CN114561391A - Clone and application of rice meiosis specific expression promoter - Google Patents
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Abstract
The invention discloses a clone and application of a rice meiosis specific expression promoter, wherein a nucleotide sequence of the rice meiosis specific expression promoter is shown as SEQ ID No.1, and a genetic engineering vector constructed by the rice meiosis specific expression promoter. The rice meiosis specific expression promoter is applied to regulation and control of expression of a specific gene, wherein the specific gene is a gene participating in both the meiosis process and the mitosis in the rice meiosis process. The invention has very important significance for the research of meiosis homologous recombination key genes and the genetic improvement of crops by cloning meiosis specific promoters.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to cloning and application of a rice meiosis specific expression promoter.
Background
Rice is one of the most important crops in the world, and rice is the staple food for about 50% of the global population. Under the conditions that the current population is rapidly increased and the cultivated land area is reduced year by year, the cultivation of high-yield rice is an important and difficult task in food production in China, and has important significance for guaranteeing food safety and sustainable development of national economy in China.
China mainly adopts a traditional crossbreeding mode, which generates genes and phenotypes with excellent characters by recombining or mutating genetic materials under natural and artificial conditions, thereby achieving the purpose of improving crop varieties. Although cross breeding can achieve integration of elite shapes within a species, it is time consuming, labor intensive, and requires years of breeding to obtain a stable phenotype. With the development of molecular biology, molecular breeding can effectively solve the problems, and particularly, the control of meiosis related genes by adopting a molecular biology technology can accelerate the integration of excellent characters more quickly, shorten the breeding period and further realize the rapid acquisition of varieties with stable excellent characters.
During meiosis, homologous recombination is a very key core event, which directly leads to recombination and exchange of genetic materials between homologous chromosomes, recombination exchange between homologous chromosomes and free combination between non-homologous chromosomes greatly increase genetic diversity of a hybrid population, improve environment adaptability of offspring and provide a genetic basis for species evolution and genetic improvement. The recombination event starts with the formation of a double-stranded break (DSB) catalyzed by the SPO11 protein. The MRN/X protein complex (MRE 11-RAD50-NBS1/XRS 2) catalyzes DSB to generate a 3 'single-chain end, and the 3' single-chain end invades into another homologous chromosome with a complete double chain under the action of RAD51 and DMC1 to form an intermediate (D-loop) with a D-loop structure. At this time, if DSB is repaired by Double Strand Break Repair pathway (DSBR), the other 3' endThe end single chain is captured by the D ring to form a dHJ (double horizontal junction) structure, and through a series of processing, a first type of exchange (CO) and a part of Non-exchange (NCO) product are finally formed, wherein the CO has interference characteristics, namely the generation of the CO at one position can inhibit the generation of another CO at an adjacent position. If DSB is repaired by relying on the MUS81-MMS4 pathway, a second class of CO with no interference characteristics can be formed. The formation of gene exchange marks the exchange of genetic material, but the number of crossovers between homologous chromosomes is tightly controlled. Only 1-2 crossovers between each pair of homologous chromosomes occur, a phenomenon known as "cross-assurance". The existence of low genetic recombination frequency and cross interference phenomenon causes the existence of close linkage relation between excellent character genes and unfavorable genes, seriously limits the polymerization between different excellent genes and brings great difficulty to the genetic breeding of crops. How to change the recombination sites to break bad linkage and improve the recombination frequency to shorten the breeding period is a great problem which troubles breeding experts for a long time. The genetic recombination frequency can be effectively improved by adopting the gene editing technology to control the meiosis process and the genetic recombination related gene, such as riceZEP1The gene knockout greatly improves the genetic recombination frequency of rice. However, some genes which are critical to the regulation of genetic recombination frequency are also involved in the regulation of mitosis process, and the knockout of the genes can affect the vegetative growth of crops and even cause the death of crop embryos. Interference (RNAi) of such genes using meiotic stage specific promoters can overcome such problems. In addition, the gene editing technology drives crop breeding into an accurate regulation era, the low editing efficiency is the biggest bottleneck of the technology in genetic breeding, and efficient gene editing can be realized in gametes by using meiosis specific promoters to drive the expression of CAS9 genes, so that a large amount of homozygous or biallelic mutants can be obtained in the T1 generation, and the problems of low rice genetic transformation efficiency and the like can be solved. Meanwhile, the meiosis specific period expression promoter is also suitable for systems such as CRISPR-activation, interference, base replacement and the like, and can realize the time-space regulation of gene editing, so that the gene editing is more accurate. Thus, cloning was reducedThe meiosis specific promoter has very important significance for researching meiosis homologous recombination key genes and improving the genetics of crops.
