CN117904139A

CN117904139A - Application of TaFPFL1-2B gene in improving drought resistance of plants

Info

Publication number: CN117904139A
Application number: CN202410251631.4A
Authority: CN
Inventors: 肖军; 王冬至; 林学磊; 周雨馨
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2024-03-05
Filing date: 2024-03-05
Publication date: 2024-04-19

Abstract

The invention discloses an application of TaFPFL1-2B gene in improving drought resistance of plants, wherein GenBank number of TaFPFL-2B gene in NCBI is CM022215.1. According to the invention, the over-expression vector of the LGY-OE-TaFPFL1-2B is constructed, the wheat field is infected by agrobacterium tumefaciens to obtain the over-expression plant, and the field planting result shows that compared with the wild field, the yield of the single plant can be increased by TaFPFL-2B under the condition of water deficiency, that is to say, the drought resistance of wheat can be improved by TaFPFL1-2B genes, so that gene resources are provided for crop water-saving drought-resistant molecular breeding.

Description

Application of TaFPFL1-2B gene in improving drought resistance of plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of TaFPFL-2B genes in improving drought resistance of plants.

Background

The world population is continuously growing, the agricultural cultivated area is reduced, and the global demand for agricultural crop products such as wheat is expected to be increased by 60% by 2050, which clearly puts a higher demand on the yield and planting of staple food crops such as wheat. On the other hand, the situations of large agricultural water consumption and water resource shortage are contradictory, and drought stress aggravated by water shortage has become the most important environmental factor affecting crop production in the global scope, thereby seriously threatening the grain production safety and the socioeconomic development. The report of the "2022 drought figure" issued by the fifteenth meeting of the united nations convention for controlling desertification "states that the number and duration of global drought occurrences has increased by 29% since 2000 and that the global economic loss due to drought is up to 1240 billions of dollars only in 1998 to 2017. The reports of the national food and agricultural organization also indicate that 83% of all economic losses due to drought occur in the agricultural field in 2006 to 2016, with losses worth up to $290 billion.

The characteristics of sessile growth enable plants to evolve various strategies to adapt to poor water-deficient drought environments, and interactions of coordination and intercommunication on morphological, physiological, cellular and molecular levels enable the plants to timely sense environmental changes so as to make adjustments in real time to maintain growth and development of the plants, and the performances of the plants can be divided into drought escape, drought avoidance, drought tolerance and drought recovery according to the respective characteristics. Drought escape is an adaptive feature of plants that grow rapidly to complete their life cycle before severe drought begins, often manifested by early heading, flowering and maturation, reduced plant height and shortened growth cycle. Drought avoidance, in turn, is primarily manifested in plants that control the absorption and loss of moisture through morphological or physiological changes in response to drought. Drought tolerance, also known as drought resistance, refers to the ability of a plant to maintain its growth, development and reproduction under drought stress conditions. Drought resistance is a complex quantitative trait, commonly controlled by multiple agronomic traits and genes, involving multiple drought response signal pathways and metabolic networks, and plants can respond to drought stress using one or more combination strategies. In terms of external visual morphology, the plants in drought state often show morphological changes, the root system configuration is remodeled along with drought stress, the plants absorb more available moisture by enhancing the root system, and the characteristics of the length, the density, the volume, the diameter and the like of the root system are all important indexes for evaluating the drought resistance of the plants, and even root hairs play an important role in adapting to the rhizosphere characteristics to maintain plant nutrition. On the other hand, a decrease in the number of leaves, a decrease in the leaf area, and a decrease in the transpiration are all important manifestations of reduced water loss. The green-keeping ability of leaves is related to the photosynthesis efficiency of plants and is also a key to maintaining plant growth. The change of stomatal conductance (stomata opening and closing) and wax accumulation are one of the self-protection mechanisms of plants, and the change of physiological and biochemical characteristics of plants is obvious during drought stress, such as osmotic protective agents like betaine, proline and the like are helpful for enhancing the stability of membranes, and the activities of enzymes like superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and the like are enhanced to establish an antioxidant defense mechanism.

In view of serious harm and great adverse effect of water deficiency or drought on agricultural production, the genetic control sites related to water conservation and drought resistance, water utilization efficiency and yield are excavated through whole genome association analysis, and a transgenic material is created through a transgenic technology and used for deeply researching the gene function and action mechanism of the excavated wheat responding to the water deficiency or drought, so that a solid research foundation is provided for creating and cultivating the water conservation and drought resistance high-yield wheat variety.

Disclosure of Invention

The invention aims to provide an application of TaFPFL1-2B genes in improving drought resistance of plants.

