CN118028307A - Application of TaNAC22-A1 gene in negative regulation of small flower number in wheat ears - Google Patents

Abstract

The invention discloses an application of TaNAC22-A1 gene in negative regulation of the small flower number in wheat ears, wherein the gene sequence numbers of the TaNAC22-A1 gene in EnsemblPlants and NCBI are TraesCS5A02G500500.1 and XM_044527981.1 respectively. According to the invention, the natural mutant is screened to obtain the TaNAC22-A1 mutant plant, and the result shows that the inflorescence morphology of the gene mutant plant is changed, the number of flowers in wheat ears is obviously increased, and the gene mutant plant plays a role in the number of flowers. Then constructing a CRISPR gene knockout vector of TaNAC22-A1, transforming wheat wild type field (WT) by using an agrobacterium inflorescence infection method to obtain a gene knockout plant, wherein an analysis result shows that the TaNAC22-A1-CR mutant strain can improve the number of flowers in wheat ears relative to the wild type under the normal growth condition, thereby providing gene resources for crop high-yield molecular breeding.

Description

Application of TaNAC22-A1 gene in negative regulation of small flower number in wheat ears

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a TaNAC22-A1 gene in negative regulation of the number of flowers in wheat ears.

Background

The number of flowers is one of the key factors in determining wheat yield during the development of wheat ears. In the growth cycle of wheat, the spike differentiation stage is a very important period, which is directly related to the fruiting and final yield of wheat. The ear development process comprises the key steps of ear differentiation, floret differentiation, pollen mother cell meiosis, pollen enrichment and the like. The number of flowers directly affects the number of grains per spike of wheat, which in turn affects overall yield. In general, the more flowers per ear, the more kernels theoretically can be formed, and thus it is possible to increase the yield per plant and unit area.

Wheat is one of the important food crops worldwide, and the yield of the wheat is directly related to the development of food safety and agricultural economy. Wheat is a major component of the people's daily diet in many countries, especially developing countries, providing a large amount of carbohydrates, proteins, and essential vitamins and minerals. Therefore, ensuring high and stable wheat yield is important to ensuring national nutrition demand and promoting social and economic stability. Wheat is also a main ration crop in China, and has important significance for ensuring high and stable yield. The ear structure of wheat has a decisive influence on the yield and quality of wheat. Improving the development structure of wheat ears can increase the yield, and improving the inflorescence structure can increase the effective number of ears and flowers per ear, thereby increasing the number of seeds per ear. This has an important meaning for improving the yield per unit area; the quality can be improved, and the inflorescence improvement can also improve the uniformity and the fullness degree of the seeds, so that the quality of wheat is improved. This is particularly important to meet different food processing requirements and to improve flour quality; the adaptability can be enhanced, the improved inflorescences can better adapt to adverse environmental conditions such as drought, high temperature and the like, the phenomenon of sterility or empty shell caused by stress is reduced, and the stability of the yield is ensured; the disease and insect resistance can be improved, the improvement of inflorescences is beneficial to improving the resistance of wheat to diseases and insect pests, the use of pesticides is reduced, the production cost is reduced, and the method is environment-friendly; meanwhile, the resource utilization efficiency can be improved, the utilization efficiency of wheat to illumination, moisture and nutrients can be improved by optimizing the inflorescence structure, the healthy growth of crops is promoted, and the sustainability of agricultural production is improved.

