CN116606864A - Rape yield regulation gene SKP1-IP15, encoding protein, expression vector and application thereof - Google Patents

Abstract

The application discloses a rape yield regulation geneSKP1‑ IP15The coded protein, the expression vector and the application thereof; the application relates to a rape homologous geneSKP1‑IP15（BnaA07G0241300ZSAndBnaC06G0264200ZS) CRISPR/Cas9 editing is carried out, and the character table of the gene on the plant height, effective branch number and the like of the rape is definedThe influence of the gene is verified and determined, so that a solid technical foundation is laid for improving the rape yield by utilizing the gene.

Description

Rape yield regulation gene SKP1-IP15, encoding protein, expression vector and application thereof

Technical Field

The application relates to the technical field of molecular breeding, in particular to a rape yield regulation geneSKP1-IP15And its coded protein, expression vector and application.

Background

Rape (rape)Brassica campestris L.) Is one of important economic crops in China, and the planting area and the yield of the economic crops are always at a higher level. In recent years, as the demand for health foods has increased, the market demand for rapeseed oil has also increased.

The main components of the oil menu plant yield mainly comprise effective branch number, fruiting rate, single pod weight and the like. The yield traits of rape are complex traits controlled by multiple genes, and research on rape yield genes has been advanced to some extent, for example, some researches have identified some genes related to rape yield, such asBnFLC.A10、BnGA20ox2AndBnSPL9etc.; however, rape yield is a complex quantitative trait, and is controlled by multiple genes, and currently obtained functional genes are not enough to fully elucidate the molecular mechanism of rape yield formation. Therefore, more rape yield related trait genes need to be further excavated and the utilization ways thereof are analyzed, so that the genetic basis for yield formation is enriched, and excellent gene resources are provided for breeding new varieties of rape.

The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

The inventor researches and discovers that the cornKWE2The gene has two homologous genes in rapeSKP1-IP15（BnaA07G0241300ZSAndBnaC06G0264200ZS) Based on the above, the inventors created the corresponding knockout mutations of the two genes, and clarified the expression of the two genes on the properties such as effective branching, plant height, leaf area and the like and the regulation and control of the yield, thereby utilizingSKP1-IP15The gene improves the rape yield and provides powerful technical support.

One of the objects of the present application is to provide a rape yield regulating geneSKP1-IP15The nucleotide sequence is at least one of the following groups:

(1) As shown in SEQ ID NO.1 or SEQ ID NO. 2;

(2) DNA molecules which are formed by one to several base substitutions and/or one to several base insertions and/or deletions and large fragment nucleotide sequence insertions/deletions/shifts/inversions on the basis of SEQ ID No.1 or SEQ ID No.2 and can influence the phenotype of the yield-related traits.

In another aspect of the present disclosure, there is provided a rape yield regulating geneSKP1-IP15The amino acid sequence of the encoded protein is at least one of the following groups:

(1) A protein consisting of the amino acid sequence encoded by SEQ ID NO.1 or SEQ ID NO. 2;

(2) As shown in SEQ ID NO.3 or SEQ ID NO. 4;

(3) A protein which is obtained by substituting, deleting and/or adding one or a plurality of amino acid residues from SEQ ID NO.3 or SEQ ID NO.4 and has the property of influencing yield.

In a third aspect of the present disclosure, there is provided a gene for regulating yield of rape comprising the sameSKP1-IP15The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacterium.

In a fourth aspect of the present disclosure, there is provided a method for identifying a rape yield-controlling geneSKP1-IP15A mutant primer pair having the sequence:

Fbox-sg123F：5’- ACCCGCCGCAAACGAACG -3’

Fbox-sg123R：5’- TGAGCCATCGATTCTCGTC -3’。

in a fifth aspect of the present disclosure, the gene is expressed asSKP1-IP15The primer is applied to rape yield trait improvement and dominant variety/strain breeding.

For example, silencing/down-regulating expression of the canola geneSKP1-IP15So as to increase the plant height, effective branch number, pod length and other characters of the rape.

The transformable plants of the present application may be monocots and dicots including, but not limited to, canola, corn, wheat, barley, rye, cotton, sweet potato, sunflower, potato, beans, peas, sorghum, switchgrass, alfalfa, arabidopsis, and the like.

One or more technical solutions provided in the embodiments of the present application at least have any one of the following technical effects or advantages:

1. gene regulating rape yieldSKP1-IP15CRISPR/Cas9 editing is carried out to obtain homozygous CRISPR-Cas9 mutant, which makes clearSKP1-IP15Influence of the gene on the plant height, effective branch number and other character expression of rape; determining the regulation and control effect of the compound on rape yield; thereby for utilization ofSKP1-IP15The gene improves the rape yield and provides a strong technical support.

2. By utilizing the gene and the method disclosed by the application, the creation of high-yield breeding materials for crops such as rape, corn and wheat can be facilitated, the breeding period of new varieties of crops is shortened, the breeding cost is reduced, and the breeding efficiency is improved.

