CN109486839B

CN109486839B - Application of arabidopsis MAPKKK kinase in breeding

Info

Publication number: CN109486839B
Application number: CN201910060341.0A
Authority: CN
Inventors: 刘鹰高; 陈沫先; 武茹茹; 朱福远
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2018-11-09
Filing date: 2019-01-22
Publication date: 2021-05-11
Anticipated expiration: 2039-01-22
Also published as: CN109486839A

Abstract

The invention discloses application of arabidopsis MAPKKK kinase in breeding, and relates to the technical field of agricultural breeding. The breeding comprises the creation of drought-resistant germplasm and early-flowering germplasm of cruciferous plants by a transgenic technology. The cruciferous plants include rape and cauliflower. There are four shears for the MAPKKK kinase, named AS1, AS2, AS3, and AS 4; the overexpression plant AS1-OE of the AS1 and the mutant CRISPRAS3-2 with the AS3 deletion show an drought-resistant phenotype; the AS3-OE plant and the AS 1-deleted mutant CRISPRAS1-1 both show drought-resistant phenotype; the AS1-OE plants and AS2/4-OE plants exhibited an early flowering phenotype and the AS3-OE plants exhibited a late flowering phenotype. The research can provide theoretical basis for the transgenic breeding work of cruciferous plants.

Description

Application of arabidopsis MAPKKK kinase in breeding

Technical Field

The invention relates to the technical field of agricultural breeding, in particular to application of arabidopsis MAPKKK kinase in breeding.

Background

Arabidopsis genus is Brassicaceae, angiosperma, dicotyledonous class. Biennial herbs with height of 7-40 cm. Basal leaves have a lotus-shaped handle and an inverted oval or spoon shape; the cauline leaves have no stem and are in the shape of needles or threads. The raceme is terminal and the petals are 4 pieces and white. The arabidopsis thaliana has the advantages of small plant, short generation time, more knots and strong vitality. The genome of arabidopsis thaliana is 1/80 of the currently known plant genome, which makes it relatively easy to clone its related genes.

The studies of Arabidopsis thaliana include forward genetics and reverse genetics. Forward genetics follows a thought from phenotypic analysis of mutants to genetic function recognition, which focuses first on mutants with certain defects. For example, if the gene regulation process involved in the drought resistance mechanism of plants is to be studied, the wild type Arabidopsis thaliana may be mutagenized chemically, physically or biologically, and then subjected to screening for mutants under drought stress. If an individual that responds differently to drought conditions than the wild type (e.g., a plant that is more or less drought-resistant than the wild type) appears in the progeny of the mutagenized population, such an individual is a mutant. The different responses of the plants to drought may be caused by the fact that a certain gene in the mutant is damaged, and the gene is necessarily related to the drought resistance mechanism of the plants. After such a mutant is obtained, the mutant gene can be located and cloned. After the gene sequence is obtained, the function of the gene can be further understood, and the form of the gene affecting the drought-resistant way of plants and the relation of other related genes in the drought-resistant way can be analyzed.

In view of the advantages of Arabidopsis in genetic operation, the method is widely applied to the research of all processes of the whole life activity of plants, and a series of important findings are obtained; however, the research on the arabidopsis protein kinase and the influence of the arabidopsis protein kinase on the growth of arabidopsis are still needed to be further researched and applied to the cultivation of arabidopsis, so that a theoretical basis is provided for the future breeding work.

Disclosure of Invention

In view of this, the embodiment of the invention provides an application of arabidopsis MAPKKK kinase in breeding.

In order to achieve the purpose, the invention mainly provides the following technical scheme:

in one aspect, the embodiment of the invention provides an application of arabidopsis MAPKKK kinase in breeding.

Preferably, the breeding comprises creating drought resistant and early flowering germplasm of crucifers by transgenic techniques.

Preferably, the cruciferous plants include rape and cauliflower.

Preferably, there are four shears for the MAPKKK kinase, designated AS1, AS2, AS3 and AS 4; the nucleotide sequence of AS1 is SEQ ID NO.1, the nucleotide sequence of AS2 is SEQ ID NO.2, the nucleotide sequence of AS3 is SEQ ID NO.3, and the nucleotide sequence of AS4 is SEQ ID NO. 4; the amino acid sequence of the protein encoded by AS1 is SEQ ID NO.5, the amino acid sequences of the proteins encoded by AS2 and AS4 are SEQ ID NO.6, and the amino acid sequence of the protein encoded by AS3 is SEQ ID NO. 6.

Preferably, the overexpression plant AS1-OE of AS1 and the mutant CRISPRAS3-2 with AS3 deletion both show a non-drought resistant phenotype; both AS3-OE plants and the AS 1-deleted mutant CRISPRAS1-1 exhibited a drought resistant phenotype.

Preferably, the AS1-OE plants and AS2/4-OE plants exhibit an early flowering phenotype and the AS3-OE plants exhibit a late flowering phenotype.

