Soybean lncRNA GmlncRNA10551 and application thereof in regulation and control of soybean grain weight
By overexpressing lncRNA GmlncRNA10551 in soybeans, the problem of increasing the weight and yield of soybeans is solved, and the effect of significantly increasing the weight of soybeans is achieved.
Patent Information
- Application Number
- CN202510198673.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively utilize long chain non-coding RNA (lncRNA) in soybeans to increase soybean grain weight and yield.
Soybean genetic transformation experiments verified and overexpressed soybean specific lncRNA GmlncRNA10551, significantly increasing the weight of soybean grains.
Overexpression of GmlncRNA10551 can significantly increase the weight of soybean grains, with an average increase of about 11.5% without affecting the plant type, thereby increasing soybean yield.
Smart Images
Figure CN120060252A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and is related to the application of plant lncRNA, and particularly relates to soybean lncRNA GmlncRNA10551 and its application in regulating soybean grain weight. Background Art
[0003] Soybean seed size is an important component factor in soybean yield formation and is a complex trait controlled by multiple genes. In previous soybean biological studies, researchers have cloned multiple coding genes that regulate soybean seed size through QTL mapping and genome-wide association analysis. However, these genes are all protein-coding genes. In fact, protein-coding genes only account for a part of the eukaryotic organism genome. The rest of the genome is constantly transcribing non-coding RNAs. These non-coding RNAs can be artificially divided into small RNAs or long non-coding RNAs (lncRNAs) according to whether their length is greater than 200 nt. lncRNAs have been proven to play important roles in plant growth and development and stress responses. For example, lncRNA MISSEN in rice can regulate the grain shape of rice grains. Therefore, exploring the functions of these lncRNAs in the genome is of great significance for crop genetic improvement.
[0004] In the invention, we verified through soybean genetic transformation experiments that an lncRNA GmlncRNA10551 is involved in positively regulating soybean seed size. Using the soybean cultivar Williams 82 as the background, we conducted overexpression experiments on GmlncRNA10551. Compared with wild-type soybeans (Williams 82), the 100-seed weight of the plants overexpressing GmlncRNA10551 increased significantly, by about 11.50%, but the plant type did not change significantly. In actual production, this lncRNA can be expected to significantly increase economic benefits without changing the field production cost. The lncRNA involved in the present invention will have broad application prospects in the cultivation of high-yield soybean varieties. Summary of the Invention
[0005] The technical problem to be solved by the present invention is how to use lncRNA to increase soybean grain weight and yield.
[0006] To solve the above technical problem, the present invention first provides lncRNA GmlncRNA10551 and its application.
[0007] The lncRNA provided by the present invention, named GmlncRNA10551, is a long non-coding RNA, derived from Glycine max (L.) Merrill of the genus Glycine, and is the application of any one of the following substances (a), (b), or (c) in soybean seed weight:
[0008] The lncRNA may be a DNA molecule of any of the following (a), (b), or (c):
[0009] (a) The DNA or RNA molecule shown in SEQ ID NO.1;
[0010] (b) A DNA or RNA molecule that hybridizes with the nucleic acid sequence defined in (a) under stringent conditions and is related to plant seed weight;
[0011] (c) A DNA or RNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence defined in (a) and is related to plant seed weight.
[0012] SEQ ID NO.1 consists of 684 nucleotides and is all the sequence of lncRNA GmlncRNA10551.
[0013] The substitutions and / or deletions and / or additions in the above (b) may be caused by natural variations or artificial mutagenesis.
[0014] The nucleic acid sequences in the above (a), (b), or (c) can be artificially synthesized, or their lncRNAs can be synthesized first and then obtained through biological expression.
[0015] In addition, recombinant vectors containing the lncRNA, overexpression vectors targeting the lncRNA, expression cassettes, transgenic cell lines, recombinant bacteria, etc. all fall within the protection scope of the present invention.
