Application of GmDWF1 protein in regulation and control of plant height

Abstract

The invention discloses an application of a GmDWF1 protein in regulating and controlling the plant height of a plant. The invention belongs to the technical field of gene engineering, and particularly relates to application of a GmDWF1 protein to regulation and control of plant height. The invention provides application of proteins GmDWF1a and GmDWF1b or substances for regulating and controlling expression of the proteins or substances for regulating and controlling activity or content of the proteins in regulation and control of plant height. Experiments prove that the protein GmDWF1a and the protein GmDWF1b can regulate and control the plant height of the soybean, the gene knockout GmDWF1a and the gene knockout GmDWF1b discover that the plant height of the soybean can be reduced and the lodging resistance can be improved after the gene knockout GmDWF1a and the gene knockout GmDWF1b are knocked out, and the gene knockout GmDWF1a and the gene knockout GmDWF1b have important theoretical significance on dominant soybean creation and soybean breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of GmDWF1 protein in regulating plant strain height. Background Art

[0002] Soybean is an important grain and oil crop, but my country's soybean production cannot meet the strong domestic demand. Plant height is an important component of crop type and one of the important agronomic traits that affect crop yield. It is related to the lodging resistance of the plant. However, with the successful use of hybrid vigor, plant height has also increased, and lodging resistance has become the key to stable yield and quality. Moderately dwarfed crop varieties not only have enhanced lodging resistance, but are also more suitable for mechanized harvesting. Therefore, the important application value of dwarf traits (genes) in crops has attracted the attention of breeders. The first "Green Revolution" that emerged in the 1950s and 1960s was a huge success. Dwarf varieties were widely promoted, significantly increasing the world's wheat and rice production.

[0003] At present, scientists have gained an in-depth understanding of the molecular mechanism of plant height regulation in crops by studying dwarf mutants of Arabidopsis, rice, wheat, peas and tomatoes. Studies have shown that plant height is jointly regulated by factors such as the environment, hormones, transcription factors, and miRNAs. There are many studies on the synthesis and signaling pathways of hormones, among which gibberellin (GA) and brassinosteroids (BR) are the two most important factors that determine plant height traits. A few plant dwarfing mutations are related to auxin (Indole-3-acetic acid, IAA), strigolactones (SL) and cell wall synthesis pathways. Dwarfing genes can directly cause changes in plant morphology or cytology, affecting the size and number of cells, thereby causing a dwarfing phenotype in plants.

[0004] Soybean is a typical pod crop. Its aboveground part is different from gramineous crops such as wheat, rice and corn. There are leaves, inflorescences and pods on each node of the main stem and branches. Semi-dwarf stalks usually reduce the number of nodes, thus affecting the number of pods and grain yield. Therefore, if soybeans want to achieve an increase in yield, it needs to coordinate between plant height and the number of main stem nodes. In addition, concentrated rainfall during the flowering and pod-setting period of soybeans in my country's soybean-growing areas often causes lodging, resulting in a decrease in yield. Plant dwarfing correspondingly enhances the lodging resistance of soybeans and achieves stable yield. However, compared with food crops such as rice, wheat and corn, the utilization of soybean dwarf mutation resources and the study of dwarf mechanisms are relatively lagging, and there are few related reports. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to reduce the height of soybean plants, enhance their lodging resistance, and obtain dwarf plants.

[0006] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in regulating plant strains.

[0007] The application provided by the present invention is the application of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in any of the following:

[0008] 1) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in regulating plant strains;

[0009] 2) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in the preparation of products for regulating plant height;

[0010] 3) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in cultivating plants with altered plant height;

[0011] 4) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for cultivating plants with altered plant height;

[0012] 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding;

[0013] The protein is any of the following:

[0014] G1) a composition consisting of a protein having an amino acid sequence of SEQ ID No. 2 and a protein having an amino acid sequence of SEQ ID No. 4;

[0015] G2) a protein whose amino acid sequence is SEQ ID No. 2 or a protein whose amino acid sequence is SEQ ID No. 4;

[0016] G3) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the proteins of G1) and G2) and having more than 80% identity with the protein shown in A1) and having a function related to regulating plant height;

[0017] G4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of G1) or G2).

[0018] The protein whose amino acid sequence is SEQ ID No. 2 is named GmDWF1a.

[0019] The protein whose amino acid sequence is SEQ ID No. 4 is named GmDWF1b.

