CN116926074A - Guide RNA for constructing gene-deleted Arabidopsis mutant plants, Arabidopsis mutant plants and preparation methods thereof - Google Patents

Abstract

The invention discloses a guide RNA for constructing a gene deletion type arabidopsis mutant plant, the arabidopsis mutant plant and a preparation method thereof, wherein the guide RNA comprises a pair of sequences shown as SEQ ID No. 1 and SEQ ID No. 2. The preparation method comprises the following steps: designing a primer pair according to the guide RNA; carrying out PCR amplification, purification and enzyme digestion by adopting the primer pair, and connecting with a vector to obtain a recombinant plasmid; transforming competent cells by adopting recombinant plasmids, screening and verifying to obtain an expression vector for knocking out genes to be knocked out in arabidopsis; and transforming agrobacterium with the obtained expression vector, soaking flowers, and screening to obtain an arabidopsis mutant plant. The technical scheme of the invention overcomes the defects of the existing plant breeding and other performances, can advance the bolting period of the arabidopsis, and provides important value for the arabidopsis in the plant breeding direction.

Description

Guide RNA and Arabidopsis mutant plants used to construct gene-deleted Arabidopsis mutant plants Variant plants and preparation methods thereof

Technical field

The present invention relates to the field of plant molecular genetics, and specifically to guide RNA for constructing gene-deleted Arabidopsis mutant plants, Arabidopsis mutant plants and preparation methods thereof.

Background technique

At present, many genetic screening systems have discovered some important proteins involved in the DNA demethylation pathway through years of research. For example, important proteins such as ROS1, IDM1, IDM2, ROS3, and MBD7 have been found in the DNA demethylation pathway. This forward genetic screening of new protein factors is inefficient and takes so long that it is difficult to further discover some new protein factors.

Therefore, searching for relevant protein factors in Arabidopsis thaliana, and constructing Arabidopsis mutant plants on this basis, and improving the performance of Arabidopsis plants in plant breeding and other directions are issues that the present invention urgently needs to solve.

Contents of the invention

In view of the above-mentioned prior art, the purpose of the present invention is to address the deficiencies in plant breeding and other performance of Arabidopsis mutant plants constructed based on related protein factors in the prior art, thereby providing a method that can advance the bolting period. Guide RNA for constructing gene-deleted Arabidopsis mutant plants, Arabidopsis mutant plants and their preparation methods provide important value in the direction of Arabidopsis plant breeding.

In order to achieve the above object, the present invention provides a guide RNA for constructing a gene-deleted Arabidopsis mutant plant, the guide RNA including a pair of sequences as shown in SEQ ID No: 1 and SEQ ID No: 2 .

The present invention also provides a method for preparing Arabidopsis thaliana mutant plants, using the guide RNA as described above to knock out the gene to be knocked out in the Arabidopsis thaliana plant.

Preferably, the preparation method includes:

S100. Design primer pairs based on guide RNA;

S200. Use plasmid pCBC-DT1T2 as a template, use the primer pair described in step S100 to perform PCR amplification, purification, and enzyme digestion, and then connect it to the vector to obtain the recombinant plasmid;

S300. Use the recombinant plasmid to transform competent cells, screen and verify, and obtain an expression vector for knocking out the gene to be knocked out in Arabidopsis;

S400. Transform the expression vector obtained in step S300 into Agrobacterium and soak the flowers, and screen to obtain Arabidopsis mutant plants.

Preferably, in step S100, the primer pair includes a first primer pair corresponding to the sequence shown in SEQ ID No: 1, and a second primer pair corresponding to the sequence shown in SEQ ID No: 2;

The sequences of the first primer pair are shown in SEQ ID No: 3 and SEQ ID No: 4;

The sequences of the second primer pair are shown in SEQ ID No: 5 and SEQ ID No: 6.

Preferably, the vector used in step S200 is pHEC401.

Preferably, in step S300, the verification process includes identifying and sequencing the screened colonies; and,

The identification uses the sequence pair shown in SEQ ID No:7 and SEQ ID No:8;

Sequencing used the sequence pair shown in SEQ ID No:7 and SEQ ID No:9.