Disclosure of Invention
The invention aims to provide the clone of the rice meiosis specific expression promoter, and the clone of the meiosis specific expression promoter has very important significance on the research of meiosis homologous recombination key genes and the genetic improvement of crops.
The invention adopts the following technical scheme for realizing the purpose, and the rice meiosis specific expression promoter is characterized in that: the nucleotide sequence of the rice meiosis specific expression promoter is shown in SEQ ID NO. 1.
The invention relates to a gene engineering vector constructed by a rice meiosis specific expression promoter.
The rice meiosis specific expression promoter is applied to regulation and control of expression of a specific gene, wherein the specific gene is a gene participating in both the meiosis process and the mitosis in the rice meiosis process.
The rice meiosis specific expression promoter disclosed by the invention specifically reduces the specific expression of a specific gene in the meiosis period without influencing the mitosis process, so that a corresponding mutant of a related gene is obtained. In the meiosis process of rice, some specific genes are involved in both meiosis and mitosis, and when a gene editing technology is adopted to construct corresponding mutants of the specific genes, the embryo of the rice can be killed, so that the research on the functions of the specific genes is greatly hindered. The specific expression promoter ProMel1 for rice meiosis period is adopted to perform RNA interference on the specific genes, the expression of the specific genes in meiosis period can be specifically reduced without influencing the mitotic process, so that corresponding mutants of related specific genes are obtained, the function research of the specific genes becomes possible, and the promoter provides convenience for the research of the functions of meiosis key genes.
The rice meiosis specific expression promoter is applied to the regulation of CAS9 gene expression, and the specific process is as follows: when the CRISPR-Cas9 system is adopted to construct rice mutants, the rice meiosis specific expression promoter ProMEL1 is utilized to drive the expression of a CAS9 gene, and efficient gene editing can be realized in gametes, so that a large number of homozygous or biallelic mutants can be obtained in T1 generations, the problem of low rice genetic transformation efficiency is effectively solved, and an effective tool is provided for a rice transgenic technology.
The rice meiosis specific expression promoter is used for a CRISPR-activating, interfering and base replacing system, and can realize the time-space regulation of gene editing, so that the gene editing is more accurate.
Compared with the prior art, the invention has the following advantages and beneficial effects: the invention clones a rice meiosis specific promoter ProMel 1. Firstly, constructing a plant binary expression vector of the promoter for driving GUS gene, transforming wild type cultivated rice Nipponbare variety, and screening positive plants. GUS staining is carried out on the root, stem, leaf and leaf sheath of the positive plant and young ear tissues in the meiosis period, and the result shows that GUS is highly expressed only in the young ear in the meiosis period, which indicates that the promoter possibly only plays a role in the meiosis process of rice. Early laboratory proofs of meiosis in riceOsRMIThe knockout of the gene leads to the death of rice, and homozygous mutants can not be obtained, the ProMel1 promoter is adopted to construct the RNAi vector of the gene, the wild type cultivated rice Nipponbare variety is transformed, the result shows,OsRMIthe gene interferes with the vegetative growth of the plant and is the same as the wild type, and the expression level of the gene is obviously reduced in young ears only in the meiosis period. The results show that ProMel1 is a meiosis period specific promoter, lays a foundation for researching meiosis key genes, and provides important gene resources for crop genetic breeding.
Drawings
FIG. 1 is a schematic diagram of the construction of the GUS vector from ProMel 1; wherein (B) to (G) result of GUS staining at different parts of rice in meiosis stage;
FIG. 2 (A) shows pCAMBIA23 2391Z construction of ProMel1 vector; (B) WT and RNAi plant phenotype profiles; (C) - (G) detection chart of gene expression quantity of different tissues of OsRMI gene RNAi plant.
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Examples
1.1 materials and methods
1.1.1 plant Material
Cultivated rice variety Nipponbare
1.1.2 strains and plasmids
E .coliStrain DH5 alpha, Agrobacterium tumefaciens (A. tumefaciens) ((R))Agrobacterium tumefaciens) An EHA 105. Plant binary expression vectors pCAMBIA2391Z, pCAMBIA23A and RNAi intermediate vector pUCCRNAi are all stored in the laboratory.