Drawings

FIG. 1 shows a diagram of a lettuce in an embodiment of the applicationSKP1-IP15Phylogenetic analysis tree of genes.

FIG. 2 is a diagram showing the comparison of phenotypic traits of canola in examples of the present application, wherein the left diagram is a transgenic control and the right diagram is canolaSKP1-IP15Two lines after gene knockout.

Detailed Description

Definitions and description of related terms:

the term "rape yield-controlling gene" refers to a nucleotide sequence having the ability to encode a protein, which specifically encodes a protein-active polypeptide having the functions of controlling plant height, effective branching number and yield.

The term "rape yield-controlling gene" also includes a gene encoding a gene having a natural biological yield-controlling functionSKP1- IP15Variant forms of the open reading frame sequences of the same functional protein; these variants include, but are not limited to: deletions, insertions and/or substitutions of 1 or several nucleotides, and additions of several (usually within 60, preferably within 30, more preferably within 10, most preferably within 5) nucleotides at the 5 'or 3' end.

The rape yield regulating gene also comprises an amino acid sequence which can translate a type of rape with the functions of regulating the plant height, effective branch number and yield of rape, and is shown as SEQ ID NO. 2. The amino acid sequence also comprises a variant form with the same function of the protein for naturally regulating and controlling the rape yield. These variants include, but are not limited to: deletion, insertion and/or substitution of 1 or several amino acids, and addition of 1 or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal. In the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein; the addition of one or several amino acids at the C-terminal and/or N-terminal will not normally alter the function of the protein either.

In addition, the nucleotide full-length sequence of the "rape yield-controlling gene" or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, the corresponding primers can be designed based on the nucleotide sequences disclosed in this example, particularly the open reading frame sequences, and amplified to the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it usually requires two or more nested PCR amplifications, and then the PCR amplification products are spliced together in the correct order.

Particularly preferred is at least one of the presently disclosed rape yield-controlling genes expressed in higher plantsSKP1- IP15Once the desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species or transferred into other varieties of the same species (including, in particular, commercial varieties) using conventional breeding techniques. The rape yield regulating genes disclosed by the applicationSKP1-IP15Is inserted into an expression cassette or is contained in a non-pathogenic self-replicating virus, and then preferably is stably integrated into the plant genome. The transformed recipient of the present application may be monocots and dicots including, but not limited to, canola, corn, wheat, barley, rye, rice, cotton, sunflower, potato, soybean, pea, switchgrass, arabidopsis, and the like. By expressing the nucleotide sequences disclosed herein in transgenic plants, the biosynthesis of functional proteins capable of enhancing the expression of the corresponding hybrid vigour is thereby promoted in the transgenic plants. In this way, transgenic plants can be produced that enhance hybrid vigour performance. In order to express the nucleotide sequences of the present application in transgenic plants, the nucleotide sequences disclosed herein may require modification and optimization, and the codons may be altered to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the present application. Moreover, high levels of expression in plants can be best achieved from coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than 50%, and most preferably greater than about 60%.

The following examples are provided to facilitate a better understanding of the present application, but it should be understood that the scope of the application is not limited to the specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The experimental methods in the embodiments of the application are all conventional methods unless otherwise specified; it will be understood by those skilled in the art that the reagents, enzymes, etc., used in the examples described below are all reagents or enzymes of analytically pure grade commercially available from the reagent company, unless otherwise specified. The materials, methods, and examples are illustrative only and not intended to be limiting.

Example one, rapeSKP1-IP15Functional verification of Gene

Based on long-term scientific research, 2 rape and corn are foundKWE2Homologous genes with relatively close genetic relationshipBnaA07G0241300ZSAndBnaC06G0264200ZS(shown as SEQ ID NO.1 and SEQ ID NO.2 respectively), which are namedSKP1-IP15(see FIG. 1). Further research and verification prove that the method has the advantages of high efficiency,SKP1-IP15gene and maizeKWE2Genes have similar gene regulatory functional properties: negative control of yield and heterosis.

To further verifySKP1-IP15Regulating and controlling effect of gene on rape yield and rapeBnaA07G0241300ZSAndBnaC06G0264200ZSCRISPR-Cas9 editing is carried out on specific sites of the genes, so that double-protruding materials of the two genes are obtained.

The specific operation is as follows:

vector construction

Two rape homologous genesBnaA07G0241300ZS、BnaC06G0264200Z3 targets are designed for each gene respectively at the A07 chromosome and the C06 chromosome, the mutation of the A07 chromosome is that the target 2 is inserted into the upstream 3 rd and 4 th bases of PAM and the A is inserted between the 3 rd and 4 th bases of PAM, and the mutation of the C06 chromosome is that the target 1 is inserted into the upstream 3 rd and 4 th bases of PAM and the C is inserted between the 3 rd and 4 th bases of PAM.

specific information for sgrnas is shown in table 1.