[0016] In the examples of the present invention, the sequence between the BamHI / XhoI restriction enzyme sites of the overexpression vector was replaced with respect to the lncRNA GmlncRNA10551 to obtain an overexpression recombinant vector. For the specific method, see Example 3.
[0017] For the convenience of identifying and screening transgenic plant cells or plants, appropriate modifications can be made to the plant expression vector to achieve the purpose of rapidly screening positive vectors and positive plants. Commonly used screening reagents for positive vectors and plants include antibiotics such as kanamycin, gentamicin, rifampicin, etc. There are also screening reagents such as herbicides, such as glyphosate. Adding some resistance marker genes to the plant vector can enable positive vectors, cells, strains or plants to survive under the screening of antibiotics or herbicides, so as to be sorted out with high efficiency.
[0018] The primer pairs for amplifying the full length or any fragment of the lncRNA also belong to the protection scope of the present invention.
[0019] The primer pairs may specifically be as follows (I):
[0020] (I) A primer pair consisting of the DNA shown in SEQ ID NO.2 and the DNA shown in SEQ ID NO.3.
[0021] (II) A primer pair consisting of the DNA shown in SEQ ID NO.4 and the DNA shown in SEQ ID NO.5.
[0022] The present invention also protects a method for cultivating transgenic plants. An overexpression vector of the lncRNA is introduced into a target plant to obtain a transgenic plant with a grain weight or grain size higher than that of the target plant. The transgenic plant is not only understood to include the transgenic contemporary plant, but also its progeny, and transferring the lncRNA to other varieties of the target plant, especially including commercial varieties, through conventional breeding techniques such as hybridization and backcrossing. Introducing the overexpression vector of the lncRNA into the target plant or other varieties will cause the lncRNA to be overexpressed in the plant, thereby improving the grain weight or grain size trait of the seeds of the target plant.
[0023] The overexpression recombinant plasmid can also be modified as follows first and then introduced into a host cell or plant to achieve a better expression effect:
[0024] 1) According to the transformation requirements, select a suitable resistance marker. Common vector resistance markers include kanamycin, hygromycin, ampicillin, etc., which helps to screen successfully recombinant vectors. In addition, in the positive screening process of transgenic plants, selecting a vector with herbicide resistance also helps to rapidly screen positive plants and save time costs;
[0025] 2) Optimize the selection of corresponding highly efficient promoters according to the type of target plant to be transformed and the specific organ to be expressed, so as to achieve the effect of overexpressing genes. Transgenic plants are often divided into monocotyledonous and dicotyledonous plants. For example, the CaMV35S promoter is often used in dicotyledonous plants; the Ubiquitin promoter is often used in monocotyledonous plants. In addition, the expression organs of transgenes need to be considered. If the specified gene is expressed in specific organs such as seeds and roots, tissue- or organ-specific promoters need to be selected; if it needs to be expressed in all stages and organs of plant growth and development, constitutive promoters need to be used.
[0026] 3) Enhancer sequences can be introduced into the vector to enhance the transcriptional efficiency of genes. Such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (such as those derived from TMV, MCMV, and AMV).
[0027] In actual operation, the specific expression of the gene of the present invention can be achieved by means of cell targeting. This process can be operated with the help of existing technologies. For example, the gene sequence targeting the organelle is fused with the target gene sequence, and then the fusion gene is introduced into plant cells to achieve specific targeted expression.
[0028] Specifically, the target gene can be introduced into the target plant through a recombinant vector. The recombinant vector carrying the target gene can be used to transform plant cells or tissues by a variety of conventional methods, including Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrotransformation, and Agrobacterium-mediated transformation, etc. The transformed plant cells or tissues can be further cultivated into complete plants.
[0029] The target plant can be either a leguminous plant or a non-leguminous plant. For example, the target plant can be a leguminous plant (such as soybean, alfalfa, lotus corniculatus, etc.); a non-leguminous plant (such as rapeseed, sunflower, corn, etc.). Arabidopsis thaliana can also be used as the research object for experiments.
