Application of maize MS2 gene
阅读说明：本技术 玉米ms2基因的应用 (Application of maize MS2 gene ) 是由 刘允军 杨丽 徐其隆 康丹 王国英 于 2020-05-28 设计创作，主要内容包括：本发明提供玉米MS2基因的应用。玉米MS2基因编码ABCG转运蛋白(SEQ ID NO:19),该基因通过控制玉米花药发育,进而调控玉米雄性不育或可育性状。本发明为基于玉米雄性不育为基础的杂交育种技术提供新的基因资源。(The invention provides application of a corn MS2 gene. The corn MS2 gene codes ABCG transport protein (SEQ ID NO:19), and the gene regulates and controls the male sterility or fertility traits of the corn by controlling the development of the corn anthers. The invention provides a new gene resource for a crossbreeding technology based on the male sterility of the corn.)
1. The following applications of the maize MS2 gene and the biological material containing the gene are as follows:
(1) controlling the development of the corn anther;
(2) controlling male sterility or fertility traits in maize;
wherein the maize MS2 gene is a gene encoding the following proteins a) or b):
a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 19; or
b) 19, protein which is derived from a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 19.
2. The use of claim 1, wherein the maize MS2 gene is subjected to site-directed mutagenesis by genetic engineering means to delete its function.
3. The use of claim 1, wherein the use comprises knocking out or reducing expression of maize MS2 gene.
4. Use according to any one of claims 1 to 3, wherein the biological material is selected from recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria or non-regenerable plant parts.
5. The construction method of the transgenic corn is characterized in that a corn gene MS2 is used as a target, a sgRNA sequence based on CRISPR/Cas9 is designed, a DNA fragment containing the sgRNA sequence is connected to a vector carrying the CRISPR/Cas to transform the corn, the site-directed mutation of the corn gene MS2 is realized, and the transgenic corn with the gene function deletion is obtained;
wherein the maize gene MS2 is as described in claim 1.
6. The method of claim 5, wherein the sgRNA site has a nucleotide sequence of 5'-TCGAAGTGGATCATCATCTG-3'.
7. Use of transgenic maize obtained according to the method of claim 5 or 6 for plant breeding.
8. Use according to claim 7, wherein the breeding purpose is the selection of plant male sterile material.
9. Use according to claim 8, characterized in that the breeding method comprises transgenesis, crossing, backcrossing, selfing or asexual propagation.
10. The method for creating the male sterile line of the corn is characterized by comprising the following steps:
1) constructing CRISPR/Cas9 system for knocking out corn gene MS2, transforming corn and harvesting T of transgenic corn plant0Seed generation;
2) seeding the above-mentioned T0Seed generation to obtain T1Plants generation, investigation of T at flowering1Selecting male sterile plants for plant fertility;
wherein the maize gene MS2 is as described in claim 1;
preferably, the CRISPR/Cas9 system of the targeted maize gene MS2, wherein the nucleotide sequence of the sgRNA site of action is 5'-TCGAAGTGGATCATCATCTG-3'.
Technical Field
The invention relates to the field of plant genetic engineering and genetic breeding, in particular to application of a corn MS2 gene.
Background
The heterosis is the first generation of hybrid produced by the mutual hybridization of parent individuals with different genotypes, is superior to the phenomenon of the parent in the aspects of yield increase, stable yield, disease resistance, stress resistance and the like, and has great application value and research significance in agricultural production practice. Corn (Zea mays L.) is an important food crop in China, is one of the most successful crops for utilizing heterosis, and is mainly used for hybrid seeds in China at present. In the corn production process, female parent emasculation is a key technical problem for restricting hybrid seed production efficiency and seed purity. In the prior corn hybrid production of China, an artificial emasculation method is mainly adopted, and a mechanical emasculation method and a chemical emasculation method are adopted in an auxiliary manner. However, manual emasculation is time-consuming and labor-consuming and affects seed purity, mechanical emasculation easily damages plants to reduce seed yield, and chemical emasculation easily generates chemical residues to affect plants and environment. The application of male sterility in the hybrid seed production process of corn can effectively solve the castration problem of female parent.
