Application of TaSPL6 gene in negative regulation of wheat yield and nitrogen use efficiency

Abstract

The present invention discloses an application of the TaSPL6 gene in negatively regulating wheat yield and nitrogen utilization efficiency. The gene encodes a SQUAMOSA promoter binding protein-like (SPL) transcription factor protein. The present invention obtains TaSPL6 RNAi transgenic plants by constructing an RNAi vector, and the results show that the TaSPL6 RNAi transgenic plants can increase the number of effective tillers per plant and increase yield under normal conditions and low-nitrogen conditions. It can be seen that the TaSPL6 gene of the present invention has potential application value in improving wheat yield and nitrogen fertilizer utilization efficiency. Crops can be genetically improved by silencing the gene, and it is expected to achieve the breeding goal of high-yield and highly efficient synergistic improvement of crops.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of TaSPL6 gene in negatively regulating wheat yield and nitrogen utilization efficiency. Background Art

[0002] Wheat (Triticum aestivum) is one of the most widely grown food crops, accounting for 25% of global cereal production. As the global population continues to increase and extreme weather caused by climate change occurs more frequently, ensuring wheat production is crucial to global food security. It is predicted that the demand for wheat production will increase by 70% by 2050. How to continuously increase wheat production is undoubtedly a huge challenge.

[0003] At present, the increase in wheat yields depends largely on the large-scale use of nitrogen fertilizers, which has caused some irreparable ecological damage. Therefore, improving the nitrogen utilization efficiency of plants while ensuring wheat yield is an urgent problem to be solved in wheat breeding and production. The number of tillers is a key trait that affects wheat yield and nitrogen response. Genes that regulate tillering by nitrogen are widely used in rice, but there are fewer genes for nitrogen-responsive tillering development in wheat and the molecular mechanism is still unclear, and even fewer related genes are actually used in breeding.

[0004] SQUAMOSA promoter binding protein-like (SPL) genes encode a type of plant-specific transcription factor. SPL plays many important roles in plant growth and development, such as participating in the establishment of plant morphology, transition of developmental periods, leaf development, flower and fruit development, GA signaling, and response to external environmental stress signals. Overexpression of TaSPL20 / 21 in rice causes an increase in the number of primary and secondary tillers, an increase in the number of panicles, and an increase in panicle length. TaSPL20 can increase grain length and width, and improve thousand-grain weight. In general, SPL transcription factors are essential for plant growth and development. Their reported functions in gramineous crops are mainly in panicle development, and there are still no reports on their response to nitrogen and nitrogen use efficiency. At present, relevant research experts have proposed a new generation of "green revolution" plant type design models for rice and wheat with "less input and more output" and a new breeding strategy for the coordinated improvement of crop high yield and nitrogen efficiency. Discovering SPL genes that respond to nitrogen will provide important genetic resources for the discovery of high-yield and high-efficiency wheat genes, the analysis of molecular mechanisms, and their timely application in wheat breeding. Summary of the invention

[0005] The purpose of the present invention is to provide the application of TaSPL6 gene in negatively regulating wheat yield and nitrogen utilization efficiency.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:

[0007] The TaSPL6 gene used in the present invention has a messenger RNA (mRNA) sequence length of 1751 bp, a coding sequence length of 1224 bp, a nucleotide sequence as shown in SEQ ID NO.1, including 407 amino acids, and an amino acid sequence as shown in SEQ ID NO.2.

[0008] The present invention also constructs a series of plant expression vectors, and the expression vectors, recombinant vectors and host cells containing the above genes also fall within the protection scope of the present invention in terms of improving wheat yield and nitrogen utilization efficiency. The host cells are microbial cells, such as fungal cells or bacterial cells, such as but not limited to Escherichia coli, Agrobacterium and yeast.

[0009] The functions of the genes protected by the present invention include not only the above-mentioned TaSPL6 gene, but also the functions of direct homologous genes in wheat that have high homology to the TaSPL6 gene in improving wheat yield and nitrogen utilization efficiency.

[0010] The biological function of the TaSPL6 gene disclosed in the present invention in improving wheat yield and nitrogen utilization efficiency is specifically manifested in that: under normal control conditions in the field, the effective tiller number and single plant yield of the TaSPL6 RNAi transgenic strain are higher than those of the wild type; under low-nitrogen conditions in the field, the total tiller number, effective tiller number and single plant yield of the TaSPL6 RNAi transgenic strain are higher than those of the wild type, and the increase is greater than that under normal control conditions in the field, and the nitrogen utilization efficiency of the TaSPL6 RNAi transgenic strain is also improved.

