CN114085846B

CN114085846B - Application of rice OsNAC2 gene in regulation of rice leaf included angle

Info

Publication number: CN114085846B
Application number: CN202111568997.7A
Authority: CN
Inventors: 姜大刚; 李流裕
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-10-04
Anticipated expiration: 2041-12-21
Also published as: CN114085846A

Abstract

The invention discloses an application of a rice OsNAC2 gene in regulation of rice leaf included angle, and belongs to the field of plant genetic engineering. The invention provides application of OsNAC2 protein or related biological materials thereof in regulation of rice leaf included angle, wherein the OsNAC2 protein is protein with an amino acid sequence of SEQ ID No. 2; the related biological material is a nucleic acid molecule capable of expressing the OsNAC2 protein, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. According to the invention, osNAC2 is applied to the leaf angle regulation of rice for the first time, and the leaf angle of a plant can be obviously reduced after the OsNAC2 is knocked out by the CRISPR/Cas9 technology, so that the regulation and control mechanism of the leaf angle can be known, and a foundation is laid for the development and improvement of an ideal plant type of rice.

Description

Application of rice OsNAC2 gene in regulation of rice leaf included angle

Technical Field

The invention relates to the field of plant genetic engineering, in particular to application of a rice OsNAC2 gene in regulation and control of rice leaf included angle.

Background

In the age with limited cultivated land area, how to more effectively utilize the limited cultivated land to improve the unit yield of crops becomes a research hotspot of breeders. In addition to increasing the yield of a single plant of a crop, increasing the planting density of a crop by improving the plant type of the crop is also an important crop improvement direction. The plant type of rice is determined by plant height, tillering, leaf length, leaf angle and spike type. The included angle of the leaves is related to the planting density of crops, the crop with a small included angle of the leaves has a compact upright plant type, and the planting density can be effectively improved, so that the unit yield of rice is improved.

The leaf angle is the inclination between the leaf and the leaf sheath and is mainly determined by the cell morphology of the leaf pillow at the junction of the leaf and the leaf sheath. Increased cell division or cell elongation on the paraxial side of the leaf pillow leads to increased leaf angle. In addition, the leaf pillow acts as the main weight-bearing organ for leaf outgrowth, and the normal growth of its cells is also critical to the inclination of the leaf. When the secondary cell walls of the leaf pillow cells cannot be normally formed, the mechanical strength of the leaf pillow becomes low, and the vertical growth of the leaves cannot be supported, so that the leaves droop, and the leaf included angle is increased.

In recent years, many leaf angle-regulating genes have been cloned and identified, such as OsDWARF4, brassinosteroid sensitive 1 (OsBRI 1), rice leaf cloning 3 (LC 3), and Austin stress factors19 (ARF 19). The mutant of the genes is mainly involved in the regulation of the rice leaf angle by regulating the content of plant hormone and signal transduction. OsDWARF4 is a key gene for synthesizing Brassinolide (BR), and the leaf included angle of a function-deficient mutant dwarf4-1 is reduced, so that the leaves are more upright. The mutant d61 of OsBRI1 has the effect of reducing the leaf angle due to the BR signal transduction obstruction in vivo. LC3 can interact with LC3-interacting protein 1 (LIP 1) protein to inhibit the expression of OsIAA12 and auxin metabolism related gene OsGH3.2, thereby influencing the signal transduction of auxin and down-regulating the content of the auxin to inhibit the growth of cells on the paraxial side of the leaf pillow so as to reduce the leaf angle. Many studies indicate that brassinolide can promote the elongation of cells on the paraxial side of a leaf pillow to positively regulate the leaf angle of rice. The auxin can regulate the growth and division of cells at the leaf pillow to negatively regulate the size of a leaf included angle, and the auxin also participates in the formation of secondary cell walls at the leaf pillow to regulate the mechanical strength of the leaf pillow. However, phytohormone regulation of leaf angle is a complex process involving many genes and multiple hormones.

The OsNAC2 transcription factor belongs to the NAC transcription factor family. The rice genome contains 151 NAC genes, which is one of the largest transcription factor families in rice, and the NAC transcription factor family is widely involved in various processes of rice growth and development, including phytohormone signal transduction, leaf senescence, some abiotic stress responses and the like. However, the function of most OsNAC transcription factors is not clear and remains to be studied.

Disclosure of Invention

The invention aims to provide an application of rice OsNAC2 gene in regulation of rice leaf angle, so as to solve the problems in the prior art, the invention applies OsNAC2 gene to regulation of rice leaf angle for the first time, and the OsNAC2 gene is knocked out by CRISPR/Cas9 technology, so that the leaf angle of a plant can be obviously reduced, the regulation and control mechanism of the leaf angle can be favorably understood, and the foundation is laid for development and improvement of an ideal plant type of rice.

