CN116926074A - Guide RNA for constructing gene-deleted arabidopsis mutant plant, arabidopsis mutant plant and preparation method 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 for constructing gene-deleted arabidopsis mutant plant, arabidopsis mutant plant and preparation method thereof

Technical Field

The invention relates to the field of plant molecular genetics, in particular to a guide RNA for constructing a gene deletion type arabidopsis mutant plant, the arabidopsis mutant plant and a preparation method thereof.

Background

Many genetic screening systems have been studied for many years to find some important proteins involved in the DNA demethylation pathway, e.g., ROS1, IDM2, ROS3, MBD7, etc. are found in the DNA demethylation pathway. This forward genetic screening of new protein factors is inefficient and long enough that it is difficult to further discover some new protein factors.

Therefore, searching related protein factors in arabidopsis, constructing an arabidopsis mutant plant on the basis, and improving the performance of the arabidopsis plant in the plant breeding direction and the like is a problem to be solved urgently.

Disclosure of Invention

Aiming at the prior art, the invention aims at overcoming the defects in the performances of the prior art, such as plant breeding and the like, of the arabidopsis mutant plant constructed based on the related protein factors, so as to provide the guide RNA for constructing the gene-deleted arabidopsis mutant plant, the arabidopsis mutant plant and the preparation method thereof, wherein the guide RNA can advance the bolting period and provide important value for the plant breeding direction of the arabidopsis.

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

The invention also provides a preparation method of the arabidopsis mutant plant, which adopts the guide RNA to knock out the gene to be knocked out in the arabidopsis plant.

Preferably, the preparation method comprises the following steps:

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.

Preferably, 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.

Preferably, the vector used in step S200 is pHEC401.

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

Preferably, the variety of the arabidopsis plant with the gene to be knocked out is Col.

The invention also provides an arabidopsis mutant plant, which is obtained by adopting the preparation method.

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 100bp.

Through the technical scheme, the invention provides a pair of guide RNAs for knocking out genes in arabidopsis, and an arabidopsis mutant plant is constructed based on the guide RNAs, so that the arabidopsis mutant plant not only antagonizes transgenic silencing, but also causes the change of whole genome DNA methylation. The bolting period of the arabidopsis mutant plant can be advanced, and important value is provided for the research of the arabidopsis in the aspect of plant breeding.

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:

FIG. 1 is a graph of the sequencing results of positive transgenic seedlings in verification example 1;

FIG. 2 is a fluorescent imaging of positive transgenic seedlings, positive controls, and negative controls in verification example 2;

FIG. 3 is a graph showing the results of fluorescent quantitative PCR detection of positive transgenic seedlings, positive controls and negative controls in verification example 3;

FIG. 4 is a comparative plot of the growth of positive transgenic seedlings in application example and conventional Arabidopsis plant Col-0;

FIG. 5 shows the whole genome DNA methylation sequencing comparison results of the positive transgenic seedlings, positive control and negative control in verification example 4.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

Wherein pCBC-DT1T2 is consistent with pCBC-DT1T2 as used in the university of agricultural university of China, proc. Institute of technology, chen Jijun laboratory, under the name "A CRISPR/Cas9 toolkit for multiplex genome editing in plants" (BMC Plant biol.2014Nov29;14:327.Pii: s12870-014-0327-y.doi:10.1186/s12870-014-0327-y. PubMed PMID: 25432517.). Of course, any conventional commercial pCBC-DT1T2 deemed suitable by those skilled in the art may be used herein, and the invention is not limited to the pCBC-DT1T2 used in this particular embodiment.

The primer pairs are synthesized and sequenced by the Kirschner biotechnology company; plasmid miniprep kit is a conventional commercial product of Jierui biology company.

The preparation method of the agrobacteria competence is as follows: streaking the GV3101 agrobacterium strain in a YEP solid culture medium containing rifampicin resistance (25 mg/L), and reversely culturing at a constant temperature of 28 ℃ for 2-3d in a dark place until single colonies grow on the surface of the culture medium; picking an agrobacterium single colony, inoculating the agrobacterium single colony into 5mL of YEP liquid culture medium containing rifampicin resistance, and culturing at 28 ℃ with 220g shaking overnight; 3-4mL of overnight culture was removed and incubated overnight with 220g shaking at 28℃in a sterile Erlenmeyer flask containing 20mL of liquid containing rifampicin antibiotics YEP; after the bacteria preservation, pouring the whole rest culture solution into a sterile conical flask containing 250mL of liquid containing rifampicin antibiotics YEP, and culturing 220g in a shaking way until the OD value is between 0.5 and 0.6; transferring the culture into a sterile centrifuge tube in an ultra-clean bench, standing on ice at 4 ℃ for 10min, centrifuging for 5min to precipitate bacteria, and collecting 4000 g; bacteria were resuspended in 50mL of 0.15mol/L NaCL solution pre-chilled on ice, then centrifuged at 4℃for 5min at 4000g, and the supernatant discarded; 5mL of CaCl 20mM was added pre-chilled on ice ₂ The bacteria were resuspended in solution, centrifuged at 4000g for 5min at 4℃and then re-added with 5mL of pre-chilled CaCl 20mM on ice ₂ The solution was resuspended, and the suspension was packaged in 100. Mu.L each tube and stored in a-80℃refrigerator in liquid nitrogen.

