CN112921051B - Method for creating male sterile breeding quality of watermelons through gene editing technology - Google Patents
Method for creating male sterile breeding quality of watermelons through gene editing technology Download PDFInfo
- Publication number
- CN112921051B CN112921051B CN202110195588.0A CN202110195588A CN112921051B CN 112921051 B CN112921051 B CN 112921051B CN 202110195588 A CN202110195588 A CN 202110195588A CN 112921051 B CN112921051 B CN 112921051B
- Authority
- CN
- China
- Prior art keywords
- seq
- male sterile
- gene editing
- sequence
- watermelons
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for creating a new male sterile germplasm of a watermelon by a gene editing technology, belonging to the technical field of plant genetic engineering. The invention firstly utilizes a CRISPR/Cas9 gene editing system to carry out gene editing knockout on the gene ClATM1 or a conserved domain thereof, so that the gene is in a function deletion or mutation, thereby forming a recessive nucleus male sterile phenotype. Compared with the normal material, the male sterile line created by the method has no visible phenotype change except that the fertility of male flowers is changed, and other tissues such as female flowers, leaves, tendrils, stems, roots, growth vigor and the like; if the created male sterile new germplasm is used for hybrid production or population improvement, the labor cost is greatly reduced, the breeding efficiency is improved, and the method has important production application potential.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a method for creating a new male sterile germplasm of a watermelon by a genetic editing technology.
Background
Watermelon (Citrullus lanatus), which is a Cucurbitaceae (curbitaceae) genus of watermelon (Citrullus), is an important Cucurbitaceae crop which is widely planted in the world, and is a fruit type cash crop, and is evaluated as the fifth largest fruit in the world due to the planting area and annual consumption. In production, watermelons have obvious heterosis, and complex procedures such as artificial bagging isolation, pollination and the like are still adopted in the conventional seed production process at present, so that the production cost is high, and the purity of seeds is difficult to completely guarantee.
Male sterility of plants is a common phenomenon in nature, and is not only an important tool for researching utilization of hybrid vigor of crops, but also an ideal material for researching plant development function. Currently, 5 watermelon male sterility mutant genes have been reported: smooth hairless male sterile gene (mgs); plant dwarf male gene (ms-dw), male sterile gene ms-1, male sterile gene ms-2 and male sterile gene ms-3. However, the above genes have not been precisely located and cloned at present, and the application of the gene editing technology to the creation of male sterile breeds has been limited.
Therefore, it is a urgent need for those skilled in the art to provide a method for creating a new male sterile germplasm of a watermelon by gene editing technology.
Disclosure of Invention
In view of this, the present invention provides a method for creating a new male sterile germplasm of a watermelon by gene editing technology.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the fine positioning of the sterile gene ClATM1 in the male sterile mutant Se18 of the watermelon is completed by the traditional gene positioning technology, and the gene is found to belong to the bHLH transcription factor family and contains the conserved domain bHLH by sequence analysis. Based on the gene, the invention creates the watermelon male sterile material by using CRISPR/Cas9 technology, and further is applied to the production process of watermelon hybrid seeds, thereby greatly improving seed production efficiency, hybrid seed purity and reducing production cost.
A method for creating a new male sterile germplasm of a watermelon by a gene editing technology comprises the following specific steps:
(1) Designing Target sites for gene editing according to CDS sequences and Target sites of ClATM1, designing two sets of editing sites, wherein each set comprises two Target points, the first set comprises Target1 and Target2, and the second set comprises Target3 and Target4;
a first set of:
target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3'; SEQ ID NO.2;
target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3'; SEQ ID NO.3;
and a second set:
target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3'; SEQ ID NO.4;
target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3'; SEQ ID NO.5;
(3) Construction of CRISPR/Cas9 editing vector
(1) Using an intermediate vector pCBC-DT1T2 as a template, respectively using a primer Target1F/Target2R and a primer Target3F/Target4R for PCR amplification, and respectively recovering a Target fragment 1 and a Target fragment 2;
(2) performing enzyme digestion on the CRISPR/Cas9 vector pBSE402 by using restriction enzyme BsaI-HF, and recovering the digested vector pBSE402;
(3) carrying out homologous recombination connection on the target fragment 1 and the target fragment 2 recovered in the step (1) and the vector pBSE402 subjected to enzyme digestion in the step (2) respectively to obtain a connection product 1 and a connection product 2;
(4) respectively converting the ligation product 1 and the ligation product 2 into escherichia coli competent DH5 alpha, and extracting the recombinant plasmid 1 and the recombinant plasmid 2 after correct sequencing; respectively transformed into agrobacterium competent cells EHA105;
(3) Genetic transformation of watermelons
After verification, the plant is used for genetic transformation of watermelons to obtain gene editing plants atm1_1 and atm1_2.
