CN110218810B - Promoter for regulating and controlling maize tassel configuration, molecular marker and application thereof - Google Patents
Promoter for regulating and controlling maize tassel configuration, molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a promoter for regulating and controlling a maize tassel configuration, a molecular marker and application thereof. The key promoter or the mutant thereof for regulating the maize tassel configuration can regulate the expression of TSH4 gene in the maize tassel, can be applied to improve the maize tassel branch number and plant type, and can also be further applied to culture new maize varieties. The invention further provides a detection primer for detecting the TSH4 gene promoter variation condition and a detection primer for detecting the TSH4 gene expression quantity in the corn, and the primers can be applied to directionally improving the tassel branch number of the corn and have application potential for corn dense planting resistance and high-yield breeding.
Description
Technical Field
The invention relates to a DNA sequence related to the number of tassel branches of corn, in particular to a promoter sequence for regulating and controlling the tassel configuration of the corn, a mutant, a molecular marker and a detection primer thereof, and further relates to application of the promoter sequence and the mutant in corn molecular breeding, belonging to the fields of a promoter for regulating and controlling the tassel configuration of the corn, a mutant thereof and application thereof.
Background
Tassels are important reproductive organs of corn, which affect the canopy composition of corn and the distribution of photosynthetic products, thereby affecting yield. The tassel grows at the top of the corn plant, and the size of the tassel directly influences the ventilation and light transmission conditions in the corn population (the shielding illumination can reach 6.5-28.8%); moreover, excessive pollen scattering (especially, too large tassel) caused by tassel can cause dust on the lower leaf to adhere to affect photosynthesis and supply of "source". In addition, the tassel is also an important "storehouse" organ, and develops earlier than the ear, and is obviously superior to the ear in nutrition competition, and the two competition directly influences the harvest yield of the corn. In general, the size of the tassel is in negative correlation with yield, the correlation coefficient reaches-0.65, and the effect of the tassel in the total variation of yield can reach 37.4%; the production practice also proves that the corn yield can be obviously improved by 5.3-16.0% after the corn is subjected to emasculation after the corn is subjected to pollen scattering, and the important relation between tassels and the yield is reflected.
The tassel configuration affects the close-planting tolerant breeding of corn. Research finds that the improvement of the yield per plant and the heterosis of the corn is not obvious in the corn breeding process in the past decades, and the increase of the yield per plant is more due to the continuous increase of the planting density and the variety tightness. Therefore, the key to improve the density of the variety and the planting density is to improve the yield per unit of the corn.
Under the condition of close planting, the distance between corn plants is closer and the competition is more intense. The large tassels tend to shade, influence photosynthesis, aggravate nutrition competition with the female tassels, waste resources and directly influence yield. Meanwhile, the gravity center of the corn plant must be increased by the large tassel, so that the phenomenon of heavy head and light feet is caused, the lodging risk of the corn is increased, and the yield is indirectly influenced. In the past decades, the size of the tassel of the corn is subjected to continuous high-strength selection, particularly in a commercial breeding unit with a faster breeding process, the tassel is reduced by even more than 36%, and the significance of the improvement of the tassel size on the corn tight-planting-resistant breeding is revealed.
The existing research shows that TSH4 is a key gene for regulating the development of maize tassels and female ears, but the mutant of the gene brings many unfavorable traits and cannot be directly applied to the breeding practice of maize. TSH4 was first cloned and reported by the Sarah Hake laboratory in Cold spring harbor, USA. TSH4 encodes a SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor under the control of miR 156. The gene plays a key role in the establishment of the boundaries of floral organs and the determination of the fate of the boundary meristem. The gene can obviously reduce the branchnumber of the maize tassel after mutation, but also can reduce the small number of tassel flowers, coat bracts of the small flowers, cluster bracts at the bases of the tassel and the female ear, disorder of the ear row number of the female ear, reduction of the ear row number and the ear grain number and other phenotypes which are unfavorable for maize production. It has been found that the regulation of expression of key genes, rather than a change in function, serves to fine-tune specific genes to achieve improved target traits without adverse effects. The corn is subjected to long-term natural and artificial selection, and is likely to contain the variant resource of the expression regulation, and finding out the variant and developing the corresponding molecular marker can play a great help role in specifically improving the tassel branch number of the corn (but not influencing other agronomic traits).