Further description will be given below by way of specific examples.

Example 1 construction of expression vector for knockout of Gene to be knocked out in Arabidopsis thaliana

Designing a pair of gRNA sequences shown as SEQ ID No. 1 and SEQ ID No. 2, and constructing a first primer pair shown as SEQ ID No. 3 and SEQ ID No. 4 and a second primer pair shown as SEQ ID No. 5 and SEQ ID No. 6 according to the gRNA sequences. Wherein, the second primer pair is obtained by reversely adding gRNA shown in SEQ ID No. 2.

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 the 25-fold diluted pCBC-DT1T2 as a template and the first primer pair and the second primer pair as primers, wherein the concentration of the primers shown in SEQ ID No. 3 and SEQ ID No. 6 was 10. Mu. Mol/L and the concentration of the primers shown in SEQ ID No. 4 and SEQ ID No. 5 was 1. Mu. Mol/L.

The PCR product was recovered by purification, and a cleavage-ligation system was established according to the system shown in Table 1 to obtain a recombinant plasmid.

TABLE 1

After purifying the obtained recombinant plasmid, 1 mu L of transformed escherichia coli competent cells are taken, kanamycin is adopted for screening, the screened colony is identified by adopting a sequence pair shown as SEQ ID No. 7 and SEQ ID No. 8, and the sequence pair shown as SEQ ID No. 7 and SEQ ID No. 9 is adopted for sequencing, so that the expression vector for knocking out the genes to be knocked out in arabidopsis is obtained.

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

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

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

The specific operation process of the transformation of the competent cells of the escherichia coli is as follows: coli competent cells were removed from the-80 ℃ refrigerator and inserted on ice until the competent cells were completely thawed. After the competent cells are melted, the recombinant plasmid (namely enzyme-linked product) is added, and the mixture is gently blown and evenly mixed by a gun head, and the ice bath is carried out for about 30 minutes. Heat shock is carried out for 90s in a water bath kettle at 42 ℃, and the water is quickly inserted into ice for 2-5min. Adding 500 μl of LB liquid medium (without antibiotics), mixing, shaking at 37deg.C, and culturing for 40-60min. After recovery culture, pouring competent cell fluid into LB solid culture medium containing corresponding antibiotics in an ultra-clean workbench, uniformly smearing with sterilized 1mL gun head, completely drying, sealing the culture medium, and inversely culturing at 37 ℃ for 14-16h.

In the present invention, plasmid extraction uses a plasmid miniprep kit. Here, the plasmid extraction is an operation used in the purification of the PCR product, and is aimed at the recovery of the aforementioned PCR product and pHEC401 vector by enzymatic cleavage purification. The adopted mode is DNA adsorption column balance treatment, which comprises the following steps: (1) adding 200 mu L of CBS buffer to the adsorption column for standby. (2) Taking 2-4mL of the bacterial liquid which is well shaken overnight according to the growth condition of the bacterial liquid, centrifuging 8000g for 1min, and discarding the supernatant. (3) mu.L of Soulization I was added, and the cells were sufficiently suspended by vortexing to form a uniform mixture. (4) After adding 250. Mu.L of Soulition II treated at 37℃and immediately and gently and sufficiently mixing up and down for 4-6 times, the cells were thoroughly lysed until a clear solution of protein was formed, the lid was opened with obvious stringiness, indicating that plasmids were present, and the process was not advisable for more than 5min. (5) Add 350. Mu.L of Soulization III, gently and sufficiently reverse upside down for 8-10 times, place at room temperature for 3min, centrifuge 12000g for 5-10min, and centrifuge 12000g for 1min on the column with CBS buffer as prepared in step (1). (6) The supernatant was sucked into an equilibrated DNA adsorption column, centrifuged at 6000g for 1min at room temperature, and the waste liquid was discarded. (7) The DNA adsorption column was replaced in a 2mL collection tube, 500. Mu.L of W1 Soulization was added, 12000g was centrifuged for 1min, and the waste liquid was discarded. (8) The DNA adsorption column was replaced in a 2mL collection tube, 500. Mu.L of Wash soulation was added, 12000g was centrifuged for 1min, and the waste liquid was discarded. This procedure was repeated once. (9) The DNA adsorption column was replaced in a 2mL collection tube, 12000g was air-separated for 1min, and Wash Solution was thoroughly removed. (10) The DNA adsorption column was placed in a clean 1.5mL centrifuge tube, dried at room temperature, 70. Mu.L of an Elutation Buffer was added, incubated for 5min at 37℃and centrifuged at 12000g for 1min to elute the plasmid. The eluate may be re-aspirated back into the DNA adsorption column and the process repeated once. The liquid is the solution containing the target plasmid, and the solution is put into a refrigerator at the temperature of minus 20 ℃ for preservation.