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

the invention provides an application of OsNAC2 protein or related biological materials thereof in regulation of rice leaf included angle,

the OsNAC2 protein is a protein with an amino acid sequence of SEQ ID No. 2;

the related biological material is a nucleic acid molecule capable of expressing the OsNAC2 protein, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

Further, the higher the expression quantity and/or activity of the OsNAC2 protein or the coding gene thereof in the rice is, the larger the leaf angle of the rice is; the lower the expression quantity and/or activity of the OsNAC2 protein or the coding gene thereof in the rice is, the smaller the leaf angle of the rice is.

Furthermore, the coding gene of the OsNAC2 protein is a DNA molecule shown in SEQ ID No. 1.

The invention also provides a method for cultivating rice with smaller leaf angle, which comprises the step of reducing the expression quantity and/or activity of OsNAC2 protein in receptor rice; the OsNAC2 protein is a protein with an amino acid sequence of SEQ ID No. 2.

Further, the method comprises the following steps: carrying out suppression expression on the coding gene of OsNAC2 protein in receptor rice to obtain transgenic rice; compared with the receptor rice, the transgenic rice has smaller blade included angle.

The invention also provides a method for cultivating rice with larger leaf included angle, which comprises the step of improving the expression quantity and/or activity of OsNAC2 protein in receptor rice; the OsNAC2 protein is a protein with an amino acid sequence of SEQ ID No. 2.

Further, the method comprises the following steps: introducing a nucleic acid molecule capable of expressing OsNAC2 protein into receptor rice to obtain transgenic rice; the transgenic rice has a larger leaf angle than the recipient rice.

Furthermore, the nucleic acid molecule capable of expressing the OsFD2 protein is a coding gene of the OsNAC2 protein and is a DNA molecule shown in SEQ ID No. 1.

The invention also provides an application of the OsNAC2 gene or the protein expressed by the OsNAC2 gene in rice breeding, wherein the OsNAC2 gene is used for improving the rice leaf angle character, improving the rice plant type and adjusting the rice planting density; the nucleotide sequence of the OsNAC2 gene is shown as SEQ ID No.1, and the sequence of the expressed protein is shown as SEQ ID No. 2.

Further, the OsNAC2 gene overexpression vector is used for transforming rice plants to obtain transgenic rice with large included angle of rice leaves; or the OsNAC2 gene knockout vector is used for transforming rice plants to obtain the transgenic rice with the rice leaf included angle reduced.

The invention discloses the following technical effects:

the invention realizes the overexpression of OsNAC2 in the transformed rice by constructing an OsNAC2 overexpression vector, the obtained plant leaves droop, and the included angle of the leaves is increased; the plant leaf included angle is reduced and the leaf is upright by constructing a CRISPR/Cas9 gene knockout vector of OsNAC2 and converting the CRISPR/Cas9 gene knockout vector into No. 11 rice, so that the planting density of rice can be improved, and the potential value of improving the population photosynthetic efficiency and the yield of rice is realized.

According to the invention, osNAC2 is applied to the leaf angle regulation of rice for the first time, and the leaf angle of a plant can be obviously reduced after the OsNAC2 is knocked out by the CRISPR/Cas9 technology, so that the regulation and control mechanism of the leaf angle can be known, and a foundation is laid for the development and improvement of an ideal plant type of rice.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a leaf angle phenotype plot of flag leaves at heading stage of wild type mid-flower No. 11 and OsNAC2 overexpression mutants;

FIG. 2 Sword leaf angle at heading stage of wild type Mihua No. 11 and OsNAC2 overexpression mutants;

FIG. 3 real-time fluorescent quantitative PCR results at the occiput of wild type mid-flower No. 11 and OsNAC2 overexpressing mutants;

FIG. 4 shows mutation sites in OsNAC2 mutant gene knockout;

FIG. 5 is a leaf angle phenotype plot of the floral 11 and OsNAC2 knockout mutant during filling stage of flag leaf in wild type;

FIG. 6 Sword leaf angle in the filling stage of wild type Mihua No. 11 and OsNAC2 mutants.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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 only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

The rice variety to be tested is the rice japonica rice variety medium flower 11, namely the rice (Oryza sativa L.) cv. medium flower 11.

