CN116676311A - Medaka parent source effector org and sgRNA fragment thereof - Google Patents
Medaka parent source effector org and sgRNA fragment thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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Abstract
The invention discloses a medaka parent source effector org and an sgRNA fragment thereof, belonging to the technical field of molecular biology, wherein the nucleotide sequence of the medaka parent source effector org is SEQ ID NO.1, and the sgRNA fragment sequence is SEQ ID NO.2-3. The CRISPR/Cas9 technology proves that the deletion of the org gene leads to the failure of the embryo to complete the activation of an early-stage zygotic genome and the genetic control process of the transformation from female parent to zygotic, so that the early-stage embryo development is stagnated and finally dies, and the gene org is taken as a maternal effector to play an important role in the early-stage embryo development.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and designs a medaka parent source effector org and an sgRNA fragment thereof.
Background
Maternal Effector Genes (MEGs) transcribe maternal factors and accumulate in oocyte cytoplasm during the growth and development of oocyte, and the maternal factors are involved in the processes of meiosis recovery of animal oocyte, male-female prokaryotic fusion after fertilization, activation of early embryo synthon gene and the like. The maternal effector genes play specific functions in different processes, so that the normal performance of life body activities is ensured. In recent years, researchers have identified a number of maternal effectors in mammals, including the Hsf1, bnc1, figla and Nlrp5 genes in mice, whereas the mining of maternal effectors in fish has not been deep enough, particularly the maternal effector function of medaka has been studied poorly. Sea water medaka has been recognized as a model fish for ecological toxicology and environmental research in Asian areas, and has features of short reproduction period (3-4 months), spawning daily, small size (3-4 cm), transparent embryo, and easiness for large-scale culture in laboratory, which make sea water medaka a model organism for in vivo gene function test.
In recent years, the development of CRISPR/Cas9 gene editing technology has been revolutionarily successful, and the application of CRISPR/Cas9 gene editing technology in model organism medaka is also becoming mature. Albino medakas were obtained by researchers through CRISPR/Cas9 technology, providing an important platform for studying pigmentation. In addition, the donor plasmid containing the heat shock promoter and the reporter gene is transferred into the medaka embryo by a non-homologous end joining method, so that the knock-in transgenic medaka is efficiently produced, and the gene function analysis is promoted. The method provides a theoretical and experimental basis for researching gene functions by using CRISPR/Cas9 technology and using the ocean water medaka as an experimental object.
Disclosure of Invention
The invention identifies the medaka parent effector org and the sgRNA fragment thereof by CRISPR/Cas9 technology. The medaka org gene is found to be consistent with other maternal effector genes, and the deletion of the medaka org gene leads to the failure of the embryo to complete the activation of an early zygotic genome and the genetic control process of maternal-to-zygotic transformation, so that early embryo development is stopped and finally dies. Furthermore, we found that the org gene was consistent with the expression pattern of other maternal effectors in early embryo development.
The invention is realized by the following technical scheme:
in a first aspect, the invention provides a medaka parent effector gene org, wherein the nucleotide sequence of the gene is SEQ ID NO.1.
A second aspect of the invention provides the sgRNA of the org gene, SEQ ID NO.2: GGTGGGTCCAGAGCGCTCTG and SEQ ID NO.3: CTCAGCGTTTCCACCCAGGA.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the org related gene of the medaka ovum is cloned for the first time, the sgRNA of the org gene is designed and synthesized, and the validity of the sgRNA is detected. The CRISPR/Cas9 technology is utilized to successfully knock out org genes in early embryo of the medaka, and the deletion of org genes in experimental groups leads to the failure of the embryo to complete activation of early zygotic genome and genetic control process of transformation from female parent to zygotic, so that early embryo development is stopped and finally dies, which is consistent with embryo development after other maternal effector gene knockouts. In addition, we studied the expression pattern of org gene in early embryo development, consistent with other maternal effector genes, very abundant expression during zygote activation period, and degradation after completing genetic control process of maternal-to-zygote conversion. The important role of org as a maternal effector in early embryo development was demonstrated.
The invention provides a nucleotide sequence of org gene, the nucleotide sequence is SEQ ID NO.1; providing org gene target site sequences and designing synthesized sgRNA sequences SEQ ID NO.2-3, and providing primers for amplifying target site fragments, wherein the primer sequences are org-gRNAF12, org-gRNAF3 and org-gRNAR respectively. In addition, the invention provides specific steps of sea water medaka culture and embryo microinjection, and specific steps of sgRNA design and synthesis; step of extracting sea water medaka single embryo DNA and primer for RT-qPCR: O.melastingma-qF, O.melastingma-qR, O.melastingma-actin-qF, O.melastingma-actin-qR.
