CN116355068A - DNA sequence for regulating and controlling corncob row number, molecular marker and application thereof - Google Patents
DNA sequence for regulating and controlling corncob row number, molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a DNA sequence for regulating and controlling the number of corncob rows, a molecular marker and application thereof. The invention firstly discloses an application of GL15 gene in regulating and controlling the number of corncob rows, and the polynucleotide sequence is SEQ ID No: 1. The invention also provides a key molecular marker for regulating the number of the corncob lines, and the molecular marker for regulating the number of the corncob lines can regulate and control the expression of GL15 genes in female corncobs, can be applied to improving the number of the corncob lines, and can be further applied to cultivating new corn varieties.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a DNA sequence for regulating and controlling the number of corncob rows, a molecular marker and application thereof.
Background
Corn is an important food, feed and industrial raw material in the world, and is also the most productive cereal crop in the world. Its adequate supply is critical to ensuring grain safety worldwide. Improving corn yield per unit through breeding is a key technical measure for guaranteeing corn supply.
The ear line number is one of the important indexes for measuring the corn kernel yield. The number of the ear lines determines the yield of corn ears on the premise that the grain size and the weight are unchanged and the number of the lines is also unchanged. As a factor influencing the corn yield, the improvement of the ear line number is significant in realizing the corn yield increase. Previous studies have found that genes reported in maize that regulate variation in ear line numbers mainly include genes of the gene family RAMOSA, CLAVATA-WUSCHEL, SPL, PIN, such as GIF1, UB3, RA3, WUS1, BIF2, KN1, PIN1, and the like. The frame shift, early termination or other mutants with altered protein functions of the genes can cause alteration of female ear development, and simultaneously can bring adverse effects such as plant height abnormality, tassel dysplasia and/or leaf organ dysplasia, and the like, so the genes cannot be directly applied to corn breeding practice.
Glossy15 (GL 15) is an APETALA2 (AP 2) -like transcription factor and plays a key role in promoting young leaf recognition traits and inhibiting leaf recognition traits. However, no report has been made so far that GL15 regulates the phenotype of ear line numbers. In addition, research shows that natural variation of some non-gene coding regions only changes the expression quantity of genes, and less adverse effects are brought; especially, some natural variations which are preserved through long breeding selection have wide application prospects in breeding.
Disclosure of Invention
The invention aims to provide an application of GL15 gene in regulating and controlling the number of corncob rows.
It is a second object of the present invention to provide a molecular marker for regulating GL15 gene expression in maize females.
The GL15 gene is applied to regulating the number of corn ears, wherein the regulation of the number of corn ears is to increase the number of corn ears; the polynucleotide sequence of the GL15 gene is selected from one of the following group of sequences:
(a) As shown in SEQ ID No:1, a polynucleotide sequence shown in seq id no;
(b) The coding amino acid sequence is shown as SEQ ID No:2, a polynucleotide sequence shown in seq id no;
(c) A polynucleotide sequence capable of hybridizing to the polynucleotide sequence of (a) or (b) under stringent hybridization conditions, wherein mutation of the polynucleotide sequence has the function of changing the number of lines of corn ears;
(d) A polynucleotide sequence having at least 95% or more similarity to the polynucleotide sequence of any one of (a) to (c), wherein the mutation of the polynucleotide sequence has the function of changing the number of lines of corn ears; or (b)
(e) A polynucleotide sequence complementary to any one of sequences (a) - (d).
The method for increasing the number of the corncob rows comprises the following steps:
(a) Constructing a GL15 gene overexpression recombinant plant expression vector; transforming the constructed over-expression recombinant plant expression vector into corn;
(b) It was backcrossed into GL 15-underexpressed maize using a molecular marker closely linked to GL 15.
