.

[0009] In a third aspect, the present application provides a recombinant expression plasmid for expressing a fragment of the replication initiation site of Physcomitrella patens, comprising the above-mentioned ori-1 sequence, and the expression plasmid for expressing the ori-1 sequence is selected from PRS305 or PRS315.

[0010] In a fourth aspect, the present application provides an engineered bacterium that expresses a fragment of the replication initiation site of Physcomitrella patens, wherein the engineered bacterium contains the above-mentioned ori-1 sequence or the above-mentioned recombinant expression plasmid.

[0011] In a fifth aspect, the present application provides a kit for identifying a fragment of a replication initiation site of Physcomitrella patens, the kit comprising the above-mentioned primer pair for identifying a fragment of a replication initiation site of Physcomitrella patens.

[0012] In some embodiments, the kit comprises a yeast expression system.

[0013] In some embodiments, the kit also includes a PCR identification reaction; wherein the PCR identification reaction system includes: 10 μL of 2× high-fidelity enzyme buffer, 4 μL of 2mM dNTPs, 0.4 μL of 1.0U / μL high-fidelity enzyme, 0.5 μL of each of 10 μM upstream and downstream primers, 1 μL of DNA template, and ultrapure water supplemented to a 20 μL system.

[0014] In some embodiments, the procedure of the PCR identification reaction includes: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 68°C for 30 s, 30 cycles; repair and extension at 68°C for 5 min, and storage at 12°C.

[0015] In a sixth aspect, the present application provides a kit for identifying a fragment of the replication initiation site of Physcomitrella patens as described above, and its use in cloning a fragment of the replication initiation site of Physcomitrella patens.

[0016] In a seventh aspect, the present application provides an application of an engineered bacterium expressing the above-mentioned Physcomitrella patens replication initiation site fragment in genetic engineering, biopharmaceuticals or agriculture.

[0017] The first aspect of the present application provides a fragment of the replication initiation site of Physcomitrella patens, which is an ori-1 sequence, wherein the ori-1 sequence is as shown in Seq.No.1; based on the determined fragment of the replication initiation site of Physcomitrella patens, new ideas and important basis are provided for the study of the replication mechanism of Physcomitrella patens, and a reference is provided for exploring the replication mechanism of other eukaryotic organisms.

[0018] The second aspect of the present application provides a primer pair for identifying the replication start site fragment of Physcomitrella patens, the primer pair comprising a first primer and a second primer, wherein the sequence of the first primer is shown in Seq.No.2, and the sequence of the second primer is shown in Seq.No.3; a primer pair comprising the first primer and the second primer is provided, which can clone the replication start site fragment of Physcomitrella patens to obtain the replication start site fragment of Physcomitrella patens with replication function, so as to further study the structural characteristics and functions of the replication start site of Physcomitrella patens and deepen the understanding of the evolution of replication start sites of eukaryotic organisms.

[0019] The third aspect of the present application provides a recombinant expression plasmid that expresses the replication initiation site fragment of Physcomitrella patens. The provided recombinant plasmid includes the Physcomitrella patens replication initiation site fragment ori-1 sequence. Therefore, the provided plasmid has replication initiation activity and can initiate replication, which is beneficial for constructing a Physcomitrella patens gene expression system and laying the foundation for subsequent genetic engineering research.

[0020] The fourth aspect of the present application provides an engineered bacterium that expresses a fragment of the replication initiation site of Physcomitrella patens, and the engineered bacterium contains the above-mentioned ori-1 sequence or the above-mentioned recombinant expression plasmid; the provided engineered bacterium can utilize the replication initiation site of Physcomitrella patens for efficient and stable expression, which is conducive to the development of genetically engineered strains of Physcomitrella patens for application in the fields of biopharmaceuticals, environmental remediation, etc.

[0021] The fifth aspect of the present application provides a kit for identifying the replication start site fragment of Physcomitrella patens, the kit comprising the above-mentioned primer pair for identifying the replication start site fragment of Physcomitrella patens; the primer pair in the kit can be used to clone the replication start site fragment of Physcomitrella patens to obtain the replication start site fragment of Physcomitrella patens with replication function, which is helpful to explore the relationship between the replication start site of Physcomitrella patens and biological processes such as gene expression regulation and cell cycle.