Drawings
FIG. 1 is a sequence diagram of the target site of the org gene in example 3 of the present invention.
FIG. 2 is a graph showing the result of electrophoresis after purification of sgRNA synthesis in example 3 of the present invention.
FIG. 3 is a phenotypic chart of wild-group embryo and experimental-group embryo development in example 4 of the present invention.
FIG. 4 is a graph showing the detection peaks of the working efficiency of SEQ ID NO.2 in example 5 of the present invention.
FIG. 5 is a graph showing the detection peaks of SEQ ID NO.3 showing the working efficiency of example 5 of the present invention.
FIG. 6 is a diagram showing the expression pattern of org gene in early embryo development in example 6 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which are obtained by a worker of ordinary skill in the art without creative efforts, are within the protection scope of the present invention based on the embodiments of the present invention.
Example 1: the sea water medaka culturing and fertilized egg collecting steps comprise:
1. sea water medaka and embryo culture
The seawater medaka circulating culture system is purchased from Shanghai sea Saint biological experiment equipment Co., ltd. The water temperature of the circulating system is 26 ℃, the PH range is maintained at 7.0-8.0, the salinity range is 32-34, the dissolved oxygen is 5-8 mg/L, the photoperiod is 14 hours (8:00-22:00) of illumination and 10 hours (22:00-8:00) of darkness. According to the mating and spawning characteristics of the ocean medaka, the proportion of female fish to male fish in each fish tank is 3:2, the culture density is generally 5-10 tails/L, fresh worms are fed for 2-3 times a day, and the growth and the reproduction of the sea water medaka are ensured. Each adult sea water medaka spawns 30-50 per day, eggs are timely taken after spawning, the medaka spawns are placed in a culture dish for culture, sea water in the culture dish is replaced every day, sufficient oxygen content in the sea water is ensured, and normal development of embryos is maintained.
2. Collecting fertilized eggs of sea water medaka
The sexually mature sea water medaka is separated into cylinders after one night before microinjection, 6-8 female fishes and 6 male fishes in each fish tank are combined into one cylinder after one hour before the system lights on the day of injection, the female fishes start spawning, and the male fishes go to rear-end collision and finish fertilization after the system lights. The number of the female fish and the male fish in the experiment can be adjusted in real time according to the spawning number of the female fish and the arrangement of the later experiment.
About half an hour after fertilization, taking off the embryo hung around the reproductive hole in time, washing the embryo with circulating system water for 2-3 times, observing the embryo development period under a microscope, sucking the embryo in the single cell period by a suction tube, and arranging the embryo on a prepared embryo clamping groove, wherein the position of the embryo is adjusted in the microscope field of view, so that the animal pole and the microinjection needle are in the same straight line. The remaining embryos are placed on ice or in a refrigerator at 4 ℃ to slow down the rate of division. Embryos after the two cell period are no longer suitable for microinjection.
Example 2: the sea water medaka embryo microinjection comprises the following steps:
1. preparation of microinjection embryo clamping groove
Dissolving 2% of agar in 50mL of sterile water, heating by a microwave oven until the agar is boiled, pouring the agar into a 70mm sterile culture dish, placing one surface of an ovulation mould with ribs into the upper layer of agar solution of the culture dish, placing the agar solution into a refrigerator at 4 ℃ for accelerating solidification, taking the mould out of the agar after solidification, and then completing preparation of embryo pulling grooves, adding a incubator at 26 ℃ into the clamping groove for preheating before use, adding sterile water into the clamping groove for storage in the refrigerator at 4 ℃ after use, and recycling.
2. Microinjection needle preparation
The microinjection needle was drawn from a capillary glass tube purchased from WPI (World Precision Instruments) company with an outer diameter of 0.8mm in a horizontal needle puller (Sutter Instaument, P-1000) according to the parameters of the medaka embryo. The pull pin parameters of medaka embryos are: tension PULL:55, temperature HEAT:550, pressure:300, rate VEL:60. the capillary glass tube is fixed in a heating wire of the needle drawing instrument, and the glass tube is blown by utilizing the temperature of the heating wire and the tensile force at the two ends of the glass tube, so that the glass tube is in a closed state. Before microinjection, fixing a microinjection needle (containing 0.05% of phenol red indicator solution) in an injector, placing a glass slide on a microscope stage, adjusting the angle of the microinjection needle to form an included angle of about 40-50 degrees with the glass slide, adjusting the position of the microinjection needle in a visual field to enable the microinjection needle to be placed in the center of the visual field, slowly adjusting a nut until the needle head just contacts the glass slide, and immediately lifting the microinjection needle after the phenol red indicator solution flows out to finish needle breakage.