[0020] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0021] In the above protein, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0022] The protein described in the above application is derived from soybean (Glycine max (L.) Merr.).

[0023] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the proteins GmDWF1a and GmDWF1b.

[0024] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (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 mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0025] In the above application, the substance regulating gene expression and the substance regulating the activity or content of the protein may be a biological material related to the protein, and the biological material may be any one of the following:

[0026] c1) a nucleic acid molecule encoding the protein described above;

[0027] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0028] c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0029] 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);

[0030] 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);

[0031] c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0032] 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);

[0033] e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene mentioned above;

[0034] e2) an expression cassette containing the nucleic acid molecule described in e1);

[0035] e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0036] 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);

[0037] 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);

[0038] e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0039] 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).

[0040] In the above biological material, the nucleic acid molecule described in c1) is a DNA molecule shown in any of the following:

[0041] d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No.1;

[0042] d2) the coding region sequence is the DNA molecule shown in SEQ ID No. 5 in the sequence listing;

[0043] d3) the nucleotide sequence is the DNA molecule shown in SEQ ID No.3;

[0044] d4) The coding region sequence is the DNA molecule shown as SEQ ID No.6 in the sequence listing.

[0045] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0046] In the above e3), the recombinant vector may be a plant gene editing vector. The plant gene editing vector may be a GmDWF1-Cas9 vector.

[0047] e3) The recombinant vector is a DNA molecule whose nucleotide sequence is shown as SEQ ID NO.7.

[0048] The structure of the recombinant expression vector GmDWF1-Cas9 is described as follows: Four target gRNA expression cassettes driven by different promoters (i.e., the DNA fragments between 7344 and 9243 of SEQ ID NO: 7) were inserted between the restriction endonuclease Bsa I cleavage sites of the pYLCRISPR / Cas9P35-B vector, and the other sequences of the pYLCRISPR / Cas9P35-B vector were kept unchanged to obtain the recombinant vector GmDWF1-Cas9 ( Figure 1 ).

[0049] The nucleotide sequence of the recombinant expression vector GmDWF1-Cas9 is sequence 7. The nucleotides 1-9701 of sequence 7 are T-DNA genetic elements inserted into the genome of the transgenic material. The size and function of each element are shown in Table 1.

[0050] Table 1. T-DNA genetic elements of the genome insertion sequence of transgenic materials

[0051]

[0052]

[0053] The microorganism described in e4) above may be Agrobacterium, and the Agrobacterium is EHA105.

[0054] Those skilled in the art can easily mutate the nucleotide sequences encoding proteins GmDWF1a and GmDWF1b of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequences of proteins GmDWF1a and GmDWF1b isolated from the present invention are all derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention as long as they encode proteins GmDWF1a and GmDWF1b and have the functions of proteins GmDWF1a and GmDWF1b.

[0055] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0056] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0057] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0058] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (ie, cosmid), Ti plasmid or viral vector.

[0059] The existing plant expression vector can be used to construct a recombinant expression vector containing the GmDWF1a and GmDWF1b genes. The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as carmine synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0060] When using the GmDWF1a and GmDWF1b genes to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of corn. They can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of 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 start region can come from the transcription start region or the structural gene.

[0061] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0062] The invention also provides a method for regulating plant height.

[0063] The method for regulating plant height provided by the present invention comprises regulating plant height by regulating the expression of genes encoding proteins GmDWF1a and GmDWF1b alone or simultaneously or regulating the activity and / or content of the proteins, or regulating the activity and / or content of genes encoding the proteins.

[0064] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be down-regulation, attenuation or reduction.

[0065] The present invention also provides a method for cultivating plants with altered plant height.

[0066] The method for cultivating plants with altered plant height provided by the present invention comprises down-regulating or inhibiting or reducing the expression level of the genes encoding the proteins GmDWF1a and GmDWF1b in the target plant, or / and down-regulating or inhibiting or reducing the activity and / or content of the genes encoding the proteins, to obtain plants with altered plant height.

[0067] In the above-mentioned breeding method, the down-regulation or inhibition or reduction of the activity and / or content of the protein in the target plant, or / and the expression level of the gene encoding the protein can be obtained by introducing a recombinant expression vector containing a nucleic acid molecule that inhibits or reduces or silences the expression of the gene encoding the proteins GmDWF1a and GmDWF1b into the recipient plant, thereby obtaining a target plant with reduced plant height.