A molecular marker for regulating expression of GL15 gene in corn female ear, comprising:
(a) SEQ ID No: mutating the 115 rd nucleotide from T to C;
(b) SEQ ID No:3, mutating the 115 th nucleotide of the polynucleotide sequence with complementary sequence from A to G;
(c) SEQ ID No: 343 nucleotides between the 151 th nucleotide and the 494 th nucleotide of the 4 sequence are deleted;
(d) SEQ ID No: the 343 nucleotides are deleted between the 151 st nucleotide and the 494 th nucleotide of the polynucleotide sequence with the complementary 4 sequences;
(e) SEQ ID No: 343 nucleotides are inserted between the 151 th nucleotide and the 152 th nucleotide of 5;
(f) SEQ ID No: the polynucleotide sequence complementary to the 5 sequence inserts 343 nucleotides between the 151 th nucleotide and the 152 th nucleotide.
As a preferred embodiment, said 343 nucleotides should be SEQ ID No:6, or SEQ ID No:6, a polynucleotide sequence complementary to the sequence.
The primer for detecting the molecular marker can be used for predicting the variation of the number of corn ears by detecting whether the molecular marker exists in corn materials.
The application of the corn GL15 gene and the molecular marker in cultivating new corn varieties with increased corn ear line numbers.
The invention has the beneficial effects that: the GL15 gene, the molecular marker SV_343bp, the SNP_9_96745402_T/C and the like provided by the invention can be applied to cultivation of high-yield or close-planting-resistant new corn varieties, and are especially applied to the aspects of improving the number of corn ears and corn yield and the like. The invention relates to a method for regulating and controlling the number of corncob rows, which comprises the following steps: the GL15 gene is regulated and expressed in corn by detecting the mutation condition of SV_343bp or SNP_9_96745402_T/C. The molecular marker for regulating and controlling the corn ear line number provided by the invention can regulate and control the expression of GL15 genes in corn female ears, has important significance for improving corn ear line number and corn unit yield, and can be further applied to the breeding of new corn varieties.
Definition of terms in connection with the present invention:
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 reference nucleic acids 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, phosphoroamidites, etc.). 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 indicated. 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 bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol. Chem.260:2605-2608 (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 hybridize specifically under specific conditions to form duplex molecules. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule may be used as a probe or primer to identify another polynucleotide molecule that shares homology.
The term "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. In general, the probe hybridizes to its target sequence to a greater degree than to other sequences (e.g., at least 2-fold over background. Stringent hybridization conditions are sequence dependent and will differ under different environmental conditions, longer sequences will hybridize specifically at higher temperatures. Target sequences 100% complementary to the probe can be identified by controlling the stringency or wash conditions of hybridization. Detailed guidance for nucleic acid hybridization can be found in the relevant literature (Tijssen, techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assys.1993.) more particularly, the stringent conditions are generally selected to be below the thermal melting point (T m ) About 5-10 deg.c. T (T) m At a temperature (at a specified ionic strength, pH and nucleic acid concentration) at which a probe that is 50% complementary to the target hybridizes to the target sequence in an equilibrium state (at T because the target sequence is present in excess) m 50% of the probes are occupied in the equilibrium state). Stringent conditions may be the following conditions: 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 with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice background hybridization, optionally 10 times background hybridization. Exemplary severeHybridization conditions may be as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃; or 5 XSSC, 1% SDS, at 65℃in 0.2 XSSC and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.
The term "plurality" as used herein generally means 2 to 8, preferably 2 to 4; "substitution" refers to the substitution of one or more amino acid residues with different amino acid residues, respectively; by "deletion" is meant a reduction in the number of amino acid residues, i.e., the absence of one or more amino acid residues therein, respectively; by "insertion" is meant an alteration 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 "promoter" refers to a polynucleotide molecule that is located in its natural state upstream or 5' to the translation initiation codon of the open reading frame (or protein coding region) and is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.
The term "expression": transcription and/or translation of endogenous genes or transgenes in plant cells.
Drawings
FIG. 1A shows that a gene co-expression module which has a significant relation with the number of corn ears is found by the WGCNA method, and the gene co-expression module comprises GL15 genes; in FIG. 1, B is the correlation between the expression level of GL15 gene and the ear line number phenotype data.
