CN103525859A - Construction method of artificial rice minichromosome and applications - Google Patents

Abstract

The invention discloses a set of rice artificial minichromosome and a method for constructing the rice artificial minichromosome-application of rice telomere sequence and centriole-containing BAC clone in the construction of rice artificial minichromosome. The invention provides a technical scheme for constructing a rice artificial minichromosome containing elements such as a centriole sequence, a telomere sequence, a screening marker, and a reporter gene. The invention constructs a set of rice artificial minichromosome by assembling essential elements of chromosomes: centrioles, telomeres, screening markers, reporter genes and the like. The invention provides a new generation carrier for studying the behavior and function of centrioles, telomeres, etc., and for genetic transformation of multiple genes or large fragments of DNA, and has very important theoretical and practical significance.

Description

A kind of construction method and application of rice artificial minichromosome

technical field

The invention relates to a method for constructing a rice artificial minichromosome in the field of biotechnology. the

Background technique

As the core of modern biotechnology, genetic engineering technology has been developed for more than 30 years and has made great achievements, but there are still many problems and challenges in its application. With the continuous development of research technology, the research has become more and more in-depth, and has entered the era of post-genome research, and the interview of various animal and plant genome sequences also provides a broader information platform for genetic engineering technology, which greatly promotes the development of agriculture. The development process of animal husbandry, food, environmental protection and other fields. the

Plant genetic engineering technology is mainly to construct exogenous genes into expressible recombinant vectors, and transform them into the host genome for expression through Agrobacterium-mediated or particle bombardment, and finally change plant traits. With the continuous development of research, the limitations have become more and more obvious. In summary, there are the following aspects:

One is that in the process of genetic transformation, due to the random insertion of foreign genes (DNA) into the host genome, it may cause the insertion inactivation of its own genes; the other is that the expression of foreign genes may be affected by the upstream or downstream regulatory elements of the insertion site. thirdly, it is difficult to realize the co-transformation of multiple genes by the traditional transgenic method; fourthly, during the transgenic process, because the exogenous gene will be integrated in multiple copies, it may cause low expression, gene silencing and undesired transgenic Stability, etc.; the randomness of transformation often places transgenes at different positions on chromosomes or on different chromosomes, and it is difficult to coordinate the expression of multiple genes. the

Although insertion inactivation and position effects can be eliminated as much as possible by existing screening techniques, multigene transformation greatly limits the research on the synergy of multiple genes in quantitative trait genes and metabolic pathways. How to effectively implement gene stacking is the main challenge and goal of future transgenic research. To effectively implement gene superposition, it is necessary to find new transgenic vectors, such as expressing foreign genes through episomal virus vectors, or superimposing multiple genes through engineered organelles and artificial minichromosomes. the

Plant artificial chromosome is a kind of genetic engineering technology used to isolate the main components that control chromosome replication from plant chromosomes, including: telomeres, centromeres and autonomous replication sequences, plus artificial selection markers, foreign gene cloning sites, etc. Genetic transformation and other components are assembled to form an artificial minichromosome carrier that can autonomously replicate, independently separate, and carry large genomic DNA fragments. It has part of the function of the chromosome and also has the function of a carrier. Because the artificial chromosome is small, it cannot be paired with the host chromosome and exists in the form of an episome in the cell cycle. Plant artificial chromosomes can provide stable multi-gene expression on additional chromosomes, provide a solution to the problem of simultaneous transformation of multiple genes, and are a new generation of transgenic carriers. For example, minichromosomes can express multiple resistance genes (insect resistance, anti-bacteria, anti-fungal, herbicide resistance, stress resistance) at the same time, and can also express genes that improve crop quality. the

The research on plant artificial chromosomes started relatively late, mainly referring to the construction methods of artificial chromosomes in animals and humans. At present, there are two main strategies for the construction of plant artificial chromosomes. One is based on its own chromosomes and is produced through necessary processing, such as telomere-mediated chromosome truncation technology; the second is based on the assembly of functional elements of chromosomes. Starting from the centromere, this method is mainly based on the generation of dicentric chromosomes, and the dicentric chromosomes are formed by cloning centromere satellite DNA sequences and other host sequences combined with transgenes; in addition, by cloning centromere In the study of maize artificial minichromosomes, Carlson et al. connected marker genes, screening genes, and centromere repeats into a circle in vitro, and then bombarded this segment of chromosomes by particle bombardment. The DNA sequence is transformed into maize cells, where the assembly of the tiny chromosomes is completed, which can be stably inherited during mitosis and meiosis. Ananiev et al. connected maize centromere sequences, telomere sequences, and selectable marker genes in vitro, introduced them into host cells by particle bombardment, and finally assembled them into minichromosomes. the