[0068] The GmDWF1a and GmDWF1b genes encode the GmDWF1a and GmDWF1b proteins.

[0069] In one embodiment of the present invention, the method for cultivating plants with altered plant height comprises the following steps:

[0070] 1) constructing a recombinant expression vector comprising the DNA molecules shown in SEQ ID NO.1 and SEQ ID NO.3 for inhibiting, reducing or silencing;

[0071] 2) Transforming the recombinant expression vector constructed in step 1) into a recipient plant;

[0072] 3) Obtain transgenic plants with altered plant height through screening and identification.

[0073] The introduction refers to introduction by recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta technology, and the like.

[0074] By using any vector that can guide the expression of foreign genes in plants, the coding genes or gene fragments for knocking out proteins GmDWF1a and GmDWF1b provided by the present invention are introduced into plant cells or recipient plants, and transgenic cell lines and transgenic plants with altered plant height can be obtained. The expression vector carrying the coding genes for knocking out proteins GmDWF1a and GmDWF1b can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissues can be cultivated into plants.

[0075] The microorganism described herein may be yeast, bacteria, algae or fungi. Among them, the bacteria may be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it may be Agrobacterium EHA105.

[0076] In the present invention, the purpose of plant breeding may include cultivating plants with reduced plant height.

[0077] In the present invention, the protein and / or the biological material mentioned above also fall within the scope of protection claimed by the present invention.

[0078] In the present invention, the plant may be a dicotyledonous plant.

[0079] In the above application or method, the dicotyledonous plant may be N1) or N2) or N3) or N4):

[0080] N1) Leguminosae;

[0081] N2) Leguminosae;

[0082] N3) soybean plants;

[0083] N4) Soybeans.

[0084] In the above, the soybeans may be soybean variety Tianlong No. 2 (soybean variety TL2 for short).

[0085] By designing gRNA specifically targeting the soybean GmDWF1 gene, constructing it into a Cas9 vector, and knocking out the soybean GmDWF1 gene using the CRISPR-Cas9 system, phenotypic identification revealed that the plant height of the Gmdwf1a homozygous single mutant strain was reduced by about 17.5%, and the plant height of the Gmdwf1aGmdwf1b homozygous double mutant strain was reduced by about 30%. The results showed that the GmDWF1 gene has a regulatory effect on plant height, but has no negative impact on yield-related agronomic traits such as the number of main stem nodes, the number of pods per plant, the number of grains per plant, and the 100-grain weight, providing a new idea for breeding short-stem lodging-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Schematic diagram of GmDWF1-Cas9 vector elements.

[0087] Figure 2 The target gene Cas9 was detected by PCR for the genome of the transformed strain T0 and T1 generations. Lane M: DL2000 DNA marker; Lanes 1-2: gmdwf1-1 of T0 and T1 generations, Lanes 3-4: gmdwf1-2 of T0 and T1 generations, Lanes 5-6: gmdwf1-3 of T0 and T1 generations; Lane (+): positive control with GmDWF1-Cas9 plasmid as template; Lane (-): non-transgenic receptor control.

[0088] Figure 3The bar gene was detected by PCR amplification of the T0 generation genome of the transformed strain. Lane M: DL2000 DNA marker; Lanes 1-2: gmdwf1-1 of T0 and T1 generations, Lanes 3-4: gmdwf1-2 of T0 and T1 generations, Lanes 5-6: gmdwf1-3 of T0 and T1 generations; Lane (+): positive control with GmDWF1-Cas9 plasmid as template; Lane (-): non-transgenic receptor control.

[0089] Figure 4 The results of DNA sequence alignment of GmDWF1a and GmDWF1b between the wild-type control and the mutant gmdwf1-1 are shown. The bases in the first row are the sequences of the wild-type soybean variety TL2, and the red dotted area in the second row is the difference in the editing sites of the successfully transformed and edited plants.

[0090] Figure 5 The results of DNA sequence alignment of GmDWF1a and GmDWF1b between the wild-type control and the mutant gmdwf1-3 are shown in Figure 1. The bases in the first row are the sequence of the wild-type soybean variety TL2, and the red area in the second row is the difference in the editing site of the mutant.

[0091] Figure 6 The phenotypes of the edited mutants. The target gene GmDWF1a of dwf1-1 (middle plant) was successfully edited, and the plant height decreased compared with the wild type (abbreviated as WT). The target genes GmDWF1a and GmDWF1b of dwf1-3 (right plant) were both edited, and the plant height decreased more.

