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

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 plant drought resistance

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to application of TaFPFL1-2B gene in improving plant drought resistance.

Background technique

The world population is growing while the area of agricultural land is decreasing. It is expected that by 2050, the global demand for agricultural crop products such as wheat will increase by 60%, which undoubtedly puts a high demand on the yield and planting of staple crops such as wheat. On the other hand, the large amount of water used in agriculture and the shortage of water resources are contradictory. Drought stress exacerbated by water shortage has become the most important environmental factor affecting crop production worldwide, seriously threatening food production security and socio-economic development. The "Drought Figures 2022" report released by the 15th Conference of the Parties to the United Nations Convention to Combat Desertification pointed out that the number and duration of global droughts have increased by 29% since 2000, and the global economic losses caused by droughts reached 124 billion US dollars between 1998 and 2017 alone. The report of the Food and Agriculture Organization of the United Nations also shows that 83% of all economic losses caused by droughts between 2006 and 2016 occurred in the agricultural sector, with a loss value of up to 29 billion US dollars.

The characteristics of fixed growth have enabled plants to evolve various strategies to adapt to the adverse water-deficient and drought environment. The coordinated interactions at the morphological, physiological, cellular and molecular levels enable plants to perceive environmental changes in a timely manner and make real-time adjustments to maintain their own growth and development. Their performance can be divided into drought escape, drought avoidance, drought tolerance and drought recovery according to their respective characteristics. Drought escape is an adaptive characteristic of plants. Plants will grow rapidly before the onset of severe drought to complete their life cycle, which is often manifested as early heading, flowering and maturity, reduced plant height and shortened growth cycle. Drought avoidance is mainly manifested in plants controlling water absorption and loss through morphological or physiological changes in response to drought. Drought tolerance, also known as drought resistance, refers to the ability of plants to maintain their growth, development and reproduction under drought stress conditions. Drought resistance is a complex quantitative trait that is jointly controlled by multiple agronomic traits and genes, involving multiple drought response signaling pathways and metabolic networks. Plants will adopt one or more combined strategies to respond to drought stress. In terms of external intuitive morphology, plants in a drought state often show morphological changes. When drought stress occurs, the root system architecture is reshaped accordingly. Plants absorb more available water by strengthening the root system. Root length, density, volume and diameter are important indicators for evaluating plant drought resistance. Even root hairs play an important role in adapting to rhizosphere characteristics to maintain plant nutrition. On the other hand, the reduction in the number of leaves, the reduction in leaf area, and the weakening of transpiration are important manifestations of reducing water loss. The ability of leaves to stay green is related to the efficiency of plant photosynthesis and is also the key to maintaining plant growth. Changes in stomatal conductance (stomatal opening and closing) and wax accumulation are one of the mechanisms of plant self-protection, and plant physiological and biochemical characteristics change significantly during drought stress. For example, osmoprotectants such as betaine and proline help to enhance membrane stability, and the activities of enzymes such as superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT) are enhanced to establish antioxidant defense mechanisms.

In view of the serious harm and significant adverse effects of water deficit or drought on agricultural production, whole genome association analysis is used to explore genetic regulatory sites related to water-saving and drought resistance, water use efficiency and yield. Transgenic materials are created through transgenic technology for in-depth research on the genetic functions and mechanisms of wheat's response to water deficit or drought, providing a solid research foundation for the creation and cultivation of water-saving and drought-resistant high-yield wheat varieties.

Summary of the invention

The purpose of the present invention is to provide application of TaFPFL1-2B gene in improving drought resistance of plants.

In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:

The TaFPFL1-2B gene used in the present invention has a GenBank number of CM022215.1 in NCBI, a messenger RNA (mRNA) sequence length of the TaFPFL1-2B gene of 388 bp, a coding sequence length of the TaFPFL1 gene of 339 bp, a nucleotide sequence as shown in SEQ ID NO.1, including 112 amino acids, and an amino acid sequence as shown in SEQ ID NO.2.

The present invention also constructs a series of plant expression vectors, and the expression vectors, recombinant vectors or transgenic plant lines containing the above genes and host cells containing the vectors also fall within the protection scope of the present invention in terms of improving plant water conservation and drought resistance.

The functions of the genes protected by the present invention include not only the above-mentioned TaFPFL1-2B gene, but also the functions of homologous genes with high homology (homology up to 99%) with the TaFPFL1-2B gene in water saving and drought resistance.