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

The TaFPFL-2B gene adopted by the invention has the GenBank number of CM022215.1 in NCBI, the length of messenger RNA (mRNA) sequence of TaFPFL1-2B gene of 3838 bp, the length of coding sequence of TaFPFL1 gene of 339bp, the nucleotide sequence shown as SEQ ID NO.1, 112 amino acids, and the amino acid sequence shown as SEQ ID NO. 2.

The invention also constructs a series of plant expression vectors, recombinant vectors or transgenic plant systems containing the genes, and the functions of host cells containing the vectors in improving the water-saving drought resistance of plants also fall into the protection scope of the invention.

The functions of the genes protected by the invention not only comprise TaFPFL1-2B genes, but also comprise the functions of homologous genes with higher homology (up to 99 percent of homology) with TaFPFL1-2B genes in water-saving and drought-resisting aspects.

The invention discloses a biological function of TaFPFL-2B gene in water-saving drought-resistant of plants, which is specifically expressed in: taFPFL 1A-2B was able to increase individual yield under field water deficit conditions relative to wild type Fielder.

According to the functions, a plant resistant to salt stress can be obtained by a transgenic mode, and in particular, a transgenic plant with water saving and drought resisting capabilities higher than that of a target plant can be obtained by introducing TaFPFL1-2B genes into the target plant.

Specifically, taFPFL A-2B gene can be introduced into the plant of interest specifically by means of the recombinant expression vector. In the method, the recombinant expression vector may be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues are cultivated into plants.

In order to improve the excellent properties of plants, the invention also protects a novel plant breeding method, and plants with altered drought resistance can be obtained by a method of regulating and controlling the expression of TaFPFL1-2B genes in plants. Wherein the means of "modulating expression of TaFPFL1-2B gene in a plant" may be over-expression, silencing, gene editing or directed mutation of TaFPFL1-2B gene. Regulating the gene expression level comprises regulating the TaFPFL-2B expression by using a DNA homologous recombination technology, a virus-mediated gene silencing technology and an agrobacterium-mediated transformation system to obtain a transgenic plant line.

More specifically, the method may be the following (1) or (2) or (3):

(1) By increasing the activity of TaFPFL A1-2B protein in the target plant, a plant with water saving and drought resistance stronger than that of the target plant is obtained;

(2) By promoting the expression of TaFPFL1-2B genes in the target plant, obtaining a plant with water-saving drought resistance stronger than that of the target plant;

(3) By inhibiting the expression of TaFPFL A gene-2B gene in target plant, the plant with water-saving drought resistance lower than that of target plant is obtained.

The "promoting expression of TaFPFL a 1-2 a gene in a plant of interest" may be achieved as follows (1) or (2) or (3):

(1) Introducing TaFPFL A1-2B gene into target plant;

(2) Introducing strong promoters and/or enhancers;

(3) Other methods common in the art, such as overexpression, overexpression.

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

Meanwhile, in the process of verifying TaFPFL1-2B gene in the invention, the over-expression strain and wild type yield characteristics under normal moisture are analyzed, and the result shows that TaFPFL1-2B over-expression strain L10-1 shows better yield characteristics than wild control field, and TaFPFL1-2B gene also has the function of improving wheat yield, so that the over-expression TaFPFL1-2B gene can not only improve drought resistance of plants, but also improve yield of plants.

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 and arabidopsis are applicable to all genes with the gene or the genes homologous to the gene.

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 present invention innovatively cloned TaFPFL1-2B in wheat (Triticum aestivum l.) using genome-wide association analysis (genome-wide association studies, GWAS). The method comprises the steps of constructing TaFPFL1-2B over-expression vector LGY-OE-TaFPFL1-2B, transforming wild wheat field by using agrobacterium infection to obtain over-expression plants, and analyzing results show that under the condition of water deficiency of a field, compared with the wild wheat field, taFPFL1-2B genes can improve drought resistance of plants by increasing single plant yield, and the TaFPFL1-2B genes can provide gene resources for water-saving drought-resistant breeding of crops.

(2) The water-saving drought-resistant plant can be obtained by a transgenic mode, specifically, the TaFPFL A-2B gene is introduced into a target plant to obtain the transgenic plant, the water-saving drought-resistant property of the plant is higher than that of the target plant, and a new way is provided for water-saving drought-resistant breeding of the plant.