Preferably, the variety of the Arabidopsis plant having the gene to be knocked out is Col.

The present invention also provides an Arabidopsis mutant plant, which is obtained by the preparation method described above.

Preferably, the bolting period of the Arabidopsis mutant plant is advanced.

Preferably, the length of the deleted gene fragment in the Arabidopsis mutant plant is not less than 100 bp.

Through the above technical solution, the present invention proposes a pair of guide RNAs for knocking out genes in Arabidopsis thaliana, and constructs Arabidopsis mutant plants based on the guide RNAs, so that the Arabidopsis mutant plants not only antagonize transgene silencing, but also At the same time, it caused genome-wide DNA methylation changes. It can advance the bolting period of Arabidopsis mutant plants, providing important value for research on Arabidopsis plant breeding.

Description of the drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the following specific embodiments, but do not constitute a limitation of the present invention. In the attached picture:

Figure 1 is a diagram of the sequencing results of the positive transgenic seedlings in verification example 1;

Figure 2 is a fluorescence imaging diagram of the positive transgenic seedlings, positive control and negative control in verification example 2;

Figure 3 is a graph showing the fluorescence quantitative PCR detection results of the positive transgenic seedlings, positive controls and negative controls in Verification Example 3;

Figure 4 is a comparison of the growth of the positive transgenic seedlings and the conventional Arabidopsis plant Col-0 in the application example;

Figure 5 is a comparison of the whole-genome DNA methylation sequencing results of the positive transgenic seedlings, positive controls and negative controls in Verification Example 4.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Among them, pCBC-DT1T2 and Chen Qijun's laboratory at the School of Biology, China Agricultural University published a paper titled "A CRISPR/Cas9 toolkit for multiplex genome editing in plants" (BMC PlantBiol.2014Nov29;14:327.pii:s12870-014-0327-y .doi:10.1186/s12870-014-0327-y.PubMed PMID:25432517.) is consistent with the pCBC-DT1T2 used in. Of course, any conventional commercially available pCBC-DT1T2 deemed appropriate by those skilled in the art can be used here, and the present invention is not limited to the pCBC-DT1T2 used in this specific embodiment.

The primer pairs used were synthesized and sequenced by GenScript Biotechnology Company; the plasmid mini-extraction kit is a commercially available product of GenScript Biotechnology Company.

The preparation method of Agrobacterium competent cells is as follows: Streak the GV3101 Agrobacterium strain in YEP solid medium containing rifampicin resistance (25 mg/L), and incubate it upside down at 28°C in the dark for 2-3 days until it reaches the surface of the medium. Grow a single colony; pick a single colony of Agrobacterium and inoculate it into 5 mL of YEP liquid medium containing rifampicin resistance, and culture it overnight at 28°C and 220 g with shaking; transfer 3-4 mL of the overnight culture into a 20 mL container In a sterile Erlenmeyer flask containing rifampicin antibiotic YEP liquid, incubate overnight at 28°C with 220 g shaking; after bacteria preservation, pour all the remaining culture liquid into a sterile Erlenmeyer flask containing 250 mL of rifampicin antibiotic YEP liquid. , 28℃, 220g shaking culture until the OD value is between 0.5-0.6; move the culture to a sterile centrifuge tube in a clean bench, place it on ice at 4℃ for 10 minutes, centrifuge at 4000g for 5 minutes to precipitate the bacteria; use 50mL Resuspend the bacteria in 0.15mol/L NaCL solution pre-cooled on ice, then centrifuge at 4°C at 4000g for 5 minutes, discard the supernatant; add 5 mL of 20mM CaCl ₂ solution pre-cooled on ice to resuspend the bacteria, 4 ℃, centrifuge at 4000g for 5 minutes, then add 5mL of 20mM CaCl ₂ solution pre-cooled on ice again to resuspend, aliquot 100μL into each tube and quickly store in liquid nitrogen in a -80℃ refrigerator.

Further explanation is provided below through specific examples.