Whether it is a top-down strategy based on natural chromosome modification or a bottom-up strategy that artificially assembles cloned chromosome functional elements, great progress has been made in mammalian and human cell research, while research in plants They are all just in their infancy, especially on rice, the main food crop, and there is no successful report on the construction of rice artificial minichromosomes by De Novo. the

Although the current research on plant artificial chromosomes is far from practical application, many problems need to be solved in order to become a transgenic carrier, which is also an important development direction of plant genetic engineering in the future, but with the deepening of people's research and the development of technology, its broad The application prospect and huge commercial value will be more valued by people. the

Contents of the invention

The object of the present invention is to provide a method for constructing a rice artificial minichromosome and a set of rice artificial minichromosomes. the

The invention provides the rice telomere sequence and the application of the centriole-containing subclones in constructing rice artificial minichromosomes. the

The invention provides a technical scheme for constructing a rice artificial chromosome containing elements such as a centriole sequence, a telomere sequence, a screening marker, and a reporter gene. the

The invention constructs a set of rice artificial minichromosome by assembling essential elements of chromosomes: centrioles, telomeres, screening markers, reporter genes and the like. The invention provides a new generation carrier for studying the behavior and function of centrioles, telomeres, etc., and for genetic transformation of multiple genes or large fragments of DNA, and has very important theoretical and practical significance. the

Description of drawings

Figure 1 shows the results of high-density membrane hybridization

Figure 2 Schematic diagram of the loading process of screening marker gene Sp ^r and forward and reverse telomere sequences

Figure 3 is a schematic diagram of the splicing process of Tn5-Transposon

Figure 4 is the obtained PCR verification electrophoresis pattern of RAC1

Figure 5 is a schematic diagram of RACs

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified. the

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. the

The pUC57, pUC19, pH7WGI2D, EZ-Tn5pMODTM-2<MCS> vectors and strains can be obtained from commercial sources. the

Embodiment 1, BAC clone screening containing rice centriole

1 Preparation of Nipponbare BAC Library (AGI) High Density Hybridization Membrane

1) Use the robotic hand to place the clones of the Nipponbare BAC library stored in the 384-well culture plate on the Hybond-N ⁺ hybridization membrane.

2) Place the membrane on X-gal/IPTG/Chl LB solid selection medium, culture at 37°C for about 24 hours. the

3) The membrane was peeled off from the LB solid selection medium, and the membrane was placed on the filter paper soaked in the following solution, with the clone side facing up. the

4) After treatment, place the membrane on filter paper and air dry. the

5) Bake the film in an oven at 80°C for 2 hours. the

6) The membrane can be used for hybridization immediately or stored at 4°C for future use. the

2 Preparation of RCS1 and RCS2 probes

1) Artificially synthesized rice centromere repeat sequence RCS1 (containing the centromere-specific retrotransposon CRR (Centromere-specific retrotransposon of rice) sequence element of cultivated rice) and RCS2 (containing the centromere tandem repeat sequence CentO), cloned in Plasmid pUC57. the

2) Take 1 μg of probe plasmid DNA and denature it in a boiling water bath for 5 minutes. the

3) Add 4 μl of DIG-High prime, make up to a total volume of 20 μl with ddH ₂ O, mix well and centrifuge.

4) At 37°C, label the probe for 20 hours. the

5) Stop the enzyme reaction at 65°C for 10 minutes. the

3Southern hybridization

1) Preheat an appropriate amount of DIG Easy Hyb (10mL/100cm ² membrane) at a temperature of 37-42°C, put the membrane in a clean hybridization tube, and prehybridize the membrane in a hybridization oven for 30 minutes.

2) Denature the DIG-labeled probe in a water bath at 100°C for 5 minutes, and then quickly place it in an ice bath. Note that DIG-11-dUTP is alkali-labile, and DNA probes cannot be denatured with alkali. the

3) Add the denatured DIG-labeled probe to the preheated DIG Easy Hyb (3.5mL/100cm ² membrane), and mix well to avoid air bubbles.

4) Pour out the pre-hybridization solution, add the hybridization solution containing the probe, incubate for 4 hours or overnight, and keep gently rotating. the

4 washing film

1) Wash twice with 2×SSC and 10% SDS, each time for 5 minutes, at a temperature of 15-25°C. the

2) Wash twice with 0.5×SSC and 0.10% SDS (preheated to a washing temperature of 65-68° C.), 15 minutes each time. the

5 colors

1) After hybridization and stringent washing, wash the membrane with Washing buffer for 1-5 minutes. the

2) Incubate in 100mL Blocking solution for 30min. the

3) Incubate in 20mL Antibody solution for 30min. the

4) Wash the membrane twice with 100mL Washing buffer, 15min each time. the

5) Equilibrate in 20mL Detection buffer for 2-5 minutes. the

6) Develop the hybridization membrane in 10mL Color substrate solution (prepared for current use), avoid light, and do not shake during the color development process. the

7) When the desired point or band intensity appears, stop the reaction, wash the membrane with 50 mL of sterilized ddH ₂ O or TE-buffer for 5 min to stop the reaction, record the results and take pictures (Figure 1).