FIG. 2 shows that a mutation site SNP_9_96745402_T/C of a first exon of a GL15 gene obtained by an eQTL method and an insertion/deletion molecular marker SV_343bp of a promoter region are obviously related to the expression quantity of the GL15 gene; the molecular marker SV_343bp is an insertion/deletion molecular marker obtained by comparing genome sequences of 14 corn varieties; the arrow parallel to the X-axis in the figure represents GL15, and the upper part of the figure shows the gene structure of GL15 and the position information of the SNP_9_96745402_T/C mutation site.
FIG. 3 shows variation of 14 different types of inbred lines at the insertion/deletion molecular marker SV_343 bp; overall, two haplotypes were formed: hap1_0/0 and Hap2_SV343.
FIG. 4A shows the statistical analysis of GL15 gene expression in the female ears of two types of inbred lines, hap1_0/0 and Hap2_S343; FIG. 4B is a statistical analysis of the ear line number phenotype of two types of inbred lines, hap1_0/0 and Hap2_S343.
In FIG. 5, A is the expression change trend of GL15 gene in 3 different years in the corn breeding process; the GL15 gene is continuously increased in expression quantity in the corn breeding process; FIG. 5B is a comparison of the frequencies of the two types of genotypes of Hap1_0/0 and Hap2_S343 in different breeding years of breeding materials.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The inbred lines used in the examples below can obtain relevant information from the "chinese crop germplasm information net" and apply for obtaining the corresponding seeds.
Example 1GL15 Gene was located in a gene co-expression module markedly correlated with ear line number
We performed WGCNA analysis of 8 yield-related traits (diameter of female ear, length of female ear, weight of female ear, length of seed, weight of seed, number of ears, weight of hundred grains, and volume weight) and 22907 expressed genes of female ear weight. 2905 genes found in the blue module were significantly correlated with ear of corn line numbers (r=0.22, p-value=5×10 -4 Fig. 1A). Also contained in this module are a number of known genes that regulate ear line number and inflorescence development, such as: GIF1, UB3, WUS1, RA3, BIF2 and PIN1. Meanwhile, the module also comprises a GL15 gene, which indicates that the GL15 gene possibly participates in regulating the ear line number of corn (figure 1A). Meanwhile, the phenotype correlation analysis of the GL15 gene expression quantity and the ear line number shows that the GL15 gene expression quantity is positively correlated with the ear line number of corn (figure 1B), and the regulation and control effect of the GL15 gene on the ear line number of corn is again proved.
Example 2 mutation site of GL15 promoter region corn ear line number was controlled by controlling GL15 gene expression level
1. Development of GL15 promoter region mutation site
137 Chinese maize inbred 11,839,476 single nucleotide polymorphic molecular markers (SNPs) were mined using the published depth (> 10×) resequencing of the 350 maize inbred in combination with the published maize B73V 3 genome. And estimating the population structure and the kindred relation of 137 corn inbred lines by using the excavated molecular markers, and then carrying out expression quantitative trait locus (eQTL) association analysis by combining the expression quantity of the GL15 gene in female ears. Wherein one SNP_9_96745402_T/C on chromosome 9 was found to be significantly correlated with GL15 gene expression levels (FIG. 2). Further studies have found that this mutation site is located in the coding region of GL15 and belongs to synonymous mutation.
2. Acquisition of nucleotide sequence of genomic region in which structural variation of GL15 promoter region is located
The promoter region is a key region for gene expression regulation, and the mutation site of the SNP_9_96745402_T/C is synonymous mutation (the 115 th nucleotide of SEQ ID No:3 is mutated from T to C, or the 115 th nucleotide of the polynucleotide sequence reciprocal of SEQ ID No:3 is mutated from A to G), so that the SNP_9_96745402_T/C is presumed to be possibly linked with other mutation of the promoter region, which actually causes GL15 gene expression change. And reports indicate that structural variation is a significant cause of variation in gene expression. In order to find the structural mutation sequence linked with SNP_9_96745402_T/C, the published genomic information of 14 maize inbred lines is used for comparing the mutation situation of GL15 genes in 14 genomes, an insertion/deletion fragment SV_343bp (the nucleotide sequence of 343bp represented by SV_343bp is shown as SEQ ID No. 6, SEQ ID No. 4 and SEQ ID No. 5 respectively represent the characteristic sequences of SV_343 bp) which is obviously linked with mutation site SNP_9_96745402_T/C is found, and the nucleotide sequence of the region and the accurate information of the mutation are obtained (figure 3).