According to the difference of plant male sterile material genotypes, the plant male sterility is divided into 3 types: cytoplasmic Male Sterility (CMS), nuclear male sterility (GMS), and karyoplasms interaction male sterility (Karyoplasm) types. In early maize production, maize T-type cytoplasmic sterile line (CMS-T) was widely used, and in the seventies of the twentieth century, northern leaf blight in american maize broke out and caused a massive reduction in local maize production, resulting in CMS-T failure. Therefore, the search for new male sterile lines of maize is imperative. Therefore, the discovery of new genic male sterility genes, the cloning and research of the functions thereof, and the analysis of the molecular mechanism of the male flower development of the corn are necessary requirements for the genetic breeding application research of the corn and the improvement of the heterosis utilization efficiency.
Disclosure of Invention
The invention aims to provide application of a corn MS2 gene.
In order to achieve the object of the present invention, in a first aspect, the present invention provides any one of the following uses of maize MS2 gene and biological material containing the gene:
(1) controlling the development of the corn anther;
(2) controlling the male sterility or fertility character of corn.
In the invention, the maize MS2 gene is from maize inbred line B73, which is a gene coding for the following proteins a) or B):
a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 19 (ABCG transporter); or
b) 19, protein which is derived from a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 19.
The nucleotide sequence of the maize MS2 gene is shown as SEQ ID NO. 17, and the CDS sequence is shown as SEQ ID NO. 18.
The application comprises the step of carrying out site-directed mutagenesis on the corn MS2 gene by utilizing a genetic engineering means so as to delete the function of the gene.
Further, the use comprises knocking out or reducing expression of maize MS2 gene.
Such biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria, or non-regenerable plant parts.
In a second aspect, the invention provides a construction method of a transgenic corn, wherein a corn gene MS2 is used as a target, a sgRNA sequence based on CRISPR/Cas9 is designed, a DNA fragment containing the sgRNA sequence is connected to a vector carrying CRISPR/Cas, the corn is transformed, the site-directed mutation of the corn gene MS2 is realized, and the transgenic corn with the gene function deletion is obtained.
Preferably, the nucleotide sequence of the sgRNA action site is 5'-TCGAAGTGGATCATCATCTG-3' (SEQ ID NO: 20).
In a third aspect, the present invention provides the use of transgenic maize obtained according to the above method in plant breeding.
Wherein, the breeding aim is to breed the male sterile material of the plant.
Breeding methods include, but are not limited to, transgenic, hybrid, backcross, selfing, or vegetative propagation.
In a fourth aspect, the present invention provides a method for creating a male sterile line of maize, comprising:
1) constructing CRISPR/Cas9 system for knocking out corn gene MS2, transforming corn and harvesting T of transgenic corn plant0Seed generation;
2) seeding the above-mentioned T0Seed generation to obtain T1Plants generation, investigation of T at flowering1Male sterile plants are selected for plant fertility (including anther development).
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides a novel male sterile corn material and a preparation method thereof, MS2 gene mutation can make corn male sterile, and provides a new gene resource for a crossbreeding technology based on corn male sterility.
Drawings
FIG. 1 is a comparison of the phenotype of wild type maize and the ms2 mutant in a preferred embodiment of the invention. Wherein, A: male ear characteristics of the wild type (left) and ms2 mutant (right) at the time of pollination; b: the major axis features of the male ear in both wild type (left) and ms2 mutant (right) at the time of dusting; c: small ear characteristics of wild type (left) and ms2 mutant (right); d: anther characteristics of wild type (left) and ms2 mutant (right); e: dyeing wild type mature anther starch potassium iodide; f: ms2 mutant mature anther starch potassium iodide staining.
FIG. 2 is a diagram showing the fine mapping of the MS2 gene on chromosome 9 of maize in a preferred embodiment of the present invention.
FIG. 3 is a phylogenetic tree analysis of the maize MS2 protein and its homologous proteins in a preferred embodiment of the invention.
FIG. 4 shows the sequence of the mutation at the target site of the homozygous mutant and the phenotypic analysis according to the preferred embodiment of the present invention. Wherein, A: a mutated sequence; b: mutant phenotype.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The maize ms2 male sterile mutant used in the following examples was from the american germplasm center.