1.1.3 tool enzymes and chemical reagents
Restriction enzymes Hind III, BamH I, Sal I, Bgl II and Xho I, high fidelity Taq DNA polymerase were purchased from Dalibao Bio. The seamless recombination kit MC40201 was purchased from a Monad organism.
1.2 methods
1.2.1 ProMel1 promoter cloning and pCAMBIA2391Z ProMel1 vector construction
Extracting rice leaf genome, amplifying and recovering ProMel1 sequence through PCR amplification; then, pCAMBIA2391Z was linearized with Hind III, the fragment was recovered, and ProMel1 was cloned into Hind III linearized pCAMBIA2391Z by a seamless recombination method, and sequenced for identification.
1.2.2 OsRMIConstruction of Gene interference vector
Selecting 200bpOsRMIAnd (3) amplifying the gene specific fragment by PCR, respectively adding Bam H1 and Sal I enzyme cutting sites at two ends of the fragment, recovering the fragment, and then cloning the fragment to a pUCCRNAi vector by two times of connection to form a hairpin structure. And (3) digesting and recovering the hairpin structure by using Pst I, cloning the hairpin structure onto a Pst I digested pCAMBIA23A vector, finally cloning the ProMel1 sequence to the upstream of the hairpin structure, and sequencing and identifying.
1.2.3 transgenic Rice obtention
The transgenic experiment is finished by Hangzhou Baige biotechnology company.
1.2.4 GUS staining
The GUS staining of rice tissue adopts Beijing Kulaibo science and technology Limited company kit, and the method refers to the instruction book
1.2.5 fluorescent quantitative PCR
The rice RNA extraction, reverse transcription and fluorescence quantitative PCR all adopt products of Dalianbao bio-company, and the method is shown in the specification.
2 results of the experiment
2.1 ProMel1 promoter is a promoter specifically expressed during meiosis
Firstly, a ProMel1 is constructed, namely a GUS plant binary expression vector (A in figure 1) is consigned to Hangzhou Baige biotechnology company to transform a wild type Nipponbare variety and screen a positive plant. And (3) planting the positive seedlings in a test field, taking rice roots, stems, leaves, leaf sheaths and young ears in the meiosis period when the rice enters the meiosis period, and carrying out GUS staining on the rice roots, the stems, the leaves and the leaf sheaths, wherein the result shows that the rice roots, the stems, the leaves and the leaf sheaths have no GUS signals, and the young ears have strong GUS signals, thereby indicating a promoter specifically expressed by the ProMel1 promoter in the meiosis period.
FIG. 1: (A) ProMel1, GUS vector construction diagram; (B) rice root GUS staining results; (C) the method comprises the following steps Rice stem GUS staining results; (D) the method comprises the following steps GUS staining of rice leaves; (E) the method comprises the following steps GUS staining results of rice leaf sheaths; (F) (G): and (5) performing GUS (glucuronidase) staining on young ears of rice.
2.2 RNAi driven by ProMel1 promoter to specifically reduce gene expression during meiosis
Rice meiosis key gene cloned in early stage of laboratoryOsRMIIn order to research the effect of the gene in the meiosis process, the gene is edited by adopting a gene editing technology, and the result indicates that the mutation of the gene causes death of rice embryos and homozygous mutants cannot be obtained. Therefore, the promoter ProMel1 is adopted to construct an RNAi vector (A in figure 2) of the gene, wild rice is transformed, positive transgenic plants are screened, and the expression quantity of the gene is analyzed. The results showed that RNAi plants grew normally (B in FIG. 2) and were fluorescentThe results of the light quantitative PCR detection show that the expression level of the gene is reduced only in the young ears of the rice at the meiosis stage compared with the wild rice, and the expression level of the gene is not obviously changed in the root, stem, leaf and leaf sheath tissues compared with the wild rice (C-G in figure 2). The result further indicates that the ProMel1 promoter is a meiosis period specific promoter, and provides convenience for researching the functions of meiosis key genes.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
SEQUENCE LISTING
<110> university of south Henan university
<120> cloning and application of rice meiosis specific expression promoter
<130> 2022
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3200
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gtcacatcgg atataaggac acatatttga agtattaaat atagtctaat aataaaataa 60
attacagatt ccgccagaaa attacgagat gaatttatta agcctaatta atccaccatt 120
tattaatgtt tactatagca ccatattatc aaatcatggc gcgattaggc ttaaaagatt 180
tgtctcgaaa tttatacgta aatgtgtaat tatttttttt tcacatttaa tacctcatag 240
atgtgtccaa acatttaggt ggtgtttgga tacagggact taactttagt ccctgtattt 300
agacactaat ttagagtatt aaatatagac tacttacaaa actaattata taaatgaaag 360
ctaatttgcg agataaattt tttaagccta attaatccac aattagagaa tgtttaatgt 420
agcatcacat aggctaatca tggattaatt aggctcaata gatttgtctc gcgaattagt 480
ccaagattat gaatgggttt tattaataat ctatgtttaa tatttataat tagtgtctaa 540
acattcgata ttatagggac ttaaaagttt tagtcccatc taaacagggt cttaggggtg 600
tttgggagag aggggctaaa ctttagcccc tctctctagc ccctctctca aaaacataag 660
tccctctcgt tttgtagaga ggggctaaac tttagcccca ctttagtccc tccaaccaaa 720
caccacctta atatgatggg tgaaaagttt ttattttgag aactaaggcc tcagtctcat 780
gcaattccat ggtaggcagt cacacacacc cactcgcggt gtctcgtctg atcgtctcgc 840
aaacagagaa gcatgggttt gggttaccca ttgtatgcat ggtcacagcc tcacagcttg 900
ccagtattgc caacttaggc cagcacgatc acccaccctt ttgcttttgg ccgcggtgta 960
cgctgctcct gtctctgata cctgcagccg ctgtcccctc cccccacggt gcagcgcatc 1020
gcagcgccag agatgctttg ccgtttgcgg ctcgaaccgc tgcgtgcttc gcttgaccac 1080
cagtccacca ccaacccctc ccctacacgc tcctcgcctc cgcggtttgg tcgtagtcga 1140
atgcgatatt gcgaggttgc ggcagcagcg gcagcagaga tttgcacagg ggaggagggc 1200
gcgaggatgc caaaagcggc atgggaaaag gagcgcatcg tgtactgtac acacctcgtc 1260
gtttttacca ccagtccacc acgcatgcac tgcgcagctc ttcttctatt ttgttctgcg 1320
ctatgataaa aaacggaccg ggatagagac agtaaaattt gctgcatgca atgttcctac 1380
tcgcaggtta tattagtgat cagaaaaacc gagccgcatg tcagcagcca ctacagaata 1440
cagaacataa ttttaacccg ggcttggaga taatgttaac tgcagttaaa atcacacagt 1500
ccacacacac ttttaacccg ggtttggaga cgatcaaaaa attttgctct acctttttcc 1560
ttttgctttt cgactactgc atgcagtctg cgccgcaacg tatacatgca tggatgagct 1620
gggccgaccg cttggacgca tctgcatgtt cccatagcct cagctttcac cggaagaacg 1680
gctgctacag ccatcgaccg cctctggctc gatcgctggg tggctctcgc agccaagacg 1740
atctgatacg atctctttgc agagcgagag agagaggggc gagcagtgat ccagtttttc 1800
aaacgtgaca cgtatgccga tcgaatcgcc gatagacgat cacaccatat agtatcgttt 1860
ttccagtgta aagttctact cgatcatgat tgatcgagtt cctctctcag gagtacagag 1920
tagctagctt aatagtatat agattattta tcttacccat gattaaaact acctagccat 1980
taattaggag tgaagatatt ttcaccttgg gtacaataca gtgtgagaat gacaggtggg 2040
cccaagccgt acgagagata attccaaggc gcatgagaga gagactggct acagatgcat 2100
gcatgggtca ttcgttcctc cagaatcttc ttctttccgt ccggtgtgat cgggttgtga 2160
cttgtcttgg aggtgtggac tcatctcatc actcactgca tgcgtccacg tgctttgcct 2220
tgcacatgta tattcacatc tcaattctca aatatttcta actgcagtaa aaatcaatgt 2280
tgcctatacg cttcgtcttg accatccctt catgaaggta attagaacac gacttgattt 2340
taaatcaaac cttttctacc catagcgatc aaaatggtcc aagatagagg cattacgtgg 2400