[0092] Figure 7 For plant height statistical analysis, the wild-type control and the mutants gmdwf1-1 and gmdwf1-3 were statistically analyzed after maturity (unit: cm). DETAILED DESCRIPTION

[0093] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0094] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0095] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0096] The pYLCRISPR / Cas9-DH(B) plasmid in the following example has been recorded in: Bao A, Chen H, Chen L, Chen S, Hao Q, Guo W, Qiu D, Shan Z, Yang Z, Yuan S, Zhang C, Zhang X, Liu B, Kong F, LiX, Zhou X, Tran LP, Cao D. CRISPR / Cas9-mediated targeted mutagenesis of GmSPL9genes alters plant architecture in soybean. BMC Plant Biol. 2019, 19(1): 131. The public can obtain it from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences (i.e., the applicant) to repeat the experiment of this application and cannot be used for other purposes.

[0097] The soybean variety Tianlong No. 2 in the following embodiments was bred by our unit and its approval number is Guoshendou 2009026. The public can obtain it from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences (i.e. the applicant) or the seed market to repeat the experiment of this application.

[0098] The following examples used EXCELL statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. T-test was used for the test, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated a very significant difference.

[0099] The primer information used in the following examples is shown in Table 2.

[0100] Table 2, primer information summary of the present invention

[0101]

[0102]

[0103] Experimental Example 1: Target design of CRISPR-Cas9 for GmDWF1 gene and construction of recombinant vector GmDWF1-Cas9

[0104] The GmDWF1 gene has two copies in soybean, GmDWF1a and GmDWF1b. The genomic DNA sequences of GmDWF1a (gene ID: Glyma.12G028300, see SEQ ID No. 1) and GmDWF1b (gene ID: Glyma.11G103500, see SEQ ID No. 3) were downloaded from the soybean database (https: / / phytozome-next.jgi.doe.gov / info / Gmax_Wm82_a4_v1).

[0105] The coding sequence (CDS) of the GmDWF1a gene in soybean variety Tianlong No. 2 is SEQ ID No. 5, and the encoded amino acid sequence of the GmDWF1a protein is SEQ ID No. 2. In the genomic DNA of soybean variety Tianlong No. 2, the genomic sequence encoding the GmDWF1a protein is shown in SEQ ID No. 1 of the sequence listing.

[0106] The coding sequence (CDS) of the GmDWF1b gene in soybean variety Tianlong No. 2 is SEQ ID No. 6, and the encoded amino acid sequence of the GmDWF1b protein is SEQ ID No. 4. In the genomic DNA of soybean variety Tianlong No. 2, the genomic sequence encoding the GmDWF1b protein is shown in SEQ ID No. 3 of the sequence listing.

[0107] 1. Target design

[0108] After aligning the sequences of GmDWF1a and GmDWF1b, the PAM (proto adjacent motif) motif (NGG) was searched in the conserved region, and a sequence 19 bp upstream of the 5' end of the PAM position was used as the target sequence. The present invention designed four targets, named T1, T2, T3 and T4, and all of them were located in the first exon of the GmDWF1 gene. The specific sequences and positions are as follows:

[0109] T1: 5'-GGCTCCCTTGCGCCCTAAG-3' (target sequences are 893-911th positions of SEQ ID No. 1; 1078-1089th positions of SEQ ID No. 3 and 7465-7483th positions of SEQ ID No. 7);

[0110] T2: 5'-TGGGTTCACCGTGAGATGG-3' (the target sequences are positions 2106-2124 of SEQ ID No. 1 and positions 2291-2309 of SEQ ID No. 3; positions 8143-8161 of SEQ ID No. 7).

[0111] T3: 5'-CTTCTTCCTCTTAGGGCGCA-3' (target sequences are 901-919 of SEQ ID No. 1 and 1086-1097 of SEQ ID No. 3; 8598-8617 of SEQ ID No. 7);

[0112] T4: 5'-CCGCATCCCAACAGCAATCC-3' (the target sequence is 1150-1161 of SEQ ID No. 1; 9070-9089 of SEQ ID No. 7.

[0113] According to the T1 sequence, T2 sequence, T3 sequence and T4 sequence, the oligo DNA single-stranded forward and reverse primer sequences of the four targets DWF1-U3dT1F / DWF1-U3dT1R, DWF1-U3bT2F / DWF1-U3bT2R, DWF1-U6-1T3F / DWF1-U6-1T3R and DWF1-U6-29T4F / DWF1-U6-29T4R were synthesized as shown in Table 1.