Drawings

FIG. 1 is a TaFPFL subgenomic homologous gene CDS sequence and encoded amino acid sequence alignment analysis, as well as evolutionary conservation analysis in multiple species; FIGS. 1A and 1B show that TaFPFL.about.1-2B, taFPFL.about.1-2A and TaFPFL.about.1-2D differ in the CDS sequence and amino acid sequence at certain positions, and FIG. 1C shows that the TaFPFL gene has certain conservation (54.47%) in various species such as wheat, rice, maize, upland cotton, white mustard, tobacco, arabidopsis, and is more similar to the rice and maize sequences of monocots.

FIG. 2 is a genomic level identification of TaFPFL-2B gene-overexpressing plants and an expression level analysis of TaFPFL 1-2B;

FIG. 3 is a representation of agronomic, seed or yield traits of TaFPFL-2B overexpressing plants L10-1, L11-1 under normal water and water deficit conditions in the field, where Fielder is wild control and L10-1, L11-1 are overexpressing lines.

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 Fielder seeds are stored for laboratory;

The overexpression vector LGY-OE is stored in a laboratory;

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

primer synthesis and sequencing were performed by Rui boxing and Hexas major.

Experimental reagent

RNA extraction kits were purchased from huawyo biotechnology limited;

Reverse transcription kits and fluorescent quantification kits were purchased from nuuzan biotechnology limited;

Various endonucleases were purchased from NEW ENGLAND Biolabs biotechnology limited;

one-step cloning enzyme was purchased from full gold biotechnology limited;

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

Experimental equipment

PCR instrument was purchased from Thermo FISHER SCIENTIFIC company;

quantitative PCR instrument was purchased from Thermo FISHER SCIENTIFIC company;

The refrigerated centrifuge is purchased from Eppendorf corporation;

the normal temperature centrifuge is purchased from Eppendoff company;

The autoclave MLS-3750 was purchased from Sanyang, japan;

Nucleic acid detector Nano-300 was purchased from Thermo FISHER SCIENTIFIC company.

Example 1TaFPFL1 and sequence analysis of homologous Gene thereof and cloning of TaFPFL1-2B Gene

Wheat TaFPFL and homologous gene nucleic acid sequences and amino acid sequences thereof are obtained through WheatOmics 1.0.0 (http:// 202.194.139.32 /) websites, and TaFPFL homologous gene nucleic acid sequences and amino acid sequences in rice, corn, upland cotton, white mustard, tobacco, arabidopsis and other species are obtained through NCBI websites (https:// www.ncbi.nlm.nih.gov /). Further, the TaFPFL and homologous gene sequences thereof and the encoded protein sequences were aligned using SnpGene and clustalx software. Extracting RNA of wheat China Spring (CS), taking cDNA obtained by reverse transcription reaction as a template, designing a specific Primer through a Primer premier5.0 website, and cloning TaFPFL-2B gene fragments through PCR reaction.

The result shows that the coding sequence of wheat TaFPFL-2B gene contains 339bp base, and the coded protein contains 112 amino acids. TaFPFL 1A 1-2B and its wheat homologous gene (TaFPFL A1-2A, taFPFL A1-2D) and the encoded amino acid have a certain site difference, but the overall homology is still higher up to 90.35% (FIGS. 1A, 1B). By constructing an amino acid sequence evolutionary tree of TaFPFL genes homologous genes in wheat, rice, corn, upland cotton, white mustard, tobacco, arabidopsis and other species, the TaFPFL genes can be seen to have higher conservation (54.47%), and have more similar functions with rice and corn sequences of monocotyledonous plants (figure 1C).

Example 2 construction of vector overexpressing TaFPFL1-2B Gene and identification of transgenic plants

To further analyze TaFPFL A-2B function, the inventors constructed the TaFPFL A-2B overexpression vector LGY-OE-TaFPFL A-2B, and obtained an overexpressed wheat plant. The specific procedure is briefly described as follows.

First, a primer with a restriction enzyme BamHI cleavage site and a FLAG tag was designed as follows:

LGY-OE-TaFPFL1-2B-F：

LGY-OE-TaFPFL1-2B-2FLAG-R：

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, the LGY-OE vector is subjected to single digestion with BamHI, and the digested product is purified.

Fourthly, carrying out homologous recombination connection on the PCR amplification product and the vector after enzyme digestion to construct an LGY-OE-TaFPFL1-2B overexpression vector;

Fifthly, converting the connection product into escherichia coli DH5 alpha by adopting a heat shock conversion method, carrying out K ⁺ (kanamycin, 50 mug/mL) resistance screening, 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 competent cells GV3101 and stored at-80℃for further use.

Seventh, the agrobacterium is used to transform wild wheat field, and the successfully transformed seedlings are identified and propagated until T ₂ or even T ₃ generation over-expressed plants are obtained.