A method for regulating and controlling the size of a rice leaf included angle by regulating and controlling the expression level of a rice transcription factor OsNAC2 in rice. The coding gene of rice OsNAC2 is driven by a constitutive promoter, and the expression of OsNAC2 is up-regulated in rice by infecting rice calluses and transforming the rice calluses into the rice through agrobacterium tumefaciens, so that rice plants with increased leaf included angles are obtained. The OsNAC2 function deletion mutant is obtained by constructing a CRISPR/Cas9 gene knockout vector of rice OsNAC2 and transforming the gene into rice, so that the rice with compact leaf stand plant type is obtained. The specific method comprises the following steps:

1. an OsNAC2 gene (LOC _ Os04g 38720) is obtained from a national rice data center (https:// www.ricedata.cn /), the sequence of the OsNAC2 gene is shown as SEQ ID No.1, the amino acid sequence of the encoded protein is shown as SEQ ID No.2, and corresponding primers are designed to amplify rice cDNA so as to obtain the cDNA of the OsNAC2 gene.

The primer sequence is as follows:

OsNAC2-F:ATGCCGCAAGCCTCCTCCTCCT

OsNAC2-R:TTAGCCGTGCCCAAATTGTTGG

SEQ ID No.1 (Rice (Oryza sativa) OsNAC2 cDNA full length):

SEQ ID No.2 (Rice (Oryza sativa) OsNAC2 protein sequence):

MEQHQGQAGMDLPPGFRFHPTDEELITHYLAKKVADARFAALAVAEADLNKCEPWDLPSLAKMGEKEWYFFCLKDRKYPTGLRTNRATESGYWKATGKDKDIFRRKALVGMKKTLVFYTGRAPKGEKSGWVMHEYRLHGKLHAAALGFLHGKPASSKNEWVLCRVFKKSLVEVGAAGGKKAAVVTMEMARGGSTSSSVADEIAMSSVVLPPLMDMSGAGAGAVDPATTAHVTCFSNALEGQFFNPTAVHGHGGGDSSPFMASFTQYGQLHHGVSLVQLLESCNGYGGLVDMAASGSQLQPAACGGERERLSASQDTGLTSDVNPEISSSSGQKFDHEAALWGY.

2. construction of rice OsNAC2 overexpression vector

After cloning and amplifying in a cDNA library of rice, the OsNAC2 gene is recovered and purified by using a DNA recovery kit of GenStar company. And connecting the purified DNA fragment into a vector pox vector (stored in the laboratory), transforming E.coli DH5a competent cells, selecting a positive single colony clone-extracted plasmid, and obtaining an overexpression vector of OsNAC2 after sequencing identification.

And infecting the callus of the No. 11 plant of the rice with agrobacterium tumefaciens, inducing and differentiating into seedlings, and obtaining the transgenic plant of the transformed OsNAC2 overexpression vector. The RNA of ZH11 and wild-type WT that had been transformed with NAC2 overexpression vector was extracted by trizol extraction. Mu.g of RNA was reverse transcribed to obtain cDNA.

Design of OsNAC2 specific primers:

q-NAC2-F：GGGCCAGTTCTTTAACCCGA

q-NAC2-R：CTCTCCAGGAGTTGCACCAG

UBQ is used as an internal reference gene, cDNA obtained by reverse transcription is used as a template, and an OsNAC2 specific primer is used for carrying out real-time fluorescence quantitative PCR. Two transgenic plants with the OsNAC2 expression quantity obviously up-regulated are obtained and are marked as OE1 and OE2.

3. Construction of CRISPR/Cas9 gene knockout vector of rice OsNAC2

According to the technical principle of CRISPR/Cas9 gene editing, through database and tool website analysis, NGG is selected in a second exon of a genome nucleotide sequence of an OsNAC2 gene, a target sequence AGGACAAGGACATCTTCAGA of a specific gRNA of the OsNAC2 gene is selected at the front position of a target gene, and in order to enable a transgenic strain to carry out targeting mutation at the front position.

The adapter primers of the gRNA target sequence are:

F：AGGACAAGGACATCTTCAGA

R：CGTCGTCCTCCCTCCGCTGA

a target sequence is introduced into the downstream of the U3/U6 promoter and the upstream of the sgRNA sequence through a first round of PCR, and the promoter, the target and the sgRNA are constructed into a complete expression cassette by carrying out PCR amplification by using a second round of universal primers. And finally, assembling the sgRNA expression cassette into a CRISPR/gRNA vector (provided by Liu Yaoguang academy and stored in the laboratory) by a side-cutting ligation method, transforming E.coli DH5a competent cells, selecting positive single colony clone-extracted plasmids, and obtaining the knockout vector of OsNAC2 after sequencing identification.

The knockout vector is introduced into No. 11 rice plants through an agrobacterium-mediated method. Taking the callus of the plant of the Zhonghua No. 11 as a transformation receptor, and obtaining a complete regeneration plant through tissue culture after transformation to obtain a transgenic plant with OsNAC2 gene function deletion, namely a homozygous plant with OsNAC2 site mutation.