Example 2 transformation of Agrobacterium

A prepared agrobacterium competent cell was taken in a refrigerator at-80℃and inserted into ice, after it was slowly thawed into liquid, 1-2. Mu.L of the expression vector prepared in example 1 was added, gently mixed with a sterile gun head, and placed on ice for 30min. After 30 minutes, the centrifuge tube was placed in liquid nitrogen and frozen for 3 minutes, and then quickly transferred to a 37 ℃ water bath prepared in advance for 5 minutes. Taking out, placing on ice for 2min, adding 500mL of YEP liquid culture medium, and recovering culture at 180g in a shaker at-28deg.C for 3h. After culturing, pouring the prepared YEP solid culture medium containing the corresponding antibiotics into an ultra-clean bench, uniformly smearing the solution with a sterile blue gun head, drying the ultra-clean bench, sealing the solution, and inversely culturing the solution in a constant temperature incubator at 28 ℃ for 2-3 days. And (3) selecting a monoclonal on the plate, carrying out PCR verification on the bacterial liquid after shaking, and preserving the bacterial liquid for later use by the identified positive clone.

Example 3, flower dipping and screening of transgenic seedlings

The pollen tube channel pouring method is specifically adopted to transfect the arabidopsis. The positive clones identified in example 2 were thawed on ice and 100. Mu.L were inoculated into 5mL of YEP liquid medium containing the corresponding antibiotic and incubated overnight at 28 ℃. After 2 days, 5mL of the cloudy bacterial liquid was poured entirely into 250mL of YEP liquid medium containing the corresponding antibiotic, and the culture was performed on 180g of the medium at 28℃for 5 to 10 hours. During the culture process, the culture is carefully checked until the OD value of the agrobacterium reaches 1.0-1.2. The bacterial liquid is poured into a sterilized 250mL centrifugal bottle, and centrifuged for 10min at 4 ℃ and 4000g, and the supernatant is discarded to obtain the re-suspended agrobacterium. 200mL of an Arabidopsis thaliana Agrobacterium buffer solution is prepared in advance (wherein the Arabidopsis thaliana Agrobacterium buffer solution is prepared according to the following method, 100mL is taken as a total system, 0.237g of MS powder and 5g of sucrose are weighed and dissolved in 80mL of deionized water, the volume is fixed to 100mL after the MS powder and the 5g of sucrose are fully dissolved), a proper amount of the Arabidopsis thaliana Agrobacterium buffer solution is taken and poured into a bottle to resuspend the Arabidopsis thaliana Agrobacterium so that the OD value is between 0.8 and 1.0, and the Arabidopsis thaliana Agrobacterium initial preparation solution is obtained. Adding Silwet-77 into the Arabidopsis thaliana Agrobacterium initial preparation according to the ratio of Silwet-77 to Arabidopsis thaliana Agrobacterium initial preparation of 40 mu L/100mL to obtain Arabidopsis thaliana Agrobacterium intrusion solution. Selecting a full-bloom arabidopsis plant, and cutting off the fruit clips which are already self-pollinated and grown in advance. The inflorescence is soaked for 1min by using the arabidopsis thaliana agrobacterium infection liquid. After the dip dyeing is finished, the arabidopsis thaliana is placed into two black trays containing preservative films, and the black trays are subjected to dark treatment for 24 hours. Then the culture medium is transferred into a climatic chamber for normal culture. And the dip dyeing is carried out again after two weeks a week, so that the conversion success rate is improved. Culturing until seeds are harvested. Seeds harvested after dip-dyeing were T1 generation and spotted on MS medium containing 50mg/L hygromycin after drying. Culturing in artificial climate chamber, and observing the growth condition of seedling about 14 days, wherein the plant containing transgene germinates and grows obviously faster.