Disclosure of Invention
One of the purposes of the invention is to provide a promoter sequence for regulating and controlling the configuration of a maize tassel;
the other purpose of the invention is to provide a mutant of the promoter sequence for regulating and controlling the maize tassel configuration;
the invention also aims to provide a molecular marker for regulating the expression of the TSH4 gene in the maize tassel;
the fourth purpose of the invention is to provide a detection primer for detecting the variation condition of the molecular marker;
the fifth purpose of the invention is to provide a detection primer for detecting the TSH4 gene expression level in corn;
the sixth purpose of the invention is to apply the promoter sequence, the mutant thereof, the molecular marker, the detection primer for detecting the variation condition of the molecular marker and the detection primer for detecting the TSH4 gene expression quantity in the corn to the corn molecular breeding.
The above object of the present invention is achieved by the following technical solutions:
the invention firstly provides a promoter sequence for regulating and controlling the configuration of a maize tassel, and polynucleotides of the promoter sequence are shown in (a), (b), (c) or (d):
(a) a polynucleotide shown as SEQ ID No. 1; or
(b) A polynucleotide capable of hybridizing to the complement of SEQ ID No.1 under stringent hybridization conditions; or
(c) A polynucleotide having at least 90% or more homology with the polynucleotide represented by SEQ ID No. 1; or
(d) The mutant obtained by deletion, substitution or insertion of one or more basic groups is carried out on the basis of the polynucleotide shown in SEQ ID NO.1, and the mutant still has the function or activity of a promoter for regulating and controlling the configuration of the maize tassel.
The promoter sequence can regulate and control the expression quantity change of the TSH4 gene in the maize tassel.
In the context of the present invention, the term "mutant" is a promoter containing a variation in which one or more nucleotides of the original promoter are deleted, added and/or substituted, preferably while substantially maintaining the function of the promoter. For example, one or more base pairs may be deleted from the 5 'or 3' end of a promoter to produce a "truncated" promoter; one or more base pairs can also be inserted, deleted or substituted within the promoter. In the case of a promoter fragment, a mutant of the promoter may comprise alterations that affect transcription of the minimal promoter to which it is operably linked. Variant promoters may be generated, for example, by standard DNA mutagenesis techniques or by chemical synthesis of variant promoters or portions thereof. Mutant polynucleotides also include polynucleotides of synthetic origin, e.g., mutants obtained by site-directed mutagenesis, or by recombinant means (e.g., DNA shuffling), or by natural selection.
As a preferred embodiment of the mutant, the invention provides a mutant of a promoter for regulating the maize tassel configuration, the polynucleotide sequence of the mutant is shown as SEQ ID No.2, the mutant is lack of 1bp base insertion at Indel _7_133209283_ C/CT, the mutant can regulate the expression of TSH4 gene in maize tassels, shows reduced tassel branch number and does not bring about other unfavorable phenotypes.
Thus, Indel _7_133209283_ C/CT can be used as a molecular marker for regulating the expression of the TSH4 gene in maize tassels.
The invention further provides a detection primer for detecting the variant condition of Indel _7_133209283_ C/CT; as a preferred embodiment, the nucleotide sequences of the specific detection primers for detecting Indel _7_133209283_ C/CT variation are respectively shown as SEQ ID No.3 and SEQ ID No. 4; the specific detection primer can be used for detecting the variation condition of Indel _7_133209283_ C/CT in the corn variety, thereby being used for molecular assisted breeding of corn.