[0022] In a sixth aspect, the present application provides a kit for identifying the replication start site fragment of Physcomitrella patens as described above, which is used for cloning the replication start site fragment of Physcomitrella patens. The provided kit includes a primer pair for identifying the replication start site fragment of Physcomitrella patens, and the Physcomitrella patens replication start site fragment ori-1 sequence can be cloned to be further used in research on genetic engineering or agricultural applications.

[0023] The seventh aspect of the present application provides an application of an engineered bacterium expressing the above-mentioned fragment of the replication start site of Physcomitrella patens in genetic engineering, biopharmaceuticals or agriculture. Since the provided engineered bacterium is constructed using the replication start site of Physcomitrella patens, it has a high stability effect. Therefore, the obtained engineered bacterium can be used in research on improving crop traits, increasing crop yields and stress resistance, or in agricultural biotechnology fields such as disease and insect resistance genetic engineering and herbicide resistance genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the growth condition of the competent yeast provided in Example 1 of the present application after the vector containing the Physcomitrella patens replication origin site is introduced.

[0026] Figure 2 It is the colony count of yeast after the competent yeast provided in Example 1 of the present application is introduced into the replication origin site of Physcomitrella patens. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0028] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0029] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0030] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0031] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0032] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0033] In a first aspect, an embodiment of the present application provides a fragment of a replication initiation site of Physcomitrella patens, wherein the fragment of a replication initiation site of Physcomitrella patens is an ori-1 sequence, wherein the ori-1 sequence is shown in Seq. No.1.

[0034] The first aspect of the embodiment of the present application provides a fragment of the replication initiation site of Physcomitrella patens, and the fragment of the replication initiation site of Physcomitrella patens is the ori-1 sequence, wherein the ori-1 sequence is as shown in Seq.No.1; based on the determined fragment of the replication initiation site of Physcomitrella patens, new ideas and important basis are provided for the study of the replication mechanism of Physcomitrella patens, and a reference is provided for exploring the replication mechanism of other eukaryotic organisms.

[0035] Among them, Seq.No.1 is as follows:

[0036]

[0037] A second aspect of an embodiment of the present application provides a primer pair for identifying a fragment of a replication start site of Physcomitrella patens, the primer pair comprising a first primer and a second primer, wherein the sequence of the first primer is shown in Seq. No. 2, and the sequence of the second primer is shown in Seq. No. 3.

[0038] A second aspect of an embodiment of the present application provides a primer pair for identifying a fragment of the replication start site of Physcomitrella patens, the primer pair comprising a first primer and a second primer, wherein the sequence of the first primer is as shown in Seq.No.2, and the sequence of the second primer is as shown in Seq.No.3; a primer pair comprising the first primer and the second primer is provided, which can clone the fragment of the replication start site of Physcomitrella patens to obtain a fragment of the replication start site of Physcomitrella patens with replication function, so as to further study the structural characteristics and functions of the replication start site of Physcomitrella patens and deepen the understanding of the evolution of the replication start site of eukaryotes.

[0039] The provided primer pair is used to amplify the replication origin site sequence ori-1, and the primer pair includes a first primer that binds to the replication origin site sequence and a second primer that binds to the replication origin site sequence.

[0040] The sequence of the first primer is shown in Seq. No. 2, which is as follows: 5′-ctatagggcgaattggagctcGCTTTTGCATTTGTCTTCCGG-3′.

[0041] The sequence of the second primer is shown in Seq. No. 3, which is as follows: 5′-atcgaattcctgcagcccgggTCATTTGAACGTTGCAATTGGC-3′.

[0042] The provided primer pair is used to amplify the replication initiation site sequence ori-1. By using the primer pair, the replication initiation site sequence is cloned and then introduced into yeast competent cells to achieve expression analysis of the replication initiation site ori-1. This can quickly and accurately achieve replication regulation of yeast, provide an important basis for studying the replication initiation site of Physcomitrella patens, and lay a foundation for subsequent research on the replication mechanism of plants.