[0030] The application of the lncRNA GmlncRNA10551 of the present invention in regulating soybean seed weight is characterized in that overexpressing this lncRNA can increase soybean seed weight. Compared with the research on past coding genes, it broadens the way of available gene resources for soybean genetic breeding and can more efficiently improve the economic benefits of soybean crops.
[0031] The application of the overexpression vector, expression cassette, transgenic cell line or recombinant bacterium of the lncRNA GmlncRNA10551 of the present invention in increasing soybean seed weight.
[0032] Beneficial effects:
[0033] The present invention utilizes an overexpression recombinant vector system to achieve the overexpression of the lncRNA GmlncRNA10551 in soybeans, obtaining an overexpression transgenic soybean line of this lncRNA, and performing phenotypic statistics and comparison on wild-type control plants and transgenic lines.
[0034] The experimental results show that: in small-scale repeated field trials, overexpression of the lncRNA GmlncRNA10551 can significantly increase the soybean grain weight, with the average 100-seed weight reaching 15.4 g; compared with the wild-type control, the 100-seed weight is significantly increased by about 11.5%. In actual production, this gene has no significant effect on the plant architecture and the number of seeds, and it can be expected to achieve a significant increase in economic benefits under the condition of unchanged field production costs. The lncRNA involved in the present invention will have broad application prospects in the cultivation of high-yield plants. Brief Description of the Drawings
[0035] Figure 1 . Amplification of the nucleic acid sequence of lncRNA GmlncRNA10551.
[0036] Figure 2 . Schematic diagram of the plant overexpression vector pFGC5941-GmlncRNA 10551.
[0037] Figure 3 . Molecular identification of the soybean overexpression line GmlncRNA10551-OE1 / 2.
[0038] Figure 4 . Plant architectures of the control line and the overexpression line GmlncRNA10551-OE1 / 2.
[0039] Figure 5 . Comparison of seed length and seed width between the control line and the overexpression line GmlncRNA10551-OE1 / 2.
[0040] Figure 6 . Difference in 100-seed weight between the control line and the overexpression line GmlncRNA10551-OE1 / 2. Detailed Embodiments
[0041] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention.
[0042] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.
[0043] The test materials used in the following examples can be purchased from commercial sources unless otherwise specified. All primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.; the transgenic transformation process was completed by Jiangsu Weimi Biotechnology Co., Ltd. The percentages in the following examples are mass percentages unless otherwise specified. T 2 represents the seeds produced by self-crossing of T 1 generation and the plants grown from them, and T 3 generation represents the seeds produced by self-crossing of T 2 generation and the plants grown from them.
[0044] The experimental materials in the following examples: Williams 82 soybean seeds were purchased from Jiangsu Weimi Biotechnology Co., Ltd.; the construction of the overexpression recombinant vector was completed by this laboratory; the Agrobacterium transformation and transgenic experimental operation processes were all completed by Jiangsu Weimi Biotechnology Co., Ltd.
[0045] Example 1. Cloning of soybean lncRNA GmlncRNA 10551
[0046] I. Designing specific primers for cloning genes
[0047] The gene sequence of GmlncRNA10551 was obtained by aligning and annotating the transcriptome sequencing libraries of multiple soybean tissues, and the genomic sequence of this lncRNA was obtained. To amplify the sequence of this lncRNA, primers were designed at the transcription start and transcription termination sites, and the sequences are as follows:
[0048] GmlncRNA10551-F1: 5'-ATGAAAGTACATATITTCTGAA-3' (SEQ ID NO.2);
[0049] GmlncRNA10551-R1: 5'-TAGCAGAATITCCATATTAGTT-3' (SEQ ID NO.3);
[0050] II. Cloning the genomic sequence and non-coding sequence of GmlncRNA10551
[0051] Using the CTAB method, DNA was extracted from soybean Williams 82 leaves. Using the DNA as the amplification template, the genomic sequence of the gene was amplified by the high-purity heat-resistant DNA polymerase (Novizan, Jiangsu, China) with the primers GmlncRNA10551-F1 / R1 (SEQ ID NO.2 and SEQ ID NO.3).