After identifying T ₀ generation transgenic positive seedlings by PCR (figures 2A and 2B), planting and propagating until T ₂ generation transgenic positive seedlings and even T ₃ generation transgenic positive seedlings are obtained, and simultaneously analyzing the expression level of TaFPFL1-2B in potential transgenic plants by qRT-PCR. As a result, it was found that the expression level of TaFPFL1-2B was up-regulated as expected in TaFPFL-2B potential transgenic lines L10-1 and L11-1 (FIG. 2C). These results indicate that the TaFPFL A-2B transgenic wheat constructed is an LGY-OE-TaFPFL A-2B overexpressing plant.

Example 3 Effect of over-expressed TaFPFL A1-2B Gene on wheat agronomic and yield traits under field Water deficit conditions

2023 Spring sowing planting season (2023, 2-2023, 6) is to plant TaFPFL-2B over-expression lines L10-1 and L11-1 and their wild control material field in the field, during which normal water treatment and water deficit treatment are respectively set by controlling watering times. And (3) when the wheat is mature, researching and counting the plant height, the spike number, the spike length, the spike width, the spike number and other field phenotypes, and after the wheat is harvested and threshed, researching and counting the seed or yield characters such as grain length, grain width, thousand grain weight and the like.

The results indicate that under normal moisture, taFPFL-2B overexpressing line L10-1 exhibited better yield traits, such as significantly increased grain width, thousand kernel weight, and individual yield, than the wild-type control Fielder (fig. 3d,3e, and 3F); while the over-expressed line L11-1 was lower in plant height and spike length than the wild-type control Fielder, the final grain length and individual yield were also significantly higher than the wild-type control Fielder (FIGS. 3A-3C, 3F-3G). Under the condition of water deficiency, the over-expression strains L10-1, L11-1 and the control Fielder have overall reduced plant height compared with normal water (figures 3A and 3G), conform to the conclusion that the prior researches on reduced plant height are met, and meanwhile, the yield of a single plant is reduced compared with the overall normal water (figure 3F), so that the field drought treatment is effective and reliable. Under water deficit, the over-expressed line L10-1 increased spike length, with individual yield significantly higher than the wild control Fielder (FIGS. 3B,3F and 3G); the overexpressing strain L11-1 had reduced grain length, grain width and thousand grain weight, but the final individual yield was also significantly higher than the wild-control Fielder (FIGS. 3C-3E and 3F).

The over-expressed lines L10-1 and L11-1 were dug out contemporaneously, and the root system configuration was found to be better than that of the wild control Fielder, e.g., longer root length and more developed root system, so it was speculated that TaFPFL that higher individual yield under water deficit was possible through the growth and configuration regulation of the root system by TaFPFL-2B (FIG. 3G).

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

The application of TaFPFL1-2B gene in improving drought resistance of plants is characterized in that the nucleotide sequence of TaFPFL1-2B gene is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein drought-resistant transgenic plants are obtained by constructing TaFPFL a 1-2B overexpression vector.
3. The use according to claim 1, wherein said drought resistance is characterized by a higher individual yield of the over-expressed TaFPFL-2B gene line than the wild type under water deficit conditions.
4. Use according to any one of claims 1-3, wherein the plant is wheat.
5. The use according to claim 3, wherein the root system of the over-expressed TaFPFL-2B gene line is more developed and longer than the wild type.
6. A plant breeding method characterized in that the method is (1) or (2) or (3) below:

(1) By increasing the activity of TaFPFL A1-2B protein in the target plant, obtaining a plant with drought resistance stronger than that of the target plant;

(2) By promoting the expression of TaFPFL1-2B genes in the target plant, obtaining a plant with drought resistance stronger than that of the target plant;

(3) Obtaining a plant with drought resistance lower than that of the target plant by inhibiting the expression of TaFPFL1-2B genes in the target plant;

the nucleotide sequence of TaFPFL A gene is shown as SEQ ID NO.1, and the amino acid sequence of TaFPFL A gene is shown as SEQ ID NO. 2.
7. The plant breeding method according to claim 6, wherein the objective plant is wheat.
8. The method of plant breeding according to claim 6, wherein the expression of TaFPFL a 1-2B gene in the target plant is promoted by over-expressing TaFPFL a 1-2B gene.
9. The method of plant breeding according to claim 6, wherein the inhibition of expression of TaFPFL a 1-2B gene in the plant of interest is by silencing TaFPFL a 1-2B gene.
10. Use of TaFPFL a gene as defined in claim 1 for increasing wheat yield.