[0043] A third aspect of the embodiments of the present application provides a recombinant expression plasmid expressing a fragment of the replication initiation site of Physcomitrella patens, comprising the above-mentioned ori-1 sequence, and the expression plasmid used to express the ori-1 sequence is selected from PRS305 or PRS315.

[0044] The third aspect of the embodiment of the present application provides a recombinant expression plasmid expressing the fragment of the replication initiation site of Physcomitrella patens. The provided recombinant plasmid includes the fragment of the replication initiation site of Physcomitrella patens ori-1 sequence. Therefore, the provided plasmid has replication initiation activity and can initiate replication, which is beneficial for constructing a gene expression system of Physcomitrella patens and laying a foundation for subsequent genetic engineering research.

[0045] The expression plasmid provided is PRS305, which is a vector that has almost no replication initiation activity in yeast due to the absence of ARS. By cloning the ori-1 sequence into the PRS305 expression vector and then analyzing it, it can be tested whether ori-1 has the replication initiation function.

[0046] The provided PRS305 vector sequence is shown in Seq.No.4, which is as follows:

[0047]

[0048]

[0049]

[0050] Furthermore, a PRS315 expression vector is provided. The sequence of the PRS315 expression vector is shown in Seq. No. 5, and Seq. No. 5 is as follows:

[0051]

[0052]

[0053]

[0054] A fourth aspect of the embodiments of the present application provides an engineered bacterium that expresses a fragment of the replication initiation site of Physcomitrella patens, wherein the engineered bacterium contains the above-mentioned ori-1 sequence or the above-mentioned recombinant expression plasmid.

[0055] The fourth aspect of the embodiment of the present application provides an engineered bacterium that expresses a fragment of the replication initiation site of Physcomitrella patens, and the engineered bacterium contains the above-mentioned ori-1 sequence or the above-mentioned recombinant expression plasmid; the provided engineered bacterium can utilize the replication initiation site of Physcomitrella patens for efficient and stable expression, which is conducive to the development of genetically engineered strains of Physcomitrella patens for application in the fields of biopharmaceuticals, environmental remediation, etc.

[0056] A fifth aspect of an embodiment of the present application provides a kit for identifying a fragment of a replication initiation site of Physcomitrella patens, the kit comprising the above-mentioned primer pair for identifying a fragment of a replication initiation site of Physcomitrella patens.

[0057] The fifth aspect of an embodiment of the present application provides a kit for identifying the replication start site fragment of Physcomitrella patens, the kit comprising the above-mentioned primer pair for identifying the replication start site fragment of Physcomitrella patens; the primer pair in the kit can be used to clone the replication start site fragment of Physcomitrella patens to obtain the replication start site fragment of Physcomitrella patens with replication function, which is helpful to explore the relationship between the replication start site of Physcomitrella patens and biological processes such as gene expression regulation and cell cycle.

[0058] In some embodiments, the kit includes a yeast expression system. Yeast is a eukaryotic organism that is easy to culture and operate, has the characteristics of rapid reproduction, and its genetic tools and transformation techniques are relatively mature. In addition, the intracellular mechanism of yeast is similar to that of higher plants, and can effectively express the relevant genes of Physcomitrella patens, thereby accurately locating the replication initiation site, and is widely used in the study of identifying the replication initiation site of plants. The yeast expression system is used to obtain a yeast monoclonal strain containing ori-1, thereby identifying the replication initiation activity of ori-1.

[0059] In some embodiments, the kit also includes a PCR identification reaction; wherein the PCR identification reaction system includes: 10 μL of 2× high-fidelity enzyme buffer, 4 μL of 2mM dNTPs, 0.4 μL of 1.0U / μL high-fidelity enzyme, 0.5 μL of each of 10 μM upstream and downstream primers, 1 μL of DNA template, and ultrapure water supplemented to a 20 μL system.

[0060] In some embodiments, the procedure of the PCR identification reaction includes: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 68°C for 30 s, 30 cycles; repair and extension at 68°C for 5 min, and storage at 12°C.

