GmGAI7 gene and its application in soybean
By performing EMS mutagenesis of the soybean variety ‘Jiyu 47’ and localizing the GmGAI7 gene, the problem of difficulty in controlling the height of soybean plants is solved, and the effect of soybeans being insensitive to gibberellin and significantly reducing the height of plants is achieved, providing a more accurate and efficient method for soybean breeding.
Patent Information
- Application Number
- CN202411946206.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-08
- Estimated Expiration
- 2044-12-27
AI Technical Summary
The existing technology is difficult to effectively explore and utilize genes related to regulating dwarf in soybeans, making it difficult to control soybean plant height and affect crop yield.
By EMS mutagenesis of the soybean variety ‘Jiyu 47’, dwarf mutant jy425 was obtained, and the main effect gene GmGAI7, which regulates soybean dwarf, was localized using BSA mixed pool sequencing and map cloning methods. Mutations in this gene result in soybeans being insensitive to gibberellins, significantly reducing the plant height of soybeans.
It has successfully broadened the understanding of the "ideal plant type" of soybeans, provided important genetic resources for molecular marker-assisted breeding, and improved the accuracy, efficiency and controllability of the breeding process.
Smart Images
Figure CN119351618B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of agricultural biotechnology and specifically relates to GmG Genes and their applications in soybean. Background Art
[0002] Soybean ( Glycine max ) is one of the important food crops in my country. It not only provides humans with a large amount of edible oil and plant protein, but is also the main source of protein for animal feed.
[0003] Plant height is a key agronomic trait that affects soybean yield. Dwarf plant type can improve plant resistance to lodging, increase individual light transmittance, improve light energy utilization, and thus increase crop yield, playing an important role in crop genetic improvement. At present, although some genes that regulate soybean dwarfing have been discovered, large-scale systematic exploration and functional research have not yet been carried out. At the same time, because soybeans are ancient tetraploids, some genes have multiple copies, and plant height is a quantitative trait that is greatly affected by genetic background and environment, which increases the difficulty of discovering soybean dwarfing genes.
[0004] The present invention obtains a dwarf mutant by mutagenizing "Jiyu 47" jy425 Then, the main effect genes regulating soybean dwarfing were located by BSA mixed pool sequencing analysis and map-based cloning. GmG The protein encoded by this gene is a member of the GRAS family, among which the DELLA domain is unique to the DELLA subfamily protein in the GRAS protein family, and the function of this domain is related to GA response. Therefore, the present invention designed an exogenous gibberellin spraying experiment to treat JY47 and jy425 ,turn out jy425 It is also insensitive to gibberellins, proving GmG The mutation of the gene (T→A) makes soybean insensitive to gibberellins and significantly reduces the plant height of soybean. Summary of the invention
[0005] The object of the present invention is to provide GmG Genes and their application in soybeans, dwarf mutants were obtained by mutagenesis of "Jiyu 47" jy425, Afterwards jy425 BSA sequencing analysis and fine positioning of the hybrid progeny of ×JN18 revealed and verified that there was a SNP variation at the chr18:3281001 site in the soybean genome, which resulted in a non-synonymous amino acid mutation (gTc / gAc). The KASP method was then used to develop a molecular marker for this site. GmGThe mutation of the gene (T→A) makes soybean insensitive to gibberellins and significantly reduces the plant height of soybeans. It can broaden the understanding of the "ideal plant type" of soybeans, provide important genetic resources for molecular marker-assisted breeding, and make the breeding process more precise, efficient and controllable.
[0006] The present invention provides a SNP site associated with soybean dwarfing, wherein the SNP site is located at position 3281001 of chromosome 18 of the soybean genome, where a T / A variation exists; and the whole genome sequence version of the soybean genome is soybean genome v2.0.
[0007] The present invention provides GmG Gene, the GmG The gene comprises the SNP site described in claim 1 or 2; GmG The gene is a DNA molecule of any one of the following (a1)-(a4): (a1). A DNA molecule as shown in SEQ ID NO.1; (a2). A DNA molecule as shown in SEQ ID NO.2; (a3). A DNA molecule containing a complementary sequence of SEQ ID No.1 or SEQ ID No.2; (a4). A DNA molecule containing a reverse sequence of SEQ ID No.1 or SEQ ID No.2.
[0008] Preferably, the GmG Gene source: dwarf mutant of soybean "Jiyu 47" induced by EMS jy425 .
[0009] The present invention provides the above technical solution GmG The GmGAI7 protein encoded by the gene, the amino acid sequence of the GmGAI7 protein is shown as SEQ ID No.3 or as shown as SEQ ID No.4.
[0010] The present invention provides a biomaterial, the biomaterial comprising: GmG Recombinant expression vector or recombinant microorganism of gene.
[0011] The present invention provides the SNP sites described in the above technical solution, GmG Application of gene, GmGAI7 protein or biological material in plants; the plants include soybeans. Preferably, the application includes one or more of negatively regulating plant gibberellin signals, negatively regulating plant height and cultivating dwarf plants.
[0012] Beneficial effect: The present invention provides GmG gene, GmGAI7 protein or GmGThe present invention screened and identified the EMS-induced mutagenesis population of "Jiyu 47" (JY47), and obtained jy425 Dwarf mutants, then jy425 BSA sequencing analysis and fine positioning of the hybrid progeny of ×JN18 revealed and verified that there was a SNP variation at the chr18:3281001 site in the soybean genome, which resulted in a non-synonymous amino acid mutation (gTc / gAc). Molecular markers were developed for this site using the KASP method. GmG The mutation of the gene (T→A) makes soybean insensitive to gibberellins and significantly reduces the height of soybean plants. Therefore, the present invention will broaden the understanding of the "ideal plant type" of soybeans, provide important genetic resources for molecular marker-assisted breeding, and make the breeding process more accurate, efficient and controllable. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0014] Figure 1 The JY47 and jy425 phenotypic differences and segregation of offspring;
[0015] Figure 2 The distribution of the ED correlation value of the key dwarfing gene provided by the present invention on the chromosome; wherein the ordinate represents the Euclidean distance;
[0016] Figure 3 The dwarfing gene provided by the present invention GmG Fine positioning of
[0017] Figure 4 The JY47 and jy425 Phenotypic differences before and after exogenous gibberellin treatment. DETAILED DESCRIPTION
[0018] Unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and methods. In the present invention, the whole genome sequence version of the soybean genome is the soybean genome v2.0, which is: "Williams 82" (Glycine_max_v2.0).
[0019] Example 1 Analysis of phenotype and genetic characteristics of soybean dwarf mutants
[0020] 1. Phenotypic identification of agronomic traits related to dwarfing
[0021] Observe JY47 (Jiyu 47) and jy425 ( jy425It was produced by mutagenesis of Jiyu 47 by 5 / 1000 ethyl methanesulfonate (EMS); EMS product number is M0880-1006, produced by Sigma-Aldrich Company) seedling phenotype, JY47 and jy425 Maturation phenotype, measurement of maturity JY47 and jy425 The plant height and main stem node number are shown in Figure 1 (exist Figure 1 The left picture in the first row shows JY47 and jy425 Seedling stage phenotype; the right picture in the first row shows JY47 and jy425 Mature phenotype; the left picture in the second row shows JY47 and jy425 The right picture in the second row shows the difference in plant height between JY47 and jy425 By comparing the mature stage JY47 and jy425 Conduct testing and combine Figure 1 Discover, jy425 The dwarfing is caused by the shortening of internode length.
[0022] 2. Genetic characteristics analysis: jy425 ×JN18 (Jinong 18) was hybridized, and the phenotypic identification and segregation ratio statistics of the hybrid offspring F2 generation (a total of 243 strains) were performed. The results are shown in Table 1 (In Table 1, =1.28< 0.05 =5.99).
[0023] Table 1 Phenotypic identification results of different F2 segregating populations
[0024]
[0025] Combining the data in Table 1, we find that jy425 The segregation ratio of normal plant height: medium dwarf: very dwarf plant height in the hybrid offspring F2 of ×JN18 was 1:2:1. Therefore, it was inferred that the gene controlling soybean dwarfing was semi-dominant.
[0026] Example 2 Soybean dwarfing gene GmG Positional cloning
[0027] 1. BSA mixed pool sequencing to determine the initial positioning interval
[0028] by jy425 As the male parent, it was crossed with Jinong 18 (JN18) to establish the F2 population. Thirty offspring with extreme phenotypes (extremely tall and extremely short) in the F2 were selected for BSA mixed pool sequencing to determine the initial positioning interval. After BSA mixed pool sequencing, high-quality Indel sites were screened and the results were associated with the ED method. The results are shown in Figure 2 ; Combine Figure 2Finally, the initial positioning interval of the soybean dwarf gene was found to be 1458449-6682692 (5.22Mb) on chromosome 18.
[0029] 2. Fine positioning of dwarfing genes
[0030] The dwarfing gene was precisely located, and the location results were as follows Figure 3 As shown (in Figure 3 The left picture shows the Indel fine positioning, where the first band is M representing DL2000 Marker, and the remaining bands are the corresponding primers designed in the initial positioning interval. jy425 The progeny marked by primers that differ from JN18; the right picture shows the KASP genotyping markers, of which the first two in the first row are JN18, jy425 , followed by some offspring, blue represents the same genotype as JN18, red represents jy425 The genotypes are consistent, and green represents heterozygous) as follows:
[0031] 2.1 Screening and positioning results of Indel markers: Based on the above initial positioning interval, corresponding Indel primers were designed (see Table 2 for specific primer information), and the F2 and F2:3 (i.e., the recombinant plants found in the F2 generation, the seeds of the corresponding plants were continued to be planted) generation populations were marked, and the interval was located at 3237628-3393653 (156kb) of chromosome 18.
[0032] Table 2 Indel primer information
[0033]
[0034] 2.2 Screening and localization results of KASP markers: Based on the results of Indel molecular marker localization, KASP genotyping primers were further designed within the interval (specific primer information is shown in Table 3) to type the SNP sites, resulting in a final fine-mapping interval of 3257392-3393653 (136 kb) on chromosome 18. There was only one non-synonymous mutation in this interval, and finally the dwarfing-related gene was screened. GmG (said GmGAI7 gene The nucleotides are shown in SEQ ID NO.1 and SEQ ID NO.2).
[0035] Table 3 KASP genotyping primer information
[0036]
[0037]
[0038]
[0039] Example 3 GmG Preliminary identification of gene function
[0040] 1. Experiment of spraying exogenous gibberellin
[0041] Select seeds with full and intact kernels (JY47 and jy425 ), planted in white pots (where the white pots contained a matrix of nutrient soil and vermiculite mixed in a volume ratio of 1:1), 3 seeds were planted in each pot, regarded as three parallel replications, and conventional management was performed; JY47 and jy425 The plants were sprayed with exogenous gibberellin (the concentrations of gibberellin were 0.1 mg / L, 1.0 mg / L and 1.5 mg / L), which was recorded as the treatment group.
[0042] JY47 treatment: The treatment groups were sprayed with different concentrations of gibberellin, once every other day for one week. Each concentration of gibberellin was sprayed with one pot of seedlings (recorded as 0.1 mg / L, 1.0 mg / L and 1.5 mg / L, respectively), and there were three seedlings in each pot. A control group (recorded as CK) was set up: it was sprayed with clean water of the same quality as the treatment group, and the plant height was recorded from the first day of spraying for 7 consecutive days. The results are shown in Figure 4 A and C in ( Figure 4 A is the phenotypic difference of JY47 before and after spraying with exogenous gibberellin; C is the difference of plant height of JY47 before and after spraying with exogenous gibberellin); jy425 Processing: The difference from JY47 is that the processing object is jy425 Seedlings, results Figure 4 B and D in ( Figure 4 B in jy425 Phenotypic differences before and after spraying of exogenous gibberellin; D is jy425 The relative elongation of the plants after spraying exogenous gibberellin was statistically analyzed. Figure 4 D in;
[0043] Combination Figure 4 It can be seen that the height of JY47 plants increased with the increase of GA3 concentration, while jy425 No significant change. jy425 The gibberellin signal transduction pathway is blocked, which proves GmG The mutation of the gene (T→A) makes soybean insensitive to gibberellins and significantly reduces the plant height of soybean.
[0044] Example 4 Soybean GmG Gene cloning
[0045] 1. RNA (Trizol method) extraction and reverse transcription
[0046] Reverse transcription PCR reaction system: see Takara reagent instructions (PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time)), specifically: soybean JY47 and jy425 The stem cDNA was used as template. GmG The gene was cloned, and the PCR system is shown in Table 4. The sequence of the cloning primer GmGAI7-F is: 5'-GGTGTTCAATTGAATTCGTG-3' (SEQ ID No.55), and the sequence of GmGAI7-R is: 5'-CCATCCAGGAGCCAATTAAT-3' (SEQ ID No.56); the PCR program is: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ post-extension for 8 min.
[0047] Table 4 GmG Gene PCR system
[0048]
[0049] The amplified PCR product was subjected to agarose gel electrophoresis detection, and then connected to the cloning vector Blunt to generate Blunt-GmGAI7, which was then sequenced. The sequencing results are shown in SEQ ID No.1 and SEQ ID No.2.
[0050] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. GmG A gene characterized by Said GmG The gene includes a SNP site associated with soybean dwarfing; the SNP site is located at position 3281001 of chromosome 18 of the soybean genome, where a T / A variation exists; the whole genome sequence version of the soybean genome is soybean genome v2.0; Said GmG A gene is a DNA molecule that is any of the following (a1)-(a2): (a1). A DNA molecule as shown in SEQ ID NO.2; (a2). A DNA molecule as shown by the complementary sequence of SEQ ID No.
2.
2. according to claim 1 GmG A gene characterized by Said GmG Gene source: soybean "Jiyu 47" dwarf mutant produced by EMS mutagenesis jy425 .
3. A biomaterial, characterized in that: The biological material comprises: GmG Recombinant expression vector or recombinant microorganism of gene.
4. The method according to claim 1 or 2 GmG Gene, or use of the biological material according to claim 3 in soybean; The applications include: One or more of negatively regulating plant gibberellin signals, negatively regulating plant height and cultivating dwarf plants.