[0011] According to its function, high-yield plants with high nitrogen utilization efficiency can be obtained through genetic modification. Specifically, RNAi vectors or CRISPR / Case9 gene editing vectors containing TaSPL6 gene fragments can be introduced into excellent wheat varieties to obtain transgenic wheat. The yield and nitrogen utilization efficiency of this plant are higher than those of wild-type plants without the introduction of relevant vectors.

[0012] The TaSPL6 gene fragment can be introduced into wheat through the recombinant expression vector. In the method, the recombinant expression vector can transform wheat cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed tissues are cultivated into plants. The transformed plant host can be monocotyledonous plants such as rice, corn, and wheat, or dicotyledonous plants such as Arabidopsis, soybean, rapeseed, and cotton.

[0013] In order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, which is the following (1) or (2) or (3):

[0014] (1) by increasing the activity of TaSPL6 protein in plants or promoting the expression of TaSPL6 gene in plants, obtaining plants whose yield and yield components are lower than those of wild type plants under low nitrogen conditions;

[0015] (2) by inhibiting / blocking the transcription / expression of the TaSPL6 gene in plants, plants with higher yield and yield components than the wild type under low nitrogen conditions were obtained;

[0016] (3) By hybridizing a plant containing the TaSPL6 gene or its active fragment with another plant, an offspring plant containing the TaSPL6 gene or its active fragment is obtained, and compared with a control plant or an unhybridized plant without the introduction of the gene or its active fragment or the recombinant expression vector, the transgenic plant or the hybrid offspring plant has a reduced nitrogen utilization efficiency.

[0017] The implementation method of “promoting the expression of TaSPL6 gene in plants” may be as follows (1) or (2) or (3):

[0018] (1) Introducing the TaSPL6 gene into plants;

[0019] (2) introduction of strong promoters and / or enhancers;

[0020] (3) Other common methods in this field.

[0021] The implementation method of “inhibiting / blocking the transcription / expression of TaSPL6 gene in plants” can be as follows (1) or (2) or (3) or (4):

[0022] (1) using RNA interference technology to introduce small double-stranded TaSPL6 RNA to generate transgenic lines with reduced TaSPL6 expression;

[0023] (2) Using CRISPR / Case9 technology to construct TaSPL6 gene editing vector to obtain mutants;

[0024] (3) obtaining TaSPL6 gene mutants through chemical reagents such as EMS and / or physical approaches such as γ-rays;

[0025] (4) Other common methods in this field.

[0026] Regulating the gene expression level includes regulating the expression of TaSPL6 using DNA homologous recombination technology, virus-mediated gene silencing technology and Agrobacterium-mediated transformation system to obtain transgenic plant strains. It involves the expression vector, transgenic cell line and host cell of TaSPL6. The host cell is a microbial cell, for example, a fungal cell or a bacterial cell, such as but not limited to Escherichia coli and Agrobacterium.

[0027] Such plants include, but are not limited to, wheat, rice, Arabidopsis, corn, cotton, rapeseed or soybean.

[0028] The term "plant" as used herein includes whole plants, parent and progeny plants, and different parts of plants, including seeds, fruits, buds, stems, leaves, roots, flowers, spikes, tissues, and organs. The term "plant" as used herein also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen, and microspores.

[0029] 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 progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.

[0030] The present invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, etc. 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 present invention also relates to foods or food additives obtained from the relevant plants.

[0031] Advantages of the present invention:

[0032] (1) By analyzing TaSPL6 RNAi transgenic wheat plants, the present invention innovatively discovered the function of SPL transcription factor in improving wheat yield and nitrogen utilization efficiency, providing important gene resources for molecular breeding of high-yield and high-efficiency crops.

[0033] (2) High-yield wheat plants with high nitrogen utilization efficiency can be obtained through genetic modification. Specifically, TaSPL6 RNAi transgenic wheat can be obtained. The yield and nitrogen utilization efficiency of the wheat plants are higher than those of the wild type, providing a new approach for high-yield and high-efficiency plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the expression pattern of TaSPL6 gene in different tissues;

[0035] Figure 2This is the response of the TaSPL6 gene in the wild-type wheat material Fielder to normal nitrogen and low nitrogen;

[0036] Figure 3 is the analysis of TaSPL6 expression levels in TaSPL6 RNAi plants;

[0037] Figure 4 is the phenotypic display and data statistical analysis of TaSPL6 RNAi under normal conditions;

[0038] Figure 5 Statistical analysis of the phenotypes of TaSPL6 RNAi and wild-type Fie1der in normal control fields and low nitrogen fields. DETAILED DESCRIPTION

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Unless otherwise specified, the practice 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, transgenic methods, construction of transgenic vectors, and obtaining transgenic plants used in the present invention can be achieved by using methods disclosed in existing literature, except for the methods used in the following examples.

[0043] 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-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 these. 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.

[0044] Biomaterials

[0045] The wheat variety Fielder seeds were kept in the laboratory;

[0046] The RNAi vector empty vector and the positive control vectors PC414C and PC336 were kept in the laboratory;

[0047] Escherichia coli DH5a and Agrobacterium tumefaciens GV3101 were maintained in the laboratory;

[0048] Primer synthesis and sequencing were completed by BGI Liuhe Biotechnology Co., Ltd.

[0049] Experimental reagents

[0050] RNA extraction kit, reverse transcription kit, plasmid mini-extraction kit, and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.;

[0051] Fluorescence quantitative Mix was purchased from Novozyme Biotechnology Co., Ltd.;

[0052] Various endonucleases were purchased from Mona Biotechnology Co., Ltd.;

[0053] One-step cloning enzyme was purchased from Novozymes Biotech Co., Ltd.

[0054] Experimental equipment

[0055] The PCR instrument was purchased from Applied Biosystems;

[0056] The refrigerated centrifuge was purchased from Eppendorf;

[0057] The quantitative PCR instrument was purchased from Bio-rad;

[0058] The laser confocal microscope was purchased from Zeiss;

[0059] The high-temperature and high-pressure sterilizer MLS-3750 was purchased from Sanyo Company of Japan;

[0060] The nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific;

[0061] The normal temperature centrifuge and SpectraMax iD5 microplate reader were purchased from Thermo Scientific.

[0062] Example 1 Cloning of TaSPL6 gene and construction of RNAi vector

[0063] RNA was extracted from the young spikelets of wheat variety Fielder during the differentiation period. The corresponding cDNA obtained by reverse transcription was used as a template. Specific primers were designed using Geneious software based on the gene sequence obtained from the Ensembl Plants database. After PCR amplification, the CDS sequence of the TaSPL6 gene was cloned.

[0064] In order to explore the period and location where TaSPL6 plays a role, the inventors first analyzed the expression pattern of TaSPL6 in different tissues and parts of the wheat variety Fielder. The roots, stems, leaves, young spikes, tillering buds, stamens, pistils and seeds of Fielder grown in the field to the jointing stage and 6-day and 16-day-old development were taken, RNA was extracted and reverse transcribed into cDNA, and then the expression level of the TaSPL6 gene was detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the expression level of the TaSPL6 gene in wheat young spikes, tillering buds and pistils was high ( Figure 1 ). This result suggests that the TaSPL6 gene may be involved in regulating the growth and development of wheat spikelets and the growth of tiller buds, thereby affecting wheat yield.

[0065] Considering that the tillering ability of wheat is a key factor in determining yield, and tillering is affected by the level of nitrogen fertilizer, the application of nitrogen fertilizer and other fertilizers in production can significantly increase the number of wheat ears and thus increase wheat yield. The inventors analyzed the response of the TaSPL6 gene to nitrogen. The tillering buds of the wild-type wheat material Fielder three-leaf and one-heart period grown under normal control conditions and low nitrogen conditions were taken, RNA was extracted and reverse transcribed into cDNA, and then the expression level of the TaSPL6 gene under different nitrogen conditions was detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the relative expression level of TaSPL6 was higher under low nitrogen (LN in the figure) than that under normal control conditions ( Figure 2 ).

[0066] In order to analyze whether TaSPL6 affects wheat yield and nitrogen use efficiency, the inventors first constructed a TaSPL6 RNAi strain. The specific process is briefly described as follows.

[0067] First, the CDS sequences of TaSPL6 genes TaSPL6-A, TaSPL6-B and TaSPL6-D on three subgenomes of wheat were obtained from the EnsemblPlants database, and the three sequences were compared using Geneious software, and a relatively conservative CDS sequence was selected as the vector insertion sequence. The inventors designed primers containing corresponding restriction sites for the conserved region of 300bp of TaSPL6, and used the cDNA sample prepared in Example 1 as a template to perform PCR amplification, and purified and recovered the amplified product. At the same time, the PC414C vector was digested, and the digested product was purified. The PCR amplification product and the PC414C vector after digestion were connected by homologous recombination, and the conserved region of 300bp of TaSPL6 was cloned into the PC414C vector;

[0068] Second, the conserved region of TaSPL6 (300 bp) and the reverse complementary sequence were inserted into the RNAi vector PC336 using Gateway technology to create TaSPL6-RNAi and TaSPL6-RNAi recombinant vectors;

[0069] Third, the ligation product was transformed into Escherichia coli DH5α using the heat shock transformation method for K + (Kanamycin, 50 μg / mL) resistance screening, select positive colonies for PCR detection, amplify the colonies identified correctly by PCR, send them for sequencing, and extract plasmids from the bacterial solution with correct sequencing for use.

[0070] Fourth, the extracted plasmid was transformed into Agrobacterium strain EHA105, and the wheat variety Fielder was transformed by Agrobacterium infection. The transgenic positive lines were screened by using resistance genes and vector-specific primers.

[0071] The panicles or tillering buds of potential transgenic plants were taken, RNA was extracted and reverse transcribed into cDNA, and the expression levels of TaSPL6-A, TaSPL6-B and TaSPL6-D in the potential transgenic plants were analyzed by qRT-PCR. The results showed that the expression level of TaSPL6 was downregulated to varying degrees in the three strains ( Figure 3 ).

[0072] Example 2 Verification of the function of TaSPL6 transgenic material in wheat yield and nitrogen use efficiency

[0073] First, the inventors identified the phenotype of TaSPL6 RNAi transgenic materials under normal field conditions and found that TaSPL6 RNAi plants promoted flowering, increased spike length, decreased spikelet number, but increased number of grains per spike; increased effective tillering, decreased thousand-grain weight, and finally significantly increased single-plant yield ( Figure 4 ).

[0074] The inventors then set up a low nitrogen treatment in the field experiment. Under normal control conditions, TaSPL6 RNAi significantly increased the number of wheat tillers and yield per plant compared to the wild-type material Fielder under low nitrogen conditions. It is worth noting that under low nitrogen conditions, the total number of tillers in the TaSPL6 RNAi line increased compared to the wild-type material Fielder, and the increase in the number of tillers and yield per plant increased ( Figure 5 ). This suggests that TaSPL6 inhibits the effect of nitrogen on increasing tillering. In other words, reducing the expression of TaSPL6 will increase the number of tillers and yield of wheat, especially under relatively low nitrogen supply levels.

[0075] In summary, silencing the TaSPL6 gene increased wheat yield and nitrogen use efficiency, especially under low nitrogen conditions. It can be seen that the TaSPL6 gene plays an important role not only in regulating wheat yield but also in nitrogen use efficiency, which is of great significance for creating new wheat germplasm with efficient nitrogen use and high yield.

[0076] 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

1. TaSPL6 The application of the gene in negatively regulating nitrogen utilization efficiency of wheat is characterized by: Said TaSPL6 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the negative regulation is as follows: TaSPL6 RNAi transgenic plants, under low nitrogen conditions in the field, TaSPL6 The total tiller number, effective tiller number and single plant yield of RNAi transgenic lines were higher than those of wild type. TaSPL6 The nitrogen utilization efficiency of RNAi transgenic plants is also higher than that of the wild type; the low nitrogen conditions are specifically as follows: field soil depth 1-10 cm, total nitrogen content 0.084%; field soil depth 10-20 cm, total nitrogen content 0.084%; field soil depth 20-30 cm, total nitrogen content 0.063%; field soil depth 30-40 cm, total nitrogen content 0.043%; normal conditions are specifically as follows: field soil depth 1-10 cm, total nitrogen content 0.107%; field soil depth 10-20 cm, total nitrogen content 0.105%; field soil depth 20-30 cm, total nitrogen content 0.068%; field soil depth 30-40 cm, total nitrogen content 0.046%.

2. Containing the substance described in claim 1 TaSPL6 The invention discloses an application of an RNAi vector of a gene and a host cell containing the RNAi vector in improving nitrogen utilization efficiency of wheat.

3. A breeding method for improving nitrogen utilization efficiency of wheat, characterized in that: The method is: by inhibiting / blocking the TaSPL6 The transcription / expression of the gene is used to obtain wheat plants whose total tiller number, effective tiller number and single plant yield are higher than those of the wild type under low nitrogen conditions; TaSPL6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The breeding method according to claim 3, characterized in that Inhibit / block wheat TaSPL6 Methods for gene transcription / expression include: (1) Using RNA interference technology to introduce small double-stranded TaSPL6 RNA, produced TaSPL6 Transgenic lines with reduced expression; (2) Construction through CRISPR / Case9 technology TaSPL6 Gene editing vector to obtain mutants.

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