[0061] A sixth aspect of the embodiments of the present application provides a use of the above-mentioned kit for identifying the replication initiation site fragment of Physcomitrella patens for cloning the replication initiation site fragment of Physcomitrella patens.

[0062] In a sixth aspect of an embodiment of the present application, a kit for identifying the replication start site fragment of Physcomitrella patens as described above is provided for use in cloning the replication start site fragment of Physcomitrella patens. The provided kit includes a primer pair for identifying the replication start site fragment of Physcomitrella patens, and the Physcomitrella patens replication start site fragment ori-1 sequence can be cloned to be further used in research on genetic engineering or agricultural applications.

[0063] A seventh aspect of the present application provides an application of an engineered bacterium expressing the above-mentioned Physcomitrella patens replication initiation site fragment in genetic engineering, biopharmaceuticals or agriculture.

[0064] The seventh aspect of the embodiment of the present application provides an application of an engineered bacterium expressing the replication start site fragment of Physcomitrella patens as described above in genetic engineering, biopharmaceuticals or agriculture. Since the provided engineered bacterium is constructed using the replication start site of Physcomitrella patens, it has a high stability effect. Therefore, the obtained engineered bacterium can be used in research on improving crop traits, increasing crop yields and stress resistance, or in agricultural biotechnology fields such as disease and insect resistance genetic engineering and herbicide resistance genetic engineering.

[0065] The following describes the invention in conjunction with specific embodiments.

[0066] Example 1

[0067] Construction and analysis of the sequence of ori-1, the replication origin fragment of Physcomitrella patens

[0068] (1) Obtaining a yeast vector containing the replication initiation site ori-1 sequence

[0069] The total RNA of Physcomitrella patens was extracted by Trizol method, and the cDNA of Physcomitrella patens was obtained as a sample after reverse transcription. PCR amplification was performed by upstream and downstream primers combined with molecular sequence markers, and nucleic acids were separated by agarose gel electrophoresis (1%).

[0070] The specific process of extracting total RNA from Physcomitrella patens by Trizol method is as follows: add a small amount of liquid nitrogen to a 1.5mL RNase-free centrifuge tube, take an appropriate amount of young plant tissue and put it in liquid nitrogen, grind the tissue into powder with a pre-cooled grinding rod; add 1mL Trizol, vortex to mix well, and let it stand on ice for 20 minutes; add 200μL chloroform, vortex vigorously, and let it stand on ice to separate; after centrifugation at 12000rpm at 4℃, aspirate the supernatant into a new centrifuge tube, add an equal volume of pre-cooled isopropanol and shake well; centrifuge at 12000rpm at 4℃ to remove the supernatant, add 1mL 75% alcohol to rinse, centrifuge at 12000rpm for 5min and remove the supernatant, place it on a clean bench to blow and dry for 10min, add 50μL DEPC water, place it on ice to dissolve, flick to mix, and measure the RNA concentration.

[0071] Using TaKara PrimeScript TM RT reagent Kit with gDNA Eraser was used for reverse transcription to obtain cDNA samples as templates for subsequent PCR amplification.

[0072] During PCR amplification, a primer pair, namely a first primer and a second primer, was used for amplification, wherein the first primer was PRS305-ori-1F, and the nucleotide sequence was as shown in Seq. No. 2, specifically: 5'-ctatagggcgaattggagctcGCTTTTGCATTTGTCTTCCGG-3'; the second primer was PRS305-ori-1R, and the nucleotide sequence was as shown in Seq. No. 3, specifically: 5'-atcgaattcctgcagcccgggTCATTTGAACGTTGCAATTGGC-3'.

[0073] The PCR amplification system for amplifying the cDNA samples of Physcomitrella patens is shown in Table 1.

[0074] Table 1

[0075] Reagents Addition amount 2x Buffer for KOD-FX 10μL 2mM dNTPs 4μL KOD FX (1.0 U / μL) 0.4μL Forward Primer (10μM) 0.5μL Reverse Primer (10μM) 0.5μL Template DNA 1μL <![CDATA[ddH 2 The]]> up to 20μL

[0076] The reaction procedure of PCR amplification is shown in Table 2.