Extracting genome DNA of a transgenic plant containing hygromycin resistance, carrying out PCR amplification on the genome DNA by using a primer containing a target site OsNAC2, and carrying out sequencing verification on a PCR product.

NAC2-CT F：GGGCCAGTTCTTTAACCCGA

NAC2-CT R：CTCTCCAGGAGTTGCACCAG

The result of the comparative sequencing shows that the transformed plants of the two homozygous mutants are obtained. One mutant CR-nac2 begins to delete and insert 7 nucleotide sequences (the obtained sequence is shown as SEQ ID No. 3) in the 305 th base of the OsNAC2 gene shown as SEQ ID No. 1; another mutant CR-nac2 begins to delete 35 nucleotide sequences at the 276 th base of the OsNAC2 gene shown in SEQ ID No.1 (the obtained sequence is shown in SEQ ID No. 4).

As for the mutant CR-nac 21, osNAC2 gene, the nucleotide sequence at positions 334-336 of OsNAC2 gene can be transcribed into a stop codon because of the frame shift mutation. As for the mutant CR-nac2, osNAC2 gene, the nucleotide sequence at position 694-696 of OsNAC2 gene may be transcribed into a stop codon due to the frame shift mutation described above.

SEQ ID No.3：

SEQ ID No.4：

4. The rice OsNAC2 overexpression mutant and the function deletion mutant as well as the wild rice Zhonghua No. 11 plant are planted in a field, and the phenotypic difference between the rice OsNAC2 mutant system plant and the Zhonghua No. 11 plant in the whole growth period is observed. As shown in the graph, compared with the plants of Zhonghua No. 11 (ZH 11), both plants of the OE1 and OE2 overexpression lines of NAC2 showed a phenotype of increased leaf angle (FIGS. 1 and 2), while both plants of the OsNAC2 knockout mutant lines CR-NAC2 and CR-NAC2 (FIG. 3) showed a phenotype of decreased leaf angle (FIGS. 4 and 5). Thereby proving that the OsNAC2 gene participates in the regulation of the leaf angle of the rice.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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Application of rice OsNAC2 gene in regulation of rice leaf included angle

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Claims

Application of OsNAC2 protein or related biomaterials thereof in regulation and control of rice leaf included angles is characterized in that the OsNAC2 protein is protein with an amino acid sequence of SEQ ID No. 2;

the related biological material is a nucleic acid molecule capable of expressing the OsNAC2 protein, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.
2. The use according to claim 1, wherein the higher the expression level and/or activity of OsNAC2 protein or a gene encoding the same in the rice, the larger the leaf angle of the rice; the lower the expression quantity and/or activity of the OsNAC2 protein or the coding gene thereof in the rice is, the smaller the leaf angle of the rice is.
3. The use according to claim 1 or 2, wherein the gene encoding OsNAC2 protein is a DNA molecule represented by SEQ ID No. 1.
4. A method for breeding rice with a smaller leaf angle, which comprises the steps of reducing the expression level and/or activity of OsNAC2 protein in receptor rice; the OsNAC2 protein is a protein with an amino acid sequence of SEQ ID No. 2.
5. The method of claim 4, comprising the steps of: carrying out suppression expression on the coding gene of OsNAC2 protein in receptor rice to obtain transgenic rice; compared with the receptor rice, the transgenic rice has smaller blade included angle.
6. A method for cultivating rice with larger leaf angle is characterized by comprising the steps of improving the expression quantity and/or activity of OsNAC2 protein in receptor rice; the OsNAC2 protein is a protein with an amino acid sequence of SEQ ID No. 2.
7. The method of claim 6, comprising the steps of: introducing nucleic acid molecules capable of expressing OsNAC2 protein into receptor rice to obtain transgenic rice; the transgenic rice has a larger leaf angle than the recipient rice.
8. The method according to claim 7, wherein the nucleic acid molecule capable of expressing OsNAC2 protein is a gene encoding OsNAC2 protein and is a DNA molecule shown as SEQ ID No. 1.
The application of the OsNAC2 gene or the protein expressed by the OsNAC2 gene in rice breeding is characterized in that the OsNAC2 gene is used for improving the rice leaf included angle character, improving the rice plant type and adjusting the rice planting density; the nucleotide sequence of the OsNAC2 gene is shown as SEQ ID No.1, and the sequence of the expressed protein is shown as SEQ ID No. 2.
10. The use according to claim 9, wherein the OsNAC2 gene overexpression vector is used for transforming rice plants to obtain transgenic rice with enlarged rice leaf included angle; or the OsNAC2 gene knockout vector is used for transforming rice plants to obtain the transgenic rice with the rice leaf included angle reduced.