cagcaattcg agactcgatc gctgggaaaa tgagaatact aggcaacagt aatttcaatc 2460
ggtaggtttt caaaaaagta atttcagtct gatccttcca gctggtacgc atgcacttct 2520
agctagtttt ttttatgcat tattgatctg tatagtattt aatttgtcaa attttggaca 2580
atgtatatca caaaaaccac accaaacata tataacttca atattaatta cgagaaaaca 2640
aaacggaagg atggtgtcaa atgggatact acactagacg tacggccttt tttgccattt 2700
gcagtttaac tttaaacatt atgaattgct acactcgtac acttgactat acatatgtat 2760
aacttcacgt tcatagcttg gttctataca atatcttaac acccaaatta ataacaaaac 2820
aaactcagat taattagttc accccgatcg atgaacttaa tgacatataa ttttccttca 2880
aaaaacacaa aaaaaaaccg gacataaatt cactttctat aatgacacat aatttgtctt 2940
gggtagcaag tttgattaac cggccgccta attaatcact cttccgtacg gacaagcata 3000
cagtcaccgc gcggtaagat caccaccccc ttctccctcc caccgcgtct agctagggtt 3060
tctcctcgtc gatcaccctc cgcctataaa atccccctcc ccttccattc ccgcaaggcc 3120
gcaacatcac tcacccactc cccacccctt cccaaaccct cacctccatc tcgttgcctt 3180
cctccacctc tctctctgcc 3200
Sequence listing
<110> university of south Henan university
<120> cloning and application of rice meiosis specific expression promoter
<130> 2022
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3200
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gtcacatcgg atataaggac acatatttga agtattaaat atagtctaat aataaaataa 60
attacagatt ccgccagaaa attacgagat gaatttatta agcctaatta atccaccatt 120
tattaatgtt tactatagca ccatattatc aaatcatggc gcgattaggc ttaaaagatt 180
tgtctcgaaa tttatacgta aatgtgtaat tatttttttt tcacatttaa tacctcatag 240
atgtgtccaa acatttaggt ggtgtttgga tacagggact taactttagt ccctgtattt 300
agacactaat ttagagtatt aaatatagac tacttacaaa actaattata taaatgaaag 360
ctaatttgcg agataaattt tttaagccta attaatccac aattagagaa tgtttaatgt 420
agcatcacat aggctaatca tggattaatt aggctcaata gatttgtctc gcgaattagt 480
ccaagattat gaatgggttt tattaataat ctatgtttaa tatttataat tagtgtctaa 540
acattcgata ttatagggac ttaaaagttt tagtcccatc taaacagggt cttaggggtg 600
tttgggagag aggggctaaa ctttagcccc tctctctagc ccctctctca aaaacataag 660
tccctctcgt tttgtagaga ggggctaaac tttagcccca ctttagtccc tccaaccaaa 720
caccacctta atatgatggg tgaaaagttt ttattttgag aactaaggcc tcagtctcat 780
gcaattccat ggtaggcagt cacacacacc cactcgcggt gtctcgtctg atcgtctcgc 840
aaacagagaa gcatgggttt gggttaccca ttgtatgcat ggtcacagcc tcacagcttg 900
ccagtattgc caacttaggc cagcacgatc acccaccctt ttgcttttgg ccgcggtgta 960
cgctgctcct gtctctgata cctgcagccg ctgtcccctc cccccacggt gcagcgcatc 1020
gcagcgccag agatgctttg ccgtttgcgg ctcgaaccgc tgcgtgcttc gcttgaccac 1080
cagtccacca ccaacccctc ccctacacgc tcctcgcctc cgcggtttgg tcgtagtcga 1140
atgcgatatt gcgaggttgc ggcagcagcg gcagcagaga tttgcacagg ggaggagggc 1200
gcgaggatgc caaaagcggc atgggaaaag gagcgcatcg tgtactgtac acacctcgtc 1260
gtttttacca ccagtccacc acgcatgcac tgcgcagctc ttcttctatt ttgttctgcg 1320
ctatgataaa aaacggaccg ggatagagac agtaaaattt gctgcatgca atgttcctac 1380
tcgcaggtta tattagtgat cagaaaaacc gagccgcatg tcagcagcca ctacagaata 1440
cagaacataa ttttaacccg ggcttggaga taatgttaac tgcagttaaa atcacacagt 1500
ccacacacac ttttaacccg ggtttggaga cgatcaaaaa attttgctct acctttttcc 1560
ttttgctttt cgactactgc atgcagtctg cgccgcaacg tatacatgca tggatgagct 1620
gggccgaccg cttggacgca tctgcatgtt cccatagcct cagctttcac cggaagaacg 1680
gctgctacag ccatcgaccg cctctggctc gatcgctggg tggctctcgc agccaagacg 1740
atctgatacg atctctttgc agagcgagag agagaggggc gagcagtgat ccagtttttc 1800
aaacgtgaca cgtatgccga tcgaatcgcc gatagacgat cacaccatat agtatcgttt 1860
ttccagtgta aagttctact cgatcatgat tgatcgagtt cctctctcag gagtacagag 1920
tagctagctt aatagtatat agattattta tcttacccat gattaaaact acctagccat 1980
taattaggag tgaagatatt ttcaccttgg gtacaataca gtgtgagaat gacaggtggg 2040
