Ahfmo gene for regulating peanut seed size and kernel weight and application thereof
By combining GWAS-QTL analysis and transcriptomics verification, the AhFMO gene was screened and heterologously expressed in Arabidopsis thaliana, which solved the problem of unclear regulation of peanut seed size and grain weight, and realized the research basis for high-yield molecular breeding of peanuts and gene screening applications in other crops.
Patent Information
- Application Number
- CN202410844699.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-27
- Publication Date
- 2026-02-17
- Estimated Expiration
- 2044-06-27
AI Technical Summary
In existing technologies, the regulatory mechanisms of peanut seed size and weight are unclear, which hinders research on peanut molecular breeding and yield improvement.
The AhFMO gene, which regulates peanut seed size and weight, was screened by combining genome-wide association analysis (GWAS) and genetic mapping (QTL mapping). The function of the gene was verified by transcriptomics analysis and homology analysis. Specific primers were designed for PCR amplification, and an overexpression vector was constructed for heterologous expression in Arabidopsis thaliana to verify its regulatory effect.
Successfully regulating Arabidopsis seed size and weight provides a research basis for high-yield molecular breeding of peanuts, improves the efficiency of peanut seed size and weight, and expands the applicability of gene screening to other crops such as wheat, rice, soybeans, and corn.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and more specifically, to an AhFMO gene that regulates peanut seed size and weight and its applications. Background Technology
[0002] Cultivated peanut (Arachis hypogaea L.) is an important oilseed and economic crop in my country, ranking first among the five major oilseed crops in terms of yield per unit area, total output, export volume, and output value. my country's annual self-sufficiency rate for edible vegetable oil is only 30%, but the current competition for land between grain and oil crops makes it difficult to expand peanut planting area. Therefore, effectively elucidating the molecular mechanisms of peanut yield traits and increasing yield per unit area through genetic improvement is an effective way to alleviate the current supply shortage of peanuts. Although five major peanut variety updates have been carried out since the 1950s, resulting in a significant increase in yield, there is still room for improvement in tapping the potential of peanut yield and cultivating new ultra-high-yielding peanut varieties.
[0003] In peanut production, many factors constrain yield, such as seed size, yield per plant, number of pods per plant, and plant type, all of which directly affect yield. Among these, peanut seed traits are one of the key factors determining yield per unit area and an important indicator of its commercial characteristics. Identifying key genes in peanuts has significant theoretical and practical value for understanding the regulatory mechanisms of pod size development and for molecular breeding of high-yield and high-quality peanuts. Meanwhile, extra-large peanuts command nearly twice the price of ordinary peanuts in the international market, making the development of new peanut varieties with even larger kernels an urgent need for the peanut industry.
[0004] In recent years, the whole genome information of cultivated peanut varieties "Tifrunner," "Shitouqi," and "Fuhua" has been successively published, providing important references for the localization and functional identification of genes for important agronomic traits in peanuts. Although a number of quantitative trait loci (QTLs) related to peanut kernel traits have been reported, these QTLs have large physical regions, the key genes controlling these traits are still being explored, and the molecular mechanisms of trait formation remain unclear, hindering research on the application of molecular breeding in peanuts. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide an AhFMO gene that regulates peanut seed size and weight and its application, the gene sequence of which is SEQ ID 1.
[0006] Furthermore, the cDNA sequence of the AhFMO gene that regulates peanut seed size and weight is SEQ ID 2.
[0007] Furthermore, the protein sequence encoded by the AhFMO gene that regulates peanut seed size and weight is SEQ ID3.
[0008] The second objective of this invention is to provide a method for screening AhFMO genes that regulate peanut seed size and weight. This method is achieved through a combination of genome-wide association analysis (GWAS) and genetic linkage analysis (QTL mapping), utilizing transcriptomics analysis of differential gene expression and homology analysis.
[0009] Furthermore, the method for screening AhFMO genes that regulate peanut seed size and weight can be applied to the screening of functional genes in other crops such as peanut, wheat, rice, soybean, and corn.
[0010] The third objective of this invention is to provide an application of the AhFMO gene, which regulates peanut seed size and weight.
