CN113234700A

CN113234700A - Rice calcineurin B protein and application thereof in breeding

Info

Publication number: CN113234700A
Application number: CN202110664340.4A
Authority: CN
Inventors: 曹云英; 高聪; 陈沫先; 鲁帅; 许锦彪; 周蓉; 汪保华; 顾奕雯; 汪紫慧; 陈瑶钰
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-08-10

Abstract

The invention discloses a rice calcineurin B protein and application thereof in breeding, relating to the technical field of agricultural breeding. The breeding includes creating low germination rate germplasm of gramineous plant seeds and short germplasm with short seedling height and less root mass through transgenic technology. The gramineous plant includes rice. The rice calcineurin B protein is CBL 8; the over-expression plants L8-OE5 and L8-OE6 of the CBL8 show a phenotype of inhibiting seed germination of the plants and a phenotype of inhibiting seedling growth of the plants; the CBL 8-deleted mutants CRISPRL8-48 and CRISPRL8-51 both showed the same phenotype as the wild type, and the germination of seeds and the growth of seedlings were normal without difference from the wild type. The research can provide theoretical basis for transgenic breeding work of gramineous plants.

Description

Rice calcineurin B protein and application thereof in breeding

Technical Field

The invention relates to the technical field of agricultural breeding, in particular to a rice calcineurin B protein and application thereof in breeding.

Background

Paddy genus angiosperma, unit cotyledonary plant class, Gramineae, Oryza genus. Annual aquatic herbaceous plants, 0.5-1.5 meters high. The two leaves are intergrown and the thread shape is like a needle. The ear is a panicle and self-pollinated. The rice has the advantages of fast growth, one or even two generations of rice produced in one year, more seeds and strong vitality. The genome sequence of the indica-japonica subtype of rice is published, the proportion of functional genes in the total genome is high, for example, the proportion of functional genes in the indica-japonica subtype of rice is 92%, and the functions of a plurality of genes are unknown, so that the cloning of related genes thereof is particularly necessary for researching the functions thereof.

Research in rice includes forward genetics and reverse genetics. Forward genetics follows a thought from phenotypic analysis of mutants to genetic function recognition, focusing on mutants with certain defects. For example, if the gene regulation process involved in the plant germination mechanism is to be studied, wild-type rice may be mutagenized by chemical, physical or biological means, and then the mutants may be screened under the conditions of germination. If an individual who responds differently to germination than the wild type (e.g., has a higher or lower germination rate than the wild type, etc.) appears in the progeny of the mutagenized population, such an individual is a mutant. The different responses of the plant to the seed germination rate may be caused by the disruption of a gene in the mutant, which must be related to the germination mechanism of the plant. 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 relationship of other related genes in the germination process and the form of the gene affecting the germination process of plant seeds can be analyzed.

In view of the advantages of rice in genetic manipulation, the rice is widely applied to the research of a plurality of processes of the whole life activity of plants, and a series of important findings are obtained; however, the research on the calcineurin B protein of rice and the influence of the calcineurin B protein on the germination of rice seeds and the growth of seedlings are yet to be further researched and applied to the cultivation of rice, so that a theoretical basis is provided for the future breeding work.

Disclosure of Invention

The invention aims to provide a rice calcineurin B protein and application thereof in breeding so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a rice calcineurin B protein, which is rice CBL 8.

Preferably, the CBL8 constructs an overexpression vector (PWM101-CBL8) of the gene to obtain overexpression strains, and the expression of the overexpression strains is verified by fluorescent quantitative PCR (polymerase chain reaction), so that 6 overexpression strains are obviously higher than wild strains.

Preferably, a strain for knocking out a specific sequence of the CBL8 gene is constructed through CRISPR-Cas9, and is identified through a clone sequencing method, and partial bases are all found to be deleted.

Preferably, the over-expression plants of CBL8, CBL8-OE5 plants and CBL8-OE6 plants show a low germination phenotype, and the CBL 8-deleted mutants, CRISPRL8-48 and CRISPR L8-51 show a normal germination phenotype, and have no difference with wild type.

Preferably, the over-expression plants of CBL8, namely CBL8-OE5 plants and CBL8-OE6 plants, show the phenotypes of shorter seedling plant height, less root and shorter length, and CRISPR L8-48 and CRISPR L8-51 show the phenotype of normal seedling growth, and are not different from wild type plants.

Preferably, the application of the rice calcineurin B protein breeding comprises the steps of creating low-germination-rate germplasm and phenotypic germplasm with short seedling height, less roots and shorter length of gramineous plants through a transgenic technology; wherein the Gramineae plant is rice.

Preferably, the calcineurin B-like protein is applied to transgenic breeding work of gramineae plants.

Compared with the prior art, the invention has the beneficial effects that: aiming at the technical problems that rice breeding work can not be accurately carried out, such as the rice material with low germination rate can not be selected through gene operation or the rice material with short seedling height, small root amount and short seedling can not be selected according to actual needs, various experiments prove that rice calcineurin B protein 8 overexpression strains and deletion mutants respectively have low germination rate/normal germination rate phenotypes and short seedling height, small root amount and short/normal phenotypes.

Drawings

FIG. 1 is a diagram showing the identification of the fluorescent quantitative expression of an over-expressed strain of rice calcineurin B protein 8 according to the present invention;

FIG. 2 is a clone sequencing alignment chart of the deletion mutant of the gene CBL8 provided by the invention;

FIG. 3 is a diagram of the germination phenotype and germination rate analysis of transgenic plants related to the gene CBL8 provided by the present invention;

FIG. 4 is a comparison graph of seedling growth phenotype, plant height and root length of transgenic plants related to the gene CBL8 provided by the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides the following technical scheme: a rice calcineurin B protein, which is rice CBL 8.