Statistics of the results: Using the Nipponbare BAC library (AGI), the probes RCS1 and RCS2 were used to screen positive clones containing centrioles, and a total of 50 positive clones were obtained (Table 1) (those with weaker signals have been deleted). the

Table 1 Centromeric BACs (BAC-C1-50)

Embodiment 2, splicing of Tn5-Transposon

1 Forward and reverse telomeric sequences are loaded on both sides of the selection marker gene (spectinomycin resistance gene Sp ^r )

1) According to the known telomere sequence of rice (repetitive sequence 5′-TTTAGGG), the artificially synthesized sequence 1 telomere fragment TEL> (about 400bp) in the pUC57 clone contains Hind III and Bgl II restriction sites on the left point, the right side contains BamH I and EcoR I restriction sites and homing endonuclease site HRS (I-CeuI)), named pUC57-TEL> (forward) as shown in Figure 2 A or pUC57-<TEL (Reverse) as shown in B in Figure 2. the

Note: pUC57-TEL> (forward) and pUC57-<TEL (reverse) are the same vector. the

2) Design primers according to the sequence of the screening marker gene (spectinomycin resistance gene Spr) (sequence from pH7WGI2D), use the plasmid pH7WGI2D as a template, and obtain the full length of ^Spr gene (including promoter and terminator) by high-fidelity enzyme amplification At the same time, Hind III and Bgl II restriction sites were introduced at the 5' end, and EcoR I and BamH I restriction sites were introduced at the 3' end. The amplification primers were: Sp-F: 5'-GTAC AAGCTTAGATCT AATTCGGGCACGAACCCAG-3'( The underlined part is the restriction site of Hind III and Bgl II); Sp-R: 5'-GATC GAATTCGGATCC GTCATGCATGATATATC TCCCAAT-3' (the part of the underline is the restriction site of EcoR I and BamH I), and the amplified product was processed by Hind III and After EcoR I digestion and purification, the Sp ^r DNA fragment (1250bp) was recovered; at the same time, the intermediate vector pUC19 was digested with Hind III and EcoR I, ligated with T4 ligase, transformed into DH5a, and the target plasmid clone was obtained, and the positive cloned plasmid was subjected to PCR and sequencing After verification, the sequencing results showed that the Sp ^r gene fragment was inserted between the Hind III and EcoR I restriction sites of the vector pUC19, and the recombinant vector was named pUC19-Sp ^r (C in Figure 2).

3) pUC57-TEL> was digested with Hind III and BamH I, and TEL> was recovered from agarose gel. At the same time, pUC19-Sp ^r was digested with Hind III and Bgl II to recover the vector fragment. T4 ligase ligation, transformation of DH5a, the target plasmid clone was obtained, and the positive cloned plasmid was verified by PCR and sequencing. The sequencing results showed that a fragment TEL> was inserted between the Hind III and Bgl II restriction sites of the vector pUC19-Sp ^r , and the recombinant The vector was named pUC19-TEL>-Sp ^r (D in Figure 2). Note: Bgl II/Bam H I in the figure is the site of restriction enzyme digestion and ligation with homologous enzymes.

4) Digest pUC57-<TEL with BamH I and Bgl II, recover <TEL from agarose gel, and simultaneously digest pUC19-TEL>-Sp ^r with BamH I, remove phosphorylation at the 5' end with CIP, and recover vector fragment. Ligate with T4 ligase, transform DH5a, obtain the target plasmid clone, and carry out PCR and sequencing verification of the positive clone plasmid (select the positive clone with reverse insertion of TEL), the sequencing results show that the BamH I restriction site in the vector pUC19-TEL>-Sp ^r A fragment <TEL was inserted between the dots, and the recombinant vector was named pUC19-TEL>-Sp ^r -<TEL (E in Figure 2).

2 Fusion expression vector construction of selection marker gene (hygromycin resistance gene Hpt) and reporter gene (green fluorescent protein GFP gene)

Primers were designed according to the full length of the hygromycin resistance gene sequence (sequence from pH7WGI2D), and an attB adapter was added. The amplification primers were: Hpt-F: -5'- GGGGACAAGTTTGTACAAAAAGCAGGCTGC ATGAAAAAGCCTGAACTCACCG-3' (the underlined part is attB1); Hpt- R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT GAGTACTTCTACACAGCCATCGG-3' (the underlined part is attB2), using the plasmid pH7WGI2D as a template, the full-length Hpt gene was obtained by high-fidelity enzyme amplification, and the target DNA fragment (960bp) was recovered; cloned into pMDC43 (public Available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences, the non-patent literature documenting this material is: Mark D. Curtis and Ueli Grossniklaus. A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiol, 200, 133 (2): 462-469), the positive cloned plasmid was verified by PCR and sequencing, and the sequencing results showed that the Hpt gene was fused after the GFP gene of the carrier pMDC43, and the recombinant vector was named pMDC43-gfp6: Hpt (A in Fig. 3 ) .