[0114] The above sequence was synthesized by Wuhan Qingke Biotechnology Co., Ltd.

[0115] 2. Target linker preparation

[0116] Dissolve the adapter primer in TE to a 100 μM stock solution, take 1 μL of each and add it to 98 μL of 0.5x TE to dilute to 1 μM. Heat at about 90°C for 30 seconds, then cool to room temperature to complete annealing.

[0117] 3. Preparation and amplification of expression cassette conjugates

[0118] The adapter primer sequences of the above four targets were connected to the corresponding sgRNA expression cassettes by BsaI digestion and ligation, and then constructed into the pYLCRISPR / Cas9P35S-B vector. The specific process is briefly described as follows:

[0119] a. Cutting and ligation reaction: dilute 1 μL 10×CutSmart buffer, 20 ng pYLsgRNA plasmid, 0.5 μL adapter target sequence, 3 U BsaI-HF enzyme, 20 U T4 DNA ligase, and 0.5 μL 10×NEB T4 DNA ligase buffer to 10 μL with sterile water and then perform 10 cycles of reaction at 37°C for 5 min and 20°C for 5 min.

[0120] b. PCR amplification: Using the ligation reaction solution as a template, four different expression cassettes were PCR amplified using primer pairs T1 (Uctcg-B1' / gRctga-B2), T2 (Uctga-B2' / gRaaga-B3), T3 (Uaaga-B3' / gRgact-B4) and T4 (Ugact-B4' / BL. The amplification conditions were 95°C, 10s; 58°C, 15s; 68°C, 20s; a total of 22 cycles. The PCR products were mixed in equal amounts and purified to obtain the sgRNA expression cassette mixture.

[0121] 4. Connect the PCR-purified product into the pYLCRISPR / Cas9P35S-B vector

[0122] 1.5 μL 10×CutSmart buffer, 100 ng pYLCRISPR / Cas9P35S-B plasmid DNA, 100 ng

[0123] The sgRNA expression cassette mixture obtained in step 3, 10U BsaI-HF, 40U T4 DNA ligase, 0.5μL 10×NEB T4 DNA ligase buffer were diluted to 15μL with sterile water. Then 15 cycles of reaction were performed at 37℃5min, 10℃5min, and 20℃5min to obtain the recombinant expression vector.

[0124] 5. E. coli transformation and target detection

[0125] The recombinant expression vector constructed above was transformed into E. coli DH5α competent cells. The specific steps were as follows: ice bath for 30 minutes, gently removed, heat shock at 42°C for 60 seconds, and placed on ice for 2 minutes; 600 μl LB liquid culture medium was added, and the culture was resuscitated at 37°C and 150 rpm for 1 hour before plating, and an appropriate amount of bacterial liquid was spread on an LB plate containing kanamycin, and cultured at 37°C overnight. Single clones were picked and colony PCR was performed using primers SPL / SPR (specific primer information is shown in Table 1). Specific primer sequence SPL / SPR information is shown in Table 2.

[0126] The PCR product of the target band amplified at about 2kb was sequenced to analyze whether the target sequence exists. If all four target sequences exist in the recombinant vector, it means that the recombinant vector expressing sgRNA1, sgRNA2, sgRNA3, and sgRNA4 have been successfully transferred into pYLCRISPR / Cas9P35-B, and the recombinant expression vector constructed above is named GmDWF1-Cas9. The nucleotide sequence of the recombinant plasmid GmDWF1-Cas9 is sequence 7 in the sequence table.

[0127] The structure of the recombinant expression vector GmDWF1-Cas9 is described as follows: an expression cassette containing four targets T1, T2, T3 and T4 (i.e., a DNA fragment between 7344-9243 of sequence 7) is inserted between the restriction endonuclease Bsa I sites of the pYLCRISPR / Cas9P35-B vector, and the other sequences of the pYLCRISPR / Cas9P35-B vector remain unchanged to obtain the recombinant vector GmDWF1-Cas9. Nucleotides 1-9701 of sequence 7 are the T-DNA region, and the names, sizes and functions of the genetic elements are shown in Table 1.

[0128] 2. Genetic transformation of soybean and screening of positive transgenic plants

[0129] The CRISPR / Cas9 plasmid GmDWF1-Cas9 carrying the sg RNA expression cassette was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium tumefaciens.