Example 1. Construction of expression vector for knocking out genes to be knocked out in Arabidopsis thaliana

Design a pair of gRNA sequences as shown in SEQ ID No: 1 and SEQ ID No: 2, and construct a first primer pair as shown in SEQ ID No: 3 and SEQ ID No: 4 according to the gRNA sequence, and as The second primer pair shown in SEQ ID No:5 and SEQ ID No:6. Among them, in the second primer pair, the gRNA shown in SEQ ID No: 2 needs to be added in the reverse direction to obtain it.

TCCTATGCATTCTAACATG(SEQ ID No:1)

GAATCCCAAGTATCTAGAA(SEQ ID No:2)

DT1-BsF:ATATATGGTCTCGATTGTCCTATGCATTCTAACATGGTT(SEQ ID No:3)

DT1-F0:TGTCCTATGCATTCTAACATGGTTTTAGAGCTAGAAATAGC(SEQ ID No:4)

DT2-R0:AACTTCTAGATACTTGGGATTCCAATCTCTTAGTCGACTCT AC(SEQ ID No:5)

DT2-BsR:ATTATTGGTCTCGAAACTTCTAGATACTTGGGATTCCAA(SEQ ID No:6)

PCR amplification was performed using 25-fold diluted pCBC-DT1T2 as the template, the first primer pair and the second primer pair as primers, where the concentration of the primers shown in SEQ ID No: 3 and SEQ ID No: 6 was 10 μmol. /L, the concentration of the primers described in SEQ ID No: 4 and SEQ ID No: 5 is 1 μmol/L.

The PCR product was purified and recovered, and the enzyme digestion-ligation system was established according to the system shown in Table 1 to obtain the recombinant plasmid.

Table 1

After the obtained recombinant plasmid is purified, 1 μL is transformed into E. coli competent cells, and then kanamycin is used to screen. The screened colonies are identified using the sequence pair shown in SEQ ID No: 7 and SEQ ID No: 8, and The sequences shown in SEQ ID No: 7 and SEQ ID No: 9 are used for sequencing to obtain an expression vector for knocking out the gene to be knocked out in Arabidopsis thaliana.

U626-F:TGTCCCAGGATTAGAATGATTAGGC(SEQ ID No:7)

U629-R:AGCCCTCTTTCTTTCGATCCATCAAC(SEQ ID No:8)

U629-F:TTAATCCAAACTACTGCAGCCTGAC(SEQ ID No:9)

Among them, the specific operation process of transforming E. coli competent cells is as follows: take out the E. coli competent cells from the -80°C refrigerator and insert them on ice until the competent cells are completely melted. After the competent cells are thawed, add the recombinant plasmid (i.e. enzyme conjugate), mix gently by pipetting, and keep in ice bath for about 30 minutes. Heat shock in a 42°C water bath for 90 seconds, quickly insert into ice, and keep in ice bath for 2-5 minutes. Add 500 μL of LB liquid medium (without antibiotics), mix well, and resume culture on a 180g shaker at 37°C for 40-60 minutes. After resuming culture, pour the competent cell solution onto the LB solid culture medium containing the corresponding antibiotics on a clean workbench, apply it evenly with a sterilized 1mL pipette tip, seal the culture medium after it is completely dry, and incubate it upside down at 37°C for 14- 16h.