3. Sea water medaka embryo microinjection
(1) Firstly, a large valve of a nitrogen tank is opened, the pressure is controlled to be within 12-13 megapascals, then a pressure reducing valve is opened, an injector is opened within 0.3 megapascals, the injection pressure of the injector is regulated to be less than 16psi, and the hold pressure is controlled to be within the zero range. The switch at the Hold voltage is turned to vent, the passage is opened, and the switch at the project voltage is turned to Hold to prevent suck-back
(2) Loading and needle loading. mu.L of RNP complex reagent was aspirated with an RNase free tip at the tip of the microinjection needle, taking care not to generate bubbles.
(3) The microinjection dose was adjusted. The microinjection needle is placed at the center of the visual field, the hold pressure is firstly increased slowly, the RNA compound is formed into liquid drops to be gathered on the microinjection needle, and then the hold pressure is finely adjusted, so that the liquid drops are not increased and are not dropped.
(4) And (3) injection. Placing the embryo in single cell stage under a stereoscopic microscope, adjusting visual field definition, adjusting embryo position, enabling the microinjection needle head and embryo animal polar cells to be in the same horizontal line, rotating the spiral to enable the needle tip of the injection needle to enter the embryo animal polar in single cell stage, stepping on the pedal, and enabling the animal polar to have red liquid drops (RNP compound) and the embryo to be unbroken.
(5) After the injection is finished, the large valve of the nitrogen tank is closed, the operating system is completely exhausted, then the pressure reducing valve is closed, and the used microinjection needle is put into the waste medical box.
(6) After the injection was completed, the roe was placed in a petri dish containing seawater and cultured in an incubator at 26 ℃. The fish egg state was observed daily, the dead eggs were aspirated and water was changed.
Example 3: the sgRNA design, synthesis and purification include the following steps:
1. sgRNA design
Inquiring medaka org gene sequence information in NCBI database, extracting medaka ovary RNA and reversing into cDNA, and specifically comprises the following steps:
total RNA extraction was performed using Total RNA Purification Kit (Norgen Biotek, canada) and the following steps were performed:
(1) Female sexually mature sea water medaka is selected for ice water anesthesia, a sterilized forceps scalpel and other tools are used for dissecting sea water, about 20mg of ovarian tissue is put into a centrifuge tube of 1.5mL RNase-free, 300 mu L of Buffer RL is added, and the mixture is ground by a tissue grinder until the solution is transparent.
(2) To the well-ground solution, 300. Mu.L of Buffer RL was added to make up to 600. Mu.L, and 600. Mu.L of 70% alcohol solution was added thereto and mixed by vortexing.
(3) The mixture was transferred to RNA Binding Column, centrifuged at 13000rpm for 1min and the filtrate was discarded.
(4) And (3) repeating the step 3.
(5) RNA Binding Column put into a fresh 1.5mL centrifuge tube of RNase-free, and 50. Mu.L of Elution Solution A was added, centrifuged at 200rpm for 2min and at 14000rpm for 1min.
(6) 1. Mu.L of total RNA dissolved in Elution Solution A was used for detection of RNA concentration and purity by using a Nanodrop2000 spectrophotometer (Thermo, waltham, MA, USA), 1. Mu.L of RNA solution was used for detection of RNA integrity by 1% agarose gel electrophoresis, and the rest was stored in an ultra-low temperature refrigerator at-80 ℃.
By DNA-free TM DNA erasure is performed by Kit (ambion Inc, austin) Kit, and the specific steps are as follows:
(1) 3. Mu.g of qualified RNA was placed in a 200. Mu.L centrifuge tube of RNase-free and placed on ice, and 10 XDNase I Buffer and 1. Mu.L rDNase I were added and mixed well.
(2) The centrifuge tube was placed in a PCR apparatus and incubated at 37℃for 30min.
(3) After incubation was completed, the centrifuge tube was removed, placed on ice, DNase Inactivation Reagent added and mixed well.
(4) Standing at room temperature for 2min, and blowing for 1-2 times.