cccaagccgt acgagagata attccaaggc gcatgagaga gagactggct acagatgcat 2100
gcatgggtca ttcgttcctc cagaatcttc ttctttccgt ccggtgtgat cgggttgtga 2160
cttgtcttgg aggtgtggac tcatctcatc actcactgca tgcgtccacg tgctttgcct 2220
tgcacatgta tattcacatc tcaattctca aatatttcta actgcagtaa aaatcaatgt 2280
tgcctatacg cttcgtcttg accatccctt catgaaggta attagaacac gacttgattt 2340
taaatcaaac cttttctacc catagcgatc aaaatggtcc aagatagagg cattacgtgg 2400
cagcaattcg agactcgatc gctgggaaaa tgagaatact aggcaacagt aatttcaatc 2460
ggtaggtttt caaaaaagta atttcagtct gatccttcca gctggtacgc atgcacttct 2520
agctagtttt ttttatgcat tattgatctg tatagtattt aatttgtcaa attttggaca 2580
atgtatatca caaaaaccac accaaacata tataacttca atattaatta cgagaaaaca 2640
aaacggaagg atggtgtcaa atgggatact acactagacg tacggccttt tttgccattt 2700
gcagtttaac tttaaacatt atgaattgct acactcgtac acttgactat acatatgtat 2760
aacttcacgt tcatagcttg gttctataca atatcttaac acccaaatta ataacaaaac 2820
aaactcagat taattagttc accccgatcg atgaacttaa tgacatataa ttttccttca 2880
aaaaacacaa aaaaaaaccg gacataaatt cactttctat aatgacacat aatttgtctt 2940
gggtagcaag tttgattaac cggccgccta attaatcact cttccgtacg gacaagcata 3000
cagtcaccgc gcggtaagat caccaccccc ttctccctcc caccgcgtct agctagggtt 3060
tctcctcgtc gatcaccctc cgcctataaa atccccctcc ccttccattc ccgcaaggcc 3120
gcaacatcac tcacccactc cccacccctt cccaaaccct cacctccatc tcgttgcctt 3180
cctccacctc tctctctgcc 3200
Claims (6)
1. A rice meiosis specific expression promoter is characterized in that: the nucleotide sequence of the rice meiosis specific expression promoter is shown in SEQ ID NO. 1.
2. A genetically engineered vector constructed from the meiosis-specific expression promoter of rice according to claim 1.
3. The use of the rice meiosis-specific expression promoter according to claim 1 for regulating the expression of a specific gene, wherein the specific gene is a gene involved in both meiosis and mitosis in rice meiosis.
4. The rice meiosis-specific expression promoter of claim 1, which can specifically reduce the expression of a specific gene during meiosis without affecting mitotic processes, thereby obtaining a mutant corresponding to the gene.
5. The application of the rice meiosis-specific expression promoter of claim 1 in regulating and controlling CAS9 gene expression is characterized by comprising the following specific processes: when the CRISPR-Cas9 system is adopted to construct rice mutants, the rice meiosis specific expression promoter ProMEL1 is utilized to drive the expression of a CAS9 gene, and efficient gene editing can be realized in gametes, so that a large number of homozygous or biallelic mutants can be obtained in T1 generations, the problem of low rice genetic transformation efficiency is effectively solved, and an effective tool is provided for a rice transgenic technology.
6. The rice meiosis specific expression promoter of claim 1 is used in CRISPR-activation, interference, base substitution system, and can realize temporal and spatial regulation of gene editing, so that the gene editing is more accurate.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921774A (en) * | 2010-05-24 | 2010-12-22 | 南京大学 | Application of Dnaj-like protein and encoded gene thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921774A (en) * | 2010-05-24 | 2010-12-22 | 南京大学 | Application of Dnaj-like protein and encoded gene thereof |
Non-Patent Citations (2)
| Title |
|---|
| WEI LIU等: "Reproductive tissue-specific translatome of a rice thermosensitive genic male sterile line", JOURNAL OF GENETICS AND GENOMICS, pages 631 * |
| 李凤丽;石素华;李爱芹;王兴军;赵术珍;: "CRISPR/Cas9基因编辑技术研究进展", 山东农业科学, no. 04, pages 168 * |
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