CN112500462A

CN112500462A - Material for resisting DNA damage and preparation method thereof

Info

Publication number: CN112500462A
Application number: CN202011374861.8A
Authority: CN
Inventors: 聂文锋
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-16

Abstract

The present invention discloses that this embodiment discloses a material for resisting DNA damage, the amino acid sequence of which is shown in SEQ ID No.1. The invention has strong resistance to the stress treatment of DNA damage substances, such as methyl methanesulfonate (MMS), can play an important role in regulating phytochemical DNA damage, and can also be applied to cultivating new plant varieties.

Description

Material for resisting DNA damage and preparation method thereof

Technical Field

The invention relates to the field of transgenic plants, in particular to a material for resisting DNA damage and a preparation method thereof.

Background

Plants are often subject to chemical stress during development, resulting in mutations in the plant's genes.

Therefore, a material that can resist DNA damage is required.

Disclosure of Invention

The present invention is directed to overcoming the above problems and providing a material resistant to DNA damage and a method for preparing the same. In order to achieve the purpose, the invention adopts the following technical scheme:

a material for resisting DNA damage has amino acid sequence shown in SEQ ID No. 1.

The invention also discloses a preparation method of the material for resisting DNA damage, which comprises the following steps:

s1, EMS mutagenesis:

after EMS mutagenesis, 1mL of 35S-SUC2 seeds are sown in soil; dividing the seeds into a plurality of groups, and mixing and harvesting the seeds in each group; the seeds of M1 were sown in 1/2MS containing 2% sucrose, and the rooted seedlings were sown in soil; the seeds received by the long-rooted plantlets in each group are a mutant, M2, and are named by the sequence number; m2 was made 2 backcrosses with the wild type, 35S-SUC2, to exclude nonsense mutations;

s2, preparing a F2 hybridization material:

hybridizing the homozygote after the backcross for 2 times with another ecotype Landsbergerecta of the arabidopsis, planting F1 generation as short root, and F1 seed, and collecting the seed to obtain heterozygote of F2 for subsequent map-based cloning;

s3, map location cloning:

S3-1:

packaging 1/2MS solid culture medium into 1L glass bottles, each bottle is 800mL, adding stirrer, adding 1% agar powder, and sterilizing at 121 deg.C for 20 min; cooling the culture medium to about 50 ℃ after sterilization, and adding hygromycin according to the volume ratio of 1/2500;

S3-2:

plate inversion, 15 × 15cm petri dish;

S3-3:

sowing, wherein 150-200F2 seeds are sown in each culture dish, and 10 culture dishes are sown in each mutant; drying in a superclean workbench, sealing with a sealing film, performing vernalization at 4 deg.C for two days, and growing in a tissue culture room;

S3-4:

selecting long and short root seedlings, namely after the seedlings grow in a tissue culture room for seven days, the seedlings of F2 have long and short root separation symptoms, counting the separation ratio of the long and short roots, and determining the long and short roots as recessive genes; removing seedlings with short roots and seedlings with root tips in the air, keeping seedlings with long roots, marking the root tips of the seedlings with long roots, and allowing the seedlings to grow for two days;

S3-5：

sampling and extracting DNA, namely sampling the root-growing seedlings capable of further growing, respectively placing the root-growing seedlings in 1.5ml test tubes, and taking 96 samples of each mutant; extracting DNA by using an SDS method;

S3-6：

preliminary positioning, namely respectively taking 10ul of DNA from 96 samples of each mutant, and uniformly mixing the DNA for PCR; primary mapping was performed with 25 labeled primers on 5 chromosomes; in each PCR, F1 generation DNA hybridized with Col0, Col-0 and Ler is used as a control;

finding out corresponding linkage markers, determining the PCR amplification of a single sample, separating 4% agarose gel, and performing 200v 35 min;

S3-7:

confirming the coarse positioning result, namely the operation method is the same as S3-6, then 4% electrophoretic gel is used for separation, and the positioning interval can be reduced to 5M after the step;

S3-8：

fine positioning: designing fine positioning of the primers according to the positioning interval of S3-7, and reducing the Mapping interval to be within 1M;

S3-9：

whole genome sequencing: finding out a mutant candidate gene in a fine positioning interval according to a whole genome sequencing result;

s4, root length complementation experiment:

amplifying a DNA sequence of a corresponding gene and a self promoter of 2Kb, connecting the DNA sequence and the self promoter to a pEnter entry vector, and then connecting the DNA sequence and the self promoter to a corresponding expression vector through an LR shuttle reaction; finally, the expression vector with the target gene is transferred to GV3101 for transgenosis, and screening is carried out according to resistance; identifying protein expression amount by Western until T2 generation, after transgene homozygous, detecting expression of related marker genes by qPCR, detecting root length to determine required map clone gene, and determining whether the gene is transgene homozygous according to root length separation and resistance.

As a modification, the total volume of the 1/2MS solid medium in S3-1 is 4L, and the total volume comprises: MS basal salt 8.66 g; MES 2 g; 80g of sucrose and adjusting the pH to 5.7-5.8.

As a modification, the final concentration of hygromycin was 20mg/L after addition of hygromycin as described in S3-1.

As an improvement, the growth in the tissue culture room in S3-3 adopts an upward growth mode, and the plate is slightly inclined, so that the roots are landed.

As a modification, the SDS extract in each of the test tubes in S3-5 included: 50mmol Tris-HCl, 100mmol NaCl, 10mmol EDTA, and 1% SDS by weight of the extract.

The invention has the advantages that:

the invention has stronger resistance to the stress treatment of DNA damage substances, such as Methyl Methane Sulfonate (MMS), can play an important role in regulating and controlling the chemical DNA damage of plants, and can also be applied to the cultivation of new plant varieties.

Drawings

FIG. 1 is a genetic sequencing map of one material resistant to DNA damage of example 1;

FIG. 2 is a screening of the preliminary step of localization of a material resistant to DNA damage in example 1; the materials screened were the ARP4-2 material disclosed in example 1, Arabidopsis Wild Type (WT) and Col-0 controls;

FIG. 3 shows the phenotype of growth and development of arp4-2 of a DNA damage resistant material of example 1;

FIG. 4 is the validation of mutant material by the transgene complementation experiment in example 1;

FIG. 5 is a schematic representation of the application of the arp4-2 material in example 1 to the treatment of DNA damaging substances, Methyl Methane Sulfonate (MMS) stress.

Detailed Description

The present invention will be described in detail and specifically with reference to the following examples so as to facilitate the understanding of the present invention, but the following examples do not limit the scope of the present invention.

Example 1

This example discloses a material resistant to DNA damage, the amino acid sequence of which is shown in SEQ ID No. 1.

The preparation method of the embodiment comprises the following steps:

s1, EMS mutagenesis:

after EMS mutagenesis, 1mL of 35S-SUC2 seeds are sown in soil; dividing the seeds into a plurality of groups, and mixing and harvesting the seeds in each group; the seeds of M1 were sown in 1/2MS containing 2% sucrose, and the rooted seedlings were sown in soil; the seeds received by the long-rooted plantlets in each group are a mutant, M2, and are named by the sequence number; m2 was made 2 backcrosses with the wild type, 35S-SUC2, to exclude nonsense mutations.

S2, preparing a F2 hybridization material:

after 2 backcrosses, the homozygote is hybridized with another ecotype Landsbergerecta of the arabidopsis, the F1 generation is short root, F1 seeds are planted, and the heterozygote is obtained after the seeds are harvested, and is used for subsequent map-based cloning.

S3, map location cloning:

S3-1:

packaging 1/2MS solid culture medium into 1L glass bottles, each bottle is 800mL, adding stirrer, adding 1% agar powder, and sterilizing at 121 deg.C for 20 min; after sterilization the medium was allowed to cool to around 50 ℃ and hygromycin was added at 1/2500 volume ratio to a final hygromycin concentration of 20 mg/L.

1/2 the total volume of the MS solid culture medium is 4L, which contains: MS basal salt 8.66 g; MES 2 g; 80g of sucrose and adjusting the pH to 5.7-5.8.

S3-2:

Plate inversion, 15 × 15cm petri dish;

S3-3:

sowing, wherein 150-200F2 seeds are sown in each culture dish, and 10 culture dishes are sown in each mutant; blow-drying in a superclean workbench, sealing with a sealing film, performing vernalization at 4 ℃ for two days, and then putting into a tissue culture room for growth, wherein the tissue culture room adopts an upward growth mode, and the plate is slightly inclined, so that the roots are favorably landed.

S3-4:

Selecting long and short root seedlings, namely after the seedlings grow in a tissue culture room for seven days, the seedlings of F2 have long and short root separation symptoms, counting the separation ratio of the long and short roots, and determining the long and short roots as recessive genes; removing the seedlings with short roots and the seedlings with the root tips in the air, keeping the seedlings with long roots, marking the root tips of the seedlings with long roots, and allowing the seedlings to grow for two days.

S3-5：

Sampling and extracting DNA, namely sampling the root-growing seedlings capable of further growing, respectively placing the root-growing seedlings in 1.5ml test tubes, and taking 96 samples of each mutant; DNA was extracted using the SDS method.

SDS extracts in the tubes included: 50mmol Tris-HCl, 100mmol NaCl, 10mmol EDTA, and 1% SDS by weight of the extract.

S3-6：

corresponding linked markers were found and single samples were determined by PCR amplification, 4% agarose gel separation, 200v 35 min.

S3-7:

Confirming the coarse positioning result, the operation method is the same as S3-6, and then 4% electrophoresis gel is used for separation, after the step, the positioning interval can be reduced to 5M.

S3-8：

Fine positioning: designing fine positioning of the primers according to the positioning interval of S3-7, and narrowing the Mapping interval to be within 1M.

S3-9：

Whole genome sequencing: and (3) finding out mutant candidate genes in the fine positioning interval according to the whole genome sequencing result.

S4, root length complementation experiment:

The embodiments of the present invention have been described in detail above, but they are merely exemplary, and the present invention is not equivalent to the above described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, it is intended that all equivalent alterations and modifications be included within the scope of the invention, without departing from the spirit and scope of the invention.

Sequence listing

<110> Yangzhou university

<120> a material for resisting DNA damage and a method for preparing the same

<130> 2020

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 123

<212> DNA

<213> Arabidopsis thaliana

<400> 1

atgtacggca gaggtgggtt tctctttaaa cccttgaatt gctttgtcgt ttatcttttg 60

agacattcct gagctgtttt tttccgtggt gggttttcac agatgaagtg tcagctattg 120

tgg 123

Claims

1. A material for resisting DNA damage, characterized in that its amino acid sequence is shown in SEQ ID No.1.

2. a kind of preparation method of the material that resists DNA damage according to claim 1, is characterized in that, comprises the steps:

S1.EMS Mutagenesis:

1mL of 35S-SUC2 seeds were sown in soil after EMS mutagenesis; divided into several groups, each group was mixed and harvested; M1 seeds were sown in 1/2MS containing 2% sucrose, and the seedlings with long roots were planted in In the soil; the seed received by the long-rooted shoots in each group is a mutant, M2, and named after the sequence number; M2 was backcrossed twice with the wild type, namely 35S-SUC2, to exclude nonsense mutations;

Preparation of S2.F2 hybridization material:

After 2 backcrosses, the homozygote is crossed with another ecotype of Arabidopsis, Landsbergerecta, the F1 generation is short-rooted, the F1 seeds are planted, and the harvested seeds are the F2 heterozygotes for subsequent map-based cloning;

S3. Bitmap clone:

S3-1:

Dispense 1/2 MS solid medium into 1L glass bottles, 800mL per bottle, put in a stirrer, add 1% agar powder, sterilize at 121°C for 20min; let the medium cool to about 50°C after sterilization , add hygromycin according to the volume ratio of 1/2500;

S3-2:

The inverted plate is a 15x15cm petri dish;

S3-3:

Sow seeds, sow 150-200F2 seeds per petri dish, and plant 10 petri dishes per mutant; blow dry in an ultra-clean workbench, seal with parafilm, put them in vernalization at 4°C for two days, and put them into tissue culture room growth;

S3-4:

Select long and short root seedlings: after growing in the tissue culture room for seven days, the seedlings of F2 will have symptoms of long and short root separation, and the separation ratio of long and short roots is counted, and it is determined that it is a recessive gene; Long-rooted seedlings, and mark the root tips of the long-rooted seedlings and let them grow for another two days;

S3-5:

Sampling and DNA extraction: Sampling those long-rooted shoots that can grow further and placing them in 1.5ml test tubes, respectively, and taking 96 samples from each mutant; using SDS method to extract DNA;

S3-6:

Preliminary localization: from 96 samples of each mutant, take 10ul DNA and mix them, and do PCR; use 25 labeled primers on 5 chromosomes to carry out initial localization; when doing PCR, use Col0, Col- The F1 generation DNA hybridized with Ler is the control;

Find the corresponding linkage marker, carry out PCR amplification for a single sample to determine, separate with 4% agarose gel, 200v for 35 minutes;

S3-7:

Confirm the rough positioning result: the operation method is the same as S3-6, then use 4% electrophoresis gel to separate, after this step, the positioning interval can be reduced to 5M;

S3-8:

Fine positioning: According to the positioning range of S3-7, design the fine positioning of primers, and reduce the mapping range to within 1M;

S3-9:

Whole-genome sequencing: According to the results of whole-genome sequencing, find candidate genes for mutation in the fine mapping interval;

S4. Root length complementation experiment:

Amplify the DNA sequence of the corresponding gene and the 2Kb self-promoter, connect to the pEnter entry vector, and then connect to the corresponding expression vector through the LR shuttle reaction; finally, transfer the expression vector with the target gene into GV3101 for transgenic , screened according to the resistance; the protein expression was identified by Western, and after the T2 generation, after the transgene was homozygous, the expression of related marker genes was detected by qPCR, and the length of the root was detected to determine the required map-based cloned gene. Long segregation and resistance to determine whether the transgene is homozygous.

3. the preparation method of a kind of material that resists DNA damage according to claim 3, is characterized in that, the total volume of 1/2MS solid medium described in S3-1 is 4L, wherein contains: MS basal salt 8.66g ; MES 2g; Sucrose 80g, and adjust pH to 5.7-5.8.

4 . The method for preparing a DNA damage-resistant material according to claim 3 , wherein after adding hygromycin as described in S3-1, the final concentration of hygromycin is 20 mg/L. 5 .

5. the preparation method of a kind of material of resisting DNA damage according to claim 3, is characterized in that, described in S3-3, adopts the mode of upward growth in tissue culture chamber growth, and the plate is slightly inclined, which is conducive to root landing.

6. the preparation method of a kind of material that resists DNA damage according to claim 3, is characterized in that, the SDS extracting solution in each described test tube in S3-5 comprises: 50mmol Tris-HCl, 100mmol NaCl, 10mmol EDTA , and 1% SDS by weight of the extract.