3. Structural variation of GL15 promoter region regulates expression of GL15 gene
The sequenced 137 inbred line can be classified into two types, hap1_0/0 and Hap2_S343, using specific primers according to the genotype at the insertion/deletion fragment SV_343bp (FIG. 3). Comparison analysis of the representative inbred line expression levels and ear line number phenotypes of the two types revealed that GL15 gene in the inbred female ears of the Hap1_0/0 type was significantly highly expressed, and that Hap1_0/0 also had an effect of significantly increasing ear line number (FIG. 4).
Example 3 mutation sites of GL15 promoter region are subjected to strong artificial selection during modern maize breeding
We collected 137 parts of maize inbred material from different breeding stages in China in the early stage, including early (CN 1960&70 s), mid-stage (CN 1980&90 s) and current (CN 2000&10 s). Analysis of the expression level of the GL15 gene in these materials shows that along with the promotion of breeding period, the expression level of the GL15 gene is obviously improved in the breeding process of Chinese corn (figure 5). Analysis of the frequency distribution of the two types of GL15 Hap1_0/0 and Hap2_S343 genotypes in the materials shows that the frequency of Hap1_0/0 is obviously improved in the Chinese corn breeding process along with the advancement of the breeding period, which shows that the Hap1_0/0 genotype is strongly selected manually in the modern corn breeding process (figure 5). This further demonstrates that the variant sequence of the GL15 promoter region has important breeding value in modern maize breeding processes.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (5)
- The application of the GL15 gene in regulating the number of corncob lines is characterized in that the regulating the number of corncob lines increases the number of corncob lines; the polynucleotide sequence of the GL15 gene is selected from one of the following group of sequences:(a) As shown in SEQ ID No:1, a polynucleotide sequence shown in seq id no;(b) The coding amino acid sequence is shown as SEQ ID No:2, a polynucleotide sequence shown in seq id no;(c) A polynucleotide sequence capable of hybridizing to the polynucleotide sequence of (a) or (b) under stringent hybridization conditions, wherein mutation of the polynucleotide sequence has the function of changing the number of lines of corn ears;(d) A polynucleotide sequence having at least 95% or more similarity to the polynucleotide sequence of any one of (a) to (c), wherein the mutation of the polynucleotide sequence has the function of changing the number of lines of corn ears; or (b)(e) A polynucleotide sequence complementary to any one of sequences (a) - (d).
- 2. The use of GL15 gene according to claim 1, wherein the method for increasing the number of corn ears comprises:(a) Constructing a GL15 gene overexpression recombinant plant expression vector; transforming the constructed over-expression recombinant plant expression vector into corn;(b) It was backcrossed into GL 15-underexpressed maize using a molecular marker closely linked to GL 15.
- 3. A molecular marker for regulating expression of GL15 gene in corn female ear, comprising:(a) SEQ ID No: mutating the 115 rd nucleotide from T to C;(b) SEQ ID No:3, mutating the 115 th nucleotide of the polynucleotide sequence with complementary sequence from A to G;(c) SEQ ID No: 343 nucleotides between the 151 th nucleotide and the 494 th nucleotide of the 4 sequence are deleted;(d) SEQ ID No: the 343 nucleotides are deleted between the 151 st nucleotide and the 494 th nucleotide of the polynucleotide sequence with the complementary 4 sequences;(e) SEQ ID No: 343 nucleotides are inserted between the 151 th nucleotide and the 152 th nucleotide of 5;(f) SEQ ID No: the polynucleotide sequence complementary to the 5 sequence inserts 343 nucleotides between the 151 th nucleotide and the 152 th nucleotide.
- 4. A primer for detecting the molecular marker according to claim 3, wherein the variation in the number of lines of corn ears can be predicted by detecting the presence or absence of the molecular marker according to claim 3 in corn material.
- 5. Use of the maize GL15 gene of claim 1 and the molecular marker of claim 3 for breeding new maize variety with increased number of lines of maize ear.
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