Biological material for regulating effective tillering related protein ETN1 of wheat and application of biological material

Abstract

The invention discloses a biological material for regulating and controlling effective tillering related protein ETN1 of wheat and application of the biological material, and belongs to the technical field of biological breeding. The technical problem to be solved by the invention is how to improve the effective tiller number of plants and / or reduce the plant height. Therefore, the invention provides the application of the ETN1 protein or a substance for regulating the expression of an encoding gene of the ETN1 protein or a substance for regulating the activity or content of the ETN1 protein in regulating the tiller number or the plant height of the plant, and the ETN1 protein can be a protein with an amino acid sequence as shown in a sequence 2 in a sequence table. The gene of the ETN1 protein is finally localized through gene localization and map-based cloning. The gene can be widely applied to the plant fields of wheat genetic breeding, germplasm resource improvement, transgenosis, genome editing breeding and the like, and plays an important role in improving germplasm resources of crops such as wheat and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological breeding, and particularly relates to biomaterials related to the protein ETN1 for regulating effective tillering in wheat and their applications. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world, providing approximately 20% of the energy source for the world's population. With the continuous growth of the global population and the reduction of arable land area, ensuring food supply security has become a major challenge faced by people all over the world. At the same time, the increasing global climate change leading to frequent extreme weather has also brought more crises to the food security supply. Therefore, breeding new wheat varieties and continuously increasing wheat yield are of great significance for ensuring food security, promoting economic development and social stability. The wheat yield components include the number of effective spikes (effective tillers), the number of grains per spike, and the 1000-grain weight. Tillering is a special branching phenomenon in monocotyledonous plants. It is not only an important factor in the wheat plant type composition but also directly affects the wheat yield. At present, although several quantitative trait loci (QTLs) controlling tillering and genes regulating tillering have been mapped in wheat, only the tiller inhibition gene TIN and the tiller number gene Tn1 have been obtained by map-based cloning. TIN encodes a cellulose synthase-like protein, and Tn1 encodes a membrane protein containing four transmembrane domains. Although the mapping and cloning of these tillering control genes provide important gene resources for the breeding of new high-yield wheat varieties, there are still few genes controlling tillering and plant height traits that have been discovered and utilized at present. It is necessary to further explore and clone genes related to wheat plant type regulation, analyze their molecular mechanisms and genetic networks, enrich the genetic basis of yield formation, and have important application value for wheat plant type improvement and molecular design breeding. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: how to increase the effective tiller number of plants and / or reduce the plant height.

[0004] To solve the above technical problem, the present invention provides the application of a protein or a substance regulating the expression of the protein-encoding gene or a substance regulating the activity or content of the protein in any one of the following, and the protein can be the ETN1 protein;

[0005] A1), Application in regulating the tiller number of plants;

[0006] A2), Application in preparing a product for regulating the tiller number of plants;

[0007] A3), Application in regulating the plant height of plants;

[0008] A4), Application in preparing a product for regulating the plant height of plants;

[0009] A5) Application in plant breeding or plant-assisted breeding;

[0010] A6) Application in the preparation of products for plant breeding or plant-assisted breeding;

[0011] The ETN1 protein may be any of the following proteins:

[0012] a1) A protein with an amino acid sequence shown in SEQ ID No. 2;

[0013] a2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in a1), having more than 80% identity with the amino acid sequence shown in a1), and being related to the effective tiller number and / or plant height of plants;

[0014] a3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

[0015] Furthermore, in the above-mentioned application, the indicators of plant breeding may include tiller number and / or plant height.

[0016] Furthermore, in the above-mentioned application, the purpose of plant breeding may include cultivating plants with an increased tiller number and / or a decreased plant height.

[0017] In the present invention, the tiller number may be the number of branches that occur below or near the ground in plants such as Gramineae. It is produced on relatively swollen tiller nodes that store abundant nutrients. The tillers directly emerging from the tiller nodes at the base of the main stem are called primary tillers. New tiller buds and adventitious roots can be produced at the base of the primary tillers, forming secondary tillers. Under good conditions, tertiary and quaternary tillers can be formed.

[0018] Furthermore, in the above-mentioned application, the tiller number may be the effective tiller number.

[0019] The effective tiller number may be the number of tillers that can produce ears and set seeds in the early stage of plants such as Gramineae. The effective tiller number is directly related to the number of ears per unit area.

[0020] Furthermore, in the above-mentioned application, the plant height may be the distance measured from the base of the plant (or tiller node) to the top of the ear (excluding the awn) when the plant height of plants such as Gramineae is stable after the flowering period. The plant height is directly related to the lodging resistance ability.

[0021] Furthermore, in the above-mentioned application, the regulation may be enhancement or promotion or up-regulation.

[0022] Furthermore, in the above-mentioned application, the regulation may also be reduction or inhibition or down-regulation.

[0023] Further, in the above application, the protein may be derived from wheat.

[0024] In the present invention, SEQ ID No.2 consists of 935 amino acid residues.

[0025] The above protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0026] A3) The connection can be achieved through a peptide bond.

[0027] The protein-tag refers to a polypeptide or protein that is expressed in fusion with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a Flag protein-tag, His protein-tag, MBP protein-tag, HA protein-tag, myc protein-tag, GST protein-tag, and / or SUMO protein-tag, etc.

[0028] Further, in the above application, the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein can be a biological material, and the biological material can be any of the following:

[0029] B1), a nucleic acid molecule encoding the above ETN1 protein;

[0030] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0031] B3), a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0032] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0033] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0034] B6), a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2), or a transgenic plant tissue containing the recombinant vector described in B3);

[0035] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0036] Further, in the above application, the expression cassette in B2) refers to DNA capable of expressing the ETN1 protein in a host cell, which may not only include a promoter that initiates transcription of the ETN1 protein-encoding gene, but also include a terminator or / and enhancer sequence that terminates transcription of the ETN1 protein-encoding gene.

[0037] In one embodiment of the present invention, the recombinant vector in B3) may be the recombinant expression vector pUbi::ETN1. The structure of the recombinant expression vector pUbi::ETN1 is as follows: the fragment between the BamH I and SacI restriction enzyme recognition sites of the overexpression vector LGY-OE3 is replaced with the cDNA fragment of ETN1 (the DNA molecule shown in SEQ ID No. 3), and the other sequences of the LGY-OE3 vector are kept unchanged, resulting in the recombinant expression vector pUbi::ETN1. In the recombinant expression vector pUbi::ETN1, the cDNA fragment of ETN1 is expressed driven by the maize Ubiquitin promoter, and the ETN1 protein with the amino acid sequence shown in SEQ ID No. 2 is expressed.

[0038] Further, in the above application, the recombinant microorganism in B4) may specifically be yeast, bacteria, algae, and fungi.

[0039] Further, in the above application, the plant tissue in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0040] Further, in the above application, the transgenic plant organ in B7) may be the roots, stems, leaves, flowers, fruits, and seeds of transgenic plants.

[0041] Further, in the above application, the transgenic plant cell line, transgenic plant tissue, and transgenic plant organ may or may not include propagation materials.

[0042] Further, in the above application, the nucleic acid molecule in B1) may be a DNA molecule as described in any one of the following g1)-g3):

[0043] g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3;

[0044] g2), a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1;

[0045] g3), a DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the effective tiller number and / or plant height of plants.

[0046] Further, in the above application, the plant may be selected from monocotyledonous plants.

[0047] Further, in the above application, the monocotyledonous plant can be selected from the Gramineae plants.

[0048] Further, in the above application, the Gramineae plant can be selected from the Triticum plants.

[0049] Further, in the above application, the Triticum plant can be selected from Triticum aestivum L.

[0050] Further, in the above application, the regulation of the effective tiller number of the plant can be to increase the effective tiller number of the plant.

[0051] Further, in the above application, the regulation of the plant height can be to reduce the plant height.

[0052] The present invention also provides a method for increasing the effective tiller number of a plant and / or reducing the plant height, the method may include introducing a nucleic acid molecule encoding the above-mentioned ETN1 protein into a recipient plant to increase the effective tiller number of the plant and / or reduce the plant height of the plant.

[0053] Further, in the above method, the nucleic acid molecule can be a DNA molecule described in any one of the following g1)-g3):

[0054] g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3;

[0055] g2), a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1;

[0056] g3), a DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the effective tiller number and / or plant height of the plant.

[0057] Further, in the above method, the plant can be selected from monocotyledonous plants.

[0058] Further, in the above method, the monocotyledonous plant can be selected from the Gramineae plants.

[0059] Further, in the above method, the Gramineae plant can be selected from the Triticum plants.

[0060] Further, in the above method, the Triticum plant can be selected from Triticum aestivum L.

[0061] The present invention also provides a method for obtaining a target plant with an increased effective tiller number and / or a reduced plant height, the method may include introducing a nucleic acid molecule encoding the above-mentioned ETN1 protein into a recipient plant to increase the effective tiller number of the plant and / or reduce the plant height of the plant.

[0062] Further, in the method described above, the nucleic acid molecule may be a DNA molecule described in any one of the following g1)-g3):

[0063] g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3;

[0064] g2), a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1;

[0065] g3), a DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2), and regulating the effective tiller number and / or plant height of plants.

[0066] Further, in the method described above, the plant may be selected from monocotyledonous plants.

[0067] Further, in the method described above, the monocotyledonous plant may be selected from Gramineae plants.

[0068] Further, in the method described above, the Gramineae plant may be selected from Triticum plants.

[0069] Further, in the method described above, the Triticum plant may be selected from Triticum aestivum L.

[0070] The present invention also provides the above-mentioned protein and / or the above-mentioned biological material.

[0071] In the present invention, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences (or nucleotide 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 gapcost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0072] The above-mentioned identity of 80% or more than 80% may be 80%, 85%, 90%, or 95% or more of identity.

[0073] The identity of more than 80% may 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%. The identity of more than 85% may be an identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The identity of more than 90% may be an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The identity of more than 95% may be an identity of at least 95%, 96%, 97%, 98% or 99%.

[0074] The beneficial technical effects achieved by the present invention are as follows:

[0075] The present invention finally locates the gene of the ETN1 protein through gene mapping and map-based cloning. This gene can be used for the regulation of superior traits in wheat, such as significantly increasing the number of effective tillers and / or significantly reducing the plant height. The wheat effective tillering gene ETN1 can be widely applied in plant fields such as wheat genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and optimizing the germplasm resources of crops such as wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a phenotypic comparison of wild-type ZK331, mutant DD331 and F1 single plants. Figure 1 In (A), it is the potted growth performance of wild-type ZK331, mutant DD331 and F1 single plants, Bar = 5 cm. Figure 1 In (B), it is the comparative statistical analysis of the number of effective tillers of wild-type ZK331, mutant DD331 and F1 single plants. Figure 1 In (C), it is the comparative statistical analysis of the plant height of wild-type ZK331, mutant DD331 and F1 single plants.

[0077] Figure 2 It is the cytological observation of wild-type ZK331 and DD331. Figure 2 In (A), it is the cross-sectional view of the stems of wild-type ZK331 and mutant DD331, Bar = 100 um. Figure 2 In (B), it is the comparative statistical analysis of the stem diameters of wild-type ZK331 and mutant DD331. Figure 2 In (C), it is the longitudinal section view of the stems of wild-type ZK331 and mutant DD331, Bar = 50 um. Figure 2 In (D), it is the comparative statistical analysis of the cell lengths of the stems of wild-type ZK331 and mutant DD331. Figure 2In (E), it is the comparative statistical analysis of the stem cell width between the wild-type ZK331 and the mutant DD331.

[0078] Figure 3 It is the distribution of BSR-Seq candidate SNPs on the chromosome.

[0079] Figure 4 It is the map-based cloning of the ETN1 gene. Figure 4 In (A), it is the genetic linkage map of ETN1. Figure 4 In (B), it is the gene annotation result of the ETN1 corresponding interval in the Chinese Spring reference genome. Figure 4 In (C), it is the schematic diagram of the ETN1 gene. Figure 4 In (D), it is the protein domain of ETN1.

[0080] Figure 5 It is the functional verification of ETN1 transgenic. Figure 5 In (A), it is the phenotype of the ETN1 overexpression transgenic plants in the T1 generation. Figure 5 In (B), it is the statistical analysis of the effective tiller number of the ETN1 overexpression transgenic plants in the T1 generation. Figure 5 In (C), it is the statistical analysis of the plant height of the ETN1 overexpression transgenic plants in the T1 generation. Specific implementation manners

[0081] The present invention will be further described in detail below in combination with 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.

[0082] 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.

[0083] The quantitative tests in the following embodiments are all set with three replicates unless otherwise specified, and the results are averaged.

[0084] The primer sequences used in the following embodiments are shown in Table 1.

[0085] Table 1 Primer sequences

[0086]

[0087] The overexpression vector LGY-OE3 was kindly provided by the research group of Li Genying from the Shandong Academy of Agricultural Sciences and was disclosed in the literature "Niaz etal. (2023) Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat. Plant Biotechnology Journal. 21:979-989". The public can obtain the above biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0088] The transgenic receptor material Fielder is stored in our laboratory and was disclosed in the literature "Lu et al., A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nature Communication, 2020, 11, 680". The public can obtain the above biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0089] Example 1: Discovery, Fine Mapping and Cloning of ETN1

[0090] 1.1 Observation of the Phenotype of Wheat Multitiller Mutant DD331

[0091] The wheat multitiller mutant DD331 was obtained by screening the EMS mutant library of Zhongke 331 (ZK331). By observing the phenotypes of DD331 and ZK331, it was found that the average effective tillers of DD331 were 17.3, and the average effective tillers of ZK331 were 10.4; the average plant height of DD331 was about 41.35 cm, and the average plant height of ZK331 was about 68.97 cm. Compared with the wild type ZK331, the number of effective tillers of the mutant DD331 increased significantly, and the plant height decreased significantly ( Figure 1 ). The data was analyzed by one-way ANOVA using Excel statistical software, and "**" indicates extremely significant difference (P < 0.01).

[0092] Using paraffin section technology to observe the cross-section of the stem tissue at the jointing stage, it was found that the stem diameter of DD331 was smaller than that of ZK331 ( Figure 2)。The outer periphery of the stem of wild-type ZK331 is a relatively regular circle, while the outer shape of the stem of mutant DD331 is irregular. In addition, the arrangement of stem cells in DD331 is also tighter than that in ZK331. Longitudinal section observation of the stem tissue at the jointing stage found that the length and width of the stem tissue cells of DD331 were significantly smaller than those of ZK331's cells( Figure 2 ), indicating that the change in the length of the stem tissue cells of DD331 may be the reason for the decrease in plant height.

[0093] 1.2. Fine mapping and cloning of the wheat effective tiller gene ETN1

[0094] Two segregating populations were obtained by crossing DD331 with ZK331 and Aikang 58 (AK58) respectively. The F1 generation plants of both the DD331×ZK331 and DD331×AK58 populations showed semi-dwarf and medium tillering phenotypes compared with their parents. The 212 F2 individuals derived from self-crossing of the F1 of DD331×ZK331 included 55 individuals with phenotypes similar to DD331, 109 individuals showing semi-dwarf phenotypes, and 48 individuals similar to the wild type, and their ratio conformed to the single-gene segregation ratio of 1:2:1 (χ 2 1:2:1 = 0.63, P>0.05). Among the F 2:3 progeny derived from the F2 of DD331×ZK331, 55 families showed homozygous dwarf and multi-tillering phenotypes, 109 families showed segregation, and 48 families showed homozygous wild-type phenotypes, also conforming to the 1:2:1 segregation ratio (χ 2 1:2:1 = 0.63, P>0.05). The plant heights of the 410 F2 individuals derived from self-crossing of the F1 of DD331×AK58 showed a continuous distribution. However, among the F 2:3 families, 97 families showed homozygous multi-tillering dwarf phenotypes (45 - 50 cm), 207 families showed segregation of tiller number and plant height, and 106 families showed homozygous tall phenotypes (60 - 75 cm), and after chi-square test, it conformed to the genetic segregation ratio of 1:2:1 (Table 2). The above genetic analysis indicates that the multi-tillering and dwarf phenotypes in mutant DD331 are controlled by an incompletely dominant single gene, tentatively named ETN1 (Effective Tiller Number 1).

[0095] Table 2 Genetic analysis of the wheat multi-tillering dwarf gene ETN1

[0096]

[0097] χ 2 0.05 = 5.991, df = 2

[0098] Due to the pleiotropic effect of ETN1, which controls both wheat tillering and plant height simultaneously, to map this gene, leaves of 40 homozygous tall plants and 40 homozygous dwarf plants from the F2 population of DD331×AK58 were selected to construct the pooled DNA samples Bulk-High-3A and Bulk-dwarf-3A respectively. Transcriptome sequencing was performed on the Illumina HiSeq4000 platform. A total of 43,627,141 and 41,364,790 raw read pairs were obtained from the two pooled DNA samples respectively. After analysis, 79 candidate SNPs related to the target traits were finally obtained (allele frequency difference (AFD)>0.8 and Fisher's exact test P-value<1e-10), all of which were located on the long arm of chromosome 3A ( Figure 3 ). To develop molecular markers, primers were designed using the Chinese Spring reference sequence of the target interval (http: / / www.wheatgenome.org / ) as a template (Table 1). After screening, 9 pairs of primers (Dr1-Dr9) were found to be polymorphic between the parents and the two pooled DNA samples (Table 1). Genotyping was performed on the F2 population consisting of 3,956 individuals using the polymorphic primers. Finally, ETN1 was mapped to a genetic interval of 0.179 cM between molecular markers Dr4 and Dr6, and marker Dr5 was co-segregated with ETN1, corresponding to a physical interval of 58.42 kb on the 3AL chromosome arm of the Chinese Spring Ref Seq v1.0 version ( Figure 4 ). Gene annotation results showed that this interval contained only one high-confidence gene WAK. The coding region of the gene was 2,808 bp in length, containing 4 exons. The N-terminal of the gene contained 2 GUB_WAK_bind domains, and the C-terminal contained 1 PKc_like super family kinase domain ( Figure 4 ). Primers DW1 and Ec1 were used to amplify the DNA and cDNA of the wild-type Zhongke 331 and the mutant ETN1 respectively. Sequence comparison analysis found that compared with the wild-type, there was a G-to-A SNP mutation in the fourth exon of WAK in the mutant ETN1, resulting in the amino acid at position 565 changing from glycine to serine ( Figure 3 ). The nucleotide sequence of the ETN1 genome is SEQ ID No.1, the coding sequence of the ETN1 gene is SEQ ID No.3, and the amino acid residues of the encoded ETN1 protein are SEQ ID No.2 (Table 1).

[0099] Table 3 ETN1 genomic sequence, encoded protein, and coding sequence

[0100]

[0101]

[0102]

[0103]

[0104] Example 2: Functional Verification of the Effective Tillering Gene ETN1 in Wheat

[0105] Using the adapter-added primer WAK-OE3 and high-fidelity DNA polymerase for sequence amplification, after sequencing verification, a 2,808-bp fragment was obtained from the cDNA of the mutant ETN1. This fragment is the open reading frame (ETN1-ORF) of the ETN1 gene, and its nucleotide sequence is SEQ ID No.3. The obtained 2,808-bp fragment was constructed into the overexpression vector LGY-OE3 using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, and the obtained recombinant expression vector was named recombinant expression vector pUbi::ETN1. The structure of the recombinant expression vector pUbi::ETN1 is as follows: The fragment between the BamH I and SacI restriction enzyme recognition sites of the overexpression vector LGY-OE3 was replaced with the cDNA fragment of ETN1 (the DNA molecule with the nucleotide sequence shown in SEQ ID No.3), and the other sequences of the LGY-OE3 vector were kept unchanged, resulting in the recombinant expression vector pUbi::ETN1. In the recombinant expression vector pUbi::ETN1, the DNA molecule with the nucleotide sequence of SEQ ID No.3 is transcribed and translated driven by the maize Ubiquitin promoter to obtain the ETN1 protein with the amino acid sequence of SEQ ID No.2.

[0106] After verification, the recombinant expression vector pUbi::ETN1 was introduced into the callus of the recipient wheat cultivar Fielder using the Agrobacterium-mediated genetic transformation method. The seedlings differentiated from the callus were transferred to the growth screening medium and cultured under light at 22 - 24°C. Regenerated wheat plants transformed with pUbi::ETN1 (T0 generation transgenic plants of the ETN1 gene) were obtained. The T0 generation transgenic plants were transplanted into flower pots (20 cm × 20 cm) and transferred to the artificial climate chamber for growth. The artificial climate chamber had a 16-hour light / 8-hour dark cycle (25°C / 18°C, relative humidity 65%). The DNA of the T0 generation transgenic wheat plants was extracted. Using the DNA as a template, PCR amplification was performed with the primer pairs OE1 and OE3. The recombinant plasmid pUbi::ETN1 was used as a positive control, and the DNA of wheat Fielder was used as a negative control for PCR amplification. The expected sizes of the amplified product fragments were approximately 407 bp and 380 bp. The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 10 min. The PCR amplification products were detected by 1% agarose gel electrophoresis, photographed under ultraviolet light, and the results were recorded. A total of 3 T0 generation transgenic positive plants were obtained by PCR detection, which were named OE 1#, OE 2#, and OE 3# respectively. Compared with the control Fielder, the effective tiller number of the transgenic positive plants increased, and the plant height decreased.

[0107] The T0 generation transgenic positive plants were self-crossed respectively to obtain the T1 generation transgenic positive plants. The T1 generation transgenic positive plants were planted in flower pots, one plant per pot, and transferred to the greenhouse for growth. During the filling stage, 10 individual plants were randomly selected from each T1 generation transgenic line for genotype and phenotype identification, with the wheat cultivar Fielder as the control. Among them, 10 plants randomly selected from the T1 generation transgenic positive lines obtained by self-crossing of the T0 generation transgenic positive plant OE 1# were named OE1-N (N is a natural number, and 1 ≤ N ≤ 10). 10 plants randomly selected from the T1 generation transgenic positive lines obtained by self-crossing of the T0 generation transgenic positive plant OE 2# were named OE2-N (N is a natural number, and 1 ≤ N ≤ 10). 10 plants randomly selected from the T1 generation transgenic positive lines obtained by self-crossing of the T0 generation transgenic positive plant OE 3# were named OE3-N (N is a natural number, and 1 ≤ N ≤ 10). 10 individual plants of the wheat cultivar Fielder randomly selected were used as the control, named Fielder-N (N is a natural number, and 1 ≤ N ≤ 10).

[0108] The experiment was carried out in four groups, namely OE 1# group, OE 2# group, OE 3# group and Fielder group. The only difference among the four groups of experiments is the wheat under test, and the rest of the conditions are exactly the same. The wheat under test in the OE 1# group is the T1 generation transgenic positive line obtained by self-crossing of T0 generation OE 1#. The wheat under test in the OE 2# group is the T1 generation transgenic positive line obtained by self-crossing of T0 generation OE 2#. The wheat under test in the OE 3# group is the T1 generation transgenic positive line obtained by self-crossing of T0 generation OE 3#. The wheat under test in the Fielder group is the wheat variety Fielder. Taking the OE 1# group as an example, during the wheat filling period, the effective tiller number and plant height of the wheat under test numbered OE1-1 to OE1-10 were counted. The effective tiller number is the number of tillers that can produce ears and set seeds. The effective tiller number is directly related to the number of ears per unit area. The plant height is the distance measured from the base of the wheat to the top of the ear (excluding the awn), which is the plant height. The plant height is directly related to the lodging resistance ability.

[0109] Using the R (V4.1.2) language, Tukey tests based on analysis of variance were performed between groups to analyze the differences between groups. Different letters represent significant differences between different groups (p < 0.01). Different letters represent significant differences at the 0.05 level, and the same letters represent non-significant differences at the 0.05 level.

[0110] The results are shown in Table 4 and Figure 5 as follows. Figure 5 In the following, Fielder, OE 1#, OE 2# and OE 3# represent the Fielder group, OE 1# group, OE 2# group and OE 3# group respectively. The results show that the effective tiller number of all transgenic positive plants increased significantly, and the plant height decreased significantly.

[0111] Table 4 Phenotypic data of transgenic positive lines

[0112]

[0113] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, 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 changes, uses or improvements of the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Use of a protein, or a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein, in any of the following, wherein the protein is the ETN1 protein; A1), Use in regulating the tiller number of plants; A2), Use in preparing a product for regulating the tiller number of plants; A3), Use in regulating the plant height of plants; A4), Use in preparing a product for regulating the plant height of plants; A5), Use in plant breeding or plant-assisted breeding; A6), Use in preparing a product for plant breeding or plant-assisted breeding; The ETN1 protein is any of the following proteins: a1), A protein with an amino acid sequence shown in SEQ ID No. 2; a2), A protein obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in a1), having more than 80% identity with the amino acid sequence shown in a1), and being related to the effective tiller number and / or plant height of plants; a3), A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

2. The application according to claim 1, wherein: The substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any of the following: B1), A nucleic acid molecule encoding the ETN1 protein in the use described in claim 1; B2), An expression cassette containing the nucleic acid molecule described in B1); B3), A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4), A recombinant microorganism containing the nucleic acid molecule described in B1) or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), A transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6), A transgenic plant tissue containing the nucleic acid molecule described in B1) or a transgenic plant tissue containing the expression cassette described in B2), or a transgenic plant tissue containing the recombinant vector described in B3); B7), A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

3. According to the use described in claim 2, wherein: The nucleic acid molecule described in B1) is a DNA molecule described in any of the following g1)-g3): g1), A DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3; g2), A DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1; g3), A DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2), and regulating the effective tiller number and / or plant height of plants.

4. The application according to any one of claims 1 to 3, characterized in that: The plant is selected from monocotyledonous plants.

5. A method for increasing the effective tiller number and / or reducing the plant height of plants, the method comprising introducing a nucleic acid molecule encoding the ETN1 protein described in claim 1 into a recipient plant to increase the effective tiller number and / or reduce the plant height of plants.

6. The method according to claim 5, characterized in that: The nucleic acid molecule is a DNA molecule as described in any one of the following g1)-g3): g1) A DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3; g2) A DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1; g3) A DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the effective tiller number and / or plant height of plants.

7. A method for obtaining a target plant with an increased effective tiller number and / or a decreased plant height, the method comprising introducing a nucleic acid molecule encoding the ETN1 protein described in claim 1 into a recipient plant to increase the effective tiller number of the plant and / or decrease the plant height of the plant.

8. The method according to claim 8, characterized in that: The nucleic acid molecule is a DNA molecule as described in any one of the following g1)-g3): g1) A DNA molecule whose coding sequence of the coding strand is SEQ ID No. 3; g2) A DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 1; g3) A DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the effective tiller number and / or plant height of plants.

9. The method according to any one of claims 5-8, characterized in that: The plant is selected from monocotyledonous plants.

10. The protein and / or the biomaterial in the application described in any one of claims 1-4.

Images