[0011] This invention also provides an SNP molecular marker that regulates peanut seed size and weight. The marker is located at base 149251594 on chromosome 16 of cultivated peanut (Arachis hypogaea cv. Tifrunner v1; https: / / www.peanutbase.org). After PAMRS genotyping, the genotype results were divided into two groups. A t-test was performed based on the corresponding phenotypic numbers. The results showed that when the genotype at this locus is G, the peanut seed size and weight are larger; when the genotype at this locus is C, the peanut seed is smaller and the weight is lower. Base 149251594 of Arahy16 exhibits C / G polymorphism.
[0012] In summary, the present invention has the following beneficial effects:
[0013] First, this invention is the first to discover the AhFMO gene that regulates peanut seed size and weight, and studies have verified that this gene can regulate seed size and weight.
[0014] Secondly, this invention provides a method for screening candidate genes related to specific functions of peanuts using GWAS-QTL joint analysis, effectively discovering new genes related to important yield traits of peanuts, and providing a research basis for improving peanut yield traits and accelerating the process of high-yield molecular aggregation breeding.
[0015] Third, the gene AhFMO, which regulates seed size and weight, has been shown to increase the length, width, and weight of Arabidopsis seeds through heterologous overexpression. Attached Figure Description
[0016] Figure 1This invention presents a selection clearance analysis diagram of the XP-CLR method between developed varieties and local varieties;
[0017] Figure 2 This invention uses GWAS to analyze the size and weight traits of peanut seeds; (a) is a GWAS analysis result diagram, (a)-1-(a)-4 are decomposition diagrams of (a); (b) is a magnified diagram of chromosomes 6 and 16; (c) is a diagram of significant SNP regions and linkage disequilibrium modules of chromosomes 6 and 16.
[0018] Figure 3 These are the results of differential expression analysis of this invention;
[0019] Figure 4 Here are the AhFMO gene diagrams: a: full-length gene diagram, b: secondary structure and variant amino acids, c: tertiary structure.
[0020] Figure 5 This is a comparison of seed parameters of Arabidopsis thaliana plants. Among them, WT is wild-type Arabidopsis thaliana, and Line1 and Line2 are heterologous expression Arabidopsis thaliana lines. a: comparison of seed size, b: significance analysis of seed length, width and thousand-seed weight. Detailed Implementation
[0021] The present invention will be further described in detail below with reference to the embodiments.
[0022] Example 1: Screening of candidate genes using GWAS-QTL combined analysis
[0023] Selection clearance between local and bred varieties was detected using the cross-population composite likelihood ratio test (XP-CLR). Figure 1 As shown, the predominantly selected regions are distributed on chromosome 16.
[0024] Using a combined approach of genome-wide association analysis (GWAS) and quantitative linkage analysis (QTL mapping), such as... Figure 2 As shown, (a) shows the GWAS analysis results, with significant SNPs mainly distributed on chromosomes 6 and 16; (b) is a magnified view of chromosomes 6 and 16; and (c) shows the significant SNP regions and linkage disequilibrium blocks on chromosomes 6 and 16. Based on literature reports, peanut seed traits, including pod and kernel size, 100-pod weight, and 100-kernel weight, are mainly co-located in the region of chromosome 16 (139.001–149.501 Mb). Subsequently, transcriptomics analysis was used to analyze the differential expression of these traits at four kernel developmental stages in varieties with extreme seed size and weight values. Homology analysis was performed based on existing functional genes, ultimately identifying 36 differentially expressed candidate genes, such as... Figure 3 As shown. Among the 36 candidate genes mentioned above, the AhFMO gene involved in this invention contains a missense mutation (Arahy16_149251594: CAA(Gln)->GAA(Glu)). The PARMS genotyping results were divided into two groups, and a t-test was performed based on the corresponding phenotypic number, indicating that this mutation was significantly associated with seed size phenotypic data. Therefore, this gene is considered the primary candidate gene for subsequent overexpression validation.