The seedlings are transferred into nutrient soil, cultured in a climatic chamber for about two months, and the single plant is harvested for T2 generation. The T2 generation seeds are respectively sown on an MS culture medium containing 50mg/L hygromycin, the seeds grow normally on the culture medium, and the strains without phenotype separation are the screened positive transgenic seedlings.

Verification example 1

And carrying out PCR identification on the screened positive transgenic seedlings to obtain homozygous mutants in the arabidopsis mutant plants which are knocked out successfully, and sequencing the homozygous mutants, wherein the sequencing result is shown in figure 1. The specific procedure for obtaining homozygous mutants based on PCR identification may be carried out in a manner which can be routinely used by those skilled in the art, and will not be described in detail herein.

As can be seen from FIG. 1, the method of the present invention provides an Arabidopsis mutant plant, and in particular, the Arabidopsis mutant plant has a deleted gene fragment of 177bp in length as shown in FIG. 1. Specifically, the sequence of the deleted gene fragment of the arabidopsis mutant plant is shown as SEQ ID No. 14.

CGGCATCTTTTTGGAACCTGGAAAGGAGTCTTTCATCCGCAAACACTTCAGCTTATTGAGAAAGAACTTGGCTTTAATGCTAAAAGTGATGGTTCAGCAGCTGTTGTATCCACAGCCAGAGCTGAGCCGCAATCTCAGCGCCCACCACATAGCATCCATGTGAATCCCAAGTATCTA(SEQ ID No:14)

Verification example 2

Seeds of the positive transgenic seedlings obtained in example 3 were single-seed-sown on MS medium. After the two leaves of the seedlings were fully opened, the seedlings were cultivated under light for 12d, sprayed with substrate fluorescein (Peikin Elmer, USA) for 10min, and then fluorescent was photographed using a fluorescent imager (Tiancan 5200, tiancan, shanghai) for 30s, and high-fluorescence control Arabidopsis Col-LUC (as positive control) and low-fluorescence control Arabidopsis ros1-7 (as negative control) were set, respectively. The results obtained are shown in FIG. 2. The fluorescence of positive transgenic seedlings after knocking out specific gene fragments is obviously reduced.

Verification example 3

Whether the transgene is silenced was verified by fluorescent quantitative PCR. Real-time fluorescent quantitative PCR amplification used CFX96 real-time PCR (Bio-RAD) system and kit used iQ SYBR Green Supermix (Bio-Rad). The Arabidopsis ACTIN2 is taken as an internal reference gene, three repeats are respectively carried out on each pair of primers on the template, and each gene amplification reaction is recorded as an 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 fluorescent quantitative PCR are shown in FIG. 3, and it can be seen from FIG. 3 that the transcription amount of the knocked-out positive transgenic seedlings is obviously reduced compared with that of Arabidopsis Col-LUC plants.

Verification example 4

Seeds of positive transgenic seedlings in example 3 were sterilized with 8% sodium hypochlorite solution and sown on MS medium. After cold treatment at 4℃for two days, the mixture was placed in an intelligent illumination incubator GTOP-S00B, manufactured by Thunb tuo-Po-nong science and technology Co., ltd, which was irradiated for 16 hours (22 ℃) and darkened for 8 hours (20 ℃). After 12 days of culture, seedlings of the positive transgenic seedlings prepared by the method are sent to Shanghai Ling En technology Co., ltd for whole genome DNA methylation sequencing. The sequencing results are aligned as shown in FIG. 5. As can be seen from FIG. 5, the DNA methylation level of the Arabidopsis mutant plants of the present invention was similar to that of the ROS1 gene mutant (i.e., ROS1 function was deleted on the background of Arabidopsis Col-0 plants) compared to the conventional Arabidopsis Col-0 plants, and a dramatic change occurred.

Based on the above experiments, it can be seen that the mutant plants of the present invention exhibit changes in the level of gene silencing and DNA methylation regulation after knocking out a specific gene.

Application example

Conventional Arabidopsis plants Col-0 and Arabidopsis mutant plants obtained by the invention are planted on MS culture medium, and are transplanted into nutrient soil after being cultured for 14 days. And the arabidopsis mutant plants obtained by the invention are grown to the bolting period of the plants under the same condition, and the bolting period is obviously advanced (shown in figure 4).

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

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

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.