Application of protein TabHLH92 in regulation and control of wheat tillering

Abstract

The invention discloses an application of a protein TabHLH92 in regulation and control of wheat tillering. The invention belongs to the technical field of biology, and particularly relates to application of protein TabHLH92 to regulation and control of wheat tillering. The protein TabHLH92 is a protein with an amino acid sequence of SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No: 6, is a protein for negatively regulating the tillering ability of wheat, can be applied to regulation of the tillering ability of plants and cultivation of plants with changed tillering ability, and also has a wide application prospect in the field of plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of protein TabHLH92 in regulating wheat tillering. Background Art

[0002] The yield of cereal crops is determined by the number of effective panicles, the number of grains per panicle, and the 1000-grain weight. The tiller number of cereal crops largely determines the number of effective panicles and is also related to the ability of crops to adapt to different environmental conditions (such as the abundance or deficiency of nitrogen in the soil). Tillering is crucial for increasing leaf area, light interception, and photosynthesis, which in turn affects the number of grains, grain filling, and yield. Therefore, tillering ability is an important trait selected during the breeding process of wheat and rice.

[0003] Tillering traits are regulated by complex and interconnected regulatory networks that involve signal transduction of nitrogen (N), carbon, and phytohormones. For example, strigolactones (SLs) or SL-derived metabolites are endogenous inhibitors of tiller bud growth and can inhibit tiller bud growth by inducing the expression of the TEOSINTE BRANCHED 1 (TB1) gene. SL biosynthesis and signal transduction are crucial for controlling wheat tillering. Knocking down the expression of the SL synthesis pathway gene TaD27 in wheat leads to SL deficiency and an increase in tiller number, while overexpressing TaD27 has the opposite phenotype. Members of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family have also been shown to be involved in SL signal transduction and thus in the regulation of rice tillering. In wheat, some SPL genes have also been found to be involved in SL signal transduction and thus affect wheat tillering. Overexpressing tae-miR156 in wheat can inhibit the expression of SPL genes such as TaSPL3 / 17, resulting in an increase in tiller number and defects in spikelet development. TaSPL3 / 17 interacts with TaD53, a key factor in the SL signaling pathway, and this interaction inhibits the transcriptional activation of the TaTB1 gene by TaSPL3 / 17. TaTB1 has previously been shown to inhibit the growth of tiller buds in wheat. Recently, TaSPL3 / 17 was found to interact with the PHYTOCHROME-INTERACTING FACTOR-LIKE (PIL) gene TaPIL1, and TaPIL1 can inhibit the growth of wheat tillers by transcriptionally activating the expression of TaTB1. In addition, some genes involved in the biosynthesis and signal transduction of auxin, cytokinin, and abscisic acid (ABA) have also been found to control wheat tillering, including the deacetylase gene TaHST1L, the auxin biosynthesis gene Tryptophan Aminotransferase-Related TaTAR2.1-3A, the auxin transporter gene TaPIN1, the TERMINAL FLOWER 1 gene TaTFL1-5, the SPL transcription factor TaSPL8, and the ankyrin-repeat protein-encoding gene Tiller Number1 (TaTN1).

[0004] Nutrients such as nitrogen and sucrose interact with some hormone signals and affect tiller growth. The mobility of sucrose in plants and its accumulation level in tiller buds are crucial for promoting the growth of tiller buds. During the process of promoting tiller bud growth, sucrose synergistically acts with cytokinin but antagonistically acts with SL. For example, sucrose can promote the accumulation of D53 protein, thereby promoting rice tillering. Sucrose is also crucial for promoting wheat tillering. For example, the tiller inhibition locus tin1 located on chromosome 1A reduces the sucrose content in tiller buds, thus reducing the tiller number of wheat. Overexpressing the TaCML20 gene in wheat increases the content of water-soluble sugars in the above-ground part and also increases the tiller number of wheat.

[0005] Nitrogen is an essential mineral nutrient for plant growth and development. Applying nitrogen fertilizer can increase the tiller number and spike number of wheat, thereby increasing wheat yield. In wheat, the transcription factors TaNAC2, TaNFYA-B1, and TabZIP60 that regulate nitrogen uptake and utilization affect the tiller number and spike number of wheat; the genes TaNRT2.5, TaGS2, and TaNADH-GOGAT involved in nitrogen uptake and assimilation also affect the tiller number and spike number of wheat. However, there is no report on the regulation of wheat tiller number by the transcription factor TabHLH92. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to increase the tiller number of wheat to improve wheat yield.

[0007] To solve the problems existing in the prior art, the present invention provides the application of a protein or a substance for regulating gene expression or a substance for regulating the activity or content of the protein in regulating the tillering ability of plants.

[0008] The application of the protein or the substance for regulating gene expression or the substance for regulating the activity or content of the protein provided by the present invention is in any one of the following:

[0009] 1) Application in regulating the tillering ability of plants;

[0010] 2) Application in preparing a product for regulating the tillering ability of plants;

[0011] 3) Application in cultivating plants with altered tillering ability;

[0012] 4) Application in preparing a product for cultivating plants with altered tillering ability;

[0013] 5) Application in plant breeding;

[0014] The protein is any one of the following proteins:

[0015] a1) A protein with an amino acid sequence of SEQ ID No: 2;

[0016] a2) A protein with the amino acid sequence of SEQ ID No: 4;

[0017] a3) A protein with the amino acid sequence of SEQ ID No: 6;

[0018] a4) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence shown in any of a1)-a3) and having the same function;

[0019] a5) A protein having more than 75% identity with the amino acid sequence defined in any of a1)-a4) and having the same function;

[0020] a6) A fusion protein obtained by connecting a tag to the end of the protein defined in any of a1)-a3).

[0021] The name of the protein described in a1) above is TabHLH92.

[0022] In order to facilitate the purification or detection of the protein in a1), a tag protein can be connected to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No: 6 in the sequence listing.

[0023] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0024] The tag proteins include but are not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0025] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein TabHLH92 of the present invention by using known methods, such as directed evolution or site-directed mutagenesis. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the protein TabHLH92 isolated from the present invention, as long as they encode the protein TabHLH92 and have the function of the protein TabHLH92, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0026] The above-mentioned identity of 75% or more can be an identity of 80%, 85%, 90% or more than 95%.

[0027] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search, the identity value (%) of a pair of amino acid sequences can be calculated.

[0028] In this article, the above-mentioned identity of 80% or more can be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0029] In this article, the above-mentioned identity of 90% or more can be an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0030] In the above application, the protein is derived from wheat (Triticum aestivum L.).

[0031] In this article, the substance that regulates the activity and / or content of the protein can be a substance that regulates gene expression, and the gene encodes the protein TabHLH92.

[0032] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six regulations: 1) regulation at the gene transcription level; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the gene's mRNA from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; 6) regulation after gene translation (that is, regulation of the activity of the protein translated from the gene).

[0033] In the present invention, the regulation can be up-regulation or enhancement or increase. The regulation can also be down-regulation or attenuation or decrease.

[0034] In this article, enhancing, increasing or upregulating the expression level of the coding gene of the aforementioned protein in the recipient plant, or / and enhancing, increasing or upregulating the activity and / or content of the coding gene of the aforementioned protein is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0035] In this article, regulating the expression of the coding gene of the protein may be inhibiting, reducing or downregulating the expression of the coding gene. Inhibiting, reducing or downregulating the expression of the coding gene can be achieved by gene knockout or gene silencing.

[0036] In this article, the so-called gene knock-out refers to the phenomenon that a specific target gene is inactivated by gene editing technology. Gene knock-out inactivates a specific target gene through changes in DNA sequences, including but not limited to those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a locus in the genome containing multiple short repeat sequences. The Cas9 protein can cleave the target sequence recognized by crRNA–tracrRNA under the mediation of RNA.

[0037] In this article, the so-called gene silencing refers to the phenomenon that a gene is not expressed or is expressed at a low level without damaging the original DNA. Gene silencing enables a gene not to be expressed or to be expressed at a low level on the premise of not changing the DNA sequence. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization and position effects, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of the gene, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi) and translation inhibition mediated by microRNA (miRNA), etc.

[0038] In the above applications, the substance for regulating gene expression or regulating the activity or content of the protein may be a biological material related to the aforementioned protein, and the biological material may be any one of the following:

[0039] c1) A nucleic acid molecule encoding the aforementioned protein;

[0040] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0041] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0042] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3);

[0043] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0044] c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2);

[0045] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0046] e1) A nucleic acid molecule that inhibits or reduces or silences the expression of the protein-coding gene described above;

[0047] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0048] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0049] e4) A recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3);

[0050] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0051] e6) A transgenic plant tissue containing the nucleic acid molecule described in e1), or a transgenic plant tissue containing the expression cassette described in e2);

[0052] e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

[0053] In the above applications, the nucleic acid molecule described in c1) can be a DNA molecule shown as any of the following,

[0054] d1) A DNA molecule whose coding region sequence is the DNA molecule shown in SEQ ID No: 1 in the sequence listing;

[0055] d2) The coding region sequence is the DNA molecule shown as SEQ ID No: 3 in the sequence listing;

[0056] d3) The coding region sequence is the DNA molecule shown as SEQ ID No: 5 in the sequence listing;

[0057] d4) A DNA molecule that has 90% or more identity with the nucleotide sequence defined in any one of d1)-d3) and encodes the protein described in claim 1;

[0058] d5) A DNA molecule that hybridizes with the nucleotide sequence defined in any one of d1)-d3) under stringent conditions and encodes the protein described above.

[0059] The nucleic acid molecules described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0060] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids or viral vectors. Specifically, it can be vector LGY-OE3.

[0061] Existing plant expression vectors can be used to construct recombinant expression vectors containing the TabHLH92 gene. The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may also contain the 3′ untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3′ end of the mRNA precursor. For example, it includes, but is not limited to, the non-translated region transcribed from the Agrobacterium tumefaciens crown gall induction (Ti) plasmid gene (such as the nopaline synthase Nos gene), and the non-translated regions at the 3′ end of plant genes (such as soybean storage protein genes) that have similar functions.

[0062] When constructing a recombinant plant expression vector using the TabHLH92 gene, any one of enhancer promoters or constitutive promoters can be added before its transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize (ubiquitin), etc. They can be used alone or in combination with other plant promoters. In addition, when constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon of the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0063] In order to facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vector can be processed, such as adding genes (GUS gene, luciferase gene, etc.) encoding enzymes or luminescent compounds that can produce color changes and are expressed in plants, antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

[0064] Using any vector that can direct the expression of foreign genes in plants, introducing the TabHLH92 gene or gene fragment provided by the present invention into plant cells or recipient plants can obtain transgenic cell lines and transgenic plants with altered tillering ability. The expression vector carrying the TabHLH92 gene can transform plant cells or tissues by conventional biological methods such as using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroconductivity, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0065] Optionally, e2) the expression cassette is the expression cassette carrying the DNA molecule shown in SEQ ID No:7.

[0066] The present invention also provides a method for improving the tillering ability of plants. The method includes step M, and step M is to inhibit or reduce or silence the activity and / or content of the protein described above in the target plant, and / or inhibit or reduce or silence the expression level of the coding gene of the protein described above to improve the tillering ability of plants.

[0067] The present invention also provides a method for reducing the tillering ability of plants. The method includes step P, and step P is to enhance, increase or up-regulate the activity and / or content of the aforementioned protein in the target plant, and / or, enhance, increase or up-regulate the expression level of the coding gene of the aforementioned protein, so as to reduce the tillering ability of plants.

[0068] In the above method, reducing the expression level and / or activity of the coding gene of the protein TabHLH92 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown techniques to decrease or inactivate the activity of the coding gene of the protein TabHLH92 in the genome of the target plant.

[0069] The present invention provides a method for cultivating plants with enhanced tillering ability, including inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, and / or inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, so as to obtain plants with enhanced tillering ability.

[0070] In one embodiment of the present invention, the breeding method for cultivating plants with enhanced tillering ability includes the following steps:

[0071] (1) Construct a recombinant expression vector for inhibiting or reducing or silencing the coding gene of the aforementioned protein;

[0072] (2) Transfer the recombinant expression vector constructed in step (1) into the recipient plant to obtain a plant with a tillering ability higher than that of the recipient plant.

[0073] In the present invention, the regulation of the expression of the protein TabHLH92 specifically refers to the expression of TabHLH92 in the above-ground parts and roots of wheat seedlings.

[0074] In the present invention, the purpose of plant breeding may include cultivating plants with enhanced tillering ability.

[0075] In the present invention, the tillering ability is reflected in the growth of tillering buds and / or the number of tillers.

[0076] In the present invention, the tillering ability also includes the tillering ability under high-nitrogen or / and low-nitrogen culture conditions.

[0077] The high-nitrogen condition is 2 mM N, and the low-nitrogen condition is 0.2 mM N.

[0078] In the present invention, the plant can be any one of the following:

[0079] N1) Dicotyledonous plants:

[0080] N2) Plants of the order Poales;

[0081] N3) Gramineous plants;

[0082] N4) Triticum plants;

[0083] N5) Wheat.

[0084] In the present invention, the wheat may be wheat variety Kenong 199.

[0085] In the present invention, the tillering ability of wheat is regulated by reducing TabHLH92, and the expression of TabHLH92 in the wheat roots and above-ground parts is inhibited by a high nitrogen supply level. TabHLH92 can negatively regulate the tiller number of wheat under different nitrogen supply levels. Reducing the expression level of TabHLH92 by the RNAi method can significantly increase the tiller number under different nitrogen supply levels, thereby laying a foundation for increasing the wheat yield under different nitrogen supply levels and achieving the goals of fertilizer saving, yield increase and environmental protection. Description of the Drawings

[0086] Figure 1 It shows the response of TabHLH92 expression in the above-ground parts and roots of hydroponic wheat seedlings to the nitrogen supply level. The high nitrogen treatment has a nitrogen supply level of 2 mM N, and the low nitrogen treatment has a nitrogen supply level of 0.2 mM N. The expression level is the relative expression level relative to the internal reference TaActin, and the data are Mean±SE of 3 biological replicates. * indicates that the difference between different nitrogen treatments reaches the significant level of P<0.05.

[0087] Figure 2 It is a schematic diagram of the LGY-OE3-TabHLH92-RNAi vector structure.

[0088] Figure 3 It is the identification of the expression level of TabHLH92 in the TabHLH92-RNAi transgenic line. The experimental materials were hydroponically cultured for 2 weeks under the condition of 2 mM N, and samples were taken for expression level identification. R92-1 and R92-2 represent the TabHLH92-RNAi transgenic lines; 92RNC is the negative control; Fielder is the transgenic receptor (wild type). Wheat TaActin is the internal reference gene. The values in the figure are mean±SE of 3 replicates. * indicates that the transgenic line is significantly different from the wild type and the negative control, with P<0.05 after significant test.

[0089] Figure 4Effects of TabHLH92-RNAi on the tiller number, total nitrogen, and sucrose content of wheat. The hydroponic experimental materials were cultured for 3 weeks, and samples were taken for trait determination. Among them, A, tiller number per plant; B, fresh weight of aboveground parts; C, length of the first tiller bud at the 4-leaf stage under high nitrogen conditions (2 mM N); D, sucrose content in different tissues under high nitrogen conditions (2 mM N); E, total nitrogen content in different tissues under high nitrogen conditions (2 mM N). The data are Mean±SE of 4 biological replicates. Different lowercase letters above the columns indicate significant differences at the P<0.05 level.

[0090] Figure 5 Effects of TabHLH92-RNAi on the spike number and grain yield of wheat. Under field experimental conditions, the spike number per plant and grain yield were investigated under nitrogen application treatment and non-nitrogen application treatment. Among them, A, spike number per plant; B, grain yield per plant. The nitrogen application rate (pure nitrogen) for the nitrogen application treatment was 18 g / m 2 , and the nitrogen application rate (pure nitrogen) for the non-nitrogen application treatment was 0 g / m 2 . The data are Mean±SE of 4 biological replicates. Different lowercase letters above the columns indicate significant differences at the P<0.05 level. Detailed implementation manners

[0091] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0092] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0093] In the following quantitative experiments of the embodiments, unless otherwise specified, three repeated experiments are set up.

[0094] The pEASY-Blunt vector (product number: CB101) in the following embodiments was purchased from TransGen Biotech Co., Ltd.

[0095] The vector LGY-OE3 in the following examples has been described in: Niaz M, Zhang L, Lv G, Hu H, Yang X, Cheng Y, Zheng Y, Zhang B, Yan X, Htun A, Zhao L, Sun C, Zhang N, Ren Y, Chen F. 2023. Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat. Plant Biotechnology Journal 21, 979-989. The biological material can be obtained from the applicant and is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0096] The wheat variety Kenong 199 in the following examples was selected and bred by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with the approval number: GS Wheat 2006017, and has been described in: Li Junming, Zhang Xiangqi, Zhang Aimin, Wang Zhiguo, An Tiaoguo, Ji Jun, Wang Jing. A new high-yield and widely adaptable wheat variety - Kenong 199 [J]. Journal of Triticeae Crops, 2007(02): 368. The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences to repeat the experiments of this application and cannot be used for other purposes.

[0097] The wheat variety Fielder in the following examples is a common recipient parent for Agrobacterium-mediated wheat transformation and has been described in: Ishida Y, Hiei Y, Komari T 2015. High efficiency wheat transformation mediated by Agrobacterium tumefaciens. In: Ogihara, Y., Takumi, S., Handa, H. (eds) Advances in Wheat Genetics: From Genome to Field. Springer, Tokyo, 167-173. The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences to repeat the experiments of this application and cannot be used for other purposes.

[0098] The wheat variety 92RNC in the following examples is the negative control self-separated from the TabHLH92-RNAi transgenic line. The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences to repeat the experiments of this application and cannot be used for other purposes.

[0099] The following examples used SPSS 11.5 statistical software to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated significant difference, while P < 0.01 (**) indicated extremely significant difference.

[0100] Example 1: Cloning and Detection of TabHLH92 Sequence Expression in Common Wheat

[0101] I. CDS Sequence Cloning of TabHLH92

[0102] Total RNA of wheat variety Kenong 199 was extracted, and the RNA concentration and quality were detected.

[0103] According to the reverse transcription kit of Toyobo (ReverTra qPCR RT Master Mix with gDNA Remover, FSQ-301), reverse transcription was carried out to obtain cDNA, which was stored at -20°C or diluted 20 times for ordinary PCR amplification. The primers used for PCR amplification of the TabHLH92 sequence were:

[0104] Primer F: 5’-ATGCAGATGGACTCCTACTACT-3’;

[0105] Primer R: 5’-TCAGAACACATTTTGCACGTGTGAT-3’.

[0106] The PCR reaction system and PCR program were as follows:

[0107] 1) PCR reaction system (TOYOBO, KFX-101, containing 2×PCR buffer for KOD FX, 2 mM dNTPs and KOD FX)

[0108] cDNA (100 ng / μL): 2 μL, Primer F (10 μM): 1.5 μL, Primer R (10 μM): 1.5 μL, 2×PCR buffer for KOD FX: 25 μL, 2 mM dNTPs: 10 μL, KOD FX (1.0 U / μL): 1 μL, ddH2O: 9 μL.

[0109] 2) PCR program

[0110] 98°C, 5 min; 98°C, 30 s, 60°C, 30 s, 68°C, 2 min, 36 cycles; 68°C, 10 min, 10°C, keep warm.

[0111] After PCR amplification, the obtained PCR products were detected by 1% agarose gel electrophoresis and ligated into the pEASY-Blunt vector for sequencing.

[0112] The results showed that in the wheat CDS sequences, the coding region (CDS) of the gene TaBHLH92-5A in the fifth homeologous group of chromosomes in the A genome (corresponding to the fifth homeologous group of chromosomes in the A chromosome group) was as shown in SEQ ID No: 1, encoding the protein shown in SEQ ID No: 2; the coding region (CDS) of the gene TaBHLH92-5B in the fifth homeologous group of chromosomes in the B genome (corresponding to the fifth homeologous group of chromosomes in the B chromosome group) was as shown in SEQ ID No: 3, encoding the protein shown in SEQ ID No: 4; the coding region (CDS) of the gene TabHLH92-5D in the fifth homeologous group of chromosomes in the D genome (corresponding to the fifth homeologous group of chromosomes in the D chromosome group) was as shown in SEQ ID No: 5, encoding the protein shown in SEQ ID No: 6.

[0113] II. Method for detecting the expression level of TabHLH92 in wheat

[0114] The following nutrient solution culture experiment was carried out using the wheat variety Kenong 199 as the material to detect the expression level of TabHLH92.

[0115] 1. Nutrient solution culture experiment

[0116] 1) Nutrient solution culture method

[0117] Select plump and uniform seeds of the wheat variety Kenong 199, sterilize them with 10% H2O2 for 20 min, rinse them several times with clear water, evenly place the seeds on a culture dish lined with filter paper, add an appropriate amount of saturated CaSO4, germinate them in the dark in an incubator at 23°C for 1 day, then transfer them to a culture tray with a gauze net, culture them with tap water for 6 days, pick out the seedlings with consistent growth, remove the endosperm, and then transfer them to an 8 L hydroponic culture box for continued culture. The nutrient solution culture experiment was divided into two groups: high nitrogen level (HN) and low nitrogen treatment (LN) (the nutrient solution formula is shown in Table 1). Each treatment was set with 3 - 4 replicates, and the culture solution was changed every 2 days. Culture temperature: 20°C; photoperiod: 16 h light / 8 h dark; light intensity: 405 μmol m -2 s -1 . After culturing for the required time, samples of roots, the base of the stem, and the above-ground part (excluding the base of the stem) were collected to analyze the gene expression level.

[0118] Table 1. Nutrient solution formula for wheat hydroponic experiment

[0119] Reagent High nitrogen treatment (2 mM N) Low nitrogen treatment (0.2 mM N) <![CDATA[Ca(NO3)2]]> 0.5 0.05 <![CDATA[NH4Cl]]> 1.0 0.1 <![CDATA[MgSO4·7H2O]]> 1.0 1.0 <![CDATA[Calcium chloride]]> 2.5 2.95 <![CDATA[H3BO3]]> 0.001 0.001 <![CDATA[(NH4)6Mo7O 24 ·4H2O]]> 0.00005 0.00005 <![CDATA[CuSO4·5H2O]]> 0.0005 0.0005 <![CDATA[ZnSO4·7H2O]]> 0.001 0.001 <![CDATA[MnSO4·H2O]]> 0.001 0.001 FeEDTA 0.1 0.1 KCl 1.5 1.5

[0120] Add 0.04% MES and adjust the pH to 6.0.

[0121] 2) Detection of TaBHLH92 expression levels in different wheat tissues by fluorescence real-time quantitative PCR method

[0122] Use the fluorescence real-time quantitative PCR (qRT-PCR) method to detect the expression levels of TaBHLH92 in different wheat tissues. The specific experimental steps are as follows:

[0123] Extract the total RNA from wheat tissues and detect the RNA concentration and quality. According to the reverse transcription kit of Toyobo (ReverTra qPCR RT Master Mix with gDNA Remover, FSQ-301), perform reverse transcription to obtain cDNA, store it at -20°C or dilute it 20-fold for real-time quantitative PCR (qRT-PCR) amplification reaction.

[0124] The reaction system and reaction conditions of the qRT-PCR reaction are as follows:

[0125] 2×SYBR Green I Master 10 μL, Primer F (10 μM) 0.4 μL, Primer R (10 μM) 0.4 μL, cDNA (diluted 20-fold after reverse transcription) 4 μL, ddH20 5.2 μL, and the total volume is 20 μL.

[0126] Set three technical replicates for each sample. 2×SYBR Green I Master is (Roche, 480 SYBR Green I Master, 4707516001).

[0127] The primers for identifying the relative expression levels of the TaBHLH92 gene can simultaneously amplify the coding regions of TaBHLH92-5A, TaBHLH92-5B, and TaBHLH92-5D. The primer sequences are as follows:

[0128] BHLH92-RT-F: 5’-GCAGCAGCAGTGCGTCAA-3’;

[0129] BHLH92-RT-R: 5’-CATTCATCTCCGTCGTCAACC-3’;

[0130] The primers for the internal reference gene TaActin are:

[0131] TaActin-F: 5’-ACCTTCAGTTGCCCAGCAAT-3’;

[0132] TaAc tin-R: 5’-CAGAGTCGAGCACAATACCAGTTG-3’.

[0133] qRT-PCR reaction procedure:

[0134] 95°C, 2 min; 95°C, 15 s, 60°C, 15 s, 72°C, 20 s, 45 cycles; 95°C, 5 s, 65°C, 1 min, 40°C, 20 s.

[0135] The instrument used for Real-Time PCR is Roche, LightCycler480 II.

[0136] After the reaction, confirm the amplification curve and melting curve of qRT-PCR. The specificity of the PCR reaction can be confirmed by analyzing the melting curve. Calculate the average Ct value. Determine the relative expression level of the target gene according to the expression level of the corresponding reference gene. The formula for calculating the relative expression level of the gene is:

[0137] Relative expression level = 2 -ΔCt , △ Ct = Ct 目标基因 -Ct 内标基因 .

[0138] The results are shown in Figure 1 , compared with the high-nitrogen treatment (2 mM N), the low-nitrogen treatment (0.2 mM N) inhibited the expression of TaBHLH92 in the roots, basal stems, and aboveground parts (excluding the basal stems) to varying degrees, indicating that the expression of TaBHLH92 is regulated by the nitrogen supply level.

[0139] Example 2, Creation of TabHLH92-RNAi Transgenic Lines and Their Trait Performances

[0140] I. Creation of TabHLH92-RNAi Transgenic Lines

[0141] Amplify the target interference fragment (corresponding to nucleotides 301 to 645 of SEQ ID No: 1) with the upstream primer TabHLH92-RNAi-F1 (5′-ggatccGCCGGCGCTGTCGAGAG-3′) with a BamHI restriction site and the downstream primer TabHLH92-RNAi-R1 (5′-gaattcCCTGGTCTTGACGTTCATGCTC-3′) with an EcoRI restriction site, and recover the target fragment (TabHLH92-S1) (corresponding to nucleotides 1 to 351 of SEQ ID No: 7).

[0142] The reverse complementary sequence of the target interfering fragment (the reverse complementary sequence corresponding to positions 301 to 645 of SEQ ID No: 1) was amplified using the upstream primer TabHLH92-RNAi-F2 (5′-aagcttCCTGGTCTTGACGTTCATGCTC-3′) with a Hind III restriction site and the downstream primer TabHLH92-RNAi-R2 (5′-gagctcGCCGGCGCTGTCGAGAG-3′) with a SacI restriction site, and the target fragment (TabHLH92-S2) (corresponding nucleotide sequence is positions 527 to 883 of SEQ ID No: 7) was recovered.

[0143] The intron fragment (S3) (corresponding nucleotide sequence is positions 353 to 527 of SEQID No: 7) in the vector was recovered by digesting the vector with EcoR I and Hind III.

[0144] The vector LGY-OE3 backbone (V1) was recovered by digesting the vector with BamHI and SacI ( Figure 2 is the structural schematic diagram of the vector LGY-OE3).

[0145] Then, the LGY-OE3 backbone (V1) was mixed and ligated with TabHLH92-S1, S3, and TabHLH92-S2 to construct a wheat LGY-OE3-TabHLH92-RNAi vector (the structural schematic diagram of the vector is as shown in Figure 2 ), and it was verified by sequencing and digestion.

[0146] The LGY-OE3-TabHLH92-RNAi vector contains the following DNA molecule. The nucleotide sequence of the DNA molecule is SEQ ID No: 7, which includes the forward sequence of the target interfering fragment (corresponding nucleotide sequence is positions 7 to 351 of SEQ ID No: 7), the intron sequence (corresponding nucleotide sequence is positions 358 to 526 of SEQID No: 7), and the reverse complementary sequence of the target interfering fragment (corresponding nucleotide sequence is positions 533 to 827 of SEQ IDNo: 7).

[0147] The structure of the LGY-OE3-TabHLH92-RNAi vector is described as follows: a recombinant vector obtained by replacing the fragment between the BamH I and Sac I recognition sites of the LGY-OE3 vector with a DNA molecule having a nucleotide sequence of SEQ ID No: 7, and keeping other nucleotide sequences on the LGY-OE3 vector unchanged.

[0148] In this study, the Agrobacterium EHA105-mediated transformation method was used to transform the immature embryos of wheat variety Fielder with the LGY-OE3-TabHLH92-RNAi vector. The Agrobacterium-mediated transformation method can be found in Ishida Y, Hiei Y, Komari T 2015. High efficiency wheat transformation mediated by Agrobacterium tumefaciens. In: Ogihara, Y., Takumi, S., Handa, H. (eds) Advances in Wheat Genetics: From Genome to Field. Springer, Tokyo, 167-173.

[0149] Positive transgenic lines R92-1 and R92-2 with significantly down-regulated TabHLH92 expression were obtained by PCR detection of the target gene and its expression level ( Figure 3 ), as well as the negative control 92RNC (negative plants without transgenic components isolated from self-crossing of transgenic materials).

[0150] Detection method for TabHLH92-RNAi positive transgenic lines: PCR reaction was carried out using the forward primer 5′-CTTCATACGCTATTTATTTGC-3′ and the reverse primer 5′-AATGGTGATCATCCAGCTCTC-3′ to detect positive transgenic plants in the T0 generation. The positive plants in the T0 generation were continuously self-crossed. How to perform PCR detection on the T1 and T2 plants to screen and obtain homozygous positive transgenic lines and negative controls without transgenic components.

[0151] Using the aforementioned nutrient solution culture method, the seedlings of TabHLH92-RNAi transgenic lines, negative controls, and receptor parent Fielder germinated for 7 days were cultured for 14 days under high nitrogen treatment conditions (see Table 1 in Example 1 for the nutrient solution of high nitrogen treatment). Samples of roots, the basal part of the stem, and the above-ground part (excluding the basal part of the stem) were collected to analyze the expression level of the TabHLH92 gene, and 3 biological replicates were set. The detection method for the expression level of the TabHLH92 gene can be found in "Detection of the expression level of TabHLH92 in different wheat tissues by fluorescence real-time quantitative PCR method" in Example 1.

[0152] The results are as Figure 3 shown. The expression level of TabHLH92 in the samples of roots, the basal part of the stem, and the above-ground part (excluding the basal part of the stem) of the positive transgenic lines R92-1 and R92-2 was significantly lower than that of the negative control 92RNC and the receptor parent Fielder. This result indicates that the expression level of TabHLH92 in Fielder was significantly reduced by RNAi interference.

[0153] II. Study on Seedling Traits of TabHLH92-RNAi Transgenic Lines

[0154] To identify the seedling traits of TabHLH92-RNAi transgenic lines, using the nutrient solution culture method described in Table 1 of Example 1, the seedlings of TabHLH92-RNAi transgenic lines, negative control, and receptor parent Fielder germinated for 7 days were cultured for 21 days under high-nitrogen and low-nitrogen treatment conditions. The tiller number was counted, the fresh weight of the above-ground part was measured, and samples of roots, stem bases, and above-ground parts (excluding stem bases) were collected to measure the sucrose content and total nitrogen content therein. And the length of the first tiller bud was measured at the four-leaf stage.

[0155] 1. Method for Measuring Sucrose Content

[0156] Using the plant sucrose content detection kit (product number: AKPL006M) produced by Beijing Boxbio Science & Technology Co., Ltd., the sucrose concentration in the dried samples was measured by colorimetry according to the operation steps of the kit, and the absorbance was measured at 480 nm. The operation steps are shown in the instruction manual of kit AKPL006M.

[0157] 2. Method for Measuring Total Nitrogen Content

[0158] Adopting the agricultural industry standard of the People's Republic of China NY / T 2017-2011 "Determination of Nitrogen, Phosphorus, and Potassium in Plants", the total nitrogen concentration in the dried samples was measured using the Kjeldahl automatic method (Kjeltec TM 8400, Foss Analytical A / S, Demark).

[0159] The results are shown by Figure 4 Under high-nitrogen (2 mM N) and low-nitrogen (0.2 mM N) treatment conditions, compared with the wild-type Fielder and negative control 92RNC, the tiller number and fresh weight of the above-ground part of the TabHLH92 down-regulated expression lines R92-1 and R92-2 were significantly increased ( Figure 4 A and B in). Under high-nitrogen (2 mM N) conditions, the length of the first tiller bud at the four-leaf stage was measured, indicating that the tiller bud lengths of R92-1 and R92-2 were significantly longer than those of the wild-type Fielder and negative control 92RNC ( Figure 4 C in).

[0160] The sucrose content and total nitrogen content in the samples of roots, stem bases, and above-ground parts (excluding stem bases) were measured under high-nitrogen (2 mM N) conditions. The results showed that the sucrose content and total nitrogen content in the samples of roots, stem bases, and above-ground parts (excluding stem bases) of R92-1 and R92-2 were significantly higher than those of the wild-type Fielder and negative control 92RNC( Figure 4In (D) and (E) above. The above results indicate that reducing the expression level of TabHLH92 in wheat by RNAi interference increased the sucrose content and total nitrogen content in the roots, the basal part of the stem, and the above-ground part (excluding the basal part of the stem), promoted the growth of tiller buds, and thus increased the tiller number.

[0161] III. Study on the spike number and grain yield traits of TabHLH92-RNAi transgenic lines

[0162] To identify the spike number and grain yield traits of TabHLH92-RNAi transgenic lines, the TabHLH92-RNAi transgenic lines, the negative control, and the receptor parent Fielder were planted in the Northern Agricultural Science and Technology Park of Shijiazhuang Academy of Agriculture and Forestry Sciences (Zhao County, Hebei).

[0163] Two treatments were set up: nitrogen application treatment and non-nitrogen application treatment. Urea was used as the nitrogen fertilizer. In the nitrogen application treatment, 12 g N / m 2 was applied as the basal fertilizer, and 6 g N / m 2 was top-dressed at the jointing stage; in the non-nitrogen application treatment, no nitrogen fertilizer was applied for both the basal fertilizer and the top-dressing. Phosphorus fertilizer was applied in both treatments. Triple superphosphate was used as the phosphorus fertilizer, and the application rate was 9 g P2O5 / m 2 and applied as the basal fertilizer. In the field experiment, each experimental material was arranged randomly in sequence. In each repetition, 1 row of each experimental material was sown, the seeding spacing was 5 cm, the row spacing was 23 cm, and the seeding depth was 3 - 5 cm. The grain yield and spike number were investigated at the wheat harvest stage.

[0164] The results showed that: whether in the non-nitrogen application treatment or in the nitrogen application treatment, the spike number per plant of the TabHLH92-RNAi transgenic lines R92-1 and R92-2 was significantly higher than that of the wild type Fielder and the negative control 92RNC ( Figure 5 in (A)), and the grain yield per plant of the TabHLH92-RNAi transgenic lines R92-1 and R92-2 was significantly higher than that of the wild type Fielder and the negative control 92RNC ( Figure 5 in (B)).

[0165] The above research results indicate that the expression of TabHLH92 in the roots and above-ground parts of wheat is inhibited by high nitrogen supply levels. Combining the trait identification results of the TabHLH92-RNAi transgenic lines with reduced expression, it shows that TabHLH92 negatively regulates the tiller number, spike number, and grain yield under different nitrogen supply levels. Reducing the expression level of TabHLH92 by the RNAi method can significantly increase the tiller number, spike number, and grain yield under different nitrogen supply levels.

[0166] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in any of the following: 1) Application in regulating plant tillering ability; 2) Application in the preparation of products for regulating plant tillering ability; 3) Application in breeding plants with altered tillering ability; 4) Use in the preparation of products for cultivating plants with altered tillering ability; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 2; a2) a protein having an amino acid sequence of SEQ ID No: 4; a3) a protein having an amino acid sequence of SEQ ID No: 6; a4) a protein having the same function as the amino acid sequence shown in any one of a1) to a3) after one or more amino acid residues are substituted and / or deleted and / or added; a5) a protein having an amino acid sequence identity of 75% or more to any one of a1) to a4) and having the same function; a6) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from wheat.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown in any of the following: d1) the coding region sequence is the DNA molecule shown in SEQ ID No: 1 in the sequence listing; d2) the coding region sequence is the DNA molecule shown in SEQ ID No: 3 in the sequence listing; d3) the coding region sequence is the DNA molecule shown in SEQ ID No: 5 in the sequence listing; d4) a DNA molecule having 90% or more identity with the nucleotide sequence defined in any one of d1) to d3) and encoding the protein described in claim 1; d5) A DNA molecule which hybridizes with any one of the nucleotide sequences specified in d1) to d3) under stringent conditions and encodes the protein described in claim 1.

5. A method for improving plant tillering ability, characterized in that: The method comprises step M, wherein step M is to inhibit, reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the tillering ability of the plant.

6. A method for reducing the tillering ability of a plant, characterized in that: The method comprises step P, wherein step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described in claim 1 or 2 to reduce the tillering ability of the plant.

7. A breeding method for cultivating plants with improved tillering ability, characterized in that: The invention comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or the activity and / or content of the protein to obtain a plant with improved tillering ability, wherein the tillering ability of the plant with improved tillering ability is higher than that of the recipient plant.

8. The method according to claim 7, characterized in that: The steps include: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described in claim 1 or 2; (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant to obtain a plant having a tillering ability higher than that of the recipient plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) Dicotyledonous plants: N2) Gramineae; N3) Gramineae; N4) Triticum; N5) Wheat.

Images