The NAC gene family is a family of plant-specific transcription factors that are widely found in a variety of plants. NAC derives its name from the first three members of the family: no APICAL MERISTEM (NAM), arabidopsis Transcription Activation Factor (ATAF), and Cup-shaped cotyledon (CUC). The NAC family of genes plays an important role in plant growth, signaling, stress management, and the like. The NAC gene is involved in plant organ formation and differentiation, such as flower development, root system construction, leaf senescence, and the like. For example, the CUC gene plays a key role in plant embryogenesis and establishment of organ boundaries. NAC gene family members play an important role in plants responding to drought, salt and alkali, cold, pest and disease damage, and other biotic and abiotic stress. They help plants adapt to adverse environmental conditions by regulating the expression of downstream stress-related genes. In addition, members of the NAC gene family are also involved in plant hormone signaling pathways, such as auxins, gibberellins, ethylene, etc., that regulate plant growth and development by interacting with these hormones. Some NAC genes play a role in the immune response of plants and can enhance the resistance of plants to pathogens. These genes may increase disease resistance in plants by activating the expression of defense-related genes. Due to their multiple functions in plant physiology and stress response, the NAC gene families become important targets for plant molecular breeding and genetic engineering. By manipulating the expression of the NAC gene, scientists can develop crops with improved properties, such as increased drought resistance, increased pest protection or improved growth habit.

Thus, improvement of wheat inflorescences is key to achieving high yield, high quality, high efficiency and sustainable development of wheat, while NAC transcription factor family plays an important role therein. Thus, by studying the wheat NAC transcription factor family genes, one can better understand the important role of NAC genes in the development of wheat, especially in the development of ears. And further, by means of modern breeding technology such as molecular marker assisted selection, gene editing and the like, wheat varieties are improved more accurately, and high-yield wheat varieties with more excellent wheat inflorescence structures and more florets are obtained, so that the ever-increasing global grain requirements are met, and the ever-changing environmental challenges are met. Has great significance for the long-term stable development and safety of grain production in China, knows and finds out genes more related to wheat ear development, and has great significance for researching the functions of wheat inflorescence development from a molecular level.

Disclosure of Invention

The invention aims to provide an application of TaNAC22-A1 gene in negative regulation of the small flower number in wheat ears.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

the gene sequence numbers of the TaNAC22-A1 gene adopted by the invention in EnsemblPlants and NCBI are TraesCS5A02G500500.1 and XM_044527981.1 respectively, the length of the messenger RNA (mRNA) sequence of the TaNAC22-A1 gene is 1351bp, the length of the coding sequence of the TaNAC22-A1 gene is 1017bp, the nucleotide sequence is shown as SEQ ID NO.1, the nucleotide sequence comprises 339 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention also constructs a plant expression vector, a transgenic plant system containing the gene knockout vector of the gene and a host cell containing the vector also fall into the protection scope of the invention in terms of improving the number of wheat ears and flowers.

The functions of the genes protected by the invention not only comprise the TaNAC22-A1 gene, but also comprise the functions of homologous genes with higher homology (up to 99 percent of homology) with the TaNAC22-A1 gene in regulating and controlling the number of wheat head development florets.

The invention discloses a biological function of TaNAC22-A1 gene in regulating and controlling the small flower number in wheat ears, which is specifically expressed in: under normal growth conditions, both the TaNAC22-A1 deletion mutant and the CRISPR gene knockout transgenic plant line had higher numbers of wheat ears and flowers than the wild type.

According to the function thereof, a plant with the number of flowers in an inflorescence, particularly, a transgenic plant with the number of flowers in an inflorescence higher than that of a target plant can be obtained by knocking out or reducing the expression level of TaNAC22-A1 gene from the target plant.

Specifically, the TaNAC22-A1 gene can be introduced into the plant of interest specifically via the CRISPR-Cas9 vector. In the method, the CRISPR-Cas9 vector may be used to transform plant cells or tissues by using conventional biological methods such as plant viral vectors, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In order to improve the excellent properties of plants, the present invention also protects a novel plant breeding method, which is the following (1) or (2):

(1) Obtaining a plant with salt stress tolerance stronger than that of the target plant by knocking out the activity of TaNAC22-A1 protein in the target plant;

(2) Obtaining plants with the number of florets different from that of the target plants by regulating and controlling the expression of TaNAC22-A1 genes in the target plants;

A method of "modulating expression of the TaNAC22-A1 gene in a plant" is over-expression, silencing or directed mutation of the TaNAC22-A1 gene.