TABLE 1 sgRNA related information

。

Genetic transformation of (II) vectors

(1) The hypocotyls of the rape seedlings after 6 days of sowing are cut by sterile forceps and a dissecting knife, each length is 0.8-1 cm, the cutting effect is better in a DM liquid culture medium, and the explants are cut vertically by one knife as much as possible.

(2) The OD value of the strain is measured (preferably about 0.8 in LB, generally 16 h), and the cultured strain is centrifuged at 6000 rpm for 10min. The supernatant was decanted and resuspended in the same volume of DM as the bacterial liquid (20 mL DM dilution, performed in sterilized culture dishes).

(3) Placing the cut explant into a dish with prepared concentration, and infecting for 15-30 min (the time can not be long, otherwise the explant is easy to die). Shaking for 4-5 times at intervals, wherein 150-200 explants per dish per 20mL bacterial liquid are suitable for infection.

Note that: the bacterial liquid can be prepared first, and after all the explants are cut, the explants are transferred into the prepared bacterial liquid together, and timing is started at the moment.

(4) Transferring into M1 culture medium, placing 20-25 explants per dish at 24 ℃ in dark.

(5) After 36-48 h, the cells were transferred to M2 and incubated in light (24 ℃ C., 16h day/8 h night).

(6) Three weeks later, transferring the plant to M3, and carrying out secondary generation every 2-3 weeks until green buds appear.

(7) Transferring into M4 for rooting, and taking 2-4 weeks.

(III) identification of the corresponding mutation sites

In transgenic plantsBnaA07G0241300ZSAndBnaC06G0264200ZSthe editing condition of the gene is detected, and the sequence of the detection primer is as follows:

Fbox-sg123F ACCCGCCGCAAACGAACG；

Fbox-sg123R TGAGCCATCGATTCTCGTC。

sequencing analysis is carried out on the sequence after PCR amplification, gene editing mutation is analyzed by using a Hi-Tom method, and the result shows that the mutation of the A07 chromosome is that the target 2 is inserted into the upstream 3 rd and 4 th bases of PAM and A is inserted between the 3 rd and 4 th bases of PAM, and the mutation of the C06 chromosome is that the target 1 is inserted into the upstream 3 rd and 4 th bases of PAM and C is inserted between the 3 rd and 4 th bases of PAM.

Example two, rapeSKP1-IP15Phenotypic analysis of knockout lines

Based on the same cultivation conditions, field phenotype observation and identification are carried out on the CRISPR-Cas9 material and the transgene control thereof obtained by the previous example, and the discovery is thatBnaA07G0241300ZSAndBnaC06G0264200ZSthe double-mutant knockout rape strain height, effective branch number and fruiting rate of the double-mutant knockout rape strain are all obviously higher than those of transgenic control materials (see figure 2). Description of the applicationSKP1-IP15Negative regulation and control of effective branching number and yield of rape, and cornKWE2Genes have relatively conserved functions.

Claims (7)

1. Rape yield regulation geneSKP1-IP15The nucleotide sequence is at least one of the following groups:

(1) As shown in SEQ ID NO.1 or SEQ ID NO. 2;

(2) DNA molecules which are formed by one to several base substitutions and/or one to several base insertions and/or deletions and large fragment nucleotide sequence insertions/deletions/shifts/inversions on the basis of SEQ ID No.1 or SEQ ID No.2 and can influence the phenotype of the yield-related traits.

2. Rape yield regulation geneSKP1-IP15The amino acid sequence of the encoded protein is at least one of the following groups:

(1) A protein consisting of the amino acid sequence encoded by SEQ ID NO.1 or SEQ ID NO. 2;

(2) As shown in SEQ ID NO.3 or SEQ ID NO. 4;

(3) A protein which is obtained by substituting, deleting and/or adding one or a plurality of amino acid residues from SEQ ID NO.3 or SEQ ID NO.4 and has the property of influencing yield.

3. A gene for regulating rape yield according to claim 1SKP1-IP15The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacterium.

4. Identification of rape yield regulation geneSKP1-IP15A mutant primer pair having the sequence:

Fbox-sg123F：5’- ACCCGCCGCAAACGAACG -3’

Fbox-sg123R：5’- TGAGCCATCGATTCTCGTC -3’ 。

5. the rape yield regulating gene of claim 1SKP1-IP15The recombinant expression vector of claim 3 or the primer pair of claim 4 in regulating rapeThe application in plant height, effective branch number or/and leaf area.

6. The rape yield regulating gene of claim 1SKP1-IP15Use of the recombinant expression vector of claim 3 or the primer pair of claim 4 in rape yield trait improvement and dominant variety/line selection.

7. The use according to claim 6, wherein expression of the canola gene is silenced/down-regulatedSKP1-IP15To increase the plant height, effective branch number and/or leaf area of rape.