In the present invention, plasmid extraction uses a plasmid mini-extraction kit. The plasmid extraction here is an operation used when purifying PCR products. It is for the enzymatic digestion, purification and recovery of the aforementioned PCR products and pHEC401 vector. The method used is DNA adsorption column equilibrium treatment, which specifically includes: (1) Add 200 μL of CBS buffer to the adsorption column for later use. (2) Depending on the growth of the bacterial solution, take 2-4 mL of the bacterial solution that has been shaken overnight, centrifuge at 8000g for 1 minute, and discard the supernatant. (3) Add 250 μL of Soultion I, and use a vortex to fully suspend the bacterial cells to form a uniform mixture. (4) Add 250 μL of Soultion II treated at 37°C, and immediately mix gently and thoroughly by turning it up and down 4-6 times to fully lyse the bacteria until a translucent protein-like solution is formed. If there are obvious sticky threads when opening the lid, this indicates If there is plasmid, this process should not take more than 5 minutes. (5) Add 350 μL of Soultion III, gently and fully invert 8-10 times, place at room temperature for 3 minutes, and centrifuge at 12,000g for 5-10 minutes. At the same time, add 12,000g of the adsorption column with CBS buffer added to the standby column in step (1). Centrifuge for 1 minute. (6) Aspirate the supernatant into the equilibrated DNA adsorption column, centrifuge at 6000g for 1 minute at room temperature, and discard the waste liquid. (7) Put the DNA adsorption column back into the 2 mL collection tube, add 500 μL of W1 Soultion, centrifuge at 12000 g for 1 min, and discard the waste liquid. (8) Put the DNA adsorption column back into the 2 mL collection tube, add 500 μL of Wash Soultion, centrifuge at 12000 g for 1 min, and discard the waste liquid. Repeat this step once. (9) Put the DNA adsorption column back into the 2mL collection tube, empty it at 12000g for 1 minute, and completely remove the Wash Solution. (10) Place the DNA adsorption column into a clean 1.5mL centrifuge tube, dry it at room temperature, add 70 μL of Elution Buffer, incubate at 37°C for 5 minutes, and centrifuge at 12000g for 1 minute to elute the plasmid. The eluent can be sucked back into the DNA adsorption column and the process can be repeated once. The liquid is the solution containing the target plasmid and is stored in a -20°C refrigerator.

Example 2. Transformation of Agrobacterium

Take a tube of prepared Agrobacterium competent cells from a -80°C refrigerator and insert it into ice. After it slowly melts into a liquid, add 1-2 μL of the expression vector prepared in Example 1 and blow gently with a sterile pipette tip. Mix well and place on ice for 30 minutes. After 30 minutes, place the centrifuge tube into liquid nitrogen for quick freezing for 3 minutes, and then quickly transfer it to a prepared 37°C water bath for 5 minutes. Take it out and put it on ice for 2 minutes, add 500 mL of YEP liquid medium, and restore the culture at 180 g in a -28°C shaker for 3 hours. After culturing, pour the YEP solid culture medium containing the corresponding antibiotics prepared in advance into the clean workbench, apply it evenly with a sterile blue pipette tip, blow dry the clean workbench, seal it, and invert it in a 28°C constant temperature incubator. 2-3 days. Pick the single clone on the plate, shake the bacteria and verify the bacterial liquid by PCR. The identified positive clones will be stored in the bacterial liquid for later use.

Example 3. Flower dipping and screening of transgenic seedlings

Here, the pollen tube channel pouring method is used to transfect Arabidopsis thaliana. After the positive clones identified in Example 2 were thawed on ice, 100 μL was inoculated into 5 mL of YEP liquid culture medium containing the corresponding antibiotics, and cultured overnight on a shaker at 28°C. After 2 days, pour all 5 mL of the turbid bacterial liquid into 250 mL of YEP liquid culture medium containing the corresponding antibiotics, and culture at 180 g on a shaker at 28°C for 5-10 hours. During the cultivation process, pay attention to check in time until the OD value of Agrobacterium reaches 1.0-1.2. Pour the bacterial solution into a sterilized 250mL centrifuge bottle, centrifuge at 4°C and 4000g for 10 minutes, discard the supernatant, and obtain resuspended Agrobacterium. Prepare 200 mL of Arabidopsis Agrobacterium buffer in advance (wherein, the Arabidopsis Agrobacterium buffer is prepared as follows: use 100 mL as the total system, weigh 0.237 g of MS powder and 5 g of sucrose and dissolve them in 80 mL of deionized water. After dissolving and diluting to 100 mL), pour an appropriate amount of Arabidopsis Agrobacterium buffer solution into a bottle and resuspend the Agrobacterium until the OD value is between 0.8-1.0 to obtain a preliminary preparation of Arabidopsis Agrobacterium tumefaciens. Add Silwet-77 to the primary preparation of Agrobacterium tumefaciens in a ratio of 40 μL/100 mL between Silwet-77 and the initial preparation of Agrobacterium tumefaciens thaliana to obtain an infection solution of Agrobacterium tumefaciens . Select a blooming Arabidopsis plant and cut off the fruit clips that have grown from self-pollination in advance. Use the above-mentioned Arabidopsis Agrobacterium infection solution to soak the inflorescences for 1 min. After dip-dying, the Arabidopsis thaliana was placed in two black trays containing plastic wrap and treated in the dark for 24 hours. Then move to artificial climate chamber for normal culture. Dip it again after a week or two to improve the success rate of transformation. Continue culturing until seeds are harvested. The seeds harvested after dip-dying were of the T1 generation, dried and then spotted on MS medium containing 50 mg/L hygromycin. Put them into an artificial climate chamber for cultivation, and observe the growth of the seedlings after about 14 days. The plants containing the transgene germinate and grow significantly faster.