The invention further provides a specific amplification primer for detecting the TSH4 gene expression level and application thereof in corn breeding, wherein the sequences of the specific detection primer are respectively shown as SEQ ID No.5 and SEQ ID No.6, and the application of the specific detection primer can detect the TSH4 gene expression level of a corn variety, thereby providing reference for corn breeding.
Furthermore, the expression cassette containing the promoter shown in SEQ ID No.1 or the mutant of the promoter shown in SEQ ID No.2, the recombinant plant expression vector containing the expression cassette, the transgenic cell line and the host bacterium belong to the protection scope of the invention.
The recombinant plant expression vector is constructed by the expression cassette and a plasmid or an expression vector and can be transferred into plant host cells, tissues or organs.
The promoter or the mutant thereof of the present invention can be used for preparing transgenic plants. For example, a recombinant plant expression vector containing the promoter or a mutant thereof is introduced into a plant cell, tissue or organ by Agrobacterium-mediated or particle gun method, and the transformed plant cell, tissue or organ is cultured into a plant to obtain a transgenic plant. The starting vector for constructing the plant expression vector can be any binary vector for transforming the plant by agrobacterium or a vector for plant microprojectile bombardment and the like.
Conventional compositions and methods for making and using plant expression vectors and host cells are well known to those skilled in the art for practicing the present invention, and specific methods can be found in, for example, Sambrook et al.
The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. The marker gene comprises: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also comprises phenotypic markers, such as beta-galactosidase, fluorescent protein and the like.
In conclusion, the promoter sequence, the mutant of the promoter sequence, the molecular marker Indel _7_133209283_ C/CT, the specific detection primer for detecting the variation condition of Indel _7_133209283_ C/CT, the specific detection primer for detecting the TSH4 gene expression level and the like provided by the invention can be applied to breeding of a new high-yield or close-planting-resistant corn variety, particularly applied to the aspects of improving the male ear configuration, increasing the corn yield and the like.
For reference, the present invention provides a method for regulating tassel branch number of maize, comprising: the promoter shown in SEQ ID No.1 or the mutant of the promoter shown in SEQ ID No.2 is used for regulating and controlling the expression of the TSH4 gene in corn.
For reference, the present invention also provides a method for breeding a new variety of high-yield or tight-planting-resistant maize, comprising: (1) connecting TSH4 gene at the downstream of the mutant of the promoter shown in SEQ ID No.2 to form an expression cassette; connecting the expression cassette with a plant expression vector to construct a recombinant plant expression vector; (2) transforming the constructed recombinant plant expression vector into corn; (3) screening to obtain a new transgenic plant variety with reduced tassel branch number; or further breeding to obtain a new variety of high-yield or close planting-resistant corn.
The transformation protocol described in the present invention and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for introducing the polynucleotide or polypeptide into a plant cell include: microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment, among others. In particular embodiments, the expression cassettes of the invention can be provided to plants using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al plant Cell reports.1986.5: 81-84).
The present invention can be used to transform any plant species, including but not limited to: monocotyledonous or dicotyledonous plants, preferably maize.
The promoter or mutant thereof for regulating the maize tassel configuration can regulate the expression of the TSH4 gene in the maize tassel, has important significance for improving the maize tassel branch number and the plant type, and can be further applied to the breeding of new maize varieties. The invention further provides a molecular marker Indel _7_133209283_ C/CT used for regulating and controlling the TSH4 gene expression, a specific detection primer for detecting the variation condition of the molecular marker Indel _7_133209283_ C/CT and a specific detection primer for detecting the TSH4 gene expression quantity in the corn, which can be directly applied to directionally improving the tassel branch number of the corn and have important application potential for the corn density-resistant planting and the high-yield new variety breeding.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.
The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).
The term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide position identity (i.e., sequence similarity or identity). The term homology, as used herein, also refers to the concept of similar functional properties between different polynucleotide molecules, e.g., promoters with similar functions may have homologous cis-elements. Polynucleotide molecules are homologous when they specifically hybridize under specific conditions to form a duplex molecule. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule can be used as a probe or primer for identifying another polynucleotide molecule that shares homology.