For example, plants having a greater number of florets than the plant of interest can be obtained by reducing/deleting the expression of the TaNAC22-A1 gene in the plant of interest.

The "reduction/deletion of expression of the TaNAC22-A1 gene in the plant of interest" can be achieved as follows (1) or (2) or (3):

(1) Knocking out the TaNAC22-A1 gene from a target plant for expression;

(2) Reducing expression of the TaNAC22-A1 gene from the plant of interest;

(3) Other methods are common in the art.

Wherein the plant of interest of the present invention is wheat.

Regulating the gene expression level comprises regulating the expression of TaNAC22-A1 by utilizing a DNA homologous recombination technology, a virus-mediated gene silencing technology and an agrobacterium-mediated transformation system to obtain a transgenic plant line.

In the present invention, the plant suitable for the present invention is not particularly limited as long as it is suitable for performing a gene transformation operation such as various crops, flower plants, forestry plants, or the like. The plant may be, for example (without limitation): dicotyledonous, monocotyledonous or gymnosperm plants.

As a preferred mode, the "plant" includes, but is not limited to: wheat, any gene having the gene or a gene homologous thereto is suitable.

As used herein, the term "plant" includes whole plants, parent and progeny plants thereof, and various parts of plants, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, in which the gene or nucleic acid of interest is found. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises the gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by a method therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the sub-representations exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. And further to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to a food or food additive obtained from the relevant plant.

The invention has the advantages that:

(1) The invention adopts a transcription regulation histology method to creatively clone the NAC transcription factor TaNAC22-A1 affecting the development process of wheat ears in wheat. The TaNAC22-A1 mutant strain and the CRISPR transgenic knockout strain are obtained, and the results show that under the normal growth condition, the numbers of wheat ears and florets of the TaNAC22-A1 deletion mutant strain and the CRISPR transgenic knockout strain are higher than those of the wild type strain. Provides gene resources for wheat high-yield molecular breeding.

(2) Plants with significantly increased florets can be obtained by transgenic means, and in particular, transgenic plants can be obtained by knocking out or reducing expression of the TaNAC22-A1 gene from a target plant, and the inflorescence florets of the plants are higher than the target plant, so that a new way is provided for high-yield breeding of plants.

Drawings

FIG. 1 is a TaNAC22-A1 CDS sequence and encoded amino acid sequence analysis; FIG. 1A is a TaNAC22-A1 CDS sequence,

FIG. 1B is the amino acid sequence encoded by TaNAC 22-A1;

FIG. 2 is subcellular localization of TaNAC 22-A1;

FIG. 3 is a phenotypic comparison of wheat TaNAC22-A1 mutant plants (Tanac-A1) and wild type plants (WT) under normal growth conditions;

FIG. 4 is a comparison of early stage floriation phenotypes in wheat TaNAC22-A1 mutant plants (Tanac-A1), CRISPR knockout plants (Tanac-A1-CR) and wild type plants (WT) spikelet under normal conditions using scanning electron microscopy;

FIG. 5 is a statistical comparison of the number of flowers in the tasac 22-A1 mutant plant (Tanac-A1), CRISPR knockout plant (Tanac-A1-CR-1, taNAC 22-A1-CR-2) and wild type plant (WT) inflorescence of wheat under normal conditions.

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Biological material

Wheat seeds are preserved in a laboratory;

the TaNAC22-A1 gene CRISPR-Cas9 knockout transgenic material is from unmixed biotechnology (Jiangsu) limited;

Coli DH 5. Alpha. And Agrobacterium GV3101 were kept in the laboratory;

The subcellular localization vector pSuper-GFP is stored in a laboratory;

Primer synthesis and sequencing were performed by the biotechnology company of the Boxing family of Beijing Rui.