Furthermore, the created new male sterile germplasm of the watermelon is applied to hybrid seed production or population improvement.
The invention creates a new male sterile germplasm of watermelon by a gene editing technology, adopts CRISPR/Cas9 or other gene editing technologies to edit or knock out the gene ClATM1 or the conserved domain sequence thereof at fixed points, so that the gene protein is in a function deletion or mutation state, and a sterile phenotype of male flowers without pollen is produced. The application of the male sterile material created by the method in production can reduce the production cost of the watermelon hybrid seeds and improve the purity of the hybrid seeds and the seed production efficiency.
Compared with the prior art, the invention discloses a method for creating a new male sterile germplasm of a watermelon by using a gene editing technology, wherein a CRISPR/Cas9 gene editing system is utilized for the first time to carry out gene editing knockout on a gene ClATM1 or a conserved domain thereof, so that the gene is in a functional deletion or mutation state, and a recessive nuclear male sterile phenotype is formed. Compared with the normal material, the male sterile line created by the method has no visible phenotype change except that the fertility of male flowers is changed, and other tissues such as female flowers, leaves, tendrils, stems, roots, growth vigor and the like; if the created male sterile new germplasm is used for hybrid production or population improvement, the labor cost is greatly reduced, the breeding efficiency is improved, and the method has important production application potential.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the structure of ClATM1 gene according to the present invention;
FIG. 2 is a diagram showing pBSE402 vector of the present invention;
FIG. 3 is a schematic diagram showing the expression elements of a first recombinant plasmid sgRNA of the present invention;
wherein U6-26p and U6-29p are promoters, U6-26t is a terminator, gRNA-Sc is a gRNA skeleton, and Target1 and Target2 are Target sites;
FIG. 4 is a graph showing the comparison of YL of the present invention with atm1_1 and atm1_2 at 4 targets;
wherein, -represents a deletion; the red marked base is a PAM structure of a CRISPR/Cas9 system target spot;
FIG. 5 is a drawing of male flower phenotype of unedited and edited plants of the present invention;
wherein YL (unedited) male flowers are fertile, while the edited plants atm1_1 and atm1_2 male flowers are sterile.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 editing site selection of Gene ClATM1
The site CRISPR-P (http:// CRISPR. Hzau. Edu. Cn/CRISPR2/news. Php, V2) of the website is designed on line according to the CDS sequence (SEQ ID NO. 1) of ClATM1 and the Target site, two sets of editing sites (a first set: target1 and Target2; a second set: target3 and Target 4) are designed, and each set comprises two targets and four targets.
The structural schematic diagram of the ClATM1 gene is shown in figure 1. Wherein, target1, target2 and Target3 are all within the non-bHLH conserved domain of gene ClATM1, while Target4 is within the conserved domain (pale green). Designing two sets of targets altogether, wherein each set of targets comprises 2 targets, and the first set comprises targets 1 and 2; the second set contains Target3 and Target4.
A first set of:
target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3'; SEQ ID NO.2;
target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3'; SEQ ID NO.3;
and a second set:
target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3'; SEQ ID NO.4;
target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3'; SEQ ID NO.5;
example 2CRISPR/Cas9 editing vector construction
(1) PCR amplification
The adaptor primer is synthesized according to the two sets of target sequences, and the adaptor primer is specifically as follows:
a first set of:
target1 sequence primer:
Target1F：5’-TCGAAGTAGTGATTGCTGTTCAGGGAACTGTTCCGTTTTAGAGCTAGAAATAGC-3’；SEQ ID NO.6；
target2 sequence primer:
Target2R：5’-TTCTAGCTCTAAAACCTACGCCGTCTCCCACTACCAATCTCTTAGTCGACTCTAC-3’；SEQ ID NO.7；
and a second set:
target3 sequence primer:
Target3F：5’-TCGAAGTAGTGATTGGCCGGAGTTTATGAAGACGGTTTTAGAGCTAGAAATAGC-3’；SEQ ID NO.8；
target4 sequence primer:
Target4R：5’-TTCTAGCTCTAAAACTCACTTCTCTTAAAAGCTCCAATCTCTTAGTCGACTCTAC-3’；SEQ ID NO.9；
underlined sections are target sequences.