(5) The RNA was transferred to a new RNase-free centrifuge tube by centrifugation at 10000rpm for 1.5 min.
RNA after DNA was erased using PrimeScript by Takara TM The kit instruction of II 1st Strand cDNA Synthesis Kit carries out reverse transcription, and the specific steps are as follows:
(1) 1. Mu.g of RNA after DNA removal was placed in a 200. Mu.L centrifuge tube of RNase-Free, placed on ice, 1. Mu. L Oligo dT Primer and 1. Mu.L of dNTP mix were added, RNase Free water was added to make the total system 10. Mu.L, and the Mixture was homogenized and centrifuged instantaneously.
(2) The centrifuge tube was placed in a PCR apparatus and incubated at 65℃for 5min. Immediately after termination, the reaction was rapidly terminated by placing on ice.
(3) A new centrifuge tube was taken and 4. Mu.L of 5X PrimeScript II Buffer, 0.5. Mu. L RNase Inhibitor, 1. Mu. L PrimeScript II RTase, 4.5. Mu.L of RNase Free water was added. And transferring the product obtained in the step 1 into a new centrifuge tube, uniformly mixing and instantly centrifuging.
(4) Placing the mixed sample into a PCR instrument, reacting at 42 ℃ for 60min, reacting at 95 ℃ for 5min to obtain cDNA, and placing the product into a refrigerator at-20 ℃ for subsequent use.
Designing a primer according to the org gene sequence, and carrying out sequence verification by taking medaka ovary cDNA as a template, wherein the sequence (SEQ ID NO. 1) of the medaka org gene CDS region is: ATGGCGTCTGGACGGCGCGTGGACGCAGCGGAGCGGGGAGGAGCCTCTGAGGAGGCGGAGGGAGCCGTGGCCCCGCGGGACGGCGGGCTCCGCGCGCTGCTCCGCGGCCTCGTCTGGCCGTTCGGGCTCGTGGTTCGAGTCTGCTCCAGAGCCTGGCAGCTGCTCGGCTTCCAGGAACCGCACGCGGTCACCTCGGCTTCCGGCCCCCCGCGTCCCAGCCGTCGGAAGCGCCTGCACCGTGTCACGCGCGCGCTGCTGTCCGTGCTGCCCCGGTGGGTCCAGAGCGCTCTGGGGTTCCCGGGACCCGCCAGCATCGGGGTCTCCCTGTCCCCAGAAATCCGGAGTTCTCCCACCAAACCTCACGGGAAAGGCAGCAAGCGCAAACAGGACGACCTGGATGACGAGGAGGAGGAGGAGCATCCGTCCTGGGTGGAAACGCTGAGCCAGGAGCTGGCAGACGACGACGGCCCGGCTGAAGATCCGGACTATGAGCCCAGCTCCGTGGAGACGGACACGGAGGAGTACGCCTCCCACAACAACAGGAGAGCGACCTGGAGGCCCCAGGAGGAGGTGTGCTGATCCGGGACGTCCAGACGACACGCCCACTCTGTAACCGACCTGGACTCGTTCTACTCGGAGCTGCTTTGTTTTCATACTTTGATGGAAATCTTGCTGGAATTGAAGTCTTTTAG.
The sgRNA sequence of org gene is designed according to the following principle: sgrnas that were first above 4 thymidines (5 '-TTTT-3') were excluded because they were recognized as transcription termination signals by RNA polymerase III. Secondly, for the efficiency of gRNA, the target site position should be on the first exon as much as possible, the length of the target site sequence is 20bp, which cannot be a multiple of 3, and the GC content is generally about 60%. Three sgrnas were designed for medaka org gene sequences:
SEQ ID NO.2:GGTGGGTCCAGAGCGCTCTG；
SEQ ID NO.3:CTCAGCGTTTCCACCCAGGA；
SEQ ID NO.4: ACTCGAACCACGAGCCCGAA. In addition, the specificity of the target site sequence was checked by inputting the target site base sequence over the entire genome of medaka by BLAST tool in NCBI.
FIG. 1 shows three target sites selected according to the sgRNA design principle. The in-box sequences represent the target sequences of sgRNAs, and the underlined sequences represent the NGG protospacer adjacent motif sequences.