Compared with the prior art, the invention has the beneficial effects that:

aiming at the technical problem that the breeding work of arabidopsis can not be accurately carried out, for example, drought-resistant arabidopsis can not be selected through gene operation or early flowering or late flowering can not be selected according to actual needs, various experiments prove that the arabidopsis MAPKKKK kinase has four shearing bodies which respectively have drought resistance/drought resistance and early flowering/late flowering phenotypes, and through the research, the needed arabidopsis seeds are screened out for cultivation through transgenic research and analysis on arabidopsis seed genes, so that the seed selection is accurate, and the time is saved.

Drawings

FIG. 1 is a diagram of the four splice bodies and three protein models of an Arabidopsis based 47MAPKKK kinase provided by the invention;

FIG. 2 is a scanning electron microscope of protoplast localization of Arabidopsis thaliana with three proteins of gene 47 provided by the present invention;

FIG. 3 is a diagram of the drought resistant phenotype planting of a transgenic plant related to gene 47 provided by the present invention;

FIG. 4 is a flowering phenotype planting map of the gene 47 overexpression plant provided by the invention.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, technical solutions, features and effects according to the present invention will be given with preferred embodiments. The particular features, structures, or characteristics may be combined in any suitable manner in the embodiments or embodiments described below.

Example 1

This example 1 provides an application of arabidopsis MAPKKK kinase in breeding; wherein, the arabidopsis 47 gene is MAPKKK kinase, and 2 variable shears thereof have two phenotypes of drought resistance and early flowering respectively; 47 genes are present in four isoforms (AS shown in FIG. 1), designated AS1, AS2, AS3 and AS 4; the nucleotide sequence of AS1 is SEQ ID NO.1, the nucleotide sequence of AS2 is SEQ ID NO.2, the nucleotide sequence of AS3 is SEQ ID NO.3, and the nucleotide sequence of AS4 is SEQ ID NO. 4; the amino acid sequence of the protein coded by AS1 is SEQ ID NO.5, the amino acid sequences of the proteins coded by AS2 and AS4 are SEQ ID NO.6, the amino acid sequence of the protein coded by AS3 is SEQ ID NO.6, and the three proteins are positioned in cell nucleus and cytoplasm through GFP; the drought-resistant phenotype experiment shows that the 47 gene full-deletion mutant is not drought-resistant compared with the wild type as shown in figure 3; meanwhile, the over-expression plant AS1-OE of the AS1 and the mutant CRISPRAS3-2 with the AS3 deletion show an drought-resistant phenotype; the AS3-OE plant and the AS 1-deleted mutant CRISPRAS1-1 both show drought-resistant phenotype; the AS1-OE plants and AS2/4-OE plants exhibited an early flowering phenotype and the AS3-OE plants exhibited a late flowering phenotype, AS shown in FIG. 4; different splice bodies of a single gene have different phenotypes, which increase gene reserve for plant drought resistance and flowering phase control. The above four cDNA sequences and three protein amino acid sequences of example 1 were obtained by PCR amplification of Arabidopsis total RNA followed by sequencing.

The research of the invention can be applied to the transgenic breeding work of cruciferous plants (rape, cauliflower and the like), such as creating drought-resistant germplasm and early flowering germplasm, and providing theoretical basis for the breeding work.

The embodiments of the present invention are not exhaustive, and those skilled in the art can select them from the prior art.

The above disclosure is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and shall be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the above claims.

Sequence listing

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Claims

1. The application of an arabidopsis kinase MAPKKK in breeding is characterized in that the breeding comprises creating drought-resistant germplasm and early-flowering germplasm of cruciferous plants by a transgenic technology; there are four shears for the kinase MAPKKK, named AS1, AS2, AS3 and AS 4; the nucleotide sequence of AS1 is SEQ ID NO.1, the nucleotide sequence of AS2 is SEQ ID NO.2, the nucleotide sequence of AS3 is SEQ ID NO.3, and the nucleotide sequence of AS4 is SEQ ID NO. 4; the amino acid sequence of the protein encoded by AS1 is SEQ ID NO.5, the amino acid sequences of the proteins encoded by AS2 and AS4 are SEQ ID NO.6, and the amino acid sequence of the protein encoded by AS3 is SEQ ID NO. 7.

2. Use of the arabidopsis kinase MAPKKK in breeding according to claim 1, wherein the cruciferous plants comprise rape and cauliflower.

3. The use of an arabidopsis kinase MAPKKK in breeding according to claim 1, wherein the overexpression plant of AS1, mutant CRISPRAS3-2, lacking AS1-OE and AS3, both exhibit an drought-resistant phenotype; both AS3-OE plants and the AS 1-deleted mutant CRISPRAS1-1 exhibited a drought resistant phenotype.

4. The use of an arabidopsis kinase MAPKKK in breeding according to claim 3, wherein the AS1-OE plants and AS2/4-OE plants exhibit an early flowering phenotype and the AS3-OE plants exhibit a late flowering phenotype.