[0077] Table 2

[0078] program temperature time Number of cycles Pre-denaturation 98℃ 2min - transsexual 98℃ 10s 30 annealing 55℃ 30s 30 extend 68℃ 30s 30 Repair extension 68℃ 5min - save 12℃ - -

[0079] The PCR reaction system and reaction procedure were used to amplify the cDNA sample of Physcomitrella patens, and the PCR product of the replication initiation site sequence ori-1 was amplified, with a size of 540 bp.

[0080] Further, the PCR product and the target vector PRS305 were used to synthesize the homologous recombination enzyme Homologous recombination was performed using the Seamless Cloning and Assembly Kit to obtain the recombinant vector PRS305-ori-1.

[0081] The vector is selected from PRS305 as the vector of the recombinant plasmid. The selection of this vector for the experiment is conducive to subsequent experiments. The sequence of the PRS305 vector is shown in Seq. No. 4, specifically:

[0082]

[0083]

[0084]

[0085] The constructed recombinant plasmid PRS305-ori-1 will be transformed into Escherichia coli Trans1-T1 competent cells.

[0086] The specific process of the E. coli competent transformation method is as follows: add the recombinant plasmid PRS305-ori-1 to the thawed 20μL E. coli Trans1-T1 competent cells, ice bath for 20 minutes, heat shock at 42℃ for 90s, and ice bath for 2 minutes; add 600μL LB liquid culture medium and activate on a 37℃ constant temperature shaker for 40 minutes. After the activated competent cells are resuspended by centrifugation, they are evenly spread on LB solid culture medium (50μg / mL AmpR) with a spreader and cultured inverted in a 37℃ incubator overnight.

[0087] Observe the colony clones grown on the plate in the 37°C oven, and select several E. coli single clones for colony PCR.

[0088] During PCR amplification, primers PRS305-YZ-F and PRS305-YZ-R were used, and their nucleotide sequences are shown in Seq. No. 6 and Seq. No. 7.

[0089] Nucleotide sequence of PRS305-YZ-F (Seq. No. 6):

[0090] 5′-CACGACGTTGTAAAACGACG-3′.

[0091] Nucleotide sequence of PRS305-YZ-R (Seq. No. 7):

[0092] 5′-GGTTAATTCCGAGCTTGGCG-3′.

[0093] The colony PCR amplification system is shown in Table 3, and the reaction procedure of colony PCR amplification is shown in Table 4.

[0094] Table 3

[0095] Reagents Addition amount 2×Taq Plus Master MixⅡ 10μL Forward Primer (10μM) 0.5μL Reverse Primer (10μM) 0.5μL Bacterial liquid PCR template 1μL <![CDATA[ddH 2 The]]> up to 20μL

[0096] Table 4

[0097] program temperature time Number of cycles Pre-denaturation 95℃ 3min - transsexual 95℃ 15s 30 annealing 55℃ 20s 30 extend 72℃ 45s 30 Repair extension 72℃ 5min - save 12℃ - -

[0098] The size of the amplified band of the positive colony was 708 bp. The identified positive colonies were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and alignment. Correct alignment indicated that the recombinant plasmid PRS305-ori-1 cloning vector was successfully constructed.

[0099] The verified positive single colony was expanded and the plasmid was extracted using the TIANGEN rapid plasmid extraction kit.

[0100] The plasmid was transformed into the yeast competent Y2HGold.

[0101] The specific process of the yeast competent transformation method is as follows: add 5 μg of the extracted recombinant plasmid PRS305-ori-1: 10 μL Carrier DNA (pretreatment: 100°C, 5min, quick ice bath, repeated once), 500 μL PEG / LiAc to the thawed 100 μL yeast Y2HGold competent cells, mix by pipetting, water bath at 30°C for 30min, and heat shock at 42°C for 15min. After centrifugation and resuspension, the transformed competent cells are evenly spread on SD solid medium (lacking leucine) with a spreader, and cultured upside down in a 30°C incubator for 48h.