[0025] Table 1. PARMS typing results
[0026]
[0027] This invention discovered that at base position 149251594 of peanut chromosome 16 (Arachishypogaeacv.Tifrunner v1; https: / / www.peanutbase.org), when the genotype at this locus is G, peanut seed size and kernel weight are higher, while when the locus is C, peanut seeds are smaller and kernel weight is lower. The base position Arahy16:149251594 exhibits C / G polymorphism, which can be used for preliminary prediction of peanut seed size and kernel weight.
[0028] Compared with commonly used EMS mutagenesis screening methods for peanut mutants, the method of this invention is more targeted, the genes to be screened are more specific, and it does not require a large amount of repetitive work in screening mutants, thus improving the efficiency and accuracy of discovering important functional genes in peanuts.
[0029] The GWAS-QTL combined analysis method is applicable to a wide range of species for screening candidate genes, not just peanuts. Other crops such as wheat, rice, soybeans, and maize can also use this method for functional gene screening, demonstrating its broad applicability. This method involves a relatively small genome sequencing range and is low-cost, making it particularly suitable for broadly locating functional genes across a species' genome.
[0030] Example 2: Amplification of the AhFMO gene regulating peanut seed size and weight
[0031] The plant material was the seeds of the peanut variety "Hua 32". Total RNA was extracted from the seeds, and cDNA was obtained using a reverse transcription kit. Using the cDNA as a template, primers were designed based on the gene region sequence from the peanut database website (https: / / www.peanutbase.org / ). The specific primer sequences are as follows:
[0032] AhFMO-F: 5'-TTGTTCATTCTCCCTTGCTCT-3';
[0033] AhFMO-R:5'-GCCTCCAACAATGACACACT-3'.
[0034] The PCR amplification system for the AhFMO gene, which regulates peanut seed size and weight, was as follows: 30.5 μL ddH2O, containing Mg... 2+ The following reagents were prepared: 10 μL of 5×HF buffer; 2 μL of 2.5 mM dNTPs; 2 μL each of AhFMO-F and AhFMO-R (5 μM each); 0.6 μL of DMSO; 1 μL of Phusion enzyme; and 2 μL of cDNA template. The PCR conditions for amplifying the peanut gene AhFMO were: 98℃ pre-denaturation for 50 s; 95℃ denaturation for 15 s, 60℃ annealing for 10 s, 72℃ for 1 min, for 35 cycles; followed by extension at 72℃ for 10 min.
[0035] like Figure 4 As shown, a: full-length gene diagram, b: secondary structure and amino acid variation sites of the gene, c: tertiary structure of the gene. The gene sequence of peanut AhFMO is SEQ ID 1, its cDNA sequence is SEQ ID 2, and its encoded protein sequence is SEQ ID 3.
[0036] Example 3: Construction of overexpression vectors and heterologous expression plants and phenotypic analysis
[0037] Based on the complete cDNA sequence of the AhFMO gene, specific primers for adding enzyme digestion adapters were designed. The primer sequences are as follows:
[0038] AF-F: 5'-gagaacacgggggactctagaATGGCTTTCCAACACAATAACCAAC-3'; AF-R: 5'-cgatcggggaaattcgagctCTCAAAAATAGTAGTCTTGGCTGGTA-3'.
[0039] The restriction enzyme sites selected were BamH1 and Kpn1. After the amplified product was correctly sequenced, it was ligated into the overexpression vector SN1301 by double enzyme digestion to construct the overexpression vector AhFMO-pSN1301. Transgenic Arabidopsis plants were obtained by Agrobacterium-mediated explant injection and screened on hygromycin medium. After two consecutive generations of screening, heterologous overexpressing Arabidopsis lines were obtained and identified as transgenic Arabidopsis lines overexpressing the AhFMO gene.
[0040] like Figure 5 As shown, compared with wild-type Arabidopsis, transgenic Arabidopsis with AhFMO gene overexpression had significantly larger seed length, width, and thousand-seed weight, indicating that the peanut AhFMO gene can regulate seed size and weight.
[0041] The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of this invention.
Claims
1. A method for regulating peanut seed size and weight AhFMO The application of genes is characterized by, The AhFMO The gene sequence is SEQ ID NO.1, and it is obtained by overexpressing the gene. AhFMO Genes that increase seed length, width, and thousand-seed weight in transgenic peanut or Arabidopsis plants.