The term "stringent hybridization conditions" as used herein means low as known in the artIonic strength and high temperature conditions. In general, Probes hybridize to their target sequences to a greater extent than to other sequences under stringent conditions (e.g., at least 2-fold over background. stringent Hybridization conditions are sequence-dependent and will differ under different environmental conditions, longer sequences specifically hybridize at higher temperatures. target sequences that are 100% complementary to Probes can be identified by controlling the stringency of Hybridization or wash conditionsm) About 5-10 ℃. T ismIs the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at T because the target sequence is present in excessmAt equilibrium 50% of the probes are occupied). Stringent conditions may be as follows: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60 ℃ for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃; or 5 XSSC, 1% SDS, incubated at 65 ℃, washed in 0.2 XSSC and washed in 0.1% SDS at 65 ℃. The washing may be for 5, 15, 30, 60, 120 minutes or more.
The term "plurality" as used herein generally means 2 to 8, preferably 2 to 4; the "substitution" refers to the substitution of one or more amino acid residues with different amino acid residues, respectively; the term "deletion" refers to a reduction in the number of amino acid residues, i.e., the absence of one or more amino acid residues, respectively; by "insertion" is meant a change in the sequence of amino acid residues that results in the addition of one or more amino acid residues relative to the native molecule.
The term "coding sequence": a nucleic acid sequence transcribed into RNA.
The term "promoter" refers to a polynucleotide molecule that is located upstream or 5' of the translation initiation codon of the open reading frame (or protein coding region) in its native state and that is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.
The term "plant promoter" is a native or non-native promoter that is functional in a plant cell. Constitutive plant promoters function in most or all tissues throughout plant development. Any plant promoter can be used as a 5' regulatory element for regulating the expression of one or more specific genes operably linked thereto. When operably linked to a transcribable polynucleotide molecule, a promoter typically causes transcription of the transcribable polynucleotide molecule in a manner similar to the transcription of the transcribable polynucleotide molecule to which the promoter is typically linked. Plant promoters may include promoters produced by manipulating known promoters to produce artificial, chimeric, or hybrid promoters. Such promoters may also be combined with cis-elements from one or more promoters, for example, by adding heterologous regulatory elements to an active promoter having some or all of its own regulatory elements.
The term "cis-element" refers to a cis-acting transcriptional regulatory element that confers an aspect of overall control over gene expression. The cis-element may function to bind transcription factors, trans-acting protein factors, which regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with more than one cis-element with different affinities.
The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule, and more preferably are contiguous. For example, a promoter is operably linked to a gene of interest if it regulates or mediates transcription of the gene of interest within the cell.
The term "transcribable polynucleotide molecule" refers to any polynucleotide molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into cells in such a way that transcribable polynucleotide molecules are transcribed into functional mRNA molecules which are translated and thereby expressed as protein products. Constructs capable of expressing antisense RNA molecules can also be constructed in order to inhibit translation of a particular RNA molecule of interest.
The term "recombinant plant expression vector": one or more DNA vectors for effecting plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.
The term "conversion": a method for introducing a heterologous DNA sequence into a host cell or organism.
The term "expression": transcription and/or translation of endogenous genes or transgenes in plant cells.
The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.
Drawings
FIG. 1A shows significant associations between Indel _7_133209283_ C/CT and maize tassel branch number phenotype obtained by GWAS method; b is a gene structure schematic diagram of TSH 4; indel _7_133209283_ C/CT is located in the promoter region 2.86kb upstream of the TSH4 gene.
FIG. 2 shows the variation of the inbred line of 12 different groups at Indel _7_133209283_ C/CT.