The biological function of the TaFPFL1-2B gene disclosed in the present invention in plant water conservation and drought resistance is specifically manifested in that, compared with the wild-type Fielder, under field water deficit conditions, TaFPFL1-2B can increase the yield per plant.

According to its function, plants tolerant to salt stress can be obtained by transgenic means. Specifically, the TaFPFL1-2B gene can be introduced into the target plant to obtain a transgenic plant, which has higher water-saving and drought-resistance capabilities than the target plant.

Specifically, the TaFPFL1-2B gene can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

In order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, which can obtain plants with changed drought resistance by the method of "regulating the expression of TaFPFL1-2B gene in plants". Among them, the method of "regulating the expression of TaFPFL1-2B gene in plants" can be overexpression, silencing, gene editing or directed mutation of TaFPFL1-2B gene. Regulating the gene expression level includes using DNA homologous recombination technology, virus-mediated gene silencing technology and Agrobacterium-mediated transformation system to regulate the expression of TaFPFL1-2B to obtain transgenic plant strains.

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

(1) By increasing the activity of TaFPFL1-2B protein in the target plant, a plant with stronger water-saving and drought-resistance than the target plant is obtained;

(2) Promoting the expression of TaFPFL1-2B gene in target plants to obtain plants with stronger water-saving and drought-resistance than target plants;

(3) By inhibiting the expression of the TaFPFL1-2B gene in the target plant, plants with lower water-saving and drought-resistance than the target plant are obtained.

The implementation method of “promoting the expression of TaFPFL1-2B gene in target plants” may be as follows (1) or (2) or (3):

(1) Introducing the TaFPFL1-2B gene into the target plant;

(2) introduction of strong promoters and/or enhancers;

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

Wherein, the target plant of the present invention is wheat.

At the same time, in the process of verifying the function of TaFPFL1-2B gene in improving plant drought resistance, the yield traits of overexpression strains and wild types under normal water content were analyzed. The results showed that the TaFPFL1-2B overexpression strain L10-1 showed better yield traits than the wild control Fielder. It can be seen that the TaFPFL1-2B gene also has the function of improving wheat yield. Therefore, overexpression of the TaFPFL1-2B gene can not only improve the drought resistance of plants, but also increase the yield of plants.

In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be, for example (but not limited to): dicotyledons, monocotyledons or gymnosperms.

As a preferred embodiment, the "plant" includes but is not limited to wheat and Arabidopsis thaliana, and any plant having the gene or a gene homologous thereto is applicable.

The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene/nucleic acid.

The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method in this patent have the same characteristics.

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

Advantages of the present invention:

(1) The present invention uses the method of genome-wide association studies (GWAS) to innovatively clone TaFPFL1-2B in wheat (Triticum aestivum L.). The overexpression vector LGY-OE-TaFPFL1-2B of TaFPFL1-2B is constructed, and the wild-type wheat Fielder is transformed by Agrobacterium infection to obtain overexpression plants. The analysis results show that under field water deficit, the TaFPFL1-2B gene can increase the yield per plant compared with the wild type, indicating that the TaFPFL1-2B gene can improve the drought resistance of plants and provide gene resources for crop water-saving and drought-resistant breeding.

(2) Water-saving and drought-resistant plants can be obtained through genetic modification. Specifically, the TaFPFL1-2B gene can be introduced into the target plant to obtain a transgenic plant. The water-saving and drought-resistant properties of the plant are higher than those of the target plant, providing a new approach for water-saving and drought-resistant plant breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a comparison analysis of the CDS sequences and encoded amino acid sequences of the TaFPFL1 subgenome homologous genes, as well as an analysis of evolutionary conservation in multiple species; Figures 1A and 1B show that there are differences in certain sites in the CDS sequences and amino acid sequences of TaFPFL1-2B, TaFPFL1-2A and TaFPFL1-2D, and Figure 1C shows that the TaFPFL1 gene is conservative (54.47%) in multiple species such as wheat, rice, corn, upland cotton, mustard white mustard, tobacco, Arabidopsis, etc., and is more similar to the sequences of rice and corn in monocotyledonous plants.

FIG2 is the genome level identification of TaFPFL1-2B gene overexpressing plants and the expression level analysis of TaFPFL1-2B;

Figure 3 shows the performance of agronomic traits, seeds or yield traits of TaFPFL1-2B overexpressing plants L10-1 and L11-1 under normal water and water deficit conditions in the field. In the figure, Fielder is the wild control, and L10-1 and L11-1 are overexpressing strains.