The intermediate vector pCBC-DT1T2 is used as a template, and high-fidelity enzyme PrimeStar Max Premix (TaKaRa) is used for PCR amplification, and the amplification system is as follows: primeStarMax Premix (2X) 25. Mu.L, template 2.5. Mu.L (100 ng/. Mu.L), upstream and downstream primers 2.5. Mu.L each, ddH 2 O17.5. Mu.L. The PCR reaction procedure was: 98℃10s,58℃5s,72℃15s,38 cycles; and at 72℃for 5min. After the PCR products are detected by electrophoresis (the sizes of fragments of two sets of editing sites are 626 bp), the target fragment 1 and the target fragment 2 are obtained by respectively carrying out glue recovery.
(2) Vector cleavage and ligation
The CRISPR/Cas9 vector pBSE402 (vector map see fig. 2) was digested with restriction enzyme BsaI-HF (NEW ENGLAND biolab) in the following system: pBSE 402. Mu.g, cutSmart buffer 5. Mu.L, bsaI-HF 1. Mu.L, ddH 2 O was added to 50. Mu.L. And (3) performing enzyme digestion for 2 hours at 37 ℃, and performing gel recovery after electrophoresis detection.
PCR products were ligated to vector: and (3) carrying out homologous recombination connection on PCR amplified products (target fragment 1 and target fragment 2) and a vector pBSE402 after enzyme digestion to obtain a connection product 1 and a connection product 2. The connection system is as follows: the molar ratio of vector to insert was about 1:2; 4. Mu.L of 5 Xreaction buffer; novoRec Plus recombinase 1. Mu.L; ddH 2 O was made up to 20. Mu.L. 50℃and 10min of connection.
(3) Recombinant plasmid transformation
Mu.l of ligation product 1 and ligation product 2, respectively, were heat-shock transformed into E.coli competent DH 5. Alpha. And plated on LB solid medium plates containing 50mg/L kanamycin, and incubated overnight at 37 ℃. The colony is positively detected by using the primers U626-IDF and U629-IDR, and bacterial liquid sequencing is performed by using the primer EDIT.
The specific primer sequences of U626-IDF and U629-IDR are as follows:
U626-IDF：5’-TGTCCCAGGATTAGAATGATTAGGC-3’；SEQ ID NO.10；
U629-IDR：5’-AGCCCTCTTCTTTCGATCCATCAAC-3’；SEQ ID NO.11；
the specific primer sequences of EDIT are as follows:
EDIT：5’-GGGAATCTGAAAGAAGAGAAGCAG-3’；SEQ ID NO.12。
after sequencing correctly, two sets of recombinant plasmids (recombinant plasmid 1 and recombinant plasmid 2) are respectively extracted and respectively transformed into agrobacterium competent cells EHA105, and PCR verification (using primers U626-IDF and U629-IDR) is correct and then used for watermelon genetic transformation.
Wherein, the skeleton of the first set of recombinant plasmids is shown in figure 3; the second set of recombinant plasmid backbone replaces Target1, target2 of the first set of recombinant plasmid backbone with Target3 and Target4, respectively.
The agrobacterium transformation procedure was as follows:
1 μl of recombinant plasmid is added into competent cells of agrobacterium EHA105, the mixture is sequentially placed on ice for 5min, quickly frozen in liquid nitrogen for 5min, water-bath is carried out at 37 ℃ for 5min, finally water-bath is carried out for 5min, 400 μl of LB liquid medium without antibiotics is added into each tube for resuscitation, and the mixture is transferred to a shaking table at 200rpm and 28 ℃ for shake culture for 2-3 hours. 100 μl of resuscitated bacterial liquid is plated on LB solid medium containing antibiotic (Kan), air dried at room temperature, and after the bacterial liquid is fully absorbed, the dish is inverted and cultured in a 28 ℃ incubator for 2-3d.