[0052] The RNA was extracted from soybean Williams 82 seeds using the TriZol method and reverse transcribed to obtain the cDNA sequence of this lncRNA. Using the cDNA as the amplification template, the lncRNA sequence was amplified by the primer pair GmlncRNA10551-F1 / R1 (SEQ ID NO.2 and SEQ ID NO.3). The amplified lncRNA fragment was detected by agarose gel electrophoresis to conform to the sequence size ( Figure 1 ), which can be used for subsequent experiments. The amplified sequence was sent to the company (GenScript, Beijing, China) for Sanger sequencing to verify the correct sequence cloning.
[0053] Example 2. Function of soybean lncRNA GmlncRNA10551
[0054] Based on the seed weight phenotype data and expression data of soybean seeds, transcriptome-wide association analysis was performed on hundreds of soybean natural populations, and the candidate lncRNA GmlncRNA10551 highly associated with seed weight was screened. Therefore, this lncRNA was studied intensively.
[0055] Example 3. Construction of plant overexpression vector of GmlncRNA10551
[0056] 1. Extract the RNA from soybean variety Williams 82 seeds and reverse transcribe it into cDNA.
[0057] 2. Design the following specific primer pair containing BamH I and Xho I adapter sequences:
[0058] GmlncRNA10551-F2: 5’-mggagaggacacgctcgagATGAAAGTACATATTTTCT GAA-3’ (SEQID NO.4);
[0059] GmlncRNA10551-R2: 5’-ctctagactcacctaggatccTAGCAGAATTTCCATATTA GTT-3’ (SEQ ID NO.5).
[0060] 3. Using the cDNA in step 1 as the template, perform PCR with the specific primer pair in step 2 and recover the PCR product.
[0061] 4. Double digest the PCR product with the restriction enzymes BamH I and Xho I and recover the digested product.
[0062] 5. Double digest the pFGC5941 vector with the restriction enzymes BamH I and Xho I and recover the vector backbone.
[0063] 6. Ligate the digested product from Step 4 and the vector backbone from Step 5 to obtain a ligation product.
[0064] 7. Sequence the ligation product. The result shows that the recombinant expression vector pFGC5941-GmlncRNA10551 is obtained (the original plasmid is pFGC5941, and the DNA shown in Sequence 1 of the Sequence Listing is inserted between the BamH I and Xho I restriction sites after the CaMV 35S promoter). The recombinant expression vector pFGC5941-GmlncRNA10551 is as Figure 2 shown.
[0065] Example 4. Obtaining and Identification of GmlncRNA10551 Transgenic Soybeans
[0066] I. Transformation of Soybeans and Identification of Transgenic Plants
[0067] 1. Introduce the recombinant expression vector pFGC5941-GmlncRNA10551 into Agrobacterium tumefaciens EHA101 to obtain recombinant Agrobacterium.
[0068] 2. Transfer the recombinant Agrobacterium into Williams 82 by infecting the axillary meristems of cotyledon nodes with Agrobacterium. After growth stabilizes, transfer it to nutrient soil for cultivation. At the same time, identify the transformation efficiency, and harvest the positive single plants T 0 and conduct seed multiplication.
[0069] 3. Harvest the T 1 -generation single plants, sow the seeds of each single plant separately, and continue to screen to observe the segregation situation of the T 2 -generation. Repeat this process until the T 3 -generation to obtain the genetically stable homozygous overexpression lines GmlncRNA10551-OE1 / 2.