Wherein, the CBL8 constructs an overexpression vector (PWM101-CBL8) of the gene to obtain an overexpression strain, the expression of the overexpression strain is verified by fluorescent quantitative PCR, and 6 overexpression strains are obviously higher than wild strains; a strain for knocking out a specific sequence of the CBL8 gene is constructed through CRISPR-Cas9, and is identified through a clone sequencing method, and partial bases are all found to be deleted.

The over-expression plants of CBL8, namely CBL8-OE5 plants and CBL8-OE6 plants, show a low germination rate phenotype, and CBL 8-deleted mutants, namely CRISPRL8-48 and CRISPR L8-51, show a normal germination rate phenotype and have no difference with wild plants; the over-expression plants of CBL8, namely CBL8-OE5 plants and CBL8-OE6 plants, show the phenotypes of short seedling height, small root quantity and short length, and CRISPR L8-48 and CRISPR L8-51 show the phenotype of normal seedling growth, and have no difference with wild types.

Example case:

the embodiment provides an application of rice calcineurin B proteins (CBLs) in breeding; wherein, rice CBL8 is one of calcineurin B-like proteins, an overexpression vector (PWM101-CBL8) of the gene is constructed to obtain an overexpression strain, the expression of the overexpression strain is verified by fluorescent quantitative PCR, and 6 overexpression strains are all obviously higher than wild strains (as shown in figure 1); a strain with the knocked-out gene is constructed through CRISPR-Cas9, and is identified through a clone sequencing method, and partial bases are all deleted (shown in figure 2); the germination phenotype experiment is shown in figure 3, the germination rate (statistics at 7 days of germination) of the CBL8 gene over-expressed strain is obviously reduced, the germination of the deletion mutant and the wild type is not different, the germination number is increased along with the extension of the germination time, the germination number is rapidly increased before 3 days, the subsequent increase is slow, the wild type and the deletion mutant reach the maximum value in 4 days and do not increase any more, the over-expressed strain is slowly increased within 3-5 days and does not increase any more after 5 days; seedling phenotype experiments As shown in FIG. 4, seedlings of the over-expressed lines were shorter and smaller in root system than the wild type 14 days after germination from seed soaking, while the deletion mutants were not different from the wild type. Namely, the over-expression plants L8-OE5 and L8-OE6 of the CBL8 show a low germination phenotype; both CRISPR L8-48 and CRISPR L8-51 exhibited a normal phenotype of germination; the over-expressed plants of CBL8, L8-OE5 and L8-OE6, both showed the phenotype of shorter seedlings, less roots and shorter; both CRISPR L8-48 and CRISPR L8-51 exhibited normal phenotype of seedling growth, as shown in figure 4; the over-expression strain and deletion mutant of the gene have different phenotypes, which provides a gene reserve for controlling seed germination and seedling growth of plants.

In conclusion, aiming at the technical problems that the rice breeding work can not be accurately carried out, such as the rice material with low germination rate can not be selected through gene operation or the rice material with short seedling plant height, small root amount and short seedling plant length can not be selected according to actual needs, various experiments prove that the rice calcineurin B protein 8 overexpression strain and the deletion mutant respectively have low germination rate/normal germination rate phenotype and short seedling plant height, small root amount and short/normal phenotype, through the research, the rice gene is subjected to transgenic research and analysis, the needed rice seeds are screened and cultured, the seed selection is accurate, and the time is saved.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims

1. A rice calcineurin B protein, which is characterized in that: the calcineurin B-like protein is rice CBL 8.

2. The rice calcineurin B protein of claim 1, wherein the protein is selected from the group consisting of: the CBL8 constructs an overexpression vector (PWM101-CBL8) of the gene to obtain an overexpression strain, and the expression of the overexpression strain is verified by fluorescent quantitative PCR (polymerase chain reaction), so that 6 overexpression strains are obviously higher than wild strains.

3. The rice calcineurin B protein of claim 2, wherein the protein is selected from the group consisting of: a strain for knocking out a specific sequence of the CBL8 gene is constructed through CRISPR-Cas9, and is identified through a clone sequencing method, and partial bases are all found to be deleted.

4. The rice calcineurin B protein of claim 1, wherein the protein is selected from the group consisting of: the over-expression plants of CBL8, namely CBL8-OE5 plants and CBL8-OE6 plants, show a low germination rate phenotype, and CBL 8-deleted mutants, namely CRISPRL8-48 and CRISPR L8-51, show a normal germination rate phenotype, and are not different from wild plants.

5. The rice calcineurin B protein of claim 1, wherein the protein is selected from the group consisting of: the over-expression plants of CBL8, namely CBL8-OE5 plants and CBL8-OE6 plants, show the phenotypes of short seedling height, small root quantity and short length, and CRISPR L8-48 and CRISPR L8-51 show the phenotype of normal seedling growth, and have no difference with wild types.

6. An application of a rice calcineurin B protein in breeding is characterized in that: the breeding comprises the steps of creating low-germination-rate germplasm and phenotypic germplasm with short seedling height, small root quantity and short length of gramineous plants by a transgenic technology; wherein the Gramineae plant is rice.

7. The use of the calcineurin B protein of rice as claimed in claim 6, wherein: the calcineurin B-like protein is applied to transgenic breeding work of gramineous plants.