[0102] Observe the colony clones grown on the plate in the 30°C incubator, and select several yeast single clones for colony PCR. The specific primers, reaction system, and reaction procedure are consistent with the above (Seq. No. 6), (Seq. No. 7), Table 3, and Table 4.

[0103] The amplified band size of the positive colony was 708 bp. The correct band size indicated that the recombinant plasmid PRS305-ori-1 was successfully introduced into yeast.

[0104] (2) Yeast culture

[0105] After the vector containing the replication origin ori-1 sequence was introduced into the competent yeast, the competent yeast was selected in a synthetic leucine-deficient medium (SD medium) and cultured in a culture room for 48 hours (culture room conditions: constant temperature of 30°C, constant humidity of 80%, and darkness for 24 hours).

[0106] (3) Results analysis

[0107] The growth of PRS305-ori-1, PRS315 and PRS305 yeast was observed by taking photos. Figure 1 It can be seen that ori-1 has better growth than PRS305. ImageJ was used to quantify the yeast colonies, such as Figure 2 As shown, the results show that the number of colonies of ori-1 is more than that of prs305, but less than that of prs315. This indicates that ori-1 has replication initiation activity in yeast, but the replication initiation activity is not as strong as that of prs315. This indicates that the ori-1 sequence has potential replication initiation activity.

[0108] The present application example utilizes a yeast expression system to express the replication initiation site ori-1 of Physcomitrella patens in yeast, obtains a monoclonal colony expressing the ori-1 replication initiation site, and identifies the replication initiation site ori-1 of Physcomitrella patens, which provides an important basis for studying the replication initiation site of Physcomitrella patens and lays a foundation for subsequent research on the replication mechanism of plants.

[0109] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A fragment of a replication initiation site of Physcomitrella patens, characterized in that: The Physcomitrella patens replication initiation site fragment is an ori-1 sequence, wherein the ori-1 sequence is shown in Seq. No.

1.

2. A primer pair for identifying a fragment of the replication initiation site of Physcomitrella patens, characterized in that: The primer pair includes a first primer and a second primer, wherein the sequence of the first primer is shown in Seq. No. 2, and the sequence of the second primer is shown in Seq. No.

3.

3. A recombinant expression plasmid expressing a fragment of the replication initiation site of Physcomitrella patens, characterized in that: The ori-1 sequence of claim 1 is included, and the expression plasmid used to express the ori-1 sequence is selected from PRS305 or PRS315.

4. An engineered bacterium expressing a fragment of the replication initiation site of Physcomitrella patens, characterized in that: The engineered bacteria contains the ori-1 sequence according to claim 1 or the recombinant expression plasmid according to claim 3.

5. A kit for identifying a fragment of the replication initiation site of Physcomitrella patens, characterized in that: The kit comprises the primer pair for identifying the replication initiation site fragment of Physcomitrella patens according to claim 2.

6. The kit for identifying the replication initiation site fragment of Physcomitrella patens according to claim 5, characterized in that, The kit includes a yeast expression system.

7. The kit for identifying the replication initiation site fragment of Physcomitrella patens according to claim 5, characterized in that, The kit also includes a PCR identification reaction; wherein the system of the PCR identification reaction includes: 10 μL of 2× high-fidelity enzyme buffer, 4 μL of 2mM dNTPs, 0.4 μL of 1.0U / μL high-fidelity enzyme, 0.5 μL of each of 10 μM upstream and downstream primers, 1 μL of DNA template, and ultrapure water supplemented to a 20 μL system.

8. The kit for identifying the replication initiation site fragment of Physcomitrella patens according to claim 7, characterized in that, The procedure of the PCR identification reaction includes: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 68°C for 30 seconds, 30 cycles; repair and extension at 68°C for 5 minutes, and storage at 12°C.

9. Use of the kit for identifying a fragment of the replication initiation site of Physcomitrella patens according to any one of claims 5 to 8 for cloning a fragment of the replication initiation site of Physcomitrella patens.

10. Use of the engineered bacteria expressing the Physcomitrella patens replication initiation site fragment as claimed in claim 4 in genetic engineering, biopharmaceuticals or agriculture.

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