EXAMPLE 3 genetic transformation of watermelon
The full watermelon germplasm 'YL' seeds are taken, soaked in distilled water at 50-55 ℃ for about 30min, and then the seed shells are peeled off. Sterilizing peeled kernels in an ultra-clean workbench with 75% alcohol for about 30s, washing twice with sterile water, then soaking and sterilizing with 3% sodium hypochlorite for 15min, washing with sterile water for 5-7 times, airing, spreading in BM solid culture medium (Agar 4.43 g/L), and culturing in dark at 25 ℃ for 3d.
And after the seeds germinate, taking out the seeds, cutting off the two ends of cotyledons, and equally dividing the rest cotyledons into 8 pieces for easy infection. During this period, EHA105 with correct PCR assay was single-colony picked into LB liquid medium containing 50mg/L kanamycin and 20mg/L rifampicin, and the bacterial solution was shaken to OD 600 When=0.8, the final concentration OD was obtained by resuspension of the bacterial suspension with MS culture (MS 519 (PhytoTech Labs) 4.43g/L, sucrose 30g/L,6-BA 1.5 mg/L) 600 =0.2. The cut cotyledons were immersed in the bacterial liquid suspension for 15min, taken out and dried, and then co-cultured in a co-culture medium CM (MS 519, 4.43G/L, sucrose, 30G/L; G3251 (PhytoTech Labs), 3G/L;6-BA, 1.5 mg/L) with filter paper, at 25℃for 3d in the dark.
After co-cultivation for 3d, the cotyledon blocks are taken out, and the surface excess agrobacterium liquid is removed by sterile water (about 5-7 times of cleaning) until the sterile water is clear, and the cotyledon blocks are taken out, dried and placed on a recovery medium RM (MS 519.43G/L, sucrose 30G/L; G3251G/L; 6-BA 1.5mg/L,200mg/L Tintin (Scientific Research Special)) for recovery cultivation at 28 ℃.
After 5-7d of recovery culture, the cultured seed is recoveredLeaves were transferred to selection medium SM (MS 519.4.43G/L, sucrose 30G/L; G3251 (PhytoTech Labs) 3G/L, 1.5 mg/L6-BA, 200mg/L Timentin, 1.4mg/L Basta) for selection culture, subcultured at 28℃for 3-4 weeks, with medium change every 7 d. The explants with distinct shoot spots were then transferred to shoot elongation medium (MS 524 (PhytoTech Labs) 4.43g/L, sucrose 30g/L, G3251 g/L, inositol 1g/L, SH organic solution 500. Mu.L/L, NAA 0.01mg/L, 6-BA 0.1mg/L, timentin 200mg/L, basta 1.4mg/L, culture conditions of 28℃in the dark for 8h/d, light 16h/d, light intensity 8000Lx. The shoots selected were excised (note that the incision did not contain callus), transferred to rooting medium (MS 519.43G/L, sucrose 30G/L,6-BA 1.5mg/L, G3251G/L, IBA 0.5mg/L and Tintin) and rooting cultured at 28℃until rooting. The preparation method of the SH organic solution comprises the following steps: 5g nicotinic acid, 5g VB 1 、0.5g VB 6 Constant volume to 500ml.
Taking out from the culture flask when the regenerated seedlings root and grow to 4-5 true leaves, slowly washing off the culture medium at the root by clear water, transplanting into a substrate sterilized at high temperature and high pressure in advance, performing heat preservation and moisture preservation culture after watering thoroughly, covering the tray with water drops after 3-4 days, and gradually uncovering the cover for hardening seedlings.