FIG. 3A shows the tassel phenotype of inbred lines of different variation types at Indel _7_133209283_ C/CT; wherein Indel _1 and Indel _0 represent genotypes with and without 1bp insertion, respectively; b is the statistics of the number phenotype of tassel branches of the inbred line in FIG. 2A; c is the expression analysis of TSH4 gene in 2mm tassel of two inbred lines in FIG. 2A.
Fig. 4 is a schematic diagram of CRISPR/Cas9 editing vector of TSH4 gene.
Fig. 5A is a schematic of the genetic structure of TSH4 and the corresponding target sequence of the CRISPR/Cas9 editing vector; b is a detection schematic diagram of the tsh4-MT mutant.
FIG. 6A is a photograph of the tassel phenotype corresponding to wild type material and mutant tsh 4-MT; b is the statistics of the tassel phenotype corresponding to wild type material and mutant tsh4-MT (n > 10).
FIG. 7A is a statistical analysis of the phenotypes of two allelic variants for Indel-7-133209283-C/CT; b is selected analysis of Indel _7_133209283_ C/CT in the corn breeding process; the colors corresponding to different allelic variations are the same as those in FIG. A.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1Indel _7_133209283_ C/CT regulates maize tassel branch number by controlling gene expression of TSH4
1. Mining of Indel _7_133209283_ C/CT
Depth using 350 parts of maize inbred line (>10X) the published genome of maize B73V3 was combined with re-sequenced data to discover 25,320,664 single nucleotide polymorphism molecular markers (SNPs) and 4,319,510 Indel polymorphism molecular markers (indels). The Indel flag includes Indel _7_133209283_ C/CT. The colony structure and the genetic relationship of 350 maize inbred lines are estimated by utilizing the mined molecular markers, and then the genome-wide association analysis (GWAS) is carried out by combining the collected phenotypes of the tassel branch numbers of 4 environments. The SNPs Chr7_133305039 and Indel _7_133209283_ C/CT on chromosome 7 were found to be significantly associated with the number of tassel branches in maize (FIG. 1A). And there is a significant linkage disequilibrium (r) between these two molecular markers20.53). Further studies found that Indel _7_133209283_ C/CT is located in the promoter region of maize tassel development regulatory gene TSH4 (SEQ ID No.2) 2.8kb from the coding region of the TSH4 gene (FIG. 1B).
2. Obtaining nucleotide sequence of genomic region where Indel _7_133209283_ C/CT is located
Specific amplification primers were designed from the B73V3 genomic region of the region where Indel-7-133209283-C/CT was located:
TSH4_Indel_F：5'-CGCTGTGCAATTTCATCAGT-3'(SEQ ID No.3)
TSH4_Indel_R：5'-TATTCAATGGCATCGTCAAG-3'(SEQ ID No.4)
the nucleotide sequence of this region and accurate information of 5 this variation were obtained by amplification of 12 different populations of maize inbred lines (FIG. 2).
3. Indel _7_133209283_ C/CT regulates expression of TSH4 gene in maize tassel
The inbred line containing the 1bp insertion and the inbred line not containing the 1bp insertion at Indel _7_133209283_ C/CT are respectively planted in the field, and when the male ear growth cone grows to 2mm long (the period is the key period for the male ear to branch), sampling and quick freezing by liquid nitrogen are carried out. Every 15 tassels of 2mm were mixed into one sample, and 3 biological replicates were taken from each inbred line. Then, RNA is extracted by a TRIzol method, and the expression level of the TSH4 gene is detected by using the following specific primers:
TSH4-F：5'-GCGCGATGAACTGGTCCCTGTA-3'(SEQ ID No.5)
TSH4-R：5'-ACGGCAAGGTAAGGCGCACA-3'(SEQ ID No.6)
the detection result shows that TSH4 gene in the tassel of the inbred line containing 1bp insertion at Indel _7_133209283_ C/CT is significantly highly expressed (figure 3). Indicating that Indel _7_133209283_ C/CT can regulate the expression of TSH4 gene in maize tassels.