Detailed ways

The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" 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-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to this. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and/or include a coding sequence in a cDNA, and/or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

biomaterials

Wheat Fielder seeds are kept in the laboratory;

The overexpression vector LGY-OE was kept in the laboratory;

Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 were maintained in the laboratory;

Primer synthesis and sequencing were completed by Ruiboxingke and Liuhe BGI Biological Co., Ltd.

Experimental reagents

RNA extraction kit was purchased from Huayueyang Biotechnology Co., Ltd.;

The reverse transcription kit and fluorescence quantitative kit were purchased from Novozyme Biotechnology Co., Ltd.

Various endonucleases were purchased from NEW ENGLAND Biolabs Biotechnology Co., Ltd.;

One-step cloning enzyme was purchased from Quanshijin Biotechnology Co., Ltd.;

Plasmid miniprep kit and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.

Laboratory equipment

The PCR instrument was purchased from Thermo Fisher Scientific;

The quantitative PCR instrument was purchased from Thermo Fisher Scientific;

The refrigerated centrifuge was purchased from Eppendorf;

The normal temperature centrifuge was purchased from Eppendoff;

The high-temperature and high-pressure sterilizer MLS-3750 was purchased from Sanyo Company, Japan;

The nucleic acid detector Nano-300 was purchased from Thermo Fisher Scientific.

Example 1 Sequence analysis of TaFPFL1 and its homologous genes and cloning of TaFPFL1-2B gene

The nucleic acid sequence and amino acid sequence of wheat TaFPFL1 and its homologous genes were obtained through the WheatOmics 1.0 (http://202.194.139.32/) website, and the nucleic acid sequence and amino acid sequence of TaFPFL1 homologous genes in rice, corn, upland cotton, mustard white, tobacco, Arabidopsis thaliana and other species were obtained through the NCBI website (https://www.ncbi.nlm.nih.gov/). Furthermore, the sequences of TaFPFL1 and its homologous genes and the encoded protein sequences were compared using SnpGene and clustalx software. RNA of wheat Chinese Spring (CS) was extracted, and the cDNA obtained by reverse transcription reaction was used as a template. Specific primers were designed through the Primer Premier5.0 website, and the TaFPFL1-2B gene fragment was cloned by PCR reaction.

The results showed that the coding sequence of wheat TaFPFL1-2B gene contains 339bp bases, and the encoded protein includes 112 amino acids. There are differences in the CDS sequence and the encoded amino acids of TaFPFL1-2B and its wheat homologous genes (TaFPFL1-2A, TaFPFL1-2D), but the overall homology is still as high as 90.35% (Figure 1A, 1B). By constructing the amino acid sequence evolution tree of homologous genes of TaFPFL1 gene in wheat, rice, corn, upland cotton, mustard, tobacco, Arabidopsis and other species, it can be seen that TaFPFL1 gene has a high degree of conservation (54.47%), and is more similar to the sequence functions of rice and corn in monocotyledonous plants (Figure 1C).

Example 2 Construction of vector for overexpressing TaFPFL1-2B gene and identification of transgenic plants

In order to further analyze the function of TaFPFL1-2B, the inventors constructed the TaFPFL1-2B overexpression vector LGY-OE-TaFPFL1-2B and obtained overexpression wheat plants. The specific process is briefly described as follows.

First, design primers with restriction endonuclease BamHI cleavage site and FLAG tag, the sequence is as follows:

LGY-OE-TaFPFL1-2B-F:

LGY-OE-TaFPFL1-2B-2FLAG-R:

Then, the cDNA sample prepared in Example 1 was used as a template to perform PCR amplification, and the amplified product was purified and recovered;

Third, the LGY-OE vector was digested with BamHI and the digested product was purified.

Fourth, the PCR amplification product and the vector after restriction digestion were connected by homologous recombination to construct the LGY-OE-TaFPFL1-2B overexpression vector;

Fifth, the ligation product was transformed into Escherichia coli DH5α using the heat shock transformation method, and K ⁺ (kanamycin, 50 μg/mL) resistance screening was performed. Positive colonies were selected for PCR detection, and the correct colonies identified by PCR were amplified and sent for sequencing. The plasmid was extracted from the bacterial solution with correct sequencing for later use.

Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101 and stored at -80°C for future use.

Seventh, use Agrobacterium to transform wild-type wheat Fielder, identify and propagate the successfully transformed seedlings until _T2 or even _T3 generation overexpression plants are obtained.