2. sgRNA synthesis and purification
Taking plasmid PGL3-U6-sgRNA-PGK-puromycin (Addgene, USA) as a template, wherein a forward primer is a T7 promoter primer sequence, a base GGG+ target site sequence for improving the efficiency of T7 polymerase and 20 bases upstream of a gRNA skeleton; the reverse primer is a reverse complement downstream of the gRNA backbone. The upstream primer and the downstream primer are respectively: org-gRNAF1: TGTAATACGACTCACTATAGGAGCTCACCTGAAGAAGCGCGGTTTT AGAGCTAGAAAT; org-gRNAF2: TGTAATACGACTCACTATAGGTGGACGGCGCGTGGACGCAGGTTTT AGAGCTAGAAAT; org-gRNAF3: TGTAATACGACTCACTATAGGGGAGCGGGGAGGAGCCTCTGGTTTT AGAGCTAGAAAT; org-gRNAR AAAAGCACCGACTCGGTGCC. The PCR products were verified and purified by agarose gel electrophoresis. The purified product was used as a transcription template and T7 HighYield TranscriptionKit (Thermo Fisher) was used to carry out transcription reactions, with a template input of 0.5. Mu.g producing 150-200. Mu.g RNA.
The transcribed RNA was purified as follows:
(1) 160. Mu.L of RNase-free ddH was added 2 O dilutes the product to 180. Mu.L.
(2) 20. Mu.L of 3M sodium acetate (pH 5.2) was added to the diluted product and thoroughly mixed.
(3) 200. Mu.L of a phenol/chloroform mixture (1:1) was added for extraction, and the mixture was centrifuged at 12,000rpm for 5min at room temperature, and the upper layer solution was transferred to a new RNase-free EP tube.
(4) Chloroform was added to the aqueous phase in an equal volume for 2 extractions and the upper aqueous phase was collected.
(5) Adding 2 times volume of absolute ethanol, mixing, incubating at-20deg.C for 30min, and centrifuging at 4deg.C at 12,000rpm for 15min.
(6) The supernatant was discarded, and the RNA pellet was washed with 500. Mu.L of pre-chilled 70% ethanol, centrifuged at 12,000rpm at 4℃and the supernatant was discarded.
(7) Uncapping and drying for 2min, adding 20 μL RNase-free ddH 2 O or other buffers solubilize RNA pellet and preserve at-80 ℃.
FIG. 2 is an electrophoretogram of sgRNA after purification. The band size of the orggRNA is about 120 bp. The org sgRNA concentrations are org SEQ ID NO.2:903.76 ng/. Mu.L, org SEQ ID NO.3:872.05 ng/. Mu.L, org SEQ ID NO.4:956.63 ng/. Mu.L.
Example 4: development process of microinjection group embryo
Three sgrnas were diluted to 100 ng/. Mu.l with 0.05% sterile enzyme-free chase indicator phenol red solution, cas9mRNA was diluted to 400 ng/. Mu.l, and the sgrnas were incubated at cas9mRNA room temperature for 5-10min, respectively, to form RNP complexes. 2nL RNP complex was injected into medaka embryo animal pole at a single cell stage, about 100 fertilized eggs were injected before the two cell stage, and wild type fertilized eggs were used as controls. The embryos of the experimental group and the wild group are placed in a constant temperature incubator at 26 ℃, and dead eggs are removed in time, so that other embryos are prevented from being polluted. The development condition, such as the animal polar cell division speed, is observed according to different stages of embryo development in different periods. As shown in fig. 3, we found that there was no significant difference in embryo development between the wild group and the experimental group within 3 hours after fertilization. Whereas after 3 hours of fertilization, experimental group embryos began to develop slowly. At about 6 hours post fertilization, a significant difference in embryo development rates occurred between the wild group and the experimental group, the number of animal polar cells in the experimental group did not increase compared to the embryos at 3 hours post fertilization, and the cell morphology was abnormal. About 10 hours after fertilization, the experimental group had extremely apoptotic embryo cells, and the vitelline membrane had darkened. By about 22 hours after fertilization, the experimental group embryo vitelline membrane shrank and died. In contrast, wild-type embryos develop normally. From the embryo development process of medaka in sea water combined with our embryo development process recorded in time, we can summarize: experimental group embryos had stopped developing before the morula stage and the blastula began to die late. About 54% of embryos die completely 24 hours after fertilization, leaving about 56% of embryos to develop slightly later than the wild-type group.
Example 5: target site work efficiency detection
Collecting experimental group and wild group embryo, and extracting medaka embryo genome DNA by cleavage method.
The method comprises the following steps:
(1) The washed individual embryos were placed in 200. Mu.L microcentrifuge tubes and the embryos were broken by using RNase Free tips or toothpicks (sterilized at high temperature).