[0070] II. Molecular Identification of GmlncRNA10551 Transgenic Plants
[0071] 1. Molecular Identification of GmlncRNA10551 Transgenic Soybean Overexpression Lines (GmlncRNA10551-OE1 / 2)
[0072] Extract the leaf RNA of the T 3 -generation homozygous lines (GmlncRNA10551-OE1 / 2) and control plants at the V 2 stage, reverse transcribe it into cDNA, and use the primer pair composed of GmlncRNA10551-F3 and GmlncRNA10551-R3 for qRT-PCR identification with the cDNA as the template. The results are shown in Figure 3 . Among them, the sequences of the primers used are as follows:
[0073] GmlncRNA10551 - F3: 5’-CAAGGCCCGGACAAACAATG-3’ (SEQ ID NO.6);
[0074] GmlncRNA10551 - R3: 5’-GGCGGGGATGAGGATCTCTA-3’ (SEQ ID NO.7);
[0075] III. Phenotypic analysis of GmlncRNA10551 transgenic soybeans
[0076] The experimental samples are as follows: T 3 generation transgenic GmlncRNA10551 overexpression lines (GmlncRNA10551 - OE1 / 2) of soybeans and the control line Williams 82.
[0077] 1. Determination of seed weight phenotype
[0078] Both the transgenic lines and the control line were planted in the same field environment. At the maturity stage of soybeans, there were no significant differences in plant types between the transgenic lines and the control line ( Figure 4 ). These materials were harvested individually and placed in an oven at 45°C for 48 h for complete drying. Using an intelligent seed - testing analysis system (Zhejiang Top Cloud - Agri Technology Co., Ltd.), the number of seeds per plant, seed length, seed width, and 100 - seed weight of soybeans were statistically analyzed.
[0079] 2. Phenotypic analysis of GmlncRNA10551 transgenic soybeans
[0080] Analysis of the phenotypic data of the transgenic soybean materials and the control line found that the average 100 - seed weight of the GmlncRNA10551 overexpression transgenic lines reached 15.4 g. Compared with excellent domestic soybean varieties, the 100 - seed weight level was within the range of the seed weight of excellent varieties. Compared with the experimental control line, the 100 - seed weight of the seeds increased significantly by about 11.50%, verifying the function of this gene in regulating seed weight ( Figure 6 ). Overexpression of the gene GmlncRNA10551 found that there were no significant differences in plant types between the overexpression lines and the control line ( Figure 4 ).
[0081] Through the transgenic experiment of overexpressing GmlncRNA10551, it was proved that lncRNA GmnlncRNA10551 is related to soybean grain development and participates in the regulation of the process of seed weight formation; among them, overexpression of GmlncRNA10551 can significantly increase the seed weight without affecting the plant type, thereby increasing the yield per plant of soybeans.
Claims
1. A lncRNA GmlncRNA10551 derived from soybean (Glycine max (L.) Merri11), selected from the nucleic acid sequence shown in any one of the following (a), (b) or (c): (a) a DNA or RNA molecule represented by SEQ ID NO.1; (b) a DNA or RNA molecule that hybridizes to the nucleic acid sequence defined in (a) under stringent conditions and is associated with plant grain weight; (c) a DNA or RNA molecule that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the nucleic acid sequence defined in (a) and that is associated with plant grain weight.
2. The overexpression vector, expression cassette, transgenic cell line or recombinant bacteria of the lncRNA GmlncRNA10551 according to claim 1.
3. A primer for amplifying the full length of lncRNA GmlncRNA10551 or any fragment thereof according to claim 1, characterized in that Selected from the following (I) or (II): (I) a primer pair consisting of the DNA shown in SEQ ID NO.2 and the DNA shown in SEQ ID NO.3; (II) A primer pair consisting of the DNA shown in SEQ ID NO.4 and the DNA shown in SEQ ID NO.
5.
4. The use of the lncRNA GmlncRNA10551 according to claim 1 in regulating soybean grain weight, characterized in that Overexpression of this lncRNA can increase soybean grain weight.
5. Use of the overexpression vector, expression cassette, transgenic cell line or recombinant bacteria of the lncRNA GmlncRNA10551 according to claim 2 in increasing soybean grain weight.