After the positive transgenic seedlings of the T ₀ generation were identified by PCR (Figures 2A, 2B), the seeds were planted and propagated until the positive transgenic seedlings of the T ₂ or even T ₃ generation were obtained. At the same time, the expression level of TaFPFL1-2B in the potential transgenic plants was analyzed by qRT-PCR. The results showed that in the TaFPFL1-2B potential transgenic lines L10-1 and L11-1, the expression of TaFPFL1-2B was upregulated as expected (Figure 2C). These results indicate that the constructed TaFPFL1-2B transgenic wheat is a LGY-OE-TaFPFL1-2B overexpressing plant.

Example 3 Effects of overexpression of TaFPFL1-2B gene on agronomic and yield traits of wheat under field water deficit conditions

In the 2023 spring planting season (February 2023-June 2023), TaFPFL1-2B overexpression lines L10-1 and L11-1 and their wild control material Fielder were planted in the field. During this period, normal water treatment and water deficit treatment were set by controlling the number of watering. When the wheat matured, the field phenotypes such as plant height, number of ears, ear length, ear width, and number of spikelets were investigated and counted. After the wheat was harvested and threshed, the seed or yield traits such as grain length, grain width, and thousand-grain weight were investigated and counted.

The results showed that under normal water conditions, the TaFPFL1-2B overexpression line L10-1 showed better yield traits than the wild control Fielder, such as significantly increased grain width, 1000-grain weight and yield per plant (Figures 3D, 3E and 3F); while the overexpression line L11-1, although lower in plant height and ear length than the wild control Fielder, had significantly higher final grain length and yield per plant than the wild control Fielder (Figures 3A-3C, 3F-3G). Under water deficit, the plant height of the overexpression lines L10-1, L11-1 and the control Fielder decreased overall compared with normal water (Figures 3A, 3G), which is consistent with the conclusion of existing studies that drought reduces plant height. At the same time, the yield per plant was also reduced overall compared with normal water (Figure 3F), indicating that field drought treatment is effective and reliable. Under water deficit, the ear length of the overexpression line L10-1 increased, and the yield per plant was significantly higher than that of the wild control Fielder (Figures 3B, 3F and 3G); although the grain length, grain width and 1000-grain weight of the overexpression line L11-1 decreased, the final yield per plant was also significantly higher than that of the wild control Fielder (Figures 3C-3E and 3F).

Overexpression lines L10-1 and L11-1 were dug out at the same time, and it was found that their root architecture was better than that of the wild control Fielder, such as longer roots and more developed root systems. Therefore, it is speculated that TaFPFL1-2B may maintain a higher single-plant yield under water deficit by regulating the growth and architecture of the root system by TaFPFL1-2B (Figure 3G).

The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims (10)

1. Application of TaFPFL1-2B gene in improving plant drought resistance, characterized in that the nucleotide sequence of the TaFPFL1-2B gene is shown in SEQ ID NO.1. 2. The use according to claim 1, characterized in that drought-resistant transgenic plants are obtained by constructing a TaFPFL1-2B overexpression vector. 3. The use according to claim 1, characterized in that the drought resistance is reflected in that, under water deficit conditions, the single plant yield of the strain overexpressing the TaFPFL1-2B gene is higher than that of the wild type. 4. The use according to any one of claims 1 to 3, characterized in that the plant is wheat. 5. The use according to claim 3, characterized in that, compared with the wild type, the root system of the strain overexpressing the TaFPFL1-2B gene is more developed and longer. 6. A plant breeding method, characterized in that the method is the following (1) or (2) or (3): (1) By increasing the activity of TaFPFL1-2B protein in the target plant, a plant with stronger drought resistance than the target plant is obtained; (2) Promoting the expression of TaFPFL1-2B gene in target plants to obtain plants with stronger drought resistance than target plants; (3) obtaining plants with lower drought resistance than the target plants by inhibiting the expression of the TaFPFL1-2B gene in the target plants; The nucleotide sequence of the TaFPFL1-2B gene is shown in SEQ ID NO.1, and the amino acid sequence of the TaFPFL1-2B protein is shown in SEQ ID NO.2. 7. The plant breeding method according to claim 6, characterized in that the target plant is wheat. 8 . The plant breeding method according to claim 6 , wherein the method of promoting the expression of the TaFPFL1-2B gene in the target plant is to overexpress the TaFPFL1-2B gene. 9 . The plant breeding method according to claim 6 , wherein the method of inhibiting the expression of the TaFPFL1-2B gene in the target plant is to silence the TaFPFL1-2B gene. 10. Use of the TaFPFL1-2B gene described in claim 1 in improving wheat yield.