(2) To the centrifuge tube, 8. Mu.L of DNA lysate (containing 20mg/ml proteinase k) was added, thoroughly mixed and transiently centrifuged.
(3) The centrifuge tube was placed in a PCR apparatus and reacted at 65℃for 30min and at 95℃for 10min. Centrifuging at 12000rpm for 5min, collecting supernatant, and preserving at-20deg.C.
The primers are designed to amplify org genes by taking genomic DNA as a template, and upstream and downstream primers (sgRNA 1-2F: AGCGGAGCGGAGGAGC; sgRNA1-2R: CCCCCGCCCGCCCGCATTTTGGTT; sgRNA-3F: TGCGTCCACAGAATCC; sgRNA-3R: CCGTGTGTTGTTGTGGGAGG) are respectively designed at 400-500bp at the upstream and downstream of a target site, the product length is within 800bp, so that the sequencing accuracy is ensured. The PCR system was 1. Mu.L of forward and reverse primer, 10. Mu.L of Rapid enzyme, 7. Mu.L of ddH, and 1. Mu.L of LDNA template 2 O. The PCR products were detected by agarose gel electrophoresis, and single band products were sent to the company for Sanger sequencing to see if overlapping peaks were present near the target site, if overlapping peaks indicated successful knockdown, if single band indicated failure.
As shown in fig. 4 and 5: the org gene sequence peak patterns of the wild-type and the experimental group of the non-dead embryos are single peaks, and 23 base mutations occur in the experimental group of the dead embryos, and continuous cover peaks respectively occur at a target site 1 and a target site 2, wherein the mutation of the target site 1 occurs on an antisense strand, and the mutation of the target site 2 occurs on a sense strand. No mutation at target site 3 occurred, indicating that sgRNA1 and sgRNA2 have the effect of directing the cleavage of cas9 protein. The total of 23 embryogenic base mutations in the experimental group was counted to be 36.5% of the dead embryos (63) in the experimental group and 20.5% in the whole experimental group (112). The loss of org-knocked-out embryo development in this experiment and eventual death, and the death or retardation of org-knock-out embryos may be caused by physical damage to the embryos.
Example 6: expression pattern of org gene in early embryo development:
O.melastigma-qF：CCCTGTCCCCAGAAATCC；
we designed primers based on medaka org gene sequence:
O.melastigma-qR：CTCCTGGCTCAGCGTTTC；
O.melastigma-actin-qF：CTGAGGATTGCTGAGCCTTACAT；
O.melastigma-actin-qR：CTCAACACAGATGATGCCGTATT。
the expression level of org gene in the early embryo development process of medaka was detected by RT-qPCR. As shown in FIG. 6, the expression level of org gene was increased slightly during the single cell period to blastula, and reached the highest value during blastula period, after which the expression level of org mRNA was decreased rapidly, and the expression of org gene was hardly detected during organogenesis. In this example, we explored the expression pattern of org gene in early embryo of medaka, the expression pattern of parent effector nmp is consistent, the expression is abundant in early zygotic gene activation stage, and it is degraded rapidly in the transformation process from parent to zygotic genetic control.
The development process of the org gene knockout embryo in the invention is consistent with the development phenotype of the parent effector genes nmp and foxr1 knockout, and the expression mode of the org gene and the parent effector gene nmp2 in the early embryo development process of the medaka is consistent, so that the org gene is proved to be taken as the parent effector gene, and the normal development of the embryo is ensured by participating in the activation process of the early embryo zygote gene. The knockout of the org gene fails to activate the early zygotic genome, the org gene may be involved in the translational activation of specific maternal mRNA in early embryo, after the org gene is knocked out, maternal products cannot accumulate before blastula stage, so that embryo cannot transit from maternal control to zygotic control, the totipotent type of embryo is destroyed, and embryo death is caused.
The invention is not limited to the specific embodiments described above, which are intended only to illustrate the use of the invention in detail, but also to the production method and the technical details of the same. Indeed, those skilled in the art will be able to find different adjustment schemes as needed from the foregoing description, and such adjustments are within the scope of the claims appended hereto.
Claims (2)
1. A medaka parent source effector gene org is characterized in that the nucleotide sequence of the gene is SEQ ID NO.1.
2. The sgRNA of the medaka parent effector org of claim 1, wherein the nucleotide sequence of the sgRNA is SEQ ID No.2-3.
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