The seedlings are moved into nutrient soil and cultured in an artificial climate chamber for about two months. A single plant is harvested to form the T2 generation. Seeds of the T2 generation were sown on MS medium containing 50 mg/L hygromycin. The lines that will grow normally on the medium and have no phenotypic separation are the screened positive transgenic seedlings.

Verification example 1

The screened positive transgenic seedlings were identified by PCR, and the homozygous mutant in the successfully knocked-out Arabidopsis mutant plant was obtained, and the homozygous mutant was sequenced. The sequencing results are shown in Figure 1. The specific operations for obtaining homozygous mutants based on PCR identification can be performed in a manner that is routinely used by those skilled in the art, and will not be described in detail here.

It can be seen from Figure 1 that the method of the present invention obtains an Arabidopsis mutant plant. Specifically, as shown in Figure 1, the length of the gene fragment deleted in the Arabidopsis mutant plant is 177 bp. Specifically, the sequence of the gene fragment deleted in the Arabidopsis mutant plant is shown in SEQ ID No: 14.

CGGCATCTTTTTGGAACCTGGAAAGGAGTCTTTCATCCGCAAACACTTCAGCTTATTGAGAAAGAACTTGGCTTTAATGCTAAAAGTGATGGTTCAGCAGCTGTTGTATCCACAGCCAGAGCTGAGCCGCAATCTCAGCGCCCACCACATAGCATCCATGTGAATCCCAAGTATCTA(SEQ ID No:14)

Verification example 2

Single seeds of the positive transgenic seedlings obtained in Example 3 were sown on MS culture medium. The seedlings were cultured for 12 days. After the two leaves of the seedlings were fully opened, the substrate fluorescein (Peikin Elmer Company, USA) was sprayed and treated in the dark for 10 minutes. Then a fluorescence imager (Tianneng 5200, Tianneng Company, Shanghai) was used to capture the fluorescence. The time is 30 s, and the high fluorescence control Arabidopsis thaliana Col-LUC (as a positive control) and the low fluorescence control Arabidopsis ros1-7 (as a negative control) are set. The results obtained are shown in Figure 2. In the present invention, the fluorescence of positive transgenic seedlings after specific gene fragments are deleted is significantly reduced.

Verification example 3

Verify whether the transgene is silenced by fluorescence quantitative PCR. Real-time fluorescence quantitative PCR amplification used the CFX96 real-time PCR (Bio-RAD) system, and the kit used iQ SYBR Green Supermix (Bio-Rad). Using Arabidopsis thaliana ACTIN2 as the internal reference gene, each pair of primers on the template was repeated three times, and each gene amplification reaction was recorded as one experimental result. The primers used included SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and SEQ ID No: 13.

LUC-Q-F: CGGAAAGACGATGACGGAAA (SEQ ID No: 10);

LUC-Q-R: CGGTACTTCGTCCACAAACA (SEQ ID No: 11);

ACTIN2-Q-F:GATGATGCGCCAAGAGCTG (SEQ ID No: 12);

ACTIN2-Q-R:GCCTCATCACCTACGTAGGCAT (SEQ ID No: 13).

The results of fluorescence quantitative PCR are shown in Figure 3. It can be seen from Figure 3 that the transcription amount of the knockout positive transgenic seedlings is significantly reduced compared with the Arabidopsis Col-LUC plants.