EXAMPLE 4 detection of transgenic watermelon plants
The DNA of the regenerated seedling of watermelon with GFP fluorescence (GFP on vector pBSE 402) is extracted by CTAB method, the steps are: taking a small part of tender leaves, quickly grinding the leaves into powder in liquid nitrogen, and placing the powder into a centrifuge tube with the volume of 1.5 ml; adding 800 μl of preheated CTAB extraction buffer, and water-bathing at 65deg.C for 30min; adding equal volume of chloroform isoamyl alcohol, wherein the volume ratio of chloroform to isoamyl alcohol is 24:1, centrifuging for 10min at 8000r/min after uniform mixing; transferring the supernatant into a new centrifuge tube, adding 2/3 volume of isopropanol, and slightly mixing the supernatant upside down; centrifuging at 10000r/min for 10min; pouring out the supernatant, washing the precipitate twice with 75% ethanol, removing the residual liquid, drying for 3min, and adding 100 μl ddH 2 O (containing 0.1% RNase) was dissolved and stored at 4℃for further use.
The extracted DNA is used as a template, the primers ATM1-CRJC-F and ATM1-CRJC-R are used for carrying out PCR amplification on the sequences of two sets of four target sites respectively, the positive control is a recombinant plasmid, and the negative control is non-transgenic plant DNA.
Wherein, the specific primer sequences of the ATM1-CRJC-F and the ATM1-CRJC-R are as follows:
ATM1-CRJC-F：5’-CCATGCCCACTGCCTATACT-3’；SEQ ID NO.13；
ATM1-CRJC-R：5’-CGCCGGCGACATGGTGAAGA-3’；SEQ ID NO.14。
the amplification system is as follows: 2X Taq PCR StarMix with loading Dye. Mu.L, template 1. Mu.L, primers 1. Mu.L each, ddH 2 O7. Mu.L. The PCR reaction procedure was: 94 ℃ for 3min;94℃30s,58℃30s,72℃1min,30 cycles; and at 72℃for 5min. The PCR products were collected according to the band size, TA cloning (pClone 007 Versatile Simple Vector Kit, TSINGKE) was performed, bacterial cell PCR (using the primers ATM1-CRJC-F, ATM 1-CRJC-R) was positively detected, and then, monoclonal was selected and sent to the test for editing confirmation.
The Target sequencing result corresponding to Target1 in atm1_1 is as follows:
5’-CTTCAGGGAACTGTTCC-3’；SEQ ID NO.15；
target sequencing results corresponding to Target2 in atm1_1 are as follows:
5’-GTAGTGGGAGACTAG-3’；SEQ ID NO.16；
target sequencing results corresponding to Target3 in atm1_2 are as follows:
5’-GCCGGAGTTTATGAAGACG-3’；SEQ ID NO.17；
target sequencing results corresponding to Target4 in atm1_2 are as follows:
5’-GAGCTTTTAAGAGATGA-3’；SEQ ID NO.18；
the comparison result is shown in fig. 4, a first set of Target points obtain a gene editing plant atm1_1, and 2bp and 4bp (-representation) are respectively deleted in Target1 and Target2 Target areas; the second set of targets obtain a gene editing plant atm1_2, and 2bp (-representation) is deleted in the Target4 Target region. Both editing plants atm1_1 and atm1_2 were homozygously edited at the target site.
EXAMPLE 5 phenotypic observations of transgenic watermelon plants
The edited plants atm1_1 and atm1_2 were planted in solar greenhouse respectively, were managed normally, and after the male flowers were opened, the phenotype was observed, and the results are shown in FIG. 5. The results of fig. 5 show that: compared with the non-edited plant YL, the two plants atm1_1 and atm1_2 have smaller overall shape, petals and stamens of the male flowers, and the stamens have no pollen to be scattered and are completely sterile.