[0130] The soybean receptor variety used was Tianlong No. 2.

[0131] The recombinant Agrobacterium tumefaciens was used to transform soybean. The specific method was referred to the literature "Bao A, Chen H, Chen L, Chen S, Hao Q, Guo W, Qiu D, Shan Z, Yang Z, Yuan S, Zhang C, Zhang X, Liu B, Kong F, LiX, Zhou X, Tran LP, Cao D (2019) CRISPR / Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean. BMC Plant Biol. 19(1): 131.", and 14 T0 generation transgenic soybean plants were obtained.

[0132] The T0 generation transgenic soybean plants were preliminarily screened using Bar protein test strips (Yunao Biotechnology Co., Ltd., Lot: 20220112, for detecting glufosinate PPT resistance), and three transgenic positive mutants resistant to glufosinate were obtained, named gmdwf1-1, gmdwf1-2, and gmdwf1-3.

[0133] 3. Creation of gmdwf1a, gmdwf1b mutants

[0134] 1) Screening and detection of transformed plants

[0135] Extract T0 and T1 plant leaf DNA of wild-type soybean and glufosinate-resistant plants gmdwf1-1, gmdwf1-2, and gmdwf1-3 obtained in step 2 to detect Cas9 and Bar genes. Plasmid GmDWF1-Cas9 was used as a positive control, and wild-type Tianlong No. 2 soybean plant DNA was used as a negative control.

[0136] Cas9 gene detection primers are shown in Table 1. PCR reaction program: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 34 cycles; 72°C for 10 min.

[0137] Bar gene detection primers are shown in Table 1. PCR reaction program: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 34 cycles; 72°C for 10 min.

[0138] PCR detection was performed using Cas9-specific primers, and the length of the amplified fragment was 964 bp. The test results are as follows: Figure 2 As shown; PCR detection was performed using Bar gene specific primers, and the amplified fragment was 518bp. The detection results are as follows Figure 3 shown.

[0139] Depend on Figure 2 and Figure 3 It can be seen that gmdwf1-1, gmdwf1-2, and gmdwf1-3 are all positive transgenic plants.

[0140] The obtained T0 generation GmDWF1 gene knockout soybeans were cultured to the T3 generation, and the soybeans of each generation were self-pollinated and then identified by PCR and first-generation sequencing to screen homozygous lines. Two homozygous mutant plants (i.e., the mutations in the two homologous chromosomes were the same) were screened and named gmdwf1-1 and gmdwf1-3 lines respectively.

[0141] 2) Detection of editing sites of target genes

[0142] By designing primers upstream and downstream of the four specific target sequence sites, PCR amplification was performed on the T2 generation transgenic positive plants gmdwf1-1, gmdwf1-2, and gmdwf1-3, and the products were purified and recovered and sequenced to detect the editing sites of the GmDWF1a and GmDWF1b genes. The target detection primers are shown in Table 1.

[0143] The sequencing results of the PCR products near the target site showed:

[0144] In gmdwf1-1 (intermediate plant), only the target gene GmDWF1a was successfully edited, and 10 bases were deleted at the T1 and T3 target positions "5'-GCGCCCTAAG-3'" (corresponding to positions 902-911 of SEQ ID No.1 and positions 24-33 of SEQ ID No.5). The deletion of these 10 nucleotides caused a frameshift starting from the 9th amino acid and premature termination at the 45th amino acid. The sequence is as follows: MSDLEAPLGRRFGWTISFSFDGSLLFLWSFPSPSPFISLHTLGM*, resulting in the loss of GmDWF1a protein function, thereby knocking out the GmDWF1a gene. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 4 The GmDWF1b gene of this event did not change compared to the wild type.

[0145] Compared with the wild type, gmdwf1-3 has the following mutations in the GmDWF1 gene and the GmDWF1a gene in both homologous chromosomes: the 10 bases "5'-GCGCCCTAAG-3'" in the GmDWF1a gene are deleted (corresponding to positions 902-911 of SEQ ID No.1 and positions 24-33 of SEQ ID No.5). The deletion of these 10 nucleotides causes a frameshift, leading to premature termination of translation and loss of GmDWF1a protein function, thereby knocking out the GmDWF1a gene. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 5 (Left side). For the GmDWF1b gene, the following mutations occurred in the GmDWF1b gene in both homologous chromosomes: the "5'-GCGCCCTAAG-3'" in the GmDWF1b gene (corresponding to positions 1083-1092 of SEQ ID No.3 and positions 21-30 of SEQ ID No.6) was deleted, resulting in a frameshift starting from the 8th amino acid and premature termination at the 45th amino acid. The sequence is as follows: MSDLEAPRGRRFGWTISFSFDGSLLFLWSFPSPSPFISLHTLGM*, causing the loss of GmDWF1b protein function, thereby knocking out the GmDWF1b gene. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 5 (right). gmdwf1-3 (right plant) has both target genes GmDWF1a and GmDWF1b edited, making it a double mutant with a greater decrease in plant height ( Figure 6 ).

[0146] Since CRISPR-cas9 technology only specifically edits the target sequence without affecting other sequences on the genome, and because of the reproductive isolation of soybeans from other species and a series of safety control measures such as geographical isolation of planting areas, it will not have any impact on other species. Therefore, this genetically modified plant is relatively safe for the environment and other species.

[0147] Example 3: Phenotypic Identification of Transformed Soybean Plants

[0148] The T3 plants of the homozygous single mutant line gmdwf1-1, the double mutant line gmdwf1-3 and the wild-type soybean variety TL2 obtained in Example 2 were cultured in an artificial climate chamber under long-day conditions (14 h light / 10 h dark) and 26° C. At least 10 individual plant data were counted for each material.

[0149] At the seedling stage, it was observed that the edited plants showed two phenotypes: semi-dwarf and dwarf. Among them, the homozygous single mutant gmdwf1-1 with a 10-base deletion in GmDWF1a showed semi-dwarf, and the double mutant gmdwf1-3 with a 10-base deletion in GmDWF1a / 1b showed a greater decrease in plant height. Figure 6 and Figure 7 As shown: relative to its recipient parent soybean variety TL2, the plant height of the mutant transformed plants gmdwf1-1 and gmdwf1-3 was significantly reduced, wherein the plant height of gmdwf1-1 decreased by 37.3% compared with the wild-type control, and the plant height of gmdwf1-3 decreased by 57.1% compared with the wild-type control. It is proved that the present invention can reduce the plant height of soybean by using the CRISPR / Cas9 system to knock out the soybean gene GmDWF1a / b to obtain a functional mutant, which provides excellent germplasm resources and efficient breeding methods and theoretical technical support for soybean lodging resistance breeding.

[0150] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that: The application is any of the following: U1) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in regulating plant strains; U2) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for regulating plant height; U3) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in cultivating plants with altered plant height; U4) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for cultivating plants with altered plant height; U5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding; The protein is any of the following: G1) a composition consisting of a protein having an amino acid sequence of SEQ ID No. 2 and a protein having an amino acid sequence of SEQ ID No. 4; G2) a protein whose amino acid sequence is SEQ ID No. 2 or a protein whose amino acid sequence is SEQ ID No. 4; G3) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the proteins of G1) and G2) and having more than 80% identity with the protein shown in A1) and having a function related to regulating plant height; G4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of G1) or G2).

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

3. The use according to claim 1 or 2, characterized in that: The substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein according to claim 1; 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 gene encoding the protein according to claim 1 or 2; 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 nucleotide sequence is the DNA molecule shown in SEQ ID No.1; d2) the coding region sequence is the DNA molecule shown in SEQ ID No. 5 in the sequence listing; d3) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.3; d4) The coding region sequence is the DNA molecule shown as SEQ ID No.6 in the sequence listing.

5. The use according to claim 3, characterized in that: e3) The recombinant vector is a DNA molecule whose nucleotide sequence is shown as SEQ ID No.

7.

6. A method for regulating plant height, characterized in that: The method comprises regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and regulating the expression level of the gene encoding the protein described in claim 1 or 2 to regulate the plant height.

7. A breeding method for cultivating plants with altered plant height, comprising regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and regulating the expression level of the gene encoding the protein described in claim 1 or 2, to obtain plants with altered plant height.

8. The method according to claim 7, characterized in that: The method of regulating the activity and / or content of the protein described in claim 1 or 2, or / and the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant comprises introducing a recombinant expression vector containing a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein described in claim 1 or 2 into the recipient plant, thereby obtaining the target plant with changed plant height; the encoding gene encodes the protein described in claim 1 or 2.

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

10. The use according to any one of claims 1 to 4, 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) Leguminosae; N3) Leguminosae; N4) soybean plants; N5)Soybean.

Images