Example 2 construction and phenotypic Observation of TSH4 mutant TSH4-MT
1. Selection of CRISPR/Cas9 editing target sequence of TSH4
First, the target editing sequence of the TSH4 gene was initially searched using SnapGene Viewer software and the B73V3 gene sequence of TSH 4. The obtained sequences were then aligned with other genomic sequences of B73 from maize to remove multiple copies and sequences that are highly similar to other genomic regions. The sgRNAs were further designed based on the finally determined target sequences and by RNA Folding Form: (http://unafold.rna.albany.edu/？q＝mfold/RNA-Folding-Form2.3) The system is used for predicting the secondary structure, and finally two optimal target sequences are selected and designed for editing the TSH4 gene (figure 5).
2. Construction of CRISPR/Cas9 gene editing vector of TSH4
The hSpCas9 sequence In human was used as a commercial In-
Cloning the PCR Cloning Kit into a pCPB vector to construct a pCPB-ZmUbi hSpCas9 vector, and introducing the target sequence obtained in the step 1 into a sgRNA expression cassette by using an overlapping PCR method; then, two sgRNA expression cassettes were passed through In-
The HD Cloning Kit is inserted into HindIII enzyme cutting sites of pCPB-ZmUbi: hSpCas9, and the finally constructed CRISPR/Cas9 gene editing vector (shown in figure 4) is used for subsequent genetic transformation after being verified to be correct by PCR sequencing.
3. Obtaining of mutant tsh4-MT
And (3) transforming the CRISPR/Cas9 vector obtained in the step 2 into a maize inbred line C01 through agrobacterium-mediated transformation. Genotype detection is carried out on T0 generation transformed seedlings by using primers designed aiming at target sequences, wherein the primer sequences are as follows:
7588-11F：5’-CACGAGCCGTTGACCTACTACT-3’(SEQ ID No.7)
7588-11R：5’-AAAAGTGCTCCCTCCACATCTC-3’(SEQ ID No.8)
a DNA fragment deletion mutant TSH4-MT was obtained, which corresponds to the mutation of the TSH4 gene as shown in FIG. 5B. tsh4-MT showed phenotype such as reduction in tassel branch number, reduction in tassel floret number, small flower covered bracts, bracts tufted at tassel and ear base, and ear row number disturbance of some ears (FIG. 6). Test example 1Indel _7_133209283_ C/CT tests in which allelic variation reducing the number of tassel branches was selected in maize breeding history
350 parts of Chinese and American corn inbred line materials collected before are divided into different breeding periods according to the breeding history of China and America. Then, the rule of frequency change of Indel _7_133209283_ C/CT in materials in different breeding periods is investigated; the analysis finds that the allelic variation without 1bp insertion (namely the allelic variation corresponding to the reduction of the number of the tassel branches of the corn) is gradually improved along with the advancing frequency of the corn breeding process (figure 7), which indicates that the allelic variation is selected in the corn breeding process and reflects the application potential of the allelic variation in the corn breeding.
SEQUENCE LISTING
<110> institute of biotechnology of Chinese academy of agricultural sciences of southern China university of agriculture
<120> promoter for regulating and controlling maize tassel configuration, molecular marker and application thereof
<130> BJ-2002-190211A
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 462
<212> DNA
<213> Zea mays L.
<400> 1
tggaaagcca ggggctgcaa gttcaggaat gcttacactg ccgctgtgca atttcatcag 60
tctcaggtcg atatccaaat atatctgata catgatatgg tatgtttggt tcacctctag 120
gcaaaactgc acctgacaag gcacactgca atcagacgtt gggtttttac taaagataag 180
atccttgccc ggccggagta gacaacaact cgatagtttc atgtgtgccc acactaataa 240
gtaaacaatg tagtatcttc caactctctt ttcaacagca agtacaagtg tgtacaacca 300
aatagttata taatgtgtgg agttttaagg aagggtactt cggttccagt taggcaaaag 360
ttgtttacaa aacccatatc taaaagtctt agaataagat ccagagaaaa gcacagtact 420
tgacgatgcc attgaatact gtactagcta gctagcccct ct 462
<210> 2
<211> 461
<212> DNA
<213> Zea mays L.