Verification example 4

The seeds of the positive transgenic seedlings in Example 3 were disinfected with 8% sodium hypochlorite solution and then sown on MS culture medium. After two days of cold treatment at 4°C, it was placed in the intelligent light incubator GTOP-S00B of Zhejiang Top Yunnong Technology Co., Ltd. with 16 hours of light (22°C)/8 hours of darkness (20°C). After 12 days of cultivation, the seedlings of the positive transgenic seedlings prepared in the present invention were sent to Shanghai Lingen Technology Co., Ltd. for whole-genome DNA methylation sequencing. The comparison of the sequencing results is shown in Figure 5. As can be seen from Figure 5, compared with the conventional Arabidopsis Col-0 plant, the DNA methylation level of the Arabidopsis mutant plant in the present invention and the ROS1 gene mutant (that is, in the Arabidopsis Col-0 Similar to the background of plants with loss of ROS1 function, dramatic changes occurred.

Based on the above verification examples, it can be seen that the mutant plant of the present invention shows changes in gene silencing and DNA methylation regulation levels after knocking out specific genes.

Application examples

The conventional Arabidopsis thaliana plant Col-0 and the Arabidopsis thaliana mutant plant obtained in the present invention are planted on MS medium, cultured for 14 days and then transplanted into nutrient soil. And continued to grow to the plant bolting stage under the same conditions, the Arabidopsis mutant plant obtained by the present invention obviously bolted in advance (as shown in Figure 4).

Therefore, the Arabidopsis mutant plant obtained by the present invention can shorten the growth cycle of the plant.

The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that each of the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without conflict. In order to avoid unnecessary repetition, the present invention combines various possible combinations. The combination method will not be further explained.

In addition, any combination of various embodiments of the present invention can also be carried out. As long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (10)

1. A guide RNA for constructing a gene-deleted arabidopsis mutant plant, characterized in that the guide RNA comprises a pair of sequences as shown in SEQ ID No. 1 and SEQ ID No. 2.

2. A method for preparing an arabidopsis mutant plant, which is characterized in that the gene to be knocked out in the arabidopsis plant is knocked out by using the guide RNA according to claim 1.

3. The preparation method according to claim 2, characterized in that the preparation method comprises:

s100, designing a primer pair according to the guide RNA;

s200, taking the plasmid pCBC-DT1T2 as a template, adopting the primer pair described in the step S100 to carry out PCR amplification, purification and enzyme digestion, and connecting with a vector to obtain a recombinant plasmid;

s300, transforming competent cells by adopting recombinant plasmids, screening and verifying to obtain an expression vector for knocking out genes to be knocked out in Arabidopsis;

s400, transforming agrobacterium with the expression vector obtained in the step S300, and leaching, and screening to obtain an arabidopsis mutant plant.

4. The method according to claim 3, wherein in step S100, the primer pair comprises a first primer pair corresponding to the sequence shown in SEQ ID No. 1 and a second primer pair corresponding to the sequence shown in SEQ ID No. 2;

the sequences of the first primer pair are shown as SEQ ID No. 3 and SEQ ID No. 4;

the sequences of the second primer pair are shown as SEQ ID No. 5 and SEQ ID No. 6.

5. The method according to claim 3 or 4, wherein the carrier used in step S200 is pHEC401.

6. The method according to claim 3 or 4, wherein in step S300, the verification process comprises identifying and sequencing the colonies after screening; and, in addition, the method comprises the steps of,

identification adopts sequence pairs shown as SEQ ID No. 7 and SEQ ID No. 8;

the sequence pairs shown as SEQ ID No. 7 and SEQ ID No. 9 are adopted for sequencing.

7. The method according to any one of claims 2 to 4, wherein the variety of the Arabidopsis plant having the gene to be knocked out is Col.

8. An arabidopsis mutant plant, characterized in that it has been obtained by the preparation method according to any one of claims 2-7.

9. The arabidopsis mutant plant of claim 8, wherein the bolting period of the arabidopsis mutant plant is advanced.

10. The arabidopsis mutant plant according to claim 8 or 9, wherein the length of the deleted gene fragment in the arabidopsis mutant plant is not less than 100bp.