Acquisition of 3Tn5-Transposon DNA

1) Digest the recombinant vector pMDC43-gfp6:Hpt with Hind III and EcoR I, recover the target fragment Hind III---35S:gfp6-Hpt:NosT---EcoR I (2946bp) from agarose gel, and use Hind III, EcoR I double digestion transposon carrier EZ-Tn5pMOD ^TM -2<MCS>, recovery of vector fragments, T4 ligase ligation, transformation of DH5a, the target plasmid clone was obtained, positive cloned plasmids were verified by PCR and sequencing, and the sequencing results It shows that the fragment Hind III --- 35S: gfp6-Hpt: NosT --- EcoR I is inserted between the Hind III and EcoR I restriction sites of the vector pMoD ^TM -2<MCS>, and the recombinant vector is named as Tn5-1 (B in Figure 3).

2) Use Hind III to digest vector pUC19-TEL>-Sp ^r -<TEL, recover the target fragment TEL>-Spr-<TEL (2070bp) from agarose gel, and use Hind III to digest carrier Tn5-1, agar The carrier Tn5-1 was recovered from the sugar gel and dephosphorylated (same as above), ligated with T4 ligase, transformed into DH5a, and cloned the target plasmid, and the positive cloned plasmid was verified by PCR and sequencing. The sequencing results showed that the Hind III enzyme in the carrier Tn5-1 The segment TEL>-Spr-<TEL was inserted between the cutting sites, and the recombinant vector was named Tn5-Transposon (C in Figure 3).

3) Single enzyme digestion of Tn5-Transposon was performed with Ear I (D in Figure 3), and Tn5-Transposon DNA (5100bp) was recovered from low melting point agarose gel. the

The assembly of embodiment 3 rice artificial minichromosomes

1BAC-C1-50 plasmid extraction, large fragment plasmid extraction using OMEGA company BAC/PAC DNA extraction kit (D2156-02), detailed steps refer to the instructions. the

2Tn5-Transposon DNA inserted into BAC-C1-50 plasmid

Prepare the following reaction system (10ul):

After the reaction, the EPI300 competent cells were transformed by electroporation, plated on LB (50 mg/L Chl, Sp) solid selection medium for screening, using Sp (same as above), GFP (F: 5'-GATGGTGATGTTAATGGGCAC-3', R: 5 '-GCGCCTTTGTATAGTTCATCC-3'), 35S(F: 5'-AAGCTTGGCGTGCCTGC-3', R: 5'-GATAGTGGGATTGTGCGTCAT-3'), Hpt(F: 5'-ATGAAAAAGCCTGAACTCACCG-3', R: 5'-GAGTACTTCTACACAGCCATCGG- 3') Four pairs of primers were positively identified by PCR (Fig. 4). the

Complete the construction of the rice artificial minichromosome comprising basic elements such as telomeres, centrioles, and selectable markers, named RAC1-50, and RAC1-50 is linearized through homing endonuclease I-Ceu I (Figure 5), namely The next step is the rice artificial minichromosome used in the genetic transformation of plants by gene gun. It is very important in theory and practice to study the behavior and function of centrioles and telomeres, and to provide a new generation of vectors for genetic transformation of multiple genes or large fragments of DNA. significance. the

Claims (6)

1. build a method for paddy rice artificial chromosome, it is characterized in that several steps below of the method:

A: forward and reverse telomeric sequence is loaded into selection markers gene both sides;

B: the fusion expression vector of selectable marker gene and reporter gene builds;

The acquisition of C:Tn5-Transposon DNA

D: application transposon interpolation is inserted into Tn5-Transposon DNA to contain in Centriolar BAC clone.

2. described in table 1, BAC is cloned in the application in the artificial minichromosomes structure of paddy rice.

3. contain the application of forward and reverse recombinant vectors of DNA shown in sequence 1 in the artificial minichromosomes of paddy rice builds in ordered list.

4. the artificial minichromosomes of paddy rice that application rights 1 technological method builds.

5. according to behavior and the function of the artificial minichromosomes of the paddy rice research centre grain building in claim 3, telomere etc.

6. according to the artificial minichromosomes of paddy rice building in claim 3, in its DNA sequence dna, insert the functional element such as goal gene, reporter gene.

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