Experimental reagent

RNA extraction kits, reverse transcription kits, and fluorescent quantification kits were purchased from beijing tiangen biotechnology limited;

Common reagents such as NaCl are purchased from Soy Corp;

hygromycin is purchased from soribao biosystems;

MS media was purchased from beijing cool pacing technologies limited;

various endonucleases (NEB) were purchased from the lark biotechnology company, inc;

one-step cloning enzyme was purchased from nuuzan biotechnology limited;

Plasmid miniprep and gel recovery kits were purchased from beijing tiangen biotechnology limited.

Experimental equipment

PCR instrument was purchased from Applied Biosystems company;

refrigerated centrifuges are purchased from Eppendoff company;

quantitative PCR instrument QuantStudio instrument was purchased from Applied Biosystems company;

The confocal laser microscope SZX16 was purchased from Olumpus company;

scanning electron microscope Hitachi-3000N was purchased from Hitachi Corp

The high temperature autoclave MLS-3750 is purchased from HIRAYAMA corporation of Japan;

Nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific company;

the room temperature centrifuge was purchased from Eppendorf corporation.

EXAMPLE 1 cloning of the TaNAC22-A1 Gene and amino acid sequence analysis thereof

Extracting RNA of Chinese spring wheat growing for 15 days, taking cDNA obtained by reverse transcription reaction as a template, designing specific primers by using TaNAC22-A1 gene sequences obtained from NCBI database through Primer premier5.0, cloning coding sequences of the TaNAC22-A1 gene respectively, and converting the nucleic acid sequences into protein sequences through a trans (https:// www.expasy, org/resources/on-line tool), and further analyzing the TaNAC22-A1 gene sequences and the coded protein sequences by using Genedoc software.

The results indicate that the coding sequence of the wheat TaNAC22-A1 gene comprises 1017bp bases (fig. 1A) and the encoded protein comprises 339 amino acids (fig. 1B). The specific primers used were:

P1-F：5′-ATGGAGGTCGATCAGGACCT-3′；

P1-R：5′-TTAATATAGCAGATGGGCGT-3′；

EXAMPLE 2 TaNAC22-A1 Gene subcellular localization

In order to explore the characteristics of the TaNAC22-A1 transcription factor, the inventors constructed the gene subcellular GFP vector pSuper-GFP-TaNAC22-A1, and the specific procedure is briefly described below.

First, a primer having a restriction enzyme EcoRI cleavage site was designed, and the sequence was as follows:

P1-EcoR I-F：5′-CGGAATTCATGGAGGTCGATCAGGACCT-3′；

P1-EcoR I-R：5′-CGGAATTCTTAATATAGCAGATGGGCGT-3′；

then, PCR amplification was performed using the cDNA sample prepared in example 1 as a template, and the amplified product was purified and recovered;

Thirdly, carrying out single enzyme digestion on the pSuper-GFP vector by adopting EcoR I, and purifying enzyme digestion products;

Fourthly, carrying out homologous recombination connection on the PCR amplification product and the vector after enzyme digestion to construct a pSuper-GFP-TaNAC22-A1 subcellular localization vector;

fifthly, converting the connection product into escherichia coli DH5 alpha by adopting a heat shock conversion method, carrying out resistance screening of A (ampicillin, 50 mug/mL), selecting positive colonies for PCR detection, amplifying and sequencing correct colonies identified by the PCR detection, and extracting plasmids from bacterial liquid with correct sequencing for later use;

Sixth, the extracted plasmid is transformed into agrobacterium GV3101 to infect tobacco leaves, and after dark culture for 3 days, the tobacco leaves are observed and photographed under a fluorescence microscope.

The TaNAC22-A1 gene, which is a transcription factor, is localized in the nucleus, as shown in fig. 2. It was demonstrated that the TaNAC22-A1 gene may function as a transcription factor in the nucleus, activating or inhibiting expression of downstream genes, involved in regulating the number of florets in wheat ear development.