<400> 2
tggaaagcca ggggctgcaa gttcaggaat gcttacactg ccgctgtgca atttcatcag 60
tctcaggtcg atatccaaat atatctgata catgatatgg tatgtttggt tcacctctag 120
gcaaaactgc acctgacaag gcacactgca atcagacgtt gggtttttac taagataaga 180
tccttgcccg gccggagtag acaacaactc gatagtttca tgtgtgccca cactaataag 240
taaacaatgt agtatcttcc aactctcttt tcaacagcaa gtacaagtgt gtacaaccaa 300
atagttatat aatgtgtgga gttttaagga agggtacttc ggttccagtt aggcaaaagt 360
tgtttacaaa acccatatct aaaagtctta gaataagatc cagagaaaag cacagtactt 420
gacgatgcca ttgaatactg tactagctag ctagcccctc t 461
<210> 3
<211> 20
<212> DNA
<213> Artifical sequence
<400> 3
cgctgtgcaa tttcatcagt 20
<210> 4
<211> 20
<212> DNA
<213> Artifical sequence
<400> 4
tattcaatgg catcgtcaag 20
<210> 5
<211> 22
<212> DNA
<213> Artifical sequence
<400> 5
gcgcgatgaa ctggtccctg ta 22
<210> 6
<211> 20
<212> DNA
<213> Artifical sequence
<400> 6
acggcaaggt aaggcgcaca 20
<210> 7
<211> 22
<212> DNA
<213> Artifical sequence
<400> 7
cacgagccgt tgacctacta ct 22
<210> 8
<211> 22
<212> DNA
<213> Artifical sequence
<400> 8
aaaagtgctc cctccacatc tc 22
Claims (1)
1. Application of a promoter sequence with a nucleotide sequence shown in SEQ ID No.2 in reducing the tassel branch number of corn.
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| CN112680471B (en) * | 2019-10-17 | 2023-08-01 | 华南农业大学 | Application of ZmSPL gene in regulation and control of development of corn stigma mastoid cells |
| CN113122568B (en) * | 2019-12-31 | 2022-07-19 | 杭州瑞丰生物科技有限公司 | Method for improving corn biomass |
| CN111909936B (en) * | 2020-07-08 | 2023-02-24 | 华南农业大学 | Application of GT1 gene in regulation and control of maize male inflorescence sex determination and/or multi-spike development |
| CN112553201A (en) * | 2020-12-10 | 2021-03-26 | 深圳大学 | Promoter ZmPSP-pro specifically expressed in maize tassel and application thereof |
| CN114591967B (en) * | 2022-03-02 | 2023-05-23 | 华南农业大学 | Application of corn TCP gene in cross breeding |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106170548A (en) * | 2013-12-31 | 2016-11-30 | 美国陶氏益农公司 | Tissue specific expression and hybrid plant generate |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106170548A (en) * | 2013-12-31 | 2016-11-30 | 美国陶氏益农公司 | Tissue specific expression and hybrid plant generate |
Non-Patent Citations (5)
| Title |
|---|
| Chuck, G et al..The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries.《DEVELOPMENT》.2010,第137卷(第8期),1243-1250. * |
| GenBank: AC211179.5;Wilson,R.K. et al.;《GenBank》;20130921;ORIGIN * |
| The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries;Chuck, G et al.;《DEVELOPMENT》;20100415;第137卷(第8期);摘要，第1248页左栏最后一段-右栏第1段，第1249页右栏最后一段-第1250页左栏第1段 * |
| Wilson,R.K. et al..GenBank: AC211179.5.《GenBank》.2013, * |
| 玉米雄穗分枝数分化发育基因研究进展;代资举等;《农学学报》;20181020(第10期);第7-12页 * |
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