Application of GmGIS3a gene in regulating plant height and flowering time of soybean

Abstract

The application belongs to the technical field of biotechnology, and particularly discloses a kind of GmGIS3a Gene in the application of regulating soybean height and flowering time. The sequence of the GmGIS3a Gene is shown as SEQ ID NO.1, GmGIS3a The amino acid sequence encoded by the gene is shown as SEQ ID NO.2. The application overexpresses soybean GmGIS3a Gene by using pTF101 vector, transforms soybean cultivar Wm82, and obtains stable overexpression plants. Research finds that the overexpression plants are dwarf compared with wild type plants, and the flowering time is early, which indicates that the GmGIS3a GmGIS3a Gene is involved in the biological process of regulating plant height and flowering time. It provides a new breeding approach for cultivating dwarf and early flowering plant varieties, and has a good application prospect in the field of plant breeding technology.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving GmGIS3a The application of genes in regulating soybean plant height and flowering time, more specifically... GmGIS3a Application of genes in dwarfing soybean plant height and advancing flowering time. Background Technology

[0002] Plant architecture regulation is important for improving soybean ( Glycine max (Linn.) Merr. Plant height plays a crucial role in soybean yield. Ideal plant type is a significant representative agronomic trait of modern crop varieties, and ideal plant type breeding is a widely adopted breeding strategy for crops such as rice, corn, and soybeans. In soybean ideal plant type breeding, plant height and other important ideal plant type traits have attracted widespread attention from researchers, as plant height determines the lodging resistance and feasibility of mechanical harvesting. Plant height is often negatively correlated with stem thickness and lignin content. Tall plants have thin and weak stems, poor lodging resistance, and are prone to lodging in wind and rain, leading to damage to photosynthetic organs, pod mold, and harvesting difficulties, directly causing yield reduction. Excessively elongated plant height may be accompanied by poor root development, further weakening plant stability.

[0003] Flowering is a key indicator of the transition of plants from vegetative growth to reproductive growth. Promoting early flowering and pod formation in soybeans helps to increase yield.

[0004] soybeans GmGIS3a The gene encodes the C2H2 zinc finger structural protein. C2H2 zinc finger structural proteins have multiple functions in plants, mainly participating in processes such as growth and development regulation, hormone signal transduction and light signal regulation, and abiotic stress response. For example, they serve as important regulatory factors for plant growth and development, including plant height, flowering time regulation, and leaf morphogenesis. In rice, SIZ1 regulates plant height through GA signaling; in Arabidopsis, ZFP6 affects flowering time, but its functions in regulating plant height and flowering time have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide GmGIS3a The application of genes in regulating soybean plant height and flowering time, more specifically... GmGIS3a Application of genes in dwarfing soybean plant height and advancing flowering time.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: soybeans GmGIS3a Gene (Gene ID: Glyma.03G217900 ), having the nucleotide sequence shown in (1) or (2) or (3): (1) The nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence with equivalent function formed by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1; (3) The sequence that hybridizes with SEQ ID NO.1 under strict hybridization conditions.

[0007] The amino acid sequence of the protein encoded by this gene is as follows: (1) The amino acid sequence shown in SEQ ID NO.2; (2) The amino acid sequence shown in SEQ ID NO.2 is replaced, deleted or added with one or more amino acids to form a sequence with the same function.

[0008] The primary objective of this invention is to provide the above-mentioned GmGIS3a Application of genes in regulating soybean plant height and flowering time.

[0009] The present invention also constructs a series of plant expression vectors, and the functions of overexpression vectors, recombinant vectors or transgenic plant lines containing the above-mentioned genes, as well as host cells containing the vectors, in regulating soybean plant height and flowering time also fall within the protection scope of the present invention.

[0010] The gene functions protected by this invention include not only those described above. GmGIS3a Genes, including those related to GmGIS3a Homologous genes with high homology (i.e., above 80%; preferably above 90%; preferably above 95%; preferably above 98%) play a role in regulating soybean plant height and flowering time.

[0011] The function of this invention in regulating soybean plant height and flowering time is specifically manifested in that, compared with the wild type, overexpression GmGIS3a The gene-injected soybean plant is shorter and flowers earlier, meaning that the aforementioned GmGIS3a The gene can dwarf soybean plant height and advance flowering time, providing a new breeding approach for cultivating new plant varieties with dwarf plant type, improved lodging resistance and earlier flowering time.

[0012] Based on its function, genetic modification can be used to obtain dwarfed plants that flower earlier; specifically, it can be used to improve... GmGIS3a The expression of the gene in the target plant yields a transgenic plant, which is shorter than the target plant and flowers earlier. More specifically, the aforementioned... GmGIS3a Genes are introduced into plant cells, tissues, or organs, and the transformed plant material is then cultivated into complete plants. Plant varieties that are shorter and flower earlier are then selected.

[0013] In one embodiment of the present invention, an overexpression vector is constructed, and the recombinant vector carrying the exogenous gene is introduced into the target plant to enhance the expression of the gene in the plant. GmGIS3a Gene expression levels.

[0014] In addition, this invention also protects a breeding method for dwarfing plants or / and increasing flowering in plants, the method being for improving the flowering rate of plants. GmGIS3a The expression of genes and / or the activity of the proteins they encode, said GmGIS3a The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2. Preferably, the plant is soybean.

[0015] Preferably, increasing the concentration of plant components GmGIS3a The gene is expressed in the form of overexpression.

[0016] Overexpressing the above GmGIS3a Gene overexpression can be achieved through various methods, such as gene overexpression mediated by plant virus vectors, Agrobacterium-mediated transformation of overexpression vectors, optimization of gene coding structures, and optimization of gene promoters to achieve overexpression. The gene overexpression method of this invention is not limited to the above methods; any method that can achieve overexpression is acceptable. GmGIS3a Genes are sufficient.

[0017] The present invention GmGIS3a When a gene is constructed into a plant expression vector, any enhancing or inducible promoter can be added before its transcription initiation nucleotide. To facilitate identification and screening of transgenic plant cells or plants, the vector can be modified, such as by adding plant selectable marker genes (GUS gene, luciferase gene, flag tag gene, etc.) or antibiotic resistance markers (such as ampicillin, kanamycin, rifampin, styromycin, etc.). The transformed plant host can be either a monocotyledonous or dicotyledonous plant, such as soybean, tobacco, wheat, corn, cucumber, tomato, Arabidopsis, alfalfa, etc. [The following text appears to be a separate, unrelated section:] Carrying this invention... GmGIS3a Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plants can be cultured into plants.

[0018] The term "plant" as used in this invention includes the whole soybean plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots, flowers, and other tissues and organs. GmGIS3a Both genes and nucleic acids are overexpressed.

[0019] This invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, leaves, fruits, flowers, stems, roots, and other tissues and organs. It further relates to other derivatives of the plant after harvest, such as dried granules or powders, oils, fats and fatty acids, starches, or proteins. This invention also relates to foods or food additives obtained from the relevant plants.

[0020] Advantages of this invention: This invention has found a soybean GmGIS3a Genes, experiments have found, overexpression GmGIS3a Compared to wild-type plants, the genetically modified plants are shorter and flower earlier, indicating that... GmGIS3a Genes are involved in the biological processes that regulate plant height and flowering time. This provides a new breeding approach for cultivating new plant varieties with dwarf plant types, improved lodging resistance, and earlier flowering times, showing promising application prospects in the field of plant breeding technology. Attached Figure Description

[0021] Figure 1 Genetically modified plants GmGIS3a Gene expression and tag protein detection results; where A represents transgenic plants. GmGIS3a Gene expression analysis, B represents the result of tag protein detection.

[0022] Figure 2 for GmGIS3a A phenotypic control of stunted plant height in transgenic plants after gene overexpression; in the figure, GmGIS3a -OE indicates overexpression in plants compared to wild-type plants. GmGIS3a -OE-1、 GmGIS3a The plant height of the two overexpressing OE-2 lines was significantly shorter.

[0023] Figure 3 for GmGIS3a Phenotypic control of earlier flowering in transgenic plants after gene overexpression. Overexpressing plants. GmGIS3a -OE compared to wild-type plants GmGIS3a -OE-1、 GmGIS3a -OE-2 flowers significantly earlier. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0025] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0027] I. Gene Acquisition and Overexpression Vector Construction A soybean was identified using molecular biology techniques. GmGIS3a The gene, whose CDS sequence is shown in SEQ ID NO.1, and whose amino acid sequence encoding the protein is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1: ATGTCTGATGAGACCTGGCCACCGAAACTATTTGGTTTCCCACTAACAGAGCAGGAGGACAGAAAATTCAGATGCCACTACTGCAAACGAGTATTCGGGAACTCGCAGGCCTTAGGAGGCCATCAAAATGCCCACAAGAAAGAGCGTCAGAGGGCGAGGCGTTTCCAAATCCACACTCATAGACGCGCCATTGCAGCAGCAGCATCGATATCTTCACATGCAATAATAACATCAGTACCCTCCATTTACT TACGAGGACACCCTCCTACTCCTACTTCTCTTCTACCCATCTTCGATCCTCATCGTCAACCCAATAACACTAAGCACTTCCCATCACGCCCTACTTTCATTCCTTCTTCTTCCAACTACCACCTTACTAATTACACTCAATGCTTCCTTTTCGGCTTTATGCCTCACCCACTCATGTACTGCAATCCTCTACTCCTAAGGTCCCTCATTTTTCTGCTGAGGGGGAAGTTGATGTCCATCTCAAATTATAA SEQ ID NO.2: MSDETWPPKLFGFPLTEQEDRKFRCHYCKRVFGNSQALGGHQNAHKKERQRARRFQIHTHRRAIAAAASISSHAITSVPSIYLRGHPPPTTSLLPIFDPHRQPNNTKHFPSRPTFIPSSSNYHLTNYTQCFPFRLYASPTHVLQSSTPKVPHFSAEGEVDVHLKL* The construction process of the overexpression vector is as follows: 1. This invention provides primers for constructing overexpression vectors, used for amplification. GmGIS3a Primers for gene sequence: GIS3a -F:5'-CCTAGTATACCCTAATTCAGAG-3' GIS3a -R:5'-GAGCTCCAACTCTCCGATCAA-3' Adapter primers for amplifying the target gene fragment and ligating it into the pTF101+3flag vector for homologous recombination: pTF101-F:5'-TCGTGCCTCCACCATGTTGACCTGCAGTATCATTTTCTTCTCCTATA-3' pTF101-R:5'-AGCTTGGCGCGCCTCCCGGGACTAGTTAATTTGAGATGGACATCAAC-3'.

[0029] RNA extraction. Seeds of wild soybean ZY06 were planted in soil containing nutrient soil and vermiculite (1:1 mixture) and grown under long-day conditions until the first whorl of trifoliate leaves opened. Soybean leaves with an area approximately the size of a fingernail were taken, and RNA was extracted from the leaves using a kit method (Kangwei Century Ultrapure RNA Kit, CW0597). RNA integrity was detected by agarose gel electrophoresis, and RNA concentration was measured using a micro spectrophotometer.

[0030] cDNA was synthesized by reverse transcription. RNA was reverse transcribed into cDNA according to the method of the kit (Novizan HiScript III 1st Strand cDNASynthesis Kit, R312).

[0031] Using step 1 above GmGIS3a CDS sequence amplification primers were used, with cDNA as a template, for PCR amplification. GmGIS3a CDS sequence.

[0032] Recover PCR products. Perform agarose gel electrophoresis on the PCR products. Cut the gel containing the target fragment and recover the product using an agarose gel extraction kit (Fullmetal Alpha). EasyPure ® The Quick Gel Extraction Kit (EG101-01) was used to recover PCR products.

[0033] Add adapters to the amplified gene sequence. Using the adapter-containing primers from step 1 and the recovered PCR product as a template, perform PCR amplification to add adapters to the gene sequence.

[0034] The PCR product was ligated into the pTF101+3flag expression vector, transformed into DH5α Escherichia coli, and single colonies were selected for testing.

[0035] Propagation of DH5α Escherichia coli with accurate sequencing results was performed using a kit method (Full Gold). EasyPure ® Plasmids were extracted using the PlasmidMiniPrep Kit (EM101-02), transformed into competent Agrobacterium tumefaciens cells EHA101, single colonies were selected for PCR identification, and the identified Agrobacterium tumefaciens cells were cultured and propagated for later use.

[0036] Obtaining genetically modified soybean plants Using soybean cultivar Wm82 as the recipient, soybean hypocotyls were infected with Agrobacterium rhizogenes EHA101 and cultured to obtain T0 seedlings.

[0037] Identification of transgenic positive lines In order to determine whether the target gene sequence is integrated into and expressed in the transgenic positive plants obtained in Example 2, the positive lines need to be identified.

[0038] 1. Take leaf samples approximately the size of a fingernail and extract DNA using a kit (NuClean PlantGenomic DNA Kit, CW0531); extract total protein using cell disruption method.

[0039] 2. Confirm the integration and expression of the target gene sequence into the receptor. T0 seedlings were identified using both PCR and Western blotting techniques. Transformants showing positive results in both PCR and Western blotting were selected. The identification primers consisted of one primer each for the CDS sequence (GIS3a-test-F) and the vector sequence (Nos-R). PCR amplification was performed using DNA as a template. The primer sequences are as follows: GIS3a-test-F: 5'-CTCATCGTCAACCCAATAA-3' Nos-R: 5'-TGAAGGCGGGAAACGACAAT-3'.

[0040] 3. Screening for homozygous transformants. Positive T0 seedlings were planted under long-day conditions. After harvesting, the seeds (T1 generation) were continued to be planted under long-day conditions. After the first round of three-fold compound leaves appeared, DNA and total protein were extracted from the leaf material. The T1 seedlings were further identified using PCR and Western blotting experiments. In this example, homozygous transformants were selected from the T2 generation, ultimately obtaining... GIS3a -OE-1、 GIS3a Two homozygous single-copy plants of type OE-2 were used as experimental materials for subsequent experimental analysis.

[0041] IV. Analysis of gene expression levels in transgenic positive lines and wild-type lines, and detection of tag proteins. Transgenic positive strains and wild-type strains were planted under long-day conditions. After they grew their first round of trifoliate compound leaves, wild-type plants were harvested separately. GmGIS3a -OE-1、 GmGIS3a Leaves the size of a fingernail from the -OE-2 plant were immediately flash-frozen in liquid nitrogen, ground into powder, and RNA was extracted. The RNA was then reverse transcribed to obtain cDNA, which was subsequently analyzed by real-time quantitative PCR (qPCR). GmGIS3a Specific qPCR primers GIS3a qPCR-F and GIS3a qPCR-R were designed based on the cDNA sequence of the gene to... Tubulin The gene was used as an internal reference gene (primers RT-Tublin-F and RT-Tublin-R). Detection was performed by qPCR. GmGIS3a Genes in W82, GmGIS3a -OE-1、 GmGIS3a Expression levels in -OE-2. Primer sequences are as follows: GIS3a qPCR-F: 5'-TGTCTGATGAGACCTGGCCAC-3' GIS3a qPCR-R: 5'-GCATGTGAAGATATCGATGCTGC-3' RT-Tublin-F: 5'- GAGAAGAGTATCCGGATAGG-3' RT-Tublin-R: 5'-GAGCTTGAGTGTTCGGAAAC -3' The results show that ( Figure 1 The original expression level of W82 (A) was almost 0 (0.002), indicating an extremely low background, suggesting that this gene is basically not transcribed in wild-type leaves. The transgenic positive plant type... GmGIS3a Gene expression levels were significantly higher in the plants than in the wild-type plants. GmGIS3a -OE-1 and GmGIS3a -OE-2 increased the expression level of the target gene by approximately 10,000-fold and 5,000-fold, respectively, indicating high overexpression efficiency. GmGIS3a The expression level of -OE-1 is approximately GmGIS3a The 2.1-fold increase in -OE-2 indicates that there may be differences in insertion site, copy number, or position effect among different positive lines.

[0042] Wild-type plants were taken separately. GmGIS3a -OE-1、 GmGIS3a Proteins were extracted from leaves of the -OE-2 plant and the fusion protein was detected by Western blotting. Figure 1 (B) Based on the amino acid sequence, the theoretical molecular weight of the fusion protein was calculated. The size of the fusion protein of GmGIS3a and the 3flag tag is approximately 23 kDa. The detected size of the fusion protein is basically consistent with the prediction, indicating that the target gene and the 3flag tag are correctly fused. This proves that the transgenic line can correctly and completely express the target fusion protein, providing reliable protein-level evidence for subsequent functional studies.

[0043] V. Phenotypic Analysis of Transgenic Positive Lines and Wild Types To clarify the overexpression GmGIS3a The effects of genes on soybean plant height and flowering time: We investigated the effects of genes on wild-type soybean (W82) and two independent overexpression genes. GmGIS3a homozygous transgenic lines ( GmGIS3a -OE-1、 GmGIS3a Phenotypic observation, comparison, and analysis were conducted using the -OE-2 method. Under long-day conditions, the date when a flower opened at any node on the main stem was recorded as R1. The flowering time was the number of days elapsed from seed emergence to R1. After recording the flowering time of all plants, the flowering time was statistically analyzed. Plant height was measured after maturity. The experimental results showed that the overexpression line was more than 9.8 cm shorter in height and had a flowering time approximately 9 days earlier than the wild type. Statistical analysis showed that the differences in plant height and flowering time between the two types were statistically significant. Therefore, compared with the wild type, GmGIS3a -OE-1、 GmGIS3a -OE-2 overexpression lines showed significantly shorter plant height ( Figure 2 And the flowering period is earlier ( Figure 3 ).

[0044] In conclusion, GmGIS3a Genes can regulate soybean plant height and flowering time through overexpression. GmGIS3a The gene can dwarf soybean plant height and advance flowering time, providing a new breeding approach for cultivating new plant varieties with dwarf plant type, improved lodging resistance and earlier flowering time.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. GmGIS3a Genes or containing the above GmGIS3a The application of gene overexpression vectors in regulating soybean plant height and flowering time is characterized by, The GmGIS3a The CDS sequence of the gene is shown in SEQ ID NO.1, and the regulation is characterized by overexpression compared to the wild type. GmGIS3a The genetically modified soybean plants are shorter and flower earlier.

2. The application according to claim 1, characterized in that, The application specifically includes: [the following is a description of the application]. GmGIS3a Genes are introduced into plant cells, tissues, or organs, and the transformed plant material is then cultivated into complete plants. Plant varieties that are shorter and flower earlier are then selected.

3. A method for breeding dwarf plants, characterized in that, The method is to improve the quality of plants. GmGIS3a The expression of genes and / or the activity of the proteins they encode, said GmGIS3a The CDS sequence of the gene is shown in SEQ ID NO.1, the amino acid sequence of the protein is shown in SEQ ID NO.2, and the plant is soybean.

4. A breeding method for increasing flowering in plants, characterized in that, The method is to improve the quality of plants. GmGIS3a The expression of genes and / or the activity of the proteins they encode, said GmGIS3a The CDS sequence of the gene is shown in SEQ ID NO.1, the amino acid sequence of the protein is shown in SEQ ID NO.2, and the plant is soybean.

5. The method according to claim 3 or 4, characterized in that, Increase the concentration of plants GmGIS3a The gene is expressed in the form of overexpression.

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