Example 2 TaNAC22-A1 Gene deletion verifies its function in the number of flowers in wheat ears

To analyze whether the TaNAC22-A1 gene is involved in the process of determining the number of flowers in the development of wheat ears, the expression pattern of the TaNAC22-A1 gene was first analyzed. The TaNAC22-A1 gene is expressed in roots, stems, leaves, ears, florets and seeds of wheat, and the expression amount is highest in the ears and the florets, so that the result shows that the TaNAC22-A1 gene can be involved in determining the number of florets in the development process of the wheat ears.

To further analyze the function of TaNAC22-A1 gene in participating in the number of flowers in the development process of wheat ears, tanac-A1 mutants were screened, and the inventors identified homozygous mutants. It was found that under normal growth conditions, the Tanac-A1 mutant had slightly increased plant height, no obvious difference in inflorescence length and significantly increased flower number in the spikelet (FIG. 3). The inflorescence early-development material was dissected, and the number of the floret primordia of Tanac-A1 mutant plants was found to be about 4 more than that of the wild type plants (FIG. 4). The number of spikelet flowers of Tanac-A1 mutants was about 8-10, significantly increased compared to the wild type (about 3-5 spikelet flowers) at late inflorescence development. The TaNAC22-A1 gene plays an important role in determining the number of flowers in the development process of wheat ears.

Further, we screened the gRNA targeting TaNAC22-A1 gene, constructed CRISPR knockout transgene material, obtained Tanac-A1-CR gene editing material. Material early in the development of the anatomic Tanac-A1-CR inflorescence was found to have 3-4 more floret primordia than wild-type for Tanac-A1-CR material (FIG. 4). Further described is the involvement of the TaNAC22-A1 gene in determining the number of florets during wheat ear development.

In summary, the number of wheat spikelet florets after deletion of the TaNAC22-A1 gene is obviously increased, which indicates that the TaNAC22-A1 gene negatively regulates the number of wheat spikelet florets; after the transgenic CRISPR knocks out the TaNAC22-A1 gene, the number of the small flowers is also obviously increased, and the same shows that the TaNAC22-A1 gene negatively regulates the number of the small flowers of the wheat. Therefore, the TaNAC22-A1 gene plays an important role in regulating and controlling the number of small flowers of wheat, and has important significance for cultivating excellent inflorescence structure and high-yield wheat.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.

Claims (6)

1. The application of the TaNAC22-A1 gene in negative regulation of the small flower number in wheat ears is characterized in that the nucleotide sequence of the TaNAC22-A1 gene is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the TaNAC22-A1 deletion mutant or transgenic plant is obtained by screening for a TaNAC22-A1 natural mutant or constructing a TaNAC22-A1 CRISPR gene knockout vector, and the number of wheat ears of the obtained mutant or transgenic plant is higher than that of the wild type under normal growth conditions.
3. 3. A plant breeding method characterized in that the method is (1) or (2) below:

   (1) Obtaining a plant with a larger number of florets than the target plant by reducing the activity of the TaNAC22-A1 protein in the target plant;

   (2) Obtaining plants with the number of florets different from that of the target plants by regulating and controlling the expression of TaNAC22-A1 genes in the target plants; the nucleotide sequence of the TaNAC22-A1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the TaNAC22-A1 protein is shown as SEQ ID NO. 2.
4. 4. A plant breeding method according to claim 3, wherein expression of the TaNAC22-A1 gene in the plant of interest is regulated in such a way that the TaNAC22-A1 gene is overexpressed, silenced or directionally mutated.
5. 5. A plant breeding method according to claim 3, wherein regulating expression of the TaNAC22-A1 gene in the plant of interest can result in plants having a greater number of florets than the plant of interest by reducing/deleting expression of the TaNAC22-A1 gene in the plant of interest.
6. 6. A plant breeding method according to claim 3, wherein the plant of interest is wheat.