In summary, the invention provides a method for creating a new male sterile germplasm of a watermelon by a gene editing technology, which can rapidly obtain a recessive nuclear male complete abortive line by editing a sequence or a conserved domain bHLH of a male sterile regulatory gene ClATM1 and has important application potential in utilization of hybrid vigor and population improvement of the watermelon.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> university of agriculture and forestry science and technology in northwest
<120> a method for creating male sterile breeder of watermelon by gene editing technique
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1530
<212> DNA
<213> Artificial Sequence
<400> 1
atgtatgaac aaactgggtg ttctgatccc gattccactc taccagatcc ttcacaaaat 60
gcctttcctc atactatccc ccatatttct gttgctttgc cttcctttcc tccgccactc 120
attgccgccg ctgcccaaga aacccatccc aattttcatg acaataatct caatctttcc 180
atggaggaca tttcttatcc tcatcaccat caccatagct ccggcgctga cgctatggaa 240
cttcagtttc aacaggcgcc cgctggaggt tttgataaca gtaatatcaa ccctgatttt 300
ggtcaagaag taacttctga ttccaaccgt atggtgtgtc ttgaccaatc cgattgggtt 360
gggactcaaa ttcaagaaat ggggtttaat caccaccacc atcaccagat tcaatctcaa 420
gatcaccatc atcatccgca ccagcagcag ttttcagatt ccgccatgcc cactgcctat 480
actcaagctc ccgacctctt gaacttctta aacatgccgg cccctgccag atgccccaac 540
aactcttcca tctccttctc caatcaccat acctcaccca tgggaggctt tcttggagac 600
cttcccgccg gagacgctgg caactcatcg tcgacttcgc tttcaataca ctacgatcct 660
ctgtttcacc tgaatcttcc gccgcagcca ccgctgttca gggaactgtt ccactctctt 720
cctcatggat atggtatacc ggcggcgagt tccagaggcc gaggaggtag tttgttcccg 780
gaaggggaga taatggagac agaaggaact gccggagttt atgaagacgg ggatggaagc 840
ggtgttttgg agttcagtag agatatggcg gattgtgttg ggaaaggaag aaatgggaaa 900
atgactaaac attttaccac tgaacgccaa agaagagttc aactgaatga gaaatataat 960
gctctcaaga gtttggttcc tattcctact aagaatgata gggcatcagt tgtgggagac 1020
gccataaatt acatccaaga gcttttaaga gaagtgaagg aactgaaact gctggtggag 1080
aagaagagat gcagcagaga gaggagcaag aggcacagga cggcggagga attagaaggg 1140
ggcggcgcgt gggatgttga aagcacaaat gcaaaggcag gtggtgtagt gggagacggc 1200
gtagaggatc aaagctacaa tttgagaagc tcatggctgc agagaaagac aaaagatact 1260
gaagttgatg tgagaattgt tgatgatgaa gtaaccataa agcttgtgca gcgtaaactc 1320
aactgcttgt tgcttgtctc taaattgctt gacgatcttc agcttgatct tcaccatgtc 1380
gccggcggcc acatcggcga ttactacagc ttcttgttca ataccaagat atatgaaggt 1440
tcatcagtgt atgcaagtgc catagccaac aaggttatgg aggcagtgga cagacaatac 1500
aacaacacca ccacatccaa tacctattaa 1530
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 2
ctgttcaggg aactgttcc 19
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 3
gtagtgggag acggcgtag 19
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 4
gccggagttt atgaagacg 19
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 5
gagcttttaa gagaagtga 19
<210> 6
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 6
tcgaagtagt gattgctgtt cagggaactg ttccgtttta gagctagaaa tagc 54
<210> 7
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 7
ttctagctct aaaacctacg ccgtctccca ctaccaatct cttagtcgac tctac 55
<210> 8
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 8
tcgaagtagt gattggccgg agtttatgaa gacggtttta gagctagaaa tagc 54
<210> 9
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 9
ttctagctct aaaactcact tctcttaaaa gctccaatct cttagtcgac tctac 55
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 10
tgtcccagga ttagaatgat taggc 25
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 11
agccctcttc tttcgatcca tcaac 25
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 12
gggaatctga aagaagagaa gcag 24
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13
ccatgcccac tgcctatact 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 14
cgccggcgac atggtgaaga 20
<210> 15
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 15
cttcagggaa ctgttcc 17
<210> 16
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 16
gtagtgggag actag 15
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 17
gccggagttt atgaagacg 19
<210> 18
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 18
gagcttttaa gagatga 17
Claims (2)
1. A method for creating male sterile quality of watermelons by a gene editing technology is characterized by comprising the following specific steps:
(1) According toClATM1Designing Target sites for gene editing by designing two sets of editing sites, wherein each set of editing sites comprises two targets, the first set comprises Target1 and Target2, and the second set comprises Target3 and Target4;
a first set of:
target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3'; SEQ ID NO.2;
target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3'; SEQ ID NO.3;
and a second set:
target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3'; SEQ ID NO.4;
target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3'; SEQ ID NO.5;
(2) Construction of CRISPR/Cas9 editing vector
(1) Using an intermediate vector pCBC-DT1T2 as a template, respectively using a primer Target1F/Target2R and a primer Target3F/Target4R for PCR amplification, and respectively recovering a Target fragment 1 and a Target fragment 2;
the nucleotide sequence of Target1F/Target2R is as follows:
Target1F：5’-TCGAAGTAGTGATTGCTGTTCAGGGAACTGTTCCGTTT
TAGAGCTAGAAATAGC-3’；SEQ ID NO.6；
Target2R：5’-TTCTAGCTCTAAAACCTACGCCGTCTCCCACTACCAA
TCTCTTAGTCGACTCTAC-3’；SEQ ID NO.7；
the nucleotide sequence of Target3F/Target4R is as follows:
Target3F：5’-TCGAAGTAGTGATTGGCCGGAGTTTATGAAGACGGTT
TTAGAGCTAGAAATAGC-3’；SEQ ID NO.8；
Target4R：5’-TTCTAGCTCTAAAACTCACTTCTCTTAAAAGCTCCAA
TCTCTTAGTCGACTCTAC-3’；SEQ ID NO.9；
(2) performing enzyme digestion on the CRISPR/Cas9 vector pBSE402 by using restriction enzyme BsaI-HF, and recovering the digested vector pBSE402;
(3) carrying out homologous recombination connection on the target fragment 1 and the target fragment 2 recovered in the step (1) and the vector pBSE402 subjected to enzyme digestion in the step (2) respectively to obtain a connection product 1 and a connection product 2;
(4) respectively converting the ligation product 1 and the ligation product 2 into escherichia coli competent DH5 alpha, and extracting the recombinant plasmid 1 and the recombinant plasmid 2 after correct sequencing; respectively transformed into agrobacterium competent cells EHA105;
(3) Genetic transformation of watermelons
After verification, the plant is respectively used for genetic transformation of watermelons to obtain gene editing plants atm1_1 and atm1_2;
the saidClATM1The CDS sequence of (C) is shown as SEQ ID NO. 1.
2. Use of a method for creating male sterile watermelons by gene editing technology according to claim 1 for creating male sterile watermelons.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110195588.0A CN112921051B (en) | 2021-02-19 | 2021-02-19 | Method for creating male sterile breeding quality of watermelons through gene editing technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110195588.0A CN112921051B (en) | 2021-02-19 | 2021-02-19 | Method for creating male sterile breeding quality of watermelons through gene editing technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112921051A CN112921051A (en) | 2021-06-08 |
| CN112921051B true CN112921051B (en) | 2023-07-25 |
Family
ID=76169993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110195588.0A Active CN112921051B (en) | 2021-02-19 | 2021-02-19 | Method for creating male sterile breeding quality of watermelons through gene editing technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112921051B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395581A (en) * | 2021-12-09 | 2022-04-26 | 西北农林科技大学 | Method for creating watermelon hololimbic leaf germplasm through gene editing technology |
| CN116064575B (en) * | 2022-08-23 | 2023-08-22 | 河南师范大学 | Chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810973A (en) * | 2005-12-19 | 2006-08-02 | 西北农林科技大学 | Recessive nucleus male sterility gene sequence of water melon |
| CN108795978A (en) * | 2018-07-06 | 2018-11-13 | 江苏省农业科学院 | A kind of method and its application for formulating male sterility crop new germ plasm by gene editing |
| CN109593874A (en) * | 2019-01-04 | 2019-04-09 | 西北农林科技大学 | A kind of SSR molecular marker and application of watermelon genic male sterile close linkage |
| CN110438150A (en) * | 2019-08-22 | 2019-11-12 | 华中农业大学 | A kind of method and application for formulating rapeseed male sterility new germ plasm by gene editing |
| CN110760538A (en) * | 2019-11-18 | 2020-02-07 | 江苏省农业科学院 | Method for creating watermelon seed material with blight resistance |
| WO2021003954A1 (en) * | 2019-07-08 | 2021-01-14 | 山东省农作物种质资源中心 | Method for establishing rape nucleus male sterile line and maintainer line |
-
2021
- 2021-02-19 CN CN202110195588.0A patent/CN112921051B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810973A (en) * | 2005-12-19 | 2006-08-02 | 西北农林科技大学 | Recessive nucleus male sterility gene sequence of water melon |
| CN108795978A (en) * | 2018-07-06 | 2018-11-13 | 江苏省农业科学院 | A kind of method and its application for formulating male sterility crop new germ plasm by gene editing |
| CN109593874A (en) * | 2019-01-04 | 2019-04-09 | 西北农林科技大学 | A kind of SSR molecular marker and application of watermelon genic male sterile close linkage |
| WO2021003954A1 (en) * | 2019-07-08 | 2021-01-14 | 山东省农作物种质资源中心 | Method for establishing rape nucleus male sterile line and maintainer line |
| CN110438150A (en) * | 2019-08-22 | 2019-11-12 | 华中农业大学 | A kind of method and application for formulating rapeseed male sterility new germ plasm by gene editing |
| CN110760538A (en) * | 2019-11-18 | 2020-02-07 | 江苏省农业科学院 | Method for creating watermelon seed material with blight resistance |
Non-Patent Citations (3)
| Title |
|---|
| "Analysis of diferentially expressed genes and pathways associated with male sterility lines in watermelon via bulked segregant RNA‑seq";Yongqi Wang et al.;《3 Biotech》;第10卷;第1-15页 * |
| "西瓜bHLH转录因子家族基因的鉴定及其在非生物胁迫下的表达分析";何洁 等;《园艺学报》;第43卷(第2期);第281-294页 * |
| Ruimin Zhang et al.."Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon".《Horticulture Research》.2021,第8卷第1-12页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112921051A (en) | 2021-06-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2019345788A1 (en) | Method for knocking out BnMAX1 gene in Brassica napus L. using CRISPR-Cas9 system and application | |
| WO2020007332A1 (en) | Method of obtaining multi-leaf alfalfa material by means of mspalm1 artificial site-directed mutant | |
| CN110257421B (en) | Construction method and application of brassica napus gene mutant PTG8 | |
| CN112921051B (en) | Method for creating male sterile breeding quality of watermelons through gene editing technology | |
| CN110878302B (en) | Method for knocking out Brassica napus Bna. TT8 gene by using CRISPR/Cas9 system and application | |
| CN113583099B (en) | Method for cultivating alfalfa male sterile line and corresponding maintainer line and related biological material thereof | |
| US11365423B2 (en) | Method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants | |
| AU706650B2 (en) | Genetic modification of plants | |
| CN108165578B (en) | Efficient preparation method for mutant aiming at multiple members of same gene family of cabbage mustard simultaneously | |
| CN116144700A (en) | Application of rice OsbZIP53 gene or protein encoded by rice OsbZIP53 gene in improving rice yield | |
| CN113046377B (en) | Male sterile gene MsGAL and application thereof | |
| CN111961675B (en) | Clonotus sinensis-free Clinopodium polycephalum closed flower gene CsCly and application thereof | |
| US20230175007A1 (en) | A plant fertility-associated protein and its application thereof | |
| WO2013082865A1 (en) | Method for preparing fertility-lowered plant | |
| CN111690679A (en) | Recombinant expression vector for cultivating cucumber male sterile line and construction method and application thereof | |
| CN118147224A (en) | Method for creating male sterile breeder of watermelon by gene editing ClMMD1 | |
| CN114150014B (en) | Method for creating corn dwarf material based on ZmEMF2b/2-2 gene | |
| CN114395581A (en) | Method for creating watermelon hololimbic leaf germplasm through gene editing technology | |
| CN118147223A (en) | Method for creating male sterile breeder of watermelon by gene editing ClMS188,188 | |
| CN116814651B (en) | Application of oat flower MYB4a transcription factor in regulating and controlling plant flower column elongation | |
| CN115948451A (en) | Application of LsARF3 protein or coding gene thereof in regulation and control of high-temperature bolting performance of leaf lettuce | |
| CN105296528A (en) | Genetic transformation method of lobelia sessilifolia | |
| CN115927389A (en) | Soybean GmAP2-a gene and application thereof | |
| CN118028364A (en) | Method for creating diploid seedless watermelons through gene editing ClCKI a1 | |
| Van der Salm | Introduction of ROL genes in